CHEPON 2024 15th International Meeting on Cholinesterases 9th International Conference on Paraoxonases Brdo pri Kranju, Slovenia 15 ─ 18 September, 2024 Organised by Programme and Book of Abstracts 15 th International Meeting on Cholinesterases 9 th International Conference on Paraoxonases Brdo pri Kranju, Slovenia 15−18 September, 2024 Programme and Book of Abstracts Organised by: University of Ljubljana, Faculty of Medicine, and University of Ljubljana, Faculty of Pharmacy Edited by: Anže Meden, Damijan Knez, Aljoša Bavec, Marko Goličnik, and Samo Mahnič-Kalamiza ISBN 978-961-95462-3-9 Published by: Institute of biochemistry and molecular genetics Faculty of Medicine, University of Ljubljana Cover photo sourced from brdo.si. 2 Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 207658243 ISBN 978-961-95462-3-9 (PDF) Book of Abstracts 15th International Meeting on Cholinesterases and 9th International Conference on Paraoxonases Brdo pri Kranju, Slovenia 15−18 September, 2024 Organised by: University of Ljubljana, Faculty of Medicine University of Ljubljana, Faculty of Pharmacy Editors: Aljoša Bavec, Marko Goličnik, Damijan Knez, Anže Meden, Samo Mahnič-Kalamiza Scientific Committee Aljoša Bavec, Marko Goličnik, Stanislav Gobec, Jure Stojan Organizing Committee Janez Smerkolj, Boštjan Petrič, Uroš Prešern, Damjan Knez, Anže Meden Reviewed by: Jure Stojan Stanislav Gobec Published by: Institute of biochemistry and molecular genetics Faculty of Medicine, University of Ljubljana URL: https://www.chepon2024.com/wpcontent/uploads/2024/09/CHEPON2024_Book_of_Abstracts.pdf Publication is free of charge. Ljubljana, 2024 Foreword It is a great pleasure to welcome you to the 15 th International Meeting on Cholinesterases and the 9 th International Conference on Paraoxonases at the Brdo Congress Centre in Slovenia, organized by the University of Ljubljana, Faculty of Medicine, and Faculty of Pharmacy. We are honored to host this meeting in Slovenia again after more than four decades, following the 2 nd International Meeting on Cholinesterases held nearby in Bled in 1983. The program offers an excellent opportunity to present your scientific achievements on the most current topics in the field of both enzymes, strengthen collaborations with existing partners, and establish new connections with scientists from the region and around the world. Established researchers, those at the beginning of their scientific careers, as well as graduate and undergraduate students, will have the chance to present their research findings through oral presentations and poster sessions. In addition to a dynamic scientific program, we encourage you to enjoy our social events. On the first evening, we will host a welcome party. On Tuesday afternoon, you are invited to a tour of Bled and Bled Castle, followed by a gala dinner at the Brdo Estate in the evening. We are confident that the Brdo Congress Centre will meet your expectations, and you will enjoy its beautiful green surroundings. We wish you a productive and enjoyable meeting and a pleasant stay in Slovenia. Sincerely yours, The Organizing Committee 4 Information for Participants Registration and Information Desk Registration will take place at the registration desk in the lobby of the Elegans Hotel on Sunday, 15 September 2024, starting from 14:00 till 16:30, and in the lobby of the Brdo Congress Centre from 16:30 till 19:00. The certificate of attendance will be provided at the registration desk. The information desk will be located in the lobby of the Brdo Congress Centre on the following days/hours: Monday, 16 September 2024, 8:15 – 11:00 Tuesday, 17 September 2024, 8:15 – 11:00 Name Badges All participants are kindly requested to wear badges at all times during the conference. The badges are necessary for the admission to all conference events (lectures, posters, welcome party, gala dinner, lunches and coffee breaks etc). Oral presentations Lectures will be held in the Grandis Hall of the Brdo Congress Centre. Speakers are kindly requested to deliver their presentations on an USB device to the computer technician in the Grandis Hall at least half an hour before the start of the session. Slides should be prepared in either MS PowerPoint or PDF format, and should come either together with any supporting materials (e.g. videos) in the same folder (alert the technical staff to copy those to the presenters’ computer as well!) or should be embedded into the PowerPoint presentation so they will be at your disposal at the time of your presentation. Technical assistance will be provided on-site to help you with the download/upload to provided laptop PCs with Windows OS, Office and Acrobate Reader software. Apple Mac computer users are asked to export their presentations to a PC-compatible format (recommended PDF files) to avoid compatibility issues. The use of presenters’ own computers will not be allowed. The projected screen will be in a wide, 16:9 (width:height) format. Please prepare your slides accordingly! All presenters are asked to respect the time limits when giving their talks. Wi-Fi and computers with wire internet access will be available at the conference hall. 5 Poster presentations There will be two poster sessions: The Poster session I will take place on Monday, 16 September 2024 from 18:15 till 19:30. The Poster session II will take place on Tuesday, 17 September 2024 from 11:00 till 12:15. Complete list of posters with poster board numbers can be found further down in the Programme section of this Book. Poster presenters are asked to mount their posters before 8:30 in the morning and remove them in the evening on the same day of the session. All presenters should look-up the numbers as-signed to posters in the Poster Sessions and pin up the posters on the display boards with the corresponding number. The authors of posters are kindly asked to stand by posters for the duration of the session. Poster format and dimensions The recommended size/format for poster is the A0 print format, i.e. 841 x 1189 mm (width x height). Please consider using the recommended format, although you can also choose other size but up to a maximum size of 900 x 1300 mm. In any case, your poster must be oriented vertically. Social Events Social programme includes Welcome party in the lobby of Brdo Congress Centre, 15 September 2024, and Gala dinner on the Duck Island in the Brdo Park, 17 September 2024. Bled half-day trip will be organized for the participants who show the interest on the registration form. 6 Committees Scientific Committee Aljoša Bavec, Ljubljana (Slovenia) Marko Goličnik, Ljubljana (Slovenia) Stanislav Gobec, Ljubljana (Slovenia) Jure Stojan, Ljubljana (Slovenia) Organizing Committee Janez Smerkolj, Ljubljana (Slovenia) Boštjan Petrič, Ljubljana (Slovenia) Uroš Prešern, Ljubljana (Slovenia) Damjan Knez, Ljubljana (Slovenia) Anže Meden, Ljubljana (Slovenia) Advisory Committee Hermona Soreq, Jerusalem (Israel) Carlo Cervellati, Ferrara (Italy) Israel Silman, Rehovot (Israel) Joel L. Sussman, Rehovot (Israel) Zrinka Kovarik, Zagreb (Croatia) Kamil Kuča, Hradec Králové (Czech Republic) Palmer Taylor, San Diego (USA) Eugenio Vilanova-Gisbert, Elche (Spain) Florian Nachon, Brétigny-sur-Orge (France) Stanislav Gobec, Ljubljana (Slovenia) Karl W.K. Tsim, Hong Kong (China) Jure Stojan, Ljubljana (Slovenia) 7 P R O G R A M M E Plenary and other Distinguished Lectures Page Sunday - Opening Session, Sunday, Sep 15 2024, 18:00-19:30 Session: Plenary and other Distinguished Lectures 27 Chairs: Stanislav Gobec, Jure Stojan, Aljoša Bavec and Marko Goličnik 18:00 A single nucleotide polymorphism in the human AChE gene alters miR-608 27 PL-1 levels, anxiety and inflammatory status Hermona Soreq, Nimrod Madrer, Yonat Tzur, Adi Bar, Estelle Bennett, Alon Simchovitz, David Greenberg 18:45 PON1, as a common denominator of neurodegenerative and cardiovas- 27 PL-2 cular diseases: proactive player or static biomarker? Carlo Cervellati, Raffealla Riccetti, Gianmarco Mola, Valentina Rosta, Alessandro Trentini Wednesday - Memorial Lecture, Wednesday, Sep 18 2024, 16:00-16:30 Session: Plenary and other Distinguished Lectures 28 Chair: Eugenio Vilanova 16:00 Memorial Lecture 28 PL-3 Eugenio Vilanova Wednesday - Closing Lecture, Wednesday, Sep 18 2024, 16:30-17:15 Session: Plenary and other Distinguished Lectures 28 Chair: Jure Stojan 16:30 Structural Characterization of Orphan and Taxonomically Restricted Pro- 28 PL-4 teins Israel Silman 11 Oral Presentations Page Monday - Morning Lectures, Monday, Sep 16 2024, 8:30-10:20 Session: Session I - Structure and dynamics of cholinesterases, paraoxonases and phosphotriesterases 31 Chair: Joel L. Sussman 8:30 An Unusual α/ β Hydrolase Fold Enzyme from an Antarctic Bacterium 31 KN-01 Joel L. Sussman 9:00 Phosphotriesterase catalyzed synthesis of chiral precursors to antiviral 31 OR-01 prodrugs Frank Raushel 9:20 Disentangling the mechanism underlying the covalent MSF-AChE adduct 32 OR-02 formation and evolvement: mechanistic insights into an aged-like inact- ive complex susceptible to reactivation by a combination of nucleophiles Jure Stojan, Alessandro Pesaresi, Anže Meden, Doriano Lamba 9:40 Acetylcholinesterase dynamics determine the potency and selectivity of 32 OR-03 inhibitors targeting disease-transmitting mosquitoes Anna Linusson Jonsson, Rashmi Kumari, Cecilia Lindgren, Rajendra Kumari, Nina Forsgren, David Andersson, Fredrik Ekström 10:00 Structure and dynamics of hAChE and oxime interactions in structure- 33 OR-04 based design of novel uncharged bis-oxime reactivators Andrey Kovalevsky, Oksana Gerlits, Thibault Alle, Carlo Ballatore, Zoran Radic Monday - Late morning Lectures, Monday, Sep 16 2024, 10:45-12:35 Session: Session II - Interactions of cholinesterases (AChE and BChE) with substrates, inhibitors and reactivators 34 Chair: Zrinka Kovarik 10:45 Evaluating cholinesterase’s interactions with inhibitors and potent react- 34 KN-02 ivators towards efficient treatment in organophosphate poisoning Zrinka Kovarik, Dora Kolić, Tena Cadez, Goran Šinko, Nikolina Maček Hrvat 11:15 Towards the fourth generation of effective uncharged bis-oxime react- 34 OR-05 ivators of organophosphate-inhibited human AChE. Andrey Kovalevsky, Thibault Alle, Oksana Gerlits, Nikolina Maček Hrvat, Carlo Ballatore, Zrinka Kovarik, Zoran Radic 12 11:35 Development and therapeutic potential of uncharged cholinesterase re- 35 OR-06 activators José Dias, Ludovic Jean, André-Guilhem Calas, Nicolas Probst, Julien De Sousa, Nicolas Lamassiaude, Christophe Landry, Ophélie Da Silva, Anissa Braiki, Camille Voros, Romulo Araoz, Caroline Coisne, Charlotte Courageux, Pierre Warnault, Franck Razafindrainibe, Anne-Julie Gastellier, Julien Gasnot, Yerri Jagadeesh, Marilène Trancart, Anne-Sophie Hanak, Fabien Gosselet, Xavier Brazzolotto, Marie-Pierre Dehouck, Florian Nachon, Denis Servent, Pierre-Yves Renard, Rachid Baati 11:55 Direct Determination of the hydrolysis of a poor substrate by human 36 OR-07 plasma Jure Stojan 12:15 Inhibition of human butyrylcholinesterase by A-series nerve agents, func- 36 OR-08 tional and structural characterization Charlotte Courageux, Anne-Julie Gastellier, Milica Denic, Fabrice Modeste, Nicolas Taudon, José Dias, Florian Nachon, Xavier Brazzolotto 12:25 Inhibition kinetics of cholinesterases from various species by the organo- 37 OR-09 phosphate CBDP in vitro Gabriele Horn, Franz Worek Monday - Afternoon Lectures, Monday, Sep 16 2024, 14:00-15:50 Session: Session III - Post exposure organo phosphates strategies and toxicology 37 Chair: Ondrej Soukup 14:00 Novichok class of organophosphorus compounds and the efficacy of cur- 37 KN-03 rent countermeasures against A-234 Ondrej Soukup, Daniel Jun 14:30 How to increase bioavailability and follow effect of charged oximes in the 38 OR-10 central nervous system? Kamil Musilek, David Malinak, Eliska Prchalova, Rudolf Andrys, Adam Skarka, Zbynek Heger, Zenon Starcuk 14:50 Novel Strategies for Optimizing the Treatment of Organophosphate Pois- 39 OR-11 oning in a Mouse Model Marilène Trancart, Anne-Sophie Hanak, Karine Thibault, Grégory Dal Bo, Alexandre Champault, Méliati Madi, Gwladys Meesemaecker, André-Guilhem Calas 15:10 Choline-O-acetyltransferase as a potential therapeutic target for nerve 39 OR-12 agent poisoning: unveiling an ion sensitive regulatory mechanism Fredrik Ekström, Nina Forsgren, Frida Jonsson, Cecilia Engdahl, Tomas Bergström 13 15:30 Investigation of neuroprotection conferred by a new therapeutic 40 OR-13 strategy after organophosphorus exposure Alexandre Champault, Julie Knoertzer, Armelle Rancillac, Grégory Dal Bo, Karine Thibault 15:40 Blood-brain barrier controlled-opening with ultrasound to improve 40 OR-14 neuroprotection after nerve agent exposure Lucie Lépinard, Sarah Leterrier, Laurène Jourdain, Karine Thibault, Anthony Novell, Grégory Dal Bo Monday - Late afternoon Lectures, Monday, Sep 16 2024, 16:15-18:05 Session: Session IV - Cholinesterase pharmacology, inhibitors and antidotes 41 Chair: Palmer Taylor 16:15 Balancing central and peripheral nervous system antidotal actions of 41 KN-04 oxime reactivators to acetylcholinesterase inhbition Palmer Taylor, Kwok-Yiu Ho, Zoran Radic 16:45 Development of the post-exposure RS194B oxime against lethal sarin va- 42 OR-15 pour and organophosphate insecticides in macaques Yvonne Rosenberg, Dennis Sullivan, Zoran Radic, Palmer Taylor 17:05 Detection of selective inhibitors of BChE in structurally and functionally 42 OR-16 different groups of molecules Anita Bosak, Ana Matošević, Marija Bartolić, Ines Primožič, Alma Ramić, Xavier Brazzolotto, Florian Nachon, Nikola Maraković 17:25 Bifunctional compounds serving as versatile cholinesterase reactivators 43 OR-17 Lukas Gorecki, Martina Hrabinova, Vendula Hepnarova, Jana Zdarova Karasova, Jan Korabecny 17:45 Attenuating organophosphate-induced neuroinflammation in mice by 43 OR-18 oxime therapy Nikolina Maček Hrvat, Katarina Ilić, Dora Kolić, Borna Puljko, Palmer Taylor, Kristina Mlinac Jerković, Svjetlana Kalanj Bognar, Zrinka Kovarik 17:55 Design and Evaluation of Acetylcholinesterase Reactivators: A study of 44 OR-19 reactive oxime- and peripheral site-moieties Cecilia Springer Engdahl, Norman Hoster, Cecilia Lindgren, Nina Forsgren, Frida Jonsson, Timo Wille, Franz Worek, Anna Linusson Jonsson, Fredrik Ekström 14 Tuesday - Morning Lectures, Tuesday, Sep 17 2024, 8:30-10:30 Session: Session V - Paraoxonase, butyrylcholinesterase and phosphotriesterase role in detoxication, biotechnology and diseases 45 Chair: Eugenio Vilanova 8:30 The thin line between substrates or inhibitors: OP-enzymes interactions 45 KN-05 for biology, toxicity, diagnosis or applications. Eugenio Vilanova, Cermen Estevan, Antonio Monroy-Noyola, Miguel A Sogorb, Jorge Estévez 9:00 The influence of single nucleotide polymorphisms and substrate type on 45 OR-20 individual enzyme-kinetic rate constants for human plasma PON1 Boštjan Petrič, Aljoša Bavec, Marko Goličnik 9:20 Albumin-copper complex hydrolyses chiral organophosphate com- 46 OR-21 pounds as a true A-esterase Antonio Monroy-Noyola, Laura Ramirez Gonzalez, Damianys Almenares-Lopez, Elizabeth Undiano, Eugenio Vilanova 9:40 Special behavior of cholinesterase interactions among thiocholine and 47 OR-22 phenyl carboxyl ester as substrates and inhibitors: Implication for re- search applications. Jorge Estévez 10:00 PON-1 arylesterase activity in older patients with mild cognitive impair- 47 OR-23 ment, late onset Alzheimer’s disease or vascular dementia Gianmarco Mola, Raffaella Riccetti, Valentina Rosta, Alessandro Trentini, Carlo Cervellati 10:10 Regulatory mechanism and functional impact of post-translational 48 OR-24 modifications on human paraoxonase 2 Nagendra sai kumar Achanta, Eros A. Lampitella, Maria Marone, Elena porzio, Giuliana Catara, Giuseppina Lacerra, Giuseppe Manco 10:20 Validation and application of two AChE-targeted environmental neuro- 48 OR-25 toxic pollutants detection systems Ruihong Zhu, Jiahui An, Heidi QH Xie, Antonio Zandona, Tena Cadez, Zrinka Kovarik, Yangsheng Chen, Li Xu, Bin Zhao 15 Wednesday - Morning Lectures, Wednesday, Sep 18 2024, 8:30-10:20 Session: Session VI - Translational research of inhibitors of cholinesterases and paraoxonases 49 Chair: Florian Nachon 8:30 Pharmaceutical development of plasma butyrylcholinesterase: a break- 49 KN-06 through in the treatment of nerve agent intoxications Florian Nachon, Anne Christine Mendes, Aurélie Nervo, Xavier Brazzolotto, Chloé Reymond, Nicolas Doisne, Moussa Kenawi, Janek Bzdrenga, Fabien Chantegreil, Méliati Madi, Thomas Soirot, Nicolas Taudon, Nicolas Belverge, Aurelie Servonnet, Fanny Magisson, Nina Jaffré, Julien Bouix, Rachel Haus, Catherine Verret, Frédéric Dorandeu 9:00 Selective butyrylcholinesterase inhibition induces antidepressant, pro- 50 OR-26 cognitive, and antianhedonic effects in a genetic animal model of de- pression: Role of acetylcholine, ghrelin, and dopamine Brian Harvey, Nadia Olivier, Stanislav Gobec, Mohammed Shahid, Urban Košak, Simon Žakelj, Christiaan Brink 9:20 Leveraging template effects of acetylcholinesterase for the develop- 51 OR-27 ment of new anti-Alzheimer drug candidates with multiple mechanisms Anna Sampietro, Wawrzyniec Haberek, Aina Bellver, Christian Griñán-Ferré, Belén Pérez, Carmen Pérez de la Lastra Aranda, Valle Palomo, Marina Naldi, Manuela Bartolini, María Isabel Loza, José Brea, Clara Bartra, Coral Sanfeliu, Christophe Morisseau, Raimon Sabate, Beste Ozaydin, Jordi Juárez-Jiménez, Bruce D. Hammock, Mercè Pallàs, Santiago Vázquez, Diego Muñoz-Torrero 9:40 A novel identification of anti-hypertensive drug spironolactone as an in- 52 OR-28 hibitor of paraoxonase-2 lactonase activity Yoko Suzumoto, Eros A. Lampitella, Maria Marone, Giovambattista Capasso, Francesco Trepiccione, Giuseppe Manco 10:00 Development of pleiotropic prodrugs to treat Alzheimer’s disease: from 52 OR-29 conception to in vivo evaluation Alice Wang, Valentin Travers–Lesage, Marc Since, Christophe Rochais 10:10 The selective butyrylcholinesterase inhibitor UW-MD-95 shows sympto- 53 OR-30 matic and neuroprotective effects in a pharmacological mouse model of Alzheimer’s disease Allison Carles, Matthias Hoffmann, Matthias Scheiner, Lucie Crouzier, Christelle Bertrand-Gaday, Arnaud Chatonnet, Michael Decker, Tangui Maurice 16 Wednesday - Late morning Lectures, Wednesday, Sep 18 2024, 10:45-12:35 Session: Session VII - Multi-target-directed ligands in Alzheimer’s disease primarily targeting cholinesterases 54 Chair: Stanislav Gobec 10:45 Multi-target-directed ligands for potential symptomatic and disease- 54 KN-07 modifying treatment of Alzheimer’s disease Svit Ferjančič Benetik, Damijan Knez, Urban Košak, Aleš Obreza, Stanislav Gobec 11:15 Advancing neocopride: a preclinical candidate multitarget directed- 54 OR-31 ligand (MTDL) for neurodegenerative disease intervention Christophe Rochais, Patrick Dallemagne 11:35 Cholinesterase-based inhibitors as multitarget small molecules for the 55 OR-32 therapy of Alzheimer’s disease José Marco Contelles, Francisco López-Muñoz 11:55 Discovery of novel BChE inhibitors for cognitive improvement 55 OR-33 Baichen Xiong, Yuanyuan Wang, Weiting Zhang, Haopeng Sun 12:15 Development of dual pharmacophore models of acetylcholinesterase 56 OR-34 and human soluble epoxide hydrolase based on local co-solvent mo- lecules distribution Weronika Bagrowska, Artur Góra 12:25 Pleiotropic prodrugs for both symptomatic and disease-modifying treat- 56 OR-35 ment of Alzheimer’s disease Anže Meden, Neža Žnidaršič, Damijan Knez, Yuanyuan Wang, Ziwei Xu, Huajing Yang, Weiting Zhang, Anja Pišlar, Andrej Perdih, Simona Kranjc Brezar, Neža Grgurevič, Stane Pajk, Haopeng Sun, Stanislav Gobec Wednesday - Afternoon Lectures, Wednesday, Sep 18 2024, 14:00-16:00 Session: Session VIII - Functions of cholinesterases in different tissues 57 Chair: Karl Wah-Keung Tsim 14:00 Acetylcholinesterase regulates inflammatory responses and pigmenta- 57 KN-08 tion in skin epidermis Karl Wah-Keung Tsim 14:30 Characterizing Cardiac and Vascular Cholinesterases: From Molecular 58 OR-36 Insights to Therapeutic Prospects Dominika Dingová, Kristína Szmicseková, Matej Kučera, Lenka Bies Pivačková, Tibor Hodbod, Parsa Shafieikazerooni, Peter Křenek, Eric Krejci, Anna Hrabovska 17 14:50 Mice with inactive cholinesterases: new tools to evaluate cholinergic and 58 OR-37 non-cholinergic functions of AChE Eric Krejci 15:10 New perspectives on the effects of dioxin-like pollutants on acetylcholin- 59 OR-38 esterase Heidi QH Xie, Guanglei Yang, Ruihong Zhu, Jiahui An, Yangsheng Chen, Li Xu, Bin Zhao 15:30 The cholinergic enigma of pigmented and Müller glial cells 59 OR-39 Paul G. Layer, Gesine Bachmann, Alex Bausch, Nicola Coronato, Gopenath Thangaraj 15:50 Acetylcholinesterase Reliefs Beta-Amyloid Plaque Burden via Enhancing 60 OR-40 Glial Activation in the Brain of 5×FAD Mouse Yingjie Xia, Xiaoyang Wang, Maggie Suisui Guo, Ran Duan, Tingxia Dong, Karl Wah-Keung Tsim 18 Poster Presentations Page Monday - Poster Session, Monday, Sep 16 2024, 18:05-19:30 Session: Poster Session 63 PO-01 Target-guided synthesis of novel butyrylcholinesterase inhibitors 63 B-01 Ines Primožič, Alma Ramić, Toni Divjak, Tomica Hrenar PO-02 Capitalizing on human BChE-ligand complex structures for the design of 63 B-02 BChE-specific reactivator against nerve agent intoxication Damijan Knez, Masa Zorman, Anne-Julie Gastellier, Charlotte Courageux, Janek Bzdrenga, José Dias, Xavier Brazzolotto PO-03 Monoquaternary analogues of double charged K-oximes (K027, K048 and 64 B-03 K203) are less effective reactivators of cholinesterases inhibited by or- ganophosphates Zuzana Kohoutova, Rudolf Andrys, Kamil Musilek, David Malinak PO-04 Halogenated pralidoxime analogues are efficiently reactivating cholin- 64 B-04 esterases Sara Rademacherova, Karolina Knittelova, Adela Fuchsova, Rudolf Andrys, Kamil Musilek, David Malinak PO-05 Brominated oxime nucleophiles are efficiently reactivating cholin- 65 B-05 esterases inhibited by nerve agents Eliska Prchalova, Rudolf Andrys, Jaroslav Pejchal, Zuzana Kohoutova, Kamil Musilek, Jana Zdarova Karasova, David Malinak PO-06 Importance of the shape of the linker between two quaternary pyridinium 65 B-06 rings on reactivation process in oximes – in vitro and in silico study Tanos Celmar Costa Franca, Fernanda Georgia Figueiredo Taborda Barbosa, Joyce Sobreiro Francisco Diz de Almeida, Eugenie Nepovimova, Rafael Dolezal, Steven Laplate, Kamil Kuca PO-07 Synthesis and in vitro assessment of the reactivation profile of clinical 66 B-07 oximes on the acetylcholinesterase model inhibited by A-230 and A-242 nerve agents’ surrogates Tanos Celmar Costa Franca, Samir Cavalcante, Daniel Kitagawa, Caio Borges, Marcelo Carneiro dos Santos, Pedro Buitrago, Roberto Souza, Antonio Luis Santos Lima, Leandro Bernardo, Kamil Kuca 19 PO-08 Synthesis, modeling and in vitro assessment of the reactivation profile 66 B-08 of monocationic isatin-oximes hibrids on the acetylcholinesterase model inhibited by nerve agents’ surrogates Tanos Celmar Costa Franca, Amanda Moraes, Samir Cavalcante, Dipanjan Bhattacharyya, Steven Laplate, Joyce Sobreiro Francisco Diz de Almeida, Pat Forgione PO-09 Triazoles as potential reactivators of human acetylcholinesterase inhib- 67 B-09 ited by the nerve agents VX and Novichok A-242 Fernanda Pires, Pedro Buitrago, Tanos Celmar Costa Franca, Samir Cavalcante, Joyce Sobreiro Francisco Diz de Almeida PO-10 Beyond carbamates: N-substituted piperidine ureas as butyrylcholin- 67 B-10 esterase inhibitors Peter Mastnak-Sokolov, Urban Košak, Damijan Knez, Svit Ferjančič Benetik, Anja Pišlar, Xavier Brazzolotto, Stanislav Gobec PO-11 Piperidine-carboxamides, -sulfonamides and -carbamates as selective 68 B-11 butyrylcholinesterase inhibitors Urban Košak, Damijan Knez, Anže Meden, Simon Žakelj, Jurij Trontelj, Jure Stojan, Maja Zakošek Pipan, Kinga Sałat, Florian Nachon, Xavier Brazzolotto, Gregor Majdič, Stanislav Gobec PO-12 In vitro reactivation screening of A-234-inhibited human recombinant 69 B-12 acetylcholinesterase and butyrylcholinesterase Martina Hrabinova PO-13 Computational investigation of hardwickic acid-derived amides using 69 B-13 molecular docking and prediction of ADME/Tox properties as potential inhibitors of cholinesterase enzymes Rayssa Ribeiro, Franco Leite, Gessica Mendes, Fernanda Georgia Figueiredo Taborda Barbosa, Samir Cavalcante, Marcelo Carneiro dos Santos, Tanos Celmar Costa França, Valdir Veiga-Junior PO-14 Modeling studies and experimental evaluation of the reactivation poten- 70 B-14 tial of oximes K027, K048, K170 and K203 against the nerve agent A-242 Daniel de Jesus de Oliveira, Fernanda Diniz Botelho, Fernanda Georgia Figueiredo Taborda Barbosa, Kamil Kuca, Steven Laplate, Samir Cavalcante, Marcelo Carneiro dos Santos, Tanos Celmar Costa França PO-15 Novichok A-232: basic knowledge of biochemical and toxicological prop- 70 B-15 erties Daniel Jun, Martina Hrabinova, Lubica Muckova, Jakub Opravil, Dominik Krupka, Alzbeta Dlabkova PO-16 Reactivation potency of GB, VX and A-234-inhibited human recombinant 71 B-16 acetylcholinesterase in vitro and in silico Jakub Opravil, Jakub Fibigar, Zbyněk Večeřa 20 PO-17 Exploring drug modality switch from in situ assembly to reversibility: re- 71 B-17 versible modulators of choline O-acetyltransferase activity Nina Forsgren, Frida Jonsson, Marcus Carlsson, Robin Afshin Sander, Cecilia Springer Engdahl, Daniel Wiktelius, Christopher Öberg, Fredrik Ekström PO-18 Kinetic and structural evidence for specific DMSO interference with re- 72 B-18 versible binding of uncharged bis-oximes to hAChE and their reactivation kinetics of OP-hAChE. Dora Kolić, Nichole Joiner, Oksana Gerlits, Andrey Kovalevsky, Zoran Radic PO-19 Outlining the A-series of organophosphorus compounds – cholinesterase 73 B-19 inhibition, reactivation, cytotoxicity Nikolina Maček Hrvat, Dora Kolić, Tena Cadez, Goran Šinko, Zrinka Kovarik PO-20 Evaluation of resveratrol compounds as therapeutics in organophos- 73 B-20 phorus poisoning Tena Cadez, Milena Mlakić, Nikolina Maček Hrvat, Irena Škorić, Zrinka Kovarik PO-21 Neuroprotective role of CNS-active uncharged bis-oxime antidotes in 74 B-21 mice exposed to organophosphate compounds Dora Kolić, Nikolina Maček Hrvat, Zrinka Kovarik, Zoran Radic PO-22 Synthesis of broad-spectrum antidotes to organophosphorus neurotox- 75 B-22 ins Estelle Beaupparain, Karine Porte, Pierre-Yves Renard, Ludovic Jean PO-23 Bis-pyridinium mono-aldoxime K203: a promising prophylactic cholin- 75 B-23 esterase reactivator for organophosphate poisoning. Syed Nurulain, Zarish Riaz, Huba Kalasz, Sajid Mehmood, Kamil Kuca PO-24 A-agents: more resistant than expected? Biomarker detection in biolo- 76 B-24 gical matrices Lukáš Prchal, Alzbeta Dlabkova Tuesday - Poster Session, Tuesday, Sep 17 2024, 11:00-12:35 Session: Poster Session 76 PO-25 PON1 gene polymorphisms and inflammatory markers in organophos- 76 B-01 phate pesticides cohorts from Cameroon and Pakistan Leonel Javeres Mbah Ntepe PO-26 Screening and Characterization of Inhibitors for the Recombinant Variant 77 B-02 of Paraoxonase 1 Neja Žnidaršič, Janez Smerkolj, Jure Stojan, Aljoša Bavec, Marko Goličnik PO-27 Exploring the Impact of Lanthanide (III) Ions on the Function of Paraox- 78 B-03 onase 1 (PON1) Janez Smerkolj, Jure Stojan, Aljoša Bavec, Marko Goličnik 21 PO-28 PON1 plasma activity in the aftermath of bariatric metabolic surgery: the 78 B-04 benefits of investigating more than one substrate Boštjan Petrič, Aljoša Bavec, Tadeja Pintar, Živa Mesarič PO-29 Recombinant human paraoxonase-1 variants depict hydrolyzing capab- 79 B-05 ilities of A-series nerve agents in vitro Janek Bzdrenga, Prakash Khandave, Thomas Soirot, Nicolas Belverge, Nicolas Taudon, Florian Nachon, Xavier Brazzolotto, Abhay H. Pande PO-30 Copper-dependent stereoselective hydrolysis of O-hexyl O-2,5- 80 B-06 dichlorophenyl phosphoramidate by recombinant serum albumins Laura Ramirez Gonzalez PO-31 Update of ESTHER, the database and server dedicated to the analysis 80 B-07 of protein and nucleic acid sequences within the superfamily of cholin- esterase relative Arnaud Chatonnet, Zhou Yu, Nicolas Roche, Pascale Marchot PO-32 In silico evaluation of the anticholinesterase activity of triazole fungicides 81 B-08 Goran Šinko PO-33 Cholinesterase monitoring for nerve agent exposure 81 B-09 Nick Coe, Jennifer Dawson, Georgia Smith, Sarah Goodchild PO-34 Immobilization of cholinesterases on magnetic microparticles for en- 82 B-10 hanced stability and biosensing applications Rudolf Andrys, Charline Monnier, Veronika Mickova, Louise Nemery, Evica Antonijevic, Kamil Musilek, Lucie Zemanova PO-35 Highly potent and selective butyrylcholinesterase inhibitors for cognitive 83 B-11 improvement and neuroprotection Baichen Xiong, Zuoaoyun Song, Haopeng Sun PO-36 Detection of butyrylcholinesterase signal peptide in human brains 83 B-12 Jacek Jasiecki, Andrew Reid, Meghan Cash, Monika Targońska, Bartosz Wasąg, Sultan Darvesh PO-37 Impact of Type 1 Diabetes Mellitus on Butyrylcholinesterase Expression 84 B-13 and Activity in Rats Tibor Hodbod, Lukas Nemet, Katarina Hadova, Anna Hrabovska PO-38 Expression of cholinesterases in rats 84 B-14 Lukas Nemet, Tibor Hodbod, Monika Zelinová, Anna Hrabovska PO-39 Investigating the Link between Butyrylcholinesterase and Pulmonary 85 B-15 Vascular Disease in Rats Parsa Shafieikazerooni, Monika Zelinová, Jana Veteskova, Eva Velasova, Tibor Hodbod, Peter Krenek, Anna Hrabovska 22 PO-40 Acetylcholinesterase and muscarinic receptors control the ultraviolet- 85 B-16 mediated release of melanosomes in cultured melanoma Maggie Suisui Guo, Yingjie Xia, Jiahui Wu, Xiaoyang Wang, Gary Ka Wing Yuen, Tingxia Dong, Karl Wah-Keung Tsim PO-41 Trehalose restores the tacrine-induced endoplasmic reticulum stress in 86 B-17 cultured neuronal cells Xiaoyang Wang, Yingjie Xia, Maggie Suisui Guo, Jiahui Wu, Tingxia Dong, Karl Wah-Keung Tsim PO-42 The Muscarinic Acetylcholine Receptor in Dermal Papilla Cells Regulates 87 B-18 Hair Growth Gary Ka Wing Yuen, Tingxia Dong, Daniel Ye, Ajiaikebaier Dilidaer, Karl Wah Keung Tsim PO-43 The Regulatory Role of Gut Microbiota in Expression of AChE in Epithelial 87 B-19 Cells: a Regulator of Inflammatory Bowel Disease Ajiaikebaier Dilidaer, Yingjie Xia, Jiahui Wu, Tingxia Dong, Karl Wah-Keung Tsim PO-44 Studying the Expression and Regulation of AChE in Multiple Cancers Using 88 B-20 Data-Driven Approach Jiahui An, Heidi QH Xie, Ruihong Zhu, Guanglei Yang, Yangsheng Chen, Li Xu, Bin Zhao PO-45 Advances in the development of new drugs against Alzheimer’s disease 88 B-21 based on tacrine scaffold Martin Novák, Vajrychova Marie, Volker M. Lauschke, Ondrej Soukup PO-46 Synthesis of a multifunctional compound targeting neuroinflammation 89 B-22 and cholinergic deficit in Alzheimer’s disease David Malinak, Zuzana Kohoutova, Miroslav Psotka, Karolina Knittelova, Rudolf Andrys, Kamil Musilek, Stanislav Gobec PO-47 Dual inhibitors targeting BChE and p38 α MAPK: a novel strategy for 90 B-23 Alzheimer’s disease therapy Svit Ferjančič Benetik, Aleš Obreza, Urban Košak, Damijan Knez, Stanislav Gobec PO-48 Berberine in comparison to 7-MEOTA for Alzheimer’s treatment: a poly- 90 B-24 pharmacological approach Syed Nurulain, Areeba Kiran, Sidra Batool, Sosan Khan, Huba Kalasz, Sajid Mehmood, Kamil Kuca PO-49 Novel amiridine-based multi-target directed ligands for the Alzheimer’s 91 B-25 disease treatment Jan Konecny, Eva Mezeiova, Martin Horak, Galina Makhaeva, Jan Korabecny 23 PO-50 Investigating the effects of basketball and volleyball as team sports on 92 B-26 social cognitive function and BDNF and cholinesterase levels in young men and women Muslum Gok, Halil Ates, Ergul Cansu incegil, Serkan Aksu PO-51 Design of AChE reactivators using versatile molecular platforms and nan- 92 B-27 odiamonds Yevgen Karpichev, Illia Kapitanov, Denys Bondar, Vadym Mochalin P L E N A R Y L E C T U R E S ’ A B S T R A C T S Plenary and other Distinguished and mass spectrometry assays revealed that Lectures this 150 nucleotides-sequence interacted with the ribosomal protein L24 (RPL24), an inter- Sunday - Opening Session action that inhibited miR-608 expression. In- versely, RPL24 depletion bidirectionally altered Sep 15, 18:00 - 19:30 the levels of multiple miRs and transfer RNA fragments (tRFs) in both human and plant sys- PL-1 A single nucleotide polymorphism in the hu- tems, demonstrating an active contribution by man AChE gene alters miR-608 levels, anxi- RPL24 to the regulation of diverse small non-ety and inflammatory status coding RNAs. Discussion: Our findings extend reports that Hermona Soreq, Nimrod Madrer, Yonat Tzur, Adi Bar, Estelle Bennett, Alon Simchovitz, David Green-Arabidopsis thaliana RPL24 binds short se- berg quences 5’ to pre-miRs and regulates their ex- The Hebrew University of Jerusalem, Israel pression. Moreover, we show that RPL24’s su- pervision of the biogenesis of small regulatory Introduction: The human AChE gene har- RNAs is pan-evolutionary, and shed new light bors a single nucleotide polymorphism (SNP), on the traumatic impact of impaired choliner-rs17228616, the impact of which remained gic networks. incompletely understood. This SNP is loc- ated in an AChEmRNA region that is targeted PL-2 by microRNA (miR)-608, which also targets PON1, as a common denominator of neuro-transcripts coding for the pro-inflammatory degenerative and cardiovascular diseases: interleukin-6 (IL6) and the CDC42 kinase. proactive player or static biomarker? Therefore, impaired miR-608-AChE interac- Carlo Cervellati, Raffealla Riccetti, Gianmarco tions could elevate AChE levels and enable in-Mola, Valentina Rosta, Alessandro Trentini creased targeting of CDC42 and IL6 by miR- University of Ferrara, Italy 608, altering the CDC42 and IL6-regulated Paraoxonase 1 (PON1) is a multifunctional physiological processes, including inflamma- protein with pleiotropic proprieties. This tion and stress. calcium-dependent enzyme exhibiting lac- Methods: We studied human carriers of tonase and ester hydrolase activity that exert rs17228616 and established transgenic mice multiple defensive roles in the human body. In carrying miR-608 flanked by its human intronic particular, PON1 is able to protect from dam-250 nucleotides-long regions, which expressed age due to lipid peroxidation damage, ex-human miR-608 in several tissues. acerbated inflammation and several types of Results: Among 368 healthy people, carriers of endogenous and exogenous toxicants. This the minor rs17228616 SNP allele presented el-beneficial action can be exerted at systemic evated blood pressure and intensified anxiety, level since most of PON1 is carried by circu-and chronically stressed soldier carriers of the lating high density lipoprotein (HDL). It is now SNP presented elevated amygdala activity. widely recognized that PON1 is one of the main Nevertheless, among 98 aged ex-prisoners of contributors of the anti-atherogenic propriet-war (ePW), SNP carriers consisted of a greater ies of these particles. fraction than expected in the 26 non-PTSD It has also become increasingly apparent ePW group. Moreover, studying the transcrip- that atherosclerosis is one of the main risk tional regulation of miR-608 identified an act- factors for Alzheimer’s disease (AD). Indeed, ive promoter located in the 150 nucleotides vascular pathology plays a relevant role in the 5’ to the pre-miR-608, that elevated miR- etiology of AD by decreasing cerebral blood 608 levels by 100-fold. Surprisingly, pulldown 27 and impairing brain amyloid beta (A β, the teins in organisms further down the evolution-putative initial trigger of neurodegeneration) ary ladder. In a dramatic paradigm change, homeostasis. This potential pathogenic link it is now accepted that all organisms contain accounts, at least in part, for the growing ‘orphan’ proteins which are devoid of identifi-body of evidence showing that patients af- able ancestors. In other cases, similar proteins fected by AD have consistently lower blood occur in several related species, again without (serum/plasma) PON1 activity compared to detectable ancestors. These are known as a cognitively healthy controls. This ’peripheral taxonomically restricted. The de novo pro- scenario’ may also reverberate in the brain. In- teins are generated by expression of novel deed, recent studies on AD animal models sug- Open Reading Frames in non-coding DNA se- gest that PON1 may be transferred through quences. Since most research on Orphan and HDL to central nervous system, where it seems taxonomically restricted proteins has been to participate in AD pathogenesis. In conclu- performed by geneticists, only sparse struc- sion, despite the lack of conclusive mechan- tural data are available for them. Thus, only istic evidence, PON1 may not be merely a static three crystal structures of orphans are de-biomarker of vascular and neurological dis- posited in the Protein Data Bank. We could eases, but a proactive player involved in their identify seven additional proteins, with well- onset and progression. defined functions, which had been expressed and purified. They had undergone partial Plenary and other Distinguished physicochemical characterization, but struc- tural data were lacking. The development Lectures of AlphaFold revolutionized protein structure prediction. We utilized AlphaFold2, and two Wednesday - Memorial other algorithms, to predict the structures of Lecture the three proteins with available crystal struc- Sep 18, 16:00 - 16:30 tures, as well as the other seven. All three confirmed the crystal structures, two of which, PL-3 interestingly, displayed novel folds. Two of Memorial Lecture the other proteins, which were predicted to Eugenio Vilanova be disordered based on their sequences, were predicted to be disordered by all three al- Plenary and other Distinguished gorithms. The remaining five were predicted Lectures to be compact, with two exceptions for Al- phaFold2. All three algorithms made similar Wednesday - Closing and high-quality predictions for a large nem- Lecture atode protein. We conjecture that this is due to many homologs in its taxonomically re- Sep 18, 16:30 - 17:15 stricted family, and to the fact that several non-related proteins have similar folds. Over- PL-4 Structural Characterization of Orphan and all, orphan and taxonomically restricted pro-Taxonomically Restricted Proteins teins are often predicted to have compact 3D structures, sometimes with a novel fold that is a Israel Silman consequence of their novel sequences, which Department of Brain Sciences, Weizmann Institute are associated with appearance of new bio- of Science, Israel logical functions. Until 20 years ago, it was believed that all proteins are derived from homologous pro- 28 O R A L P R E S E N T A T I O N S ’ A B S T R A C T S Session I - Structure and protein engineering. dynamics of cholinesterases, The uniqueness of the primary structures of GLase and its close homologs is that they in- paraoxonases and clude 6 highly conserved Cys residues out of phosphotriesterases a total of 9 Cys residues in GLase but with no S-S bonds. GLase was successfully cloned, ex- Monday - Morning pressed, and purified from E. coli. It was crys- Lectures tallized, and its X-ray structure was solved at Sep 16, 8:30 - 10:20 2.95 Å resolution, revealing a canonical α/ β hydrolase fold. KN-01 An Unusual α/ β Hydrolase Fold Enzyme from OR-01 an Antarctic Bacterium Phosphotriesterase catalyzed synthesis of Joel L. Sussman chiral precursors to antiviral prodrugs Department of Chemical and Structural Biology, Frank Raushel Weizmann Institute of Science, Israel Texas A&M University, United States ***Keynote Lecture*** Nucleoside analogs are among the most We here report the discovery of a new common medications given for the treat-type of bacterial carboxylesterase. GLase ment of viral infections and cancers. The is produced by the psychrophilic bacterium, therapeutic effectiveness of nucleoside ana-Glaciecola pallidula, which inhabits Antarctic logs can be dramatically improved by phos-sea-ice habitats. Catalytic bio-scavengers, phorylation. The ProTide approach was de-e.g. bacterial phosphotriesterases (PTE) and veloped at Cardiff University using a phos- mammalian paraoxonase1 (PON1), have been phorylated nucleoside that is masked by es- developed for the detoxification of organo- terification with an amino acid and phenol phosphate (OP) pesticides and chemical war- forming a chiral phosphorus center. The FDA fare nerve agents. All native OP hydrolases has approved Sofosbuvir, Tenofovir Alafenam- (OPH) are active toward the less toxic Rp ide, and Remdesivir for use as a treatment (+) stereo-isomer of asymmetric OPs. Con- for hepatitis C, HIV, and COVID-19, respect- sequently, novel OPH variants with reversed ively. The biological activity of the ProTides stereo-selectivity and improved hydrolytic po-depends, in part, on the phosphorus stereo- tency were developed using enhanced evol- chemistry and thus it is imperative that effi-ution and protein engineering approaches. cient methods be developed for the chem-GLase, is inhibited by OPs such as paraoxon ical synthesis and isolation of diastereomer-and VX. Notably, following inhibition by (-)- ically pure ProTides. Chiral ProTides are often VX or by (-)-EMP-MeCyC, the more toxic ste- synthesized by direct displacement of a labile reoisomers of these OPs, and by paraoxon, phenol (p-nitrophenol or pentafluorophenol) the enzyme recovers spontaneously within 60-from a chiral phosphoramidate precursor with 90 minutes. Conversely, GLase inhibited by the appropriate nucleoside analog. The abil-either (+)-VX or (+)-EMP-MeCyC does not re- ity to produce these chiral products is thus dic-cover after inhibition. Thus, it is the first known tated by the synthesis of the chiral phosphor-carboxylesterase that reactivates spontan- amidate precursors. The enzyme phospho-eously after inhibition by the toxic isomers triesterase (PTE) from Pseudomonas diminuta of anti-acetylcholinesterase phosphonates. is well known for its high stereoselectivity and GLase may serve as a structural template for broadsubstrateprofile. ScreeningPTE variants developing new catalytic bio-scavengers of from enzyme evolution libraries enabled the toxic OPs by directed evolution combined with 31 identification of variants of PTE that can ste- ing to the key catalytic serine. reoselectively hydrolyze chiral phosphoramid- Kinetic and crystallographic studies of the ate precursors. The variant G60A-PTE exhib- interaction between Torpedo californica its a 165-fold preference for hydrolysis of the acetylcholinesterase and a small organosulf- RP-isomer, while the variant In1W-PTE has a onate, methanesulfonyl fluoride (MSF), indeed 1400-fold preference for hydrolysis of the SP- revealed irreversibly methylsulfonylated serine isomer. Using these variants of PTE, the SP- and 200, to be isosteric with the bound aged RP-isomers of the ProTide precursors were isol- sarin/soman analogues. The potent bulky ated on a preparative scale with no detect- reversible inhibitor 7-bis-tacrine (BTA) adopts, able contamination of the opposite isomer. in the active site of the crystal structure of This approach enabled the chemo-enzymatic the MSF-enzyme adduct, a location and an synthesis of the pure (RP)-diastereomer of Re- orientation that closely resemble the one mdesivir. This work was supported in part by being found in the crystal structure of the BTA-the National Institutes of Health. enzyme complex. Remarkably, the presence of BTA accelerates the rate of methanesulf- OR-02 onylation by a factor of two. This unexpected Disentangling the mechanism underlying result can be explained on the basis of two the covalent MSF-AChE adduct formation facts: i) the steric hindrance exerted by BTA and evolvement: mechanistic insights into to MSF in accessing the active site and ii) an aged-like inactive complex susceptible the acceleration of the MSF-enzyme adduct to reactivation by a combination of nucle- formation as a consequence of the lowering ophiles of the rotational and translational degrees Jure Stojan1, Alessandro Pesaresi2, Anže Meden3, of freedom in the proximity of the catalytic Doriano Lamba 2 serine. 1Institute of Biochemistry, Faculty of Medicine, It is well known that pralidoxime alone or University of Ljubljana, Ljubljana, Slovenia in the presence of the substrate acet- 2Institute of Crystallography, National Research ylcholine cannot reactivate the active site Council, Trieste Outstation, Trieste, Italy serine of the TcAChE-MSF adduct. We show 3Faculty of Pharmacy, University of Ljubljana, that the simultaneous presence of prali-Ljubljana, Slovenia doxime and the additional neutral oxime, 4Institute of Crystallography, National Research 2-[(hydroxyimino)methyl]-l-methylimidazol, Council, Trieste Outstation, Trieste; Interuniversity triggers the reactivation process of TcAChE Consortium ”Biostructures and Biosystems National within the hour timescale. Institute”, Roma, Italy Overall, our results pave the way toward the Chemical warfare nerve agents and likely use of a cocktail of distinctive oximes as pesticides, known as organophosphorus com- a promising recipe for an effective and fast pounds inactivate cholinesterases (ChEs) by reactivation of aged cholinesterases. phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the OR-03 Acetylcholinesterase dynamics determine course of time, phosphorylation is followed by thepotencyandselectivityofinhibitorstar-loss of an organophosphate-leaving group geting disease-transmitting mosquitoes and the bond with ChEs becomes irreversible, Anna Linusson Jonsson 1, Rashmi Kumari1, a process known as aging. Differently, struc- Cecilia Lindgren1, Rajendra Kumari1, Nina turally related irreversible catalytic poisons Forsgren2, David Andersson1, Fredrik Ekström2 bearing sulfur instead of phosphorus convert 1Umeå University, Sweden ChEs in its aged form only by covalently bind- 2CBRN Defence and Security, Swedish Defence Re- 32 search Agency, Sweden OR-04 Structure and dynamics of hAChE and oxime Vector control of mosquitoes with insect- interactions in structure-based design of icides is an important tool for preventing the novel uncharged bis-oxime reactivators spread of mosquito-borne diseases including Andrey Kovalevsky 1, Oksana Gerlits2, Thibault malaria, dengue, chikungunya, and Zika. New Alle3, Carlo Ballatore3, Zoran Radic3 active ingredients for insecticides are urgently 1Neutron Scattering Division, Oak Ridge National needed because existing active ingredients Laboratory, Oak Ridge, United States exhibit off-target toxicity and are subject to 2Department of Natural Sciences, Tennessee Wes-increasing resistance. We therefore aim to leyan University, Athens, United States develop non-covalent inhibitors of the valid- 3University of California San Diego, United States ated insecticidal target acetylcholinesterase 1 (AChE1) from mosquitoes. We have recently solved nearly a dozen X- ray structures of human acetylcholinesterase In this study, we use molecular dynamics (hAChE; EC 3.1.1.7) in complex with novel het- simulations to identify structural proper- erocyclic bis-oxime reactivators, including ties essential for the potency of reversible complexes with organophosphate (OP)- inhibitors targeting AChE1 from Anopheles inhibited, inactive hAChE. Structures provide gambiae (AgAChE1), the malaria-transmitting unique insights into dynamics of both protein mosquito, and for selectivity relative to the and small molecule oxime interactions inform- vertebrate mus musculus AChE (mAChE). We ative for structure-based design and optim-show that the main collective motions of apo ization of uncharged nucleophilic bis-oximes. AgAChE1 and mAChE differ, with AgAChE1 Our previous inelastic neutron scattering (INS) exhibiting less dynamic movements. Opening studies of native and OP-conjugated hAChE and closing of the gorge, which regulates revealed the acyl pocket loop as a primary access to the catalytic triad, is enabled by hAChE domain whose vibrational dynamics is different mechanisms in the two species, affected by POX phosphorylation. We have which could be linked to their differing amino now investigated effects of both uncharged acid sequences. Inhibitor binding reduced heterocyclic oximes as a new class of CNS-the overall magnitude of dynamics of AChE. active reactivators as well as pyridinium oxime In particular, more potent inhibitors reduced MMB4 in terms of enzyme dynamics as in-the flexibility of the Ω loop at the entrance ferred from a diversity of static conformations of the gorge. The selectivity of inhibitors for of the acyl-pocket loop in those structures. In AgAChE1 over mAChE derives from the pos- addition, various conformations of new un-itioning of the α-helix lining the binding gorge. charged heterocyclic bis-oximes reveal that single attachment point to hAChE via central Our findings emphasize the need to con- heterocycle allows for enhanced conforma- sider dynamics when developing inhibitors tional flexibility of nucleophile-bearing arms targeting this enzyme and highlight factors critical in accessing the targeted phosphorus needed to create potent and selective atom in the conjugated OP. We have shown in AgAChE1 inhibitors that could serve as active corresponding structures how non-productive ingredients to combat disease-transmitting oxime-bearing arm orientation could be mosquitoes. straightened and converted into product- ive binding conformation in 1,4-bisoximes without shifting the central heterocyclic an- choring ring. Additionally, conformational flexibility of the nucleophile-bearing arm 33 of the single bis-oxime could be captured nucleophiles such as oximes to avoid severe in both non-productive and reactivation- health effects after exposure to OP. How-productive orientation in corresponding ever, both inhibition and reactivation of both X-ray structures. Our new X-ray structures enzymes are fine-tuning chemical processes illustrate specific structural advantages of that depend on the structure of all react-uncharged heterocyclic bis-oximes compared ants. Recently, we evaluated the inhibition of to pyridinium-based oximes while allowing us cholinesterase activity with OP herbicides. It is to learn about the protein dynamics of hAChE worth pointing out that herbicides - anilofos, structural domains. bensulide and piperophos inhibit both cholin- esterases through a network of non-covalent This research was supported by the Counter- and covalent interactions, while butamifos ACT Program, NIH Office of the Director (OD), inhibits only with covalent binding to the and the NINDS, [Grant Number 1R21NS120839- catalytic serine. These findings give insight 01A2]. into the potential toxic effects of herbicides in use. Our focus was also the reactivation of OP Session II - Interactions of nerve agents-inhibited AChE with newly syn- thesized oximes that were proven to be more cholinesterases (AChE and efficient reactivators than standard oximes. BChE) with substrates, inhibitors New OPs such as Novichoks urgently ask for and reactivators an efficient medical countermeasure based on reactivators of both AChE and BChE. Monday - Late morning Supported by the European Regional De- Lectures velopment Fund (KK.01.1.1.02.0007), Next Gen- Sep 16, 10:45 - 12:35 eration EU (BioMolTox project), and Croatian Science Foundation (IP-2022-10-6685). KN-02 Evaluating cholinesterase’s interactions OR-05 with inhibitors and potent reactivators Towards the fourth generation of effect-towards efficient treatment in organo- ive uncharged bis-oxime reactivators of phosphate poisoning organophosphate-inhibited human AChE. Zrinka Kovarik, Dora Kolić, Tena Cadez, Goran Andrey Kovalevsky1, Thibault Alle2, Oksana Šinko, Nikolina Maček Hrvat Gerlits3, Nikolina Maček Hrvat4, Carlo Ballatore2, Institute for Medical Research and Occupational Zrinka Kovarik4, Zoran Radic 2 1 Health, Croatia Neutron Scattering Division, Oak Ridge National ***Keynote Lecture*** Laboratory, Oak Ridge, United States 2 The main action mechanism of organo- University of California San Diego, United States 3 phosphorus compounds (OP) is the inhibition Department of Natural Sciences, Tennessee Wes- of acetylcholinesterase (AChE) that causes leyan University, Athens, United States 4 the accumulation of the neurotransmitter Institute for Medical Research and Occupational acetylcholine leading to the paralysis of Health, Croatia cholinergic synaptic transmission. Although Guided by the X-ray structures of un- BChE is generally considered as having no charged zwitterionic monoxime RS-194B in natural physiological function, the most likely complex with native and OP-conjugated hu-function for BChE is as backup for AChE man acetylcholinesterase (hAChE; EC 3.1.1.7) and protection of synaptic AChE from man-and by in silico computational evaluation we made and naturally occurring poisons. Both developed the first generation of uncharged, enzymes should be reactivated by strong 34 heterocyclic bis-oximes with superior in vitro OR-06 reactivation, very good in vivo therapeutic Development and therapeutic potential of efficacy in organophosphate (OP)-exposed uncharged cholinesterase reactivators mice, and with favorable pharmacokinetic José Dias 1, Ludovic Jean2, André-Guilhem (PK) properties. The X-ray structures of repres- Calas1, Nicolas Probst3, Julien De Sousa4, Nicolas entative first-generation bis-oximes in com-Lamassiaude5, Christophe Landry6, Ophélie Da plex with hAChE revealed opportunities for Silva1, Anissa Braiki3, Camille Voros4, Romulo Araoz5, Caroline Coisne6, Charlotte Courageux1, Pierre their structural improvement towards better Warnault3, Franck Razafindrainibe4, Anne-Julie steric fit into geometry of the active center Gastellier1, Julien Gasnot3, Yerri Jagadeesh4, gorge of hAChE that led to synthesis of the Marilène Trancart1, Anne-Sophie Hanak1, Fa- second generation heterocyclic bis-oximes. bien Gosselet6, Xavier Brazzolotto1, Marie-Pierre The in vitro reactivation potency of the second Dehouck6, Florian Nachon1, Denis Servent5, Pierre- generation was, however, inferior to the bis- Yves Renard7, Rachid Baati4 oximes of the first generation for reasons ra- 1Institut de Recherche Biomédicale des Armées, tionalized by their corresponding X-ray struc- France tures with hAChE. That led us to design the 2Laboratoire CiTCoM, Université Paris Cité, France third generation of bis-oximes with specific- 3Université Rouen Normandie, Rouen, France ally modified central heterocyclic core result- 4ICPEES UMR CNRS 7515, Université de Strasbourg, ing in improved in vitro kinetics of reactivation Strasbourg, France of hAChE inhibited by OPs paraoxon, VX, sarin, 5Université Paris-Saclay, CEA, Département Médic- cyclosarin and fenamiphos. PK properties of aments et Technologies pour la Santé, SIMoS, representative bis-oximes of the third genera- France tion appeared reasonably good and improved 6Laboratoire de la Barrière Hémato-Encéphalique over the second generation. In expectation (LBHE), Université d’Artois, Faculté des Sciences of more detailed in vivo therapeutic charac- Jean Perrin, Lens., France terization of the third bis-oxime generation we 7Laboratoire COBRA, Université de Rouen, France are now developing a prototype fourth gener-Acetylcholinesterase (AChE), a critical ation in silico for further improved nucleophilic enzyme in the Central Nervous System (CNS), attack geometry and bioavailability, towards breaks down a vital neurotransmitter, acet-achieving a goal of highly efficient centrally ylcholine. Organophosphorus nerve agents active reactivating antidotes against OP in- (OPNAs) and pesticides irreversibly inhibit toxication. AChE, disrupting nerve communication and This research was supported by the potentially leading to death if left untreated. CounterACT Program, NIH Office of the Butyrylcholinesterase (BChE), another closely Director (OD), and the NINDS, [Grant Num- related enzyme, is crucial but has broader bers 1U01NS083451, 1R21NS120839-01A2 and functions beyond nerve signaling. Unlike 1R21NS120884-01A2], and by the UCSD Aca- AChE, BChE is found mainly in blood plasma demic Senate award BG114128. and is also inhibited by OPNAs. The French military’s current treatment for OPNA ex- posure utilizes an auto-injector containing a methanesulfonate salt of 2-PAM to reactivate AChE, alongside atropine, an anticholinergic drug, and avizafone, a prodrug of diazepam designed to mitigate convulsions. However, this treatment struggles to reach the brain effectively (limited CNS bioavailability), only 35 works against a limited range of nerve agents of plasma with an appropriate starting suc- (narrow spectrum of action), and doesn’t cinyldicholine concentration. In a subsequent always achieve optimal results. Uncharged third step, the final determination of the cata-reactivators for inhibited cholinesterases lytic and Michaelis constant is obtained by the offer a promising alternative. Our research simultaneous fit of first two data sets together group has been developing and testing new with the curve of time course of residual suc-generations of these molecules for over a cinyldicholine concentration. decade. These reactivators aim to surpass current treatments by better accessing the OR-08 brain, having a broader impact on various Inhibition of human butyrylcholinesterase nerve agents, reactivating both AChE and by A-series nerve agents, functional and BChE and potentially influencing nicotinic structural characterization receptor activity. Our upcoming presentation Charlotte Courageux, Anne-Julie Gastellier, Milica Denic, Fabrice Modeste, Nicolas Taudon, will explore these advancements in uncharged José Dias, Florian Nachon, Xavier Brazzolotto reactivators and discuss the future of this field. Institut de Recherche Biomédicale des Armées, OR-07 France Direct Determination of the hydrolysis of a Butyrylcholinesterase, a circulating homo- poor substrate by human plasma log of acetylcholinesterase, is a crucial bio- Jure Stojan scavenger against nerve agents, particularly Institute of Biochemistry, Faculty of Medicine, Uni- evident in prophylaxis but also for treatment versity of Ljubljana, Slovenia against percutaneous exposure to persist- Plasma cholinesterase is non-specific for ent nerve agents like VX. In response to the its substrates. Like butyryl(thio)choline, it can emerging threat posed by a new class of successfully hydrolyse also different other non- nerve agents, known as Novichoks or A-series physiological esters which enter the blood agents, the international authorities have re- stream as xenobiotics. Usually, their degrad- vised the OPCW Schedule 1 table to strengthen ation is significantly slower then that of bu-proliferation control. These agents, charac- tyryl(thio)choline. One of such substances is terized by their stability and potent inhibition the depolarizing skeletal muscle relaxant suc- of cholinesterases, have focused significant cinyldicholine, used mostly during emergency attention in the recent literature. surgrery. When injected intravenously it is Our investigations focused on the inhibit- normally hydrolysed by plasma butyrylcholin- ory effects of three A-series agents, namely esterse within a few minutes. However, un- A-230, A-232, and A-234, on recombinant hu- der different pathologic states, like liver faili- man butyrylcholinesterase (hBChE), determ-ure, but also in the case of atypical enzyme ining their respective inhibition constants of the paralysis may last dangerously long. Since the same order as VX, demonstrating the high direct determination of kinetic parameters bio-scavenging potential of hBChE for these for the hydrolysis of succinyldicholine is very agents. Both A-232 and A-234 possess al- tricky, we suggest a simple method, based on koxy substituents similar to other nerve agents its inhibition of butyrilthiocholine hydrolysis of or pesticides. Once the agent is covalently plasma samples. In the first experiment the in-bound to the active site serine of hBChE, hibition parameters are detrmined and in the the alkoxy substituent may undergo dealkyla- second one, the remaining concentration of tion over time, known as aging, resulting in a succinyldicholine is assesed from the degree phosphylate anionic adduct that is refract-of inhibition after different incubation times ory to the reactivation by oxime-based coun- 36 termeasures. Employing LC/MS and LC/MS2 Cynomolgus monkey, pig, mini pig, guinea pig, techniques, we probed for potential aging in mouse, and rat AChE as well as BChE by CBDP hBChE following inhibition by A-232 and A-234 were determined in the presence of substrate and did not observe discernible dealkylation to obtain the bimolecular rate constants for even after 11 days. the inhibition (ki). To elucidate the absence of aging, we Results: Human BChE was found to be most determined the crystallographic structures of sensitive towards CBDP, indicating a ki value hBChE inhibited by the A-agents. The respect- of 3.24 ×108 M- 1min- 1. The determination of ive phosphyl-adducts on the catalytic ser- the inhibition kinetics of human AChE by CBDP ine residue exhibited similarities. The amidine resulted in a ki value of 2.84 × 105 M- 1min- 1. substituent orients toward Trp82 within the In addition, markedly more pronounced inhib- choline-binding pocket. The phosphyl oxygen ition rates of BChE from the species guinea fits in the oxyanion hole while the respective al-pig, mini pig, pig, rat, Cynomolgus monkey, koxy substituents, or methyl group in the case and mouse by CBDP were found as compared of A-230, are directed toward Trp231 within the to those of AChE from the respective sources, acyl-binding pocket. Such a position of the al- indicating 2.0- to 89.6-fold higher ki values. koxy substituents away from the catalytic his- Conclusions: The inhibition kinetics of cholin-tidine, which plays a crucial role in the aging esterases from various species by CBDP mechanisms, explains the absence of aging for showed apparent species differences and A-232 and A234 in hBChE. a wide variation of the inhibition rate con- stants ki. From the results of the present study OR-09 a Cynomolgus monkey model seems most Inhibition kinetics of cholinesterases from appropriate with respect to the similarities in various species by the organophosphate the CBDP inhibition kinetics and the plasma CBDP in vitro composition in comparison with humans. Gabriele Horn, Franz Worek Bundeswehr Institute of Pharmacology and Toxico- Session III - Post exposure logy, Germany organo phosphates strategies Introduction: An increasingly reported and toxicology illness during air travel referred to as aerotoxic syndrome is accompanied by neurological Monday - Afternoon symptoms and has been associated to Lectures the exposure to tri-ortho-cresyl phosphate (TOCP), which is a toxic component included Sep 16, 14:00 - 15:50 in fumes escaping from the engine into the KN-03 bleed air of the cabin (i.e. ”fume event”). Novichok class of organophosphorus com-TOCP is converted in vivo to the metabolite pounds and the efficacy of current counter-2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin- measures against A-234 2-oxide (CBDP) by cytochrome P450. CBDP Ondrej Soukup 1, Daniel Jun2 is a known inhibitor of acetylcholinesterase 1University Hospital Hradec Kralove, Biomedical Re- (AChE) and butyrylcholinesterase (BChE). search Centre, Czech Republic In this in vitro study, species differences of 2University of Defence, Czech Republic the inhibitor CBDP were assessed, which are ***Keynote Lecture*** important for the design of animal model A-series agent belongs to a generation studies. of nerve agents that were developed during Methodology: The inhibition kinetics of human, the 1970s but got to wide public knowledge 37 just recently. The poisoning of a former Rus- charged molecules that are only poorly pensian spy Sergei Skripal and his daughter in etrating the blood-brain barrier (BBB), and 2018 by A-agent representative A-234 led to thus they have limited impact on reactiva-the inclusion A-series agents into the Chem- tion of brain acetylcholinesterase. For this ical Weapons Convention. Even though five reason, various strategies are being developed years have already passed, there is still very to overcome this drawback, i.e., uncharged little information on its chemical properties, reactivators, conjugation with molecules that biological activities, and treatment options have transporters in the BBB, bypassing the with established antidotes. We experiment- BBB through intranasal delivery, inhibition of ally assessed A-234 stability, determined its BBB efflux transporters or nanoparticle drug inhibitory and reactivation potential towards delivery systems. human acetylcholinesterase (AChE) and bu- The nanoparticle drug delivery seems to be tyrylcholinesterase (BChE). We also assessed very promising approach that can be used its toxicity in rats and therapeutic effects of even for double-charged oximes with low BBB different antidotal approaches. The results penetrability. For this reason, double-charged of spontaneous A-234 hydrolysis confirmed its oxime K027 was conjugated to BODIPY frag- stability during the first 72 h. A-234 was found ment and it was encapsulated into the hu-to be a potent inhibitor of both human ChEs man recombinant ferritin nanovehicles. The (AChE IC50 = 0.101 ± 0.003 �M and BChE IC50 oxime or encapsulated oxime were found to be = 0.036 ± 0.002 �M), whereas the five marketed bioaccumulated primarily in liver and kidneys oximes have negligible reactivation ability to- of mice, but some amount was distributed also ward A-234-inhibited HssAChE and HssBChE. to the brain, where it was detectable even The acute toxicity of A-234 is comparable to after 24 h indicating better CNS bioaccumu-that of VX and in the context of therapy, at- lation and tissue retention. In addition, oxime ropine and diazepam effectively mitigate A- K027 conjugated to DOTA fragment was en-234 lethality. Even though oxime administra- capsulated into the human recombinant fer-tion may induce minor improvements, selec- ritin nanovehicles. This conjugate seems to alted oximes (HI-6 and methoxime) do not re- low real-time observation of oxime biodistri-activate ChEs in vivo. bution in vivo via magnetic resonance imaging (MRI), i.e., the BBB penetration and bioaccu- OR-10 mulation in mice. Most recently, monocharged How to increase bioavailability and follow pyridinium reactivators with excellent reactiv-effect of charged oximes in the central ation of OP-inhibited AChE were prepared and nervous system? proved to enhanced brain reactivation of mice Kamil Musilek 1, David Malinak1, Eliska poisoned by VX agent. These approaches Prchalova1, Rudolf Andrys1, Adam Skarka1, Zbynek seem to be of benefit for crucial reactivation Heger2, Zenon Starcuk3 of acetylcholinesterase in CNS. This work was 1University of Hradec Kralove, Hradec Kralove, supported by Czech Science Foundation (no. Czech Republic GA22-14568S) and University of Hradec Kralove 2Mendel University in Brno, Czech Republic (Faculty of Science). 3Czech Academy of Sciences, Institute of Scientific Instruments, Czech Republic The cholinesterase reactivators (so called “oximes”) are used as causal antidotes in case of organophosphorus intoxications. The clin- ically used oximes have mono- or double- 38 OR-11 ing pathways associated with the CWNA pois- Novel Strategies for Optimizing the Treat- oning toxidrome or implicated in respirat-ment of Organophosphate Poisoning in a ory disorders with similar symptoms (such as Mouse Model asthma, opioid overdose, etc.) were evalu- Marilène Trancart, Anne-Sophie Hanak, Karine ated. Their efficacy in preventing ventilat- Thibault, Grégory Dal Bo, Alexandre Champault, ory abnormalities induced by VX exposure was Méliati Madi, Gwladys Meesemaecker, André- assessed by dual-chamber plethysmography. Guilhem Calas Finally, 24-hour survival tests were conduc- Institut de Recherche Biomédicale des armées, ted to confirm their therapeutic effectiveness France against VX. Organophosphate compounds (OP), such OR-12 as VX, used as chemical warfare nerve agents Choline-O-acetyltransferase as a potential (CWNA) pose significant threats due to their therapeutic target for nerve agent poison- impact on the vital physiological systems of ing: unveiling an ion sensitive regulatory the poisoned organism. A primary concern mechanism is the risk of respiratory failure resulting from Fredrik Ekström, Nina Forsgren, Frida Jonsson, cholinergic dysregulation following systemic Cecilia Engdahl, Tomas Bergström cholinesterase (ChE) enzyme inhibition. There- Swedish Defence Research Agency, Sweden fore, it is essential to have effective treatment. To treat OP poisoning promptly, several coun- This research focuses on developing anti- tries have developed self-injectable devices. dotes to counteract the devastating effects These devices typically contain a competit- of nerve agent intoxication, a pressing con-ive muscarinic receptor antagonist, atropine, cern in warfare, terrorism, and targeted as-a benzodiazepine to prevent convulsions, and sassinations. We center our investigation an oxime to reactivate inhibited ChE. However, on the enzyme Choline-O-acetyltransferase the efficacy of oximes varies depending on the (ChAT), a critical component in neurotransmit- OP, and their limited ability to cross the blood- ter production and a promising drug target. brain barrier presents significant limitations. Our study explores arylvinylpyridinium-based To address the weaknesses of the current compounds, traditionally viewed as inhibitors, therapeutic system, we focused on three areas revealing them to be substrates in a coenzyme of research: A-dependent hydrothiolation reaction that 1) Assessment of the relevance of the forms active inhibitors in-situ. This novel mech- No-Observed-Adverse-Effect-Level (NOAEL) anism results in an adduct embedded within dose as an optimized dose of oxime in the ChAT’s active site tunnel, establishing signific- treatment of CWNA poisoning. ant interactions in a hydrophobic pocket near 2) Combination of two oximes as a treat- the choline binding site. These findings pave ment for CWNA poisoning. A suitable combin-the way for the development of more potent ation could broaden the spectrum of thera- and bioactive ChAT inhibitors, potentially use-peutic efficacy and protect both peripheral ful for symptomatic treatment of nerve agent and central ChE. intoxications. Additionally, we unveil the dis- 3) Identification of the mechanisms caus- covery of a molecular switch responsive to ing respiratory failure during CWNA poison-ionic strength and regulatory phosphoryla- ing. We aimed to characterize respirat- tions that modulate ChAT’s structure and dy-ory pathophysiology in mice following sub- namics. This discovery holds broad implic- cutaneous sub-lethal VX exposure. Candid- ations for understanding and manipulating ate compounds targeting cholinergic signal- the functions of the choline/carnitine enzyme 39 family across various diseases. Moreover, we the onset and recurrence of epileptic seizures. highlight the critical role of X-ray synchrotron We demonstrated the beneficial effects of radiation in fragment-based screening. This Mix treatment on the long-term behaviour of advanced technique provides unique oppor- mice and its ability to attenuate neuroinflam-tunities for discovering compounds targeting mation. These results showed that RM048 ChAT and other challenging drug targets, sig- combined to HI-6, provided a better neuro- nificantly enhancing drug discovery efforts. protection after VX exposure. Finally, patch- clamp recording on mouse brain slices showed OR-13 that RM048 modulates neuronal activity by in- Investigation of neuroprotection conferred creasing the amplitude and the duration of by a new therapeutic strategy after organ- hyperpolarization of pyramidal neurons. ophosphorus exposure Conclusion: Alexandre Champault 1, Julie Knoertzer1, HI-6/RM048 association improved mice Armelle Rancillac2, Grégory Dal Bo1, Karine central and peripheral recovery after VX ex- Thibault1 posure at short and long term. Our study sug- 1Institut de Recherche Biomédicale des Armées, gests that the use of RM048 is very promising Brétigny sur Orge, France for improving the medical care of NA expos- 2CIRB, CNRS UMR7241/INSERM U1050, Collège de ures. France, Paris, France Introduction: OR-14 Organophosphate (OP) nerve agent (NA) Blood-brain barrier controlled-opening like VX are irreversible acetylcholinesterase with ultrasound to improve neuroprotection (AChE) inhibitors. Acute NA exposure leads to after nerve agent exposure a cholinergic syndrome, due to acetylcholine Lucie Lépinard1, Sarah Leterrier2, Laurène Jourdain2, Karine Thibault1, Anthony Novell2, accumulation in synapses, manifested by fas- Grégory Dal Bo 1 ciculation, tremors, muscle paralysis and a res- 1Institut de Recherche Biomédicale des Armées, piratory distress leading to death. Antidote France treatment includes a muscarinic cholinergic 2BioMaps, CEA, CNRS, Inserm, France receptor antagonist (atropine) and an oxime able to reactivate OP-inhibited AChE. In cent- Introduction: ral nervous system, oximes action is limited due Organophosphate compounds (OP) found to their weak ability to cross the blood brain in the most toxic chemical warfare agents barrier (BBB). The goal of this study was to test (such as VX), are also widely used as pesti-therapeutic potential of a new oxime (RM048) cides in several parts of the world. These highly designed to cross the BBB, alone or in associ-lipophilic chemical compounds cross easily the ation with another oxime, HI-6 unable to cross blood brain barrier (BBB) and irreversibly in-the BBB. hibit cholinesterase (ChE), resulting in an in- Methodology: crease in acetylcholine (ACh) concentration at To evaluate the potential antidote effect cerebral and systemic levels. Acute exposure of RM048, nine-week-old male Swiss mice ex- to OP causes an acute cholinergic toxidrome posed to a lethal dose of VX were used and re- that can lead to death if not treated. The cur-ceived therapy containing atropine with either rent antidote therapy combines a muscarinic HI-6, RM048 or both oximes (Mix). receptor antagonist (atropine sulfate) to limit Results: ACh signal, and oximes (pralidoxime or HI-6) to Using EEG recordings, we showed that reactivate the inhibited ChE. However, these RM048 alone and Mix treatments prevented oximes are unable to cross the BBB and there-40 fore cannot reactivate brain ChE, which can ***Keynote Lecture*** result in long-term neurological dysfunctions. Achieving antidotal actions of oximes in the Methodology: treatment of toxicity from irreversible cholin- The objective of this study was to assess esterase inhibitors, such as the organophos- the potential of focalized ultrasound (FUS)- phates (OPs), may well require separate dos-induced BBB opening to enhance the passage ing schedules depending on whether toxicity is of oximes (pralidoxime or HI-6) into the brain due to acute or chronic exposures. These en-and optimize neuroprotection in VX-exposed counters may arise from longer term exposures mice. to pesticides, an acute exposure from a lar- Results: ger dose of the toxic pesticide and from dis- Our results showed a significant reactiva- tinct modes of exposure. To investigate these tion of cerebral ChE in mice exposed to a sub-differences, we have carried out systematic lethal dose of VX when the therapy associated studies beginning with exposure to the par-FUS and HI-6 compared to the animals that ticular organophosphate under consideration received HI-6 without FUS. Conversely, prali- and subsequent treatment with the reactivat-doxime demonstrated no capacity to reactiv- ing oxime. We have also distinguished the time ate, regardless of the presence or absence of intervals between exposure and oxime reactiv-FUS. The beneficial effects of FUS + HI-6 were ator treatment. We began these studies with then evaluated in animals exposed to a lethal isolated and purified human cholinesterases, dose of VX. The results demonstrated that the expressed in tissue culture cell after transfec- animal recovery was significantly improved by tion of the human acetylcholinesterase gene the FUS/HI-6 association, with an AChE react- sequence into mammalian cells. Such initial ivation in the main cerebral structures and a studies also require measurements of the ex- significant reduction of neuroinflammation at tent and rate of inhibition. They were fol- different time points after the intoxication. Fi- lowed by studies in mice after parenteral in- nally, we report improved 24h survival with FUS jection or oral exposure to the organophos- + HI-6 after lethal VX-exposure. phate. Mice carry the advantages of purpose Conclusion: of the experiments to get a range of dosages The combination of FUS and oxime has the of both the organophosphate and antidotal potential to significantly enhance the medical oxime, and estimates of duration of action re- care of individuals exposed to OP. lated to re-synthesis active enzyme, as well as the initial pharmacokinetic parameters of the Session IV - Cholinesterase oxime. Also, mice carry the advantages of do- ing statistics on animal number and costs as pharmacology, inhibitors and well as the availability of genetic knock-out antidotes stains. However, as a small rodent species, they fall short of allowing studies to proceed Monday - Late afternoon to humans. Lectures Our session is devoted dealing with the com- Sep 16, 16:15 - 18:05 plexities of an antidotal reactivating agent, where both toxicity of the offending toxic or- KN-04 ganophosphate and the antidote should be Balancing central and peripheral nervous considered in terms of pharmacokinetics and system antidotal actions of oxime reactiv- pharmacodynamics. Studies have been sup-ators to acetylcholinesterase inhbition ported by the NIH, CounterACT program. Palmer Taylor, Kwok-Yiu Ho, Zoran Radic University of California San Diego, United States 41 OR-15 and BChE reactivation in severely intoxicated Development of the post-exposure RS194B macaques. RS194B can potentially be de-oxime against lethal sarin vapour and or- livered to military and civilian personnel using ganophosphate insecticides in macaques an autoinjector favoured by the US military or Yvonne Rosenberg 1, Dennis Sullivan2, Zoran more appropriately delivered orally as a pill in Radic3, Palmer Taylor3 rural clinics and hospitals in the case of agri- 1PlantVax Inc, United States cultural workers following insecticides due to 2IIT Research Institute, United States the slower onset of symptoms. 3University of California San Diego, United States OR-16 Introduction: Deliberate nerve agent Detection of selective inhibitors of BChE releases as well as occupational and self- in structurally and functionally different inflicted insecticide exposures resulting in groups of molecules fatalities, emphasize the need for organo- Anita Bosak 1, Ana Matošević1, Marija Bartolić1, phosphate (OP) countermeasures for both Ines Primožič2, Alma Ramić2, Xavier Brazzolotto3, military and civilian populations. Therapeutic Florian Nachon3, Nikola Maraković1 countermeasures against OP neurotoxins 1Institute for Medical Research and Occupational involve several strategies: (i) preventing OP Health, Croatia poisoning through administering pre-exposure 2Faculty of Science, Croatia bioscavenger treatments that scavenge OPs 3Institut de Recherche Biomédicale des Armées, before they inhibit their physiological AChE France targets in the brain and in the periphery (ii) post-exposure oxime plus atropine that can Selective inhibition of butyrylcholinesterase rapidly reactivate OP-inhibited AChE and (BChE) has proven to be a very promising dir- reduce severe symptoms or (iii) a combination ection in the development of drugs against of both. Alzheimer’s disease (AD) based on the hypo- Methodology: Macaques were exposed to thesis of cholinergic dysfunction. Although lethal OP doses of sarin vapor (49.6ug/kg) and AD is one of the most common neurodegen- paraoxon (100ug/kg) by inhalation or orally erative disorders, accounting for about 60-to the phosphorothioate insecticides chlor- 80% of all dementia cases, its pathogenesis pyrifos and parathion (50mg/kg). When clinis complex and not fully understood. In re- ical symptoms were severe, macaques were cent years, therapies for AD primarily focused injected IM with 50-80mg/kg of a new low on beta-amyloid and tau have received more MW zwitterionic oxime RS194B (developed at attention. However, various beta-amyloid- UCSD) plus low-dose atropine and monitored and tau-targeting agents have failed in clin-clinically and for AChE and BChE activity. ical trials, leaving improvement of cholinergic Results: The simplified structure and neutral neurotransmission, achieved mainly by inhib-nature of post-exposure RS194B enabled rapid ition of acetylcholinesterase (AChE), still the blood-brain barrier (BBB) passage and resul- most effective therapy for AD. In the brain of ted in rapid reactivation of OP-inhibited RBC- healthy adults, AChE is responsible for 80% of AChE and circulating BChE with dramatic re- the ACh activity, nearly 1013-fold more act-versal both early and advanced clinical OP ive than BChE. However, since during AD pro- symptoms. gression, AChE activity levels decline by up to Conclusions: Centrally acting RS194B is cur- 85% and the BChE/AChE ratio can change rently the most efficacious post OP expos- from 1:5 to 11:1, selective inhibition of BChE ure treatment developed ; requiring only a has emerged as a prudent strategy to elev- single IM injection for rapid recovery and AChE ate the ACh levels within the brain, improving 42 the cognitive and memorial functions of AD polar” agent and can have either a product- patients at mild and moderate stages. In our ive or non-productive orientation. In our work, project to discover ChE inhibitors with addi- we decided to prepare several series of hybrids tional action on other AD hallmarks, new se- with two reactivating moieties. These were lective BChE inhibitors were identified. Three designed to be active against both cholin-groups of compounds with diverse structural esterases, AChE and butyrylcholinesterase. As and functional cores were evaluated: a group a result, we also have an active ingredient that with carbamates structural similar to bam-would be effective against a variety of organ- buterol, a prodrug of bronchodilator terbu- ophosphate inhibitors. We used specific frag-taline, and two groups of ligands whose struc- ments known for their different selectivity and ture resemble that of tacrine (derivatives of 4-activity and followed different synthetic ap- aminoquinolines), and compounds with oxime proaches to cover as many structural possib-moiety (quinuclidinium-based O-alkyloximes). ilities as possible. Interactions with amino acids from the BChE We will discuss the synthesis of seven sym- active site and structural features important metric uncharged bis-oximes, eight asymmet-for selective inhibition of BChE were analyzed ric uncharged bis-oximes, and five asymmet-by molecular docking, SAR analysis, and crystal ric permanently charged compounds as re-structure of its complex with human BChE. activators to counteract organophosphorus ACKNOWLEDGEMENTS: The authors poisoning. The structure-activity relationship would like to thank the CSF (Grant no. IP- is presented in detail, showing in vitro reactiv- 2020-02-9343) and the ERDF project no. ation capabilities on both cholinesterases. We KK.01.1.1.02.0007. have also evaluated our compounds in human, mouse and rat enzymes. This has allowed us OR-17 to better correlate in vivo experiments with Bifunctional compounds serving as versatile potential human applications. In addition, cholinesterase reactivators our lead molecules were also evaluated for A- Lukas Gorecki 1, Martina Hrabinova1, Ven- agents (”novichok”) inhibition. Finally, we will dula Hepnarova1, Jana Zdarova Karasova1, Jan present our latest in vivo data on several high- Korabecny2 lighted candidates, including MTD-estimated 1Department of Toxicology and Military Pharmacy, toxicity, pharmacokinetics, and pharmacody-Military Faculty of Medicine, University of Defence, namics in sarin and VX-poisoned mice. Our Hradec Kralove, Czech Republic lead candidate has demonstrated superior ef- 2Biomedical Research Centre, University Hospital ficacy over standard clinical antidotes both in Hradec Kralove, Hradec Kralove, Czech Republic vitro and in vivo according to our results. Protection against the most toxic chem- ical weapons of mass destruction is still in- OR-18 adequate. Current causal antidotes are Attenuating organophosphate-induced based on reactivation of acetylcholinesterase neuroinflammation in mice by oxime ther- (AChE), which is covalently inhibited by these apy organophosphorus compounds. Most clinical Nikolina Maček Hrvat 1, Katarina Ilić2, Dora Kolić1, Borna Puljko3, Palmer Taylor4, Kristina Mlinac and experimental reactivators consist of two Jerković3, Svjetlana Kalanj Bognar3, Zrinka Kovarik1 parts. One part has an oxime group respons- 1Institute for Medical Research and Occupational ible for reactivation and the other part serves Health, Croatia as an anchor to the enzyme. These properties 2Kingś College London, United Kingdom are essential for sufficient antidotal efficacy. 3School of Medicine, University of Zagreb, Croatia However, such molecule also acts as a ”bi-4University of California San Diego, United States 43 search Agency, Sweden Therapy in case of organophosphate (OP) 2Umeå University, Sweden exposure requires the use of an oxime react- 3Bundeswehr Institute of Pharmacology and Toxic-ivator of OP-inhibited cholinesterases (ChE). ology, Germany However, not all approved oximes are equally effective in reactivating both ChE or, for dif- Organophosphorus nerve agents (OPNAs) ferent OPs. Additionally, they do not pen- and organophosphorus pesticides (OPPs) etrate the blood-brain barrier (BBB) in ad- are toxic chemicals that covalently inhibit equate concentrations to reactivate synaptic the neurotransmitter-regulator acetylcholin-acetylcholinesterase (AChE). Therefore, novel esterase (AChE), thereby causing a rapid centrally active oximes such as RS194B have shutdown of the nervous system and ulti-been developed to find a more effective ther- mately leading to death if not treated. apy. Herein we investigated the effect of Current treatment of intoxications caused the therapy with the uncharged, but ionizable by OPNAs and OPPs include antidotes that en-oxime RS194B that crosses the BBB and re- gage in a chemical reaction with the phos-activates OP-inhibited synaptic AChE on at- phorus atom of the adduct of inhibited AChE, tenuating the neuroinflammation in mice ex- liberating a functional enzyme. Current clin-posed to sarin. The levels of specific proteins ical reactive antidotes, so-called reactivat-expressed in glial and neuronal cells were de- ors, include pyridinium oximes such as 2-PAM, termined in the cortex and diencephalon of HI-6 and obidoxime. The efficiency of these sarin-exposed mice, mice treated with oxime reactivators is limited. They lack a broad- RS194B or pyridinium oxime 2PAM after sarin spectrum potency, meaning that they are exposure, and untreated control mice. Mi- not able to reactivate AChE independently of croglial response was detected with the level OPNA-species, and, due to their permanent of ionized calcium-binding adapter molecule charge, they have a low ability of penetrating 1 (IBA-1), and astrogliosis with the glial fibrillary the blood-brain barrier to reach AChE in the acidic protein (GFAP) level, whereas neuronal central nervous system. cell viability was determined following neur- In our lab we recently designed and eval- onal nuclei antigen (NeuN) immunoreactivity. uated a new reactivator candidate (1) that The results indicate the neuroprotective po-showed promising indications of broad spec- tential of RS194B oxime, demonstrating that trum activity. Candidate 1 was designed with RS194B therapy in mice reduces sarin-induced an aromatic moiety that interacts with AChE’s neurotoxicity, particularly within 1.5 hours after peripheral site (PS), a linker, and a reactive sarin exposure. oxime moiety interacting with AChE’s catalytic This research was supported by the HDTRA-19- site. In this work we have used 1 as a start-1-006-UCSD-113020, and the European Union ing point in a rational design of new react- – Next Generation EU (Class: 643-02/23- ivators and here we present the design and 01/00016, Reg. no. 533-03-23-0006). evaluation of these analogues. 26 compounds were designed, synthesised and biochemically OR-19 characterised for their potential as reactivat- Design and Evaluation of Acetylcholin- ors against OPNA-inhibited hAChE. esterase Reactivators: A study of reactive The main findings from our work: oxime- and peripheral site-moieties 1) Our study highlights the effective applic- Cecilia Springer Engdahl 1, Norman Hoster2, ation of classic medicinal chemistry principles, Cecilia Lindgren2, Nina Forsgren1, Frida Jonsson1, such as matched molecular series, in guiding Timo Wille3, Franz Worek3, Anna Linusson Jonsson2, the design of reactivators. Fredrik Ekström1 2) We found that alteration of the PS- 1CBRN Defence and Security, Swedish Defence Re- 44 binding moieties exert distinct influence on the biotic substrates or inhibitors have been iden-structure-activity relationship of AChE react- tified? Other example: about 90% of the ivators, and represent a critical building block phenyl valerate esterase activity of the soluble for potency. fraction of chicken sciatic nerve a time pro- 3) We have identified matched antidote gressive inhibition by paraoxon was observed pairs (i.e. neutral/charged) that allow us to in-by Estévez et al, and partly is due to BuChE, vestigate reactivator potency and distribution and simultaneously it is also time progress-in animal treatment models. ive reactivated, therefore paraoxon might be considered as a slow substrate of theses B- Session V - Paraoxonase, esterases. Have them a role in detoxication butyrylcholinesterase and in situ (the target tissue of toxicity)? Simil- arly, M Sogorb described that serum albumin phosphotriesterase role in was able to hydrolyze some phosphoramid- detoxication, biotechnology and ates and paraoxon by a mechanism of a tran- diseases sient phosphoryl-protein stage in the same catalytic site of the p-nitrophenyl butyrate, by Tuesday - Morning a mechanism of type B-esterases. Moreover, Lectures Monroy et al have described that, in the pres- Sep 17, 8:30 - 10:30 ence of Cu or Zn, avian albumin hydrolyzes the same compounds at a faster rate, as an A- KN-05 esterase. The complexity and diversity of po- The thin line between substrates or inhib- tential interactions, either in their biological itors: OP-enzymes interactions for biology, function or in artificial properties, open our intoxicity, diagnosis or applications. terest of research and offer possibilities of de- Eugenio Vilanova 1, Cermen Estevan1, Antonio velopment in diagnosis, therapy and biotech- Monroy-Noyola2, Miguel A Sogorb1, Jorge Estévez1 nological applications. Work partly supported 1Universidad Miguel Hernandez de Elche, Spain by Asociación Biotox, Spain. 2Universidad Autónoma del Estado de Morelos, Mexico OR-20 ***Keynote Lecture*** The influence of single nucleotide poly- Behind the classical simplified concepts of morphisms and substrate type on individual A-, and B-esterases that N Aldridge defined enzyme-kinetic rate constants for human to discriminate the activity measured with plasma PON1 p-nitrophenyl acetate that is inhibited by Boštjan Petrič, Aljoša Bavec, Marko Goličnik paraoxon or DFP (B) from that activity which Institute of Biochemistry and Molecular Genetics, is not sensitive (A), and later demonstrated to Faculty of Medicine, University of Ljubljana, Slove-be able to hydrolyze paraoxon, there is a com- nia, Slovenia plex and diverse different mechanisms and The kinetic parameters of human PON1 consequences of the OP-interaction with pro- have seldom been investigated: only a hand-teins either as substrate or inhibitors. For ex- ful of articles report Km and kcat values for ample, in the transcriptome of glioblastoma the most common substrates, and a single art-cells, we found that about 11.000 genes are icle reports the influence of the rs662 (Q192R) expressed, from which more than 2300 sites polymorphism on Km. In a recent study, cal-were detected with the consensus sequence culating kinetic parameters and determining for serin-esterases [G-x-S-x-G], and therefore genotype for blood plasma samples of 161 potential candidates to be phosphorylated by participants allowed us to additionally cla-OPS. For how many of them potential xeno- 45 rify the relations between PON1 genotype and PON1 phenotype. For each plasma sample, O-hexyl O-2,5-dichlorophenyl phosphor- we measured lactonase activity with dihydro- amidate (HDCP) and other phosphoramidate coumarin (DHC) and arylesterase activity with analogs were designed to elucidate the neur- phenylacetate (PA), as well as PON1 concen- otoxic acute effects of insecticide metham- tration with ELISA and mass spectroscopy, idophos. Studies in hens demonstrated their and calculated Km, Vmax, kcat and kcat/Km delayed neuropathic effect in hens by inhib-for both substrates. We then used the the ition the neuropathy target esterase (NTE) in Kruskal-Wallis test to calculate correlations nerve tissues. Experimental assays with HDCP between genotype and phenotype, and cal- enantiomers showed that the R(+)-HDCP is the culated Pearson correlations between differ- molecule that induces the inhibition and aging ent kinetic parameters. We showed that the of NTE. However, calcium-dependent hydro- SNP rs854560 (L55M) also has an influence on lysis studies with domestic mammal serum in- Km for both substrates, although considerably cluding human sera showed that the S(+)- weaker than that of rs662. kcat is also strongly HDCP is the isomer hydrolyzed. The racemic influenced by rs662, but not influenced at all HDCP and others chiral organophosphorus by rs854560. For both activities, we found that (OPs) have been a tool to identify new A- rs854560 influences the rate constant k1, i.e. esterase activities in biological tissues. A first the formation of the ES complex, but not k2, tests showed a copper-dependent HDCPase i.e. the breakdown of the complex into E and activity in chicken serum, which is around 20- P. rs662 has the opposite effect, it influences fold higher than its calcium-dependent activ- breakdown into E and P (k2), but not ES com- ity. This activity was stereoselective, opposite plex formation (k1). We also acquired some in- to the calcium-dependent activity observed in sight about the differences between PA and vertebrate tissues, for this reason, it was called DHC hydrolysis: k1 is more closely correlated ”antagonistic stereoselectivity”. Hydrolysis as-between PA and DHC than k2, meaning that says with different chiral OPs, divalent cations the ES complex formation stage is more sim- and metalloproteins allowed to identify the ilar for both substrates than breakdown into E chicken (CSA) and turkey serum albumin (TSA) and P. Previous similar studies on PON1 kinetics as the proteins responsible for this copper-worked with enzyme from a handful of parti- dependent hydrolysis of HDCP and trichloro- cipants or a single individual; we demonstrate nate (chiral OPs). Subsequent studies with an-the usefulness of measuring kinetic paramet- imal serum albumins showed that the highest ers for each participant in larger clinical stud- levels and stereoselectivity of this A-esterase ies. are in the avian albumin (chicken, turkey and goat). While mammal serum albumins had OR-21 low levels (10%) HDCPase activity and were Albumin-copper complex hydrolyses chiral non-stereoselective. Except for goat serum organophosphate compounds as a true A- albumin (GSA), which hydrolyzes trichloron-esterase ate at similar levels to TSA. The preincubation Antonio Monroy-Noyola 1, Laura Ramirez of CSA with aminoacid modifier compounds Gonzalez1, Damianys Almenares-Lopez2, Elizabeth (DFP, DTNB, zinc, paraoxon, and others) and Undiano1, Eugenio Vilanova3 incubation with HDCP racemic plus copper 1Universidad Autónoma del Estado de Morelos, in physiological conditions, and the species Mexico specificity, suggests the N-terminal sequence 2Universidad de la chontalpa, Mexico of avian albumin (DAEHK) as a required se- 3Universidad Miguel Hernandez de Elche, Spain quence, while the trichloronate hydrolysis by GSA and other recent computational and ex- 46 perimental data, suggest the catalytic parti-OR-23 cipation of other site in the copper-dependent PON-1 arylesterase activity in older patients A-esterase activity of albumin. with mild cognitive impairment, late onset Alzheimer’s disease or vascular dementia OR-22 Gianmarco Mola, Raffaella Riccetti, Valentina Special behavior of cholinesterase inter- Rosta, Alessandro Trentini, Carlo Cervellati actions among thiocholine and phenyl University of Ferrara, Italy carboxyl ester as substrates and inhibitors: Implication for research applications. Background: A wealth of evidence sug- gests that paraoxonase-1 (PON-1) may confer Jorge Estévez protection against inflammation and oxid- Universidad Miguel Hernández de Elche, Spain ative stress, which might be involved in the The kinetic studies of the competition pathogenesis of late onset Alzheimer’s disease between substrates supply relevant informa- (AD) and vascular dementia (VAD), as well tion about the interaction between phenyl- as of the prodromal phase of dementia, the valerate (PV) or phenylacetate (PA) and acet- so-called mild cognitive impairment (MCI). ylthiocholine (AtCh) in cholinesterases, sug- Here we extended previous findings with gesting that other(s) site(s) different to the the aim of evaluating whether serum PON-1 cholinesterase active site could be involved arylesterase activity might be associated with in the PVase and PA-esterase activities. In dementia and/or MCI, and to observe possible silico and kinetic experiments in butyrylcholin- correlations with other biological markers of esterase show a site (PV-site) that interacts inflammation and oxidative stress. with PV and this is related to the Asn298 residue, which is far from the catalytic site. Fur- Methods: Serum PON-1 arylesterase activity thermore, the thiocholine released at the act- was assessed in subjects with mild cognitive site of recombinant human acetylcholin- ive impairment (MCI, n = 511), Late-Onset esterase, cause alterations in PVase activity Alzheimer’s Disease (AD, n = 381), vascular de- and PA-activity, where AtCh could act as a mentia (VAD, n =100), mixed dementia AD-VAD Trojan horse. (MIXED, n =153), other dementia subtypes (n = New studies show that the thiocoline 30) and in older normal cognitive controls (n = interaction in the active site is permanent, 403). suggesting differences between recombinant cholinesterases and purified cholinesterases Results: Compared to controls, arylesterase from biological samples. Recombinant activity was significantly lower in MCI, LOAD, cholinesterases would interact with the acet- and MIXED (p<0.01 for all comparisons), ylcholine or another substrate for the first whereas it was comparable in VAD and time as nascent protein, however, the purified in other dementia subtypes. In the whole cholinesterases would interact as a veteran samples, there was significant and negative protein. These differences could have im- correlation between arylesterase activity and plications in the use of recombinant and serum Myeloperoxidase (MPO) activity (r= purified cholinesterases in biotechnological -0.100, p<0.013), which was driven by Controls applications. (r=-0.171, 0.016). In addition, we found a sig- nificant and negative correlation between arylesterase and serum homocysteine in MCI (r=-0.161, p<0.007) and MIXED group (r=0.099, p<0.029). 47 Conclusions: Overall, our results confirm ribosylation on PON2’s lactonase and antiox-that a decreased PON-1 arylesterase activ- idant activities in response to pyocyanin and ity is an early feature of dementia-related H2O2 treatments. Furthermore, our research diseases. Further longitudinal exploration aims to elucidate the ubiquitination of PON2, of the role of this enzyme in the onset and identify the E3 ligases involved, and explore progression of these disorders are required. potential crosstalk between ADP-ribosylation and ubiquitination. Our findings are poised OR-24 to significantly enhance the understanding of Regulatory mechanism and functional im- PON2 regulation and its multifaceted roles in pact of post-translational modifications on cellular defense mechanisms. These novel in-human paraoxonase 2 sights may pave the way for novel therapeutic Nagendra sai kumar Achanta, Eros A. Lampi- strategies targeting PON2 PTMs to modulate tella, Maria Marone, Elena porzio, Giuliana Catara, its activity in disease contexts. Giuseppina Lacerra, Giuseppe Manco National Research Council, Italy References The Human Paraoxonase (PON) family en- [1] Draganov., et al. ”PON1, PON2, and PON3 compasses three highly conserved lactonases: are lactonases with overlapping and distinct PON1, PON2 and PON3, with PON2 exhibiting substrate specificities.” Journal of lipid re-the highest lactonase activity [1]. search Predominantly located on the plasma mem- [2] Mandrich L., et al. An engineered version of brane, PON2 plays a critical role in innate PON2 opens the way to understand the role of immunity, serving as a primary defense its PTMs in modulating catalytic activity. PLoS against infections. Previous studies have One. highlighted several Post Translational modific- [3] Carusone,T. M, et al. ”WTAP and BIRC3 ations (PTMs) of PON2 including Glycosylation, are involved in the posttranscriptional mech-Ubiquitination and ADP ribosylation which anisms that impact on the expression and occur on opposite sides of the molecule. activity of the human lactonase PON2.” CDD. Notably, the rapid decrease in PON2 activ- [3] Kong, Andy T., et al. ”MSFragger: ultrafast ity in extract from HeLa cells exposed to and comprehensive peptide identification the bacterial quormone 3-Oxo-dodecanoyl in mass spectrometry–based proteomics.” Homoserine Lactone (3OC12HSL) has been Nature methods (2017) attributed to a ubiquitination at lysine 144 [2]. [4] Polasky., et al ”MSFragger-Labile ”Molecu- Our recent analysis leveraging advanced Pro- lar & Cellular Proteomics” (2023). teomic data analysis tools, have revised the previously identified ADP-ribosylation site [3] OR-25 from D124 to R101 [4]. Site-directed mutagen- Validation and application of two AChE-esis, followed by western blot analysis con- targeted environmental neurotoxic pollut- firmed R101 as the putative ADP-ribosylation ants detection systems site. Current investigations are focused on Ruihong Zhu 3, Jiahui An3, Heidi QH Xie3, Antonio Zandona1, Tena Cadez1, Zrinka Kovarik1, Yangsheng identifying the Poly ADP-Ribose Polymerases Chen3, Li Xu3, Bin Zhao3 (PARPs) involved in PON2 ADP-ribosylation, 1Institute for Medical Research and Occupational with ART5 emerging as a potential interactor Health, Croatia from interactome databases with its correct 2Institute for Medical Research and Occupational substrate specificity for Health, Croatia Arginine ADP-ribosylation [6]. Additionally, 3Research Center for Eco-Environmental Sciences, we are also examining the impact of ADP- Chinese Academy of Sciences, Beijing, China 48 Introduction: Acetylcholinesterase (AChE) Session VI - Translational is an enzyme with catalytic activity for the hy- research of inhibitors of drolysis of acetylcholine. It plays a crucial role cholinesterases and in cholinergic neurotransmission, controlling paraoxonases motor function across various species, and regulates numerous advanced brain functions Wednesday - Morning in mammals. AChE not only has important Lectures physiological functions, but it is also a target Sep 18, 8:30 - 10:20 of public-concerned organophosphorus (OP) pesticides. Therefore, AChE has been used as KN-06 a biomarker for monitoring the contamina- Pharmaceutical development of plasma tion of OP pesticides and screening out emer- butyrylcholinesterase: a breakthrough in ging contaminants with potential neurotoxic the treatment of nerve agent intoxications effects. Methodology: In this study, two ready-Florian Nachon 1, Anne Christine Mendes2, to-use enzymatic colorimetric assay systems Aurélie Nervo1, Xavier Brazzolotto1, Chloé were established and validated for electric eel Reymond1, Nicolas Doisne1, Moussa Kenawi1, AChE (eAChE) and human recombinant AChE Janek Bzdrenga1, Fabien Chantegreil1, Méliati (hAChE) to study the species differences of Madi1, Thomas Soirot1, Nicolas Taudon1, Nicolas selected known AChE inhibitory compounds. Belverge1, Aurelie Servonnet1, Fanny Magisson1, Nina Jaffré1, Julien Bouix3, Rachel Haus3, Catherine We evaluated the repeatability and reprodu- Verret3, Frédéric Dorandeu3 cibility of the system using BW 284c51. Us- 1Institut de Recherche Biomédicale des Armées, ing this detection system, the concentration- France effect curves of phosalone, chlorpyrifos, fe- 2Centre de Transfusion Sanguine des Armées, namiphos, methamidophos and ethoprophos France on eAChE and hAChE were plotted, and IC50 3Direction de la Formation de la Recherche et de values were obtained and compared. Fi- l’Innovation, Académie de Santé, France nally, eight dioxin-like (DL-) and non-dioxin 4Direction de la Formation de la Recherche et de (NDL-) polychlorinated biphenyls (PCBs) were l’Innovation, Académie de Santé, France screened for the effects of hAChE activity us- ***Keynote Lecture*** ing the validated detection system. Results: Organophosphorus nerve agents (OPNA) The system has good repeatability and re- irreversibly inhibit cholinesterases, leading to producibility. We obtained IC50 values for 5 an accumulation of acetylcholine and an OP pesticides and found subtle species differ- acute cholinergic crisis. This can cause death ences in their inhibitory effects on eAChE and if untreated. Therapies and prophylaxis based hAChE. We also screened out three NDL-PCBs on pharmacological organic molecules exist with mild inhibition of hAChE� including PCB52, but have various efficacy against the broad PCB138, and PCB153�that can be considered spectrum of OPNA. Decades of research in dif-as novel environmental AChE ligands for fur- ferent animal models have shown that inject-ther neurotoxicology studies.Conclusions: We able human butyrylcholinesterase (BChE) can have established reliable eAChE and hAChE neutralize OPNA and prevent or limit endo-detection systems that have the potential to genous cholinesterase inhibition. It also im-study species differences among AChE dis- proves the fate of victims if used as a post-ruptors/ligands, and can be used to discover exposure treatment for OPNA with a delayed new AChE inhibitors or ligands in emerging en-toxicokinetic profile. However, BChE has never vironmental contaminants. been fielded to date. We decided to remedy this situation by developing plasma-derived 49 human BChE for the advanced treatment of rent treatments. Addressing MDD-associated OPNA intoxication. anhedonia and cognitive deficits are espe- The pharmaceutical development of this cially problematic. Consequently, innovative countermeasure embraces the GMP produc- approaches and new neurobiological targets tion of plasma BChE, preclinical safety and for antidepressants are needed. Pre-clinical pharmacokinetics animal studies, preclinical findings describing the role of acetylcholine efficacy studies to justify the therapeutic dose, (ACh) and ghrelin in MDD have been prom- and a Phase I clinical trial for safety assess- ising. However, it is their interaction with one ment for granting a special-use authorization another and the role of butyrylcholinesterase by the health authorities. (BChE) that warrants investigation. We stud- GMP batches of highly purified plasma ied the possible antidepressant-like behavi- hBChE at the g-scale were obtained from the oural and neurobiological effects of a novel Cohn fraction IV-4 of human plasma frac- butyrylcholinesterase inhibitor (BChEI) versus a tioning using a Hupresin™ affinity chromato- reference antidepressant, the selective sero-graphy. The preclinical and efficacy stud- tonin reuptake inhibitor (SSRI), escitalopram. ies were performed on Göttingen minipigs, a Methodology: Using a genetic rat model of standard large animal model in OPNA intoxic- MDD, the Flinders Sensitive Line rat, and doses ation and countermeasures studies. Evidence of BChEI (30, 60mg/kg) and escitalopram of effectiveness was shown against supra- (10, 20mg/kg), despair-related behaviour and lethal percutaneous doses of VX, with a 1-locomotor activity, anhedonia, and cognitive h delayed IV bolus of BChE associated with function were assessed in the forced swim test late RSDL® decontamination but without any (FST), open field test (OFT), sucrose preference additional pharmacological therapy. The test (SPT) and novel object recognition test clinical trial Phase I on human volunteers (NORT), respectively. In addition, cortico- demonstrated the safety of the pharmaceut- hippocampal levels of ACh, monoamines, ical product at the anticipated human thera- brain derived neurotrophic factor (BDNF), as peutic dose with a blood half-life of 10 days. well as serum acyl and desacyl ghrelin, growth hormone, and BChE, were assayed using OR-26 liquid chromatography-mass spectrometry Selective butyrylcholinesterase inhibition (LC-MS) or enzyme-linked immunosorbent induces antidepressant, pro-cognitive, and assay (ELISA). antianhedonic effects in a genetic animal Results: Both BChEI and escitalopram model of depression: Role of acetylcholine, demonstrated significant and compar-ghrelin, and dopamine able antidepressant-like effects in the FST. Brian Harvey 1, Nadia Olivier1, Stanislav Gobec2, Moreover, the BChEI significantly reduced Mohammed Shahid3, Urban Košak2, Simon Žakelj2, anhedonic behaviour in the SPT and im- Christiaan Brink1 proved cognition in the NORT, surpassing 1North-West University, South Africa that of escitalopram. BChEI-treated rats 2University of Ljubljana, Faculty of Pharmacy, Slov- exhibited elevated acyl-to-desacyl ghrelin enia and ACh-to-choline ratios. These ratios pos- 3MS4Pharma Ltd, United Kingdom itively correlated with antidepressant-like Introduction: Despite new treatments be- and cognitive-enhancing effects in a dose- coming available, major depressive disorder dependent manner. Moreover, increased (MDD) poses significant challenges due to ghrelin positively correlated with increased a delayed onset of action, adverse effects, dopamine concentrations, known for its and the limited overall effectiveness of cur- association with reward and/or hedonic behaviour. 50 Conclusions: The investigated selective that multisite inhibitors, designed to hit both BChEI shows promising, dose-dependent the active and peripheral aromatic sites (CAS, antidepressant-like actions that involve ACh, PAS), are structurally well suited to hit other ghrelin, and dopamine. It is especially its biological targets of interest for Alzheimer’s antianhedonic and pro-cognitive qualities disease (AD) treatment. They include soluble that warrant investigation. epoxide hydrolase (sEH), another enzyme with a large active site cavity, involved in OR-27 neuroinflammation, and the aggregation of Leveraging template effects of acet- amyloidogenic proteins with key pathogenic ylcholinesterase for the development of roles in several neurodegenerative diseases. new anti-Alzheimer drug candidates with Here we show the structure-activity and multiple mechanisms structure-DMPK property studies of a new Anna Sampietro1, Wawrzyniec Haberek1, Aina class of compounds that were designed as Bellver1, Christian Griñán-Ferré1, Belén Pérez5, multisite inhibitors of AChE and sEH. These Carmen Pérez de la Lastra Aranda4, Valle Palomo4, compounds display very potent inhibit- Marina Naldi7, Manuela Bartolini7, María Isabel ory activity on human AChE, sEH and bu- Loza6, José Brea6, Clara Bartra8, Coral Sanfeliu8, Christophe Morisseau2, Raimon Sabate1, Be- tyrylcholinesterase (BChE) as well, with IC50 ste Ozaydin1, Jordi Juárez-Jiménez1, Bruce D. values in the nanomolar or subnanomolar Hammock2, Mercè Pallàs1, Santiago Vázquez1, range. They also inhibit the aggregation of Diego Muñoz-Torrero 1 β-amyloid peptide (A β 42) and tau protein 1Faculty of Pharmacy and Food Sciences, University in intact Escherichia coli cells that overex-of Barcelona, Spain press these proteins, with potencies in the 2Department of Entomology and Nematology and low micromolar range. TAR DNA-binding Comprehensive Cancer Center, University of Cali- protein (TDP-43) is another aggregation- fornia Davis, United States prone protein, which has a key pathogenic 3Pharmacology and Toxicology Section, Faculty of role in amyotrophic lateral sclerosis and Pharmacy and Food Sciences, University of Bar-frontotemporal dementia, and also in AD, in celona, Spain; Institute of Neurosciences (UBneuro), which it aggravates A β 42 and tau patholo- University of Barcelona, Spain, Spain gies. Interestingly, several compounds of this 4Instituto Madrileño de Estudios Avanzados en family have been found to inhibit the ethac-Nanociencia (IMDEA Nanociencia), Madrid, Spain rynic acid-induced aggregation of TDP-43 5Department of Pharmacology, Therapeutics and in SH-SY5Y cells and in the CL6049 strain of Toxicology, Autonomous University of Barcelona, Caenorhabditis elegans, which overexpresses Spain human TDP-43. A lead compound (ASP45) 6BioFarma Research Group, Centro Singular de In- with very interesting activity profile, brain vestigación en Medicina Molecular y Enfermedades permeability, favorable aqueous solubility and Crónicas (CIMUS), Universidade de Santiago de microsomal stability, and devoid of neurotox- Compostela, Spain icity, has been selected for further preclinical 7Department of Pharmacy and Biotechnology, Uni- studies. versity of Bologna, Italy Acknowledgements: This work was suppor- 8Institute of Biomedical Research of Barcelona, ted by grants PID2020-118127RB-I00, funded CSIC and Institut d’Investigacions Biomèdiques Au-by MICIU/AEI/10.13039/501100011033, and gust Pi i Sunyer (IDIBAPS), Barcelona, Spain 2021SGR00357, funded by AGAUR. The particular architecture of the catalytic gorge of acetylcholinesterase (AChE) and its composition, rich in aromatic residues, make 51 OR-28 of 250 �M spironolactone, Vmax of PON2 A novel identification of anti-hypertensive decreased by approximately 45 %, while KM drug spironolactone as an inhibitor of showed 4-fold enhancement. These results paraoxonase-2 lactonase activity indicate the inhibition of rPON2 activity by Yoko Suzumoto 1, Eros A. Lampitella1, Maria spironolactone may be a mixed type en- Marone1, Giovambattista Capasso2, Francesco zyme inhibition, with different binding of Trepiccione3, Giuseppe Manco1 spironolactone to free PON2 with respect to 1Institute of Biochemistry and Cell Biology, National PON2-substrate complex. Docking analyses Research Council of Italy, Italy demonstrated that binding sites for the inhib- 2Biogem, Biology and Molecular Genetics Institute, itor and substrate are partially overlapping in Italy the proximity of active site, which may indic- 3Department of Translational Medical Sciences, ate a competition between two compounds. University of Campania ‘Luigi Vanvitelli’, Italy Conclusions: Our study demonstrated that Introduction: Paraoxonase 2 (PON2) is anti-hypertensive drug spironolactone inhibits an antioxidant lactonase showing hydrolytic PON2 lactonase activity presumably through activity against 3-oxo-C12 homoserine lac- binding near the active site of PON2. Further tone (HSL). Previous report demonstrated study is required to confirm the type of in-PON2 expressed in distal nephron in kidneys, hibition and unveil the role of PON2 in blood where PON2 may play a role on blood pressure pressure regulation involving aldosterone, regulation through epithelial sodium channel ENaC and spironolactone. (ENaC). Recently, we have discovered that OR-29 PON2 in rat renal cortex is upregulated at so- Development of pleiotropic prodrugs to dium restricted condition in parallel with ENaC treat Alzheimer’s disease: from conception activation. These regulations are dependent to in vivo evaluation on steroid hormone aldosterone since pre-Alice Wang, Valentin Travers–Lesage, Marc treatment with mineralocorticoid receptor Since, Christophe Rochais antagonist spironolactone prevented these Centre d’Etudes et de Recherche sur le Médicament effects of sodium restriction (unpublished de Normandie (CERMN), Université de Caen Nor-data). Since widely-used anti-hypertensive mandie, Caen, France , France drug spironolactone contains a lactone residue, we hypothesized there might be a Introduction: Alzheimer’s disease (AD) is a direct interaction between spironolactone multifactorial disease which involved several and PON2 which may subsequently affects pathogenic pathways. Some treatments slow PON2 function. the progression of AD, but the development Methodology: Human recombinant PON2 of effective drugs remains a challenge. Thus, (rPON2) was expressed in E.coli and purified to develop news drugs against AD, the Multi- from inclusion bodies. PON2 kinetic paramet- Target Directed Ligands (MTDLs) approach ers were measured using 3-oxo-C12 HSL as seems promising. In this context, the design substrate in the presence of spironolactone of novels pleiotropic prodrugs, capable of as putative inhibitor. In order to integrate with inhibiting cholinesterases (AChE or BuChE) kinetic data, molecular dockings using PON2 according to the same mechanism as rivastig- model with spironolactone and 3-oxo-C12 mine and then releasing an active metabolite HSL were performed. to modulate 5-HT4R, is a promising strategy to Results: Kinetic measurements utilizing purified treat AD. rPON2 revealed that spironolactone inhibits PON2 lactonase activity. In the presence Methodology: Preliminary studies have led 52 to the discovery of an original benzisoxazole carbamate-based compound acting as a po-core with partial agonist activity on 5-HT4R. tent pseudo-irreversible BChE inhibitor, with Subsequently, we synthesized a series of novel high selectivity vs AChE, and showing prom-carbamylated prodrugs on this specific scaf- ising protective potentials in AD. We charac-fold. Biological evaluations were performed terized the neuroprotective activity of UW-on ChE, 5-HT4R and cellular model. Then, the MD-95 in mice treated intracerebroventricu- modification of the carbamate substituents larly with oligomerized A β 25-35 peptide using has enabled us to perform a first SAR study behavioral, biochemical and immunohisto-aiming to increase the selectivity on BuChE. chemical approaches. When injected acutely 30 min before the behavioral tests (spontan- Results: Twelve carbamylated prodrugs eous alternation in the Y-maze, object recog-were synthesized and tested. Some have nition or passive avoidance), UW-MD-95 (0.3-3 shown interesting activity, in the nM order, mg/kg) showed anti-amnesic effects in A β 25-selective on BuChE over AChE and 5-HT4R. 35-treated mice. When injected once-a-day The inhibition mechanism of cholinesterases over 7 days, it prevented A β 25-35-induced and the effect of prodrugs on cellular model memory deficits. This effect was lost in BChE were evaluated. Permeability and druggability knockout mice. Moreover, the compound pre- parameters were also determined to identify vented A β 25-35-induced oxidative stress (as-prodrugs administration route. sessed by lipid peroxidation or cytochrome c release), neuroinflammation (IL-6 and TNF� Conclusions: These studies provided initial levels or GFAP and IBA1 immunoreactivity) in structure-activity relationship (SAR) data for the hippocampus and cortex, and apoptosis the three targets, enabling us to identify the (Bax level). Moreover, UW-MD-95 significantly most promising candidate for in vivo studies in reduced the increase in soluble A β 1-42 level in mice through intranasal administration. the hippocampus induced by A β 25-35. UW- MD-95 appeared as a potent neuroprotective OR-30 compound in the A β 25-35 model of AD, with The selective butyrylcholinesterase inhib- potentially an impact on A β 1-42 accumula-itor UW-MD-95 shows symptomatic and tion that could suggest a novel mechanism of neuroprotective effects in a pharmacolo- neuroprotection. gical mouse model of Alzheimer’s disease Allison Carles1, Matthias Hoffmann2, Matthias Scheiner2, Lucie Crouzier1, Christelle Bertrand- Gaday1, Arnaud Chatonnet1, Michael Decker2, Tan- gui Maurice 1 1Université de Montpellier, Montpellier, France 2Julius Maximilians University Würzburg, Germany Alzheimer’s disease (AD) is a devastat- ing dementia characterized by extracellular amyloid- β (A β) protein aggregates and intracellular tau protein deposition. Clinically avail- able drugs mainly target acetylcholinesterase (AChE) and indirectly sustain cholinergic neur- onal tonus. Butyrylcholinesterase (BChE) also controls acetylcholine (ACh) turnover and is involved in the formation of Aß aggregates and senile plaques. UW-MD-95 is a novel 53 Session VII - ergic degeneration, eventually culminating in Multi-target-directed ligands in overall cognitive impairment. Examination of the binding sites of BChE and p38 Alzheimer’s disease primarily α MAPK revealed a structural similarity between the targeting cholinesterases hinge and hydrophobic region I of p38 α MAPK and the acyl-binding pocket and peripheral Wednesday - Late morning aromatic site of BChE. This formed the start- Lectures ing point for the development of dual BChE/ Sep 18, 10:45 - 12:35 p38 α MAPK inhibitors using structure-based drug discovery approaches. Two series of mul- KN-07 tifunctional inhibitors will be presented that Multi-target-directed ligands for potential have the potential not only to alleviate the symptomatic and disease-modifying treat- symptoms of Alzheimer’s disease but also to ment of Alzheimer’s disease address its underlying aetiology. Svit Ferjančič Benetik, Damijan Knez, Urban Košak, Aleš Obreza, Stanislav Gobec OR-31 University of Ljubljana, Faculty of Pharmacy, Slove- Advancing neocopride: a preclinical can- nia didate multitarget directed-ligand (MTDL) ***Keynote Lecture*** for neurodegenerative disease intervention The enzymatic activity of butyrylcholin- Christophe Rochais, Patrick Dallemagne esterase (BChE) in the brain increases with Université de Caen Normandie, Centre d’Etudes et the progression of Alzheimer’s disease, making de Recherche sur le Médicament de Normandie BChE a promising target for the treatment of (CERMN), Caen, France advanced stages of the disease. In addition The targeting of multiple molecular causes to cholinergic hypofunction, neuroinflamma- implicated in the pathogenesis of Alzheimer’s tion is a characteristic pathological change in disease (AD) with a single drug is now re- Alzheimer’s disease. However, the simultan- garded as a priority by numerous scientists. eous targeting of both pathologies by a single One strategy in polypharmacology is the de- molecule is not addressed by any of the drugs velopment of ”multi-target-directed ligands” currently in use or in clinical trials, highlighting (MTDLs), which have the potential to benefit a critical gap in therapeutic approaches. We multiple targets implicated in the complex AD. propose that the simultaneous targeting of In this context, we have developed pioneering BChE and mitogen-activated protein kinase work in this field, which involved the modula- p38 α (p38 α MAPK) by dual-acting small motion of a RS67,333, a reference 5-HT4R partial lecule inhibitors represents a novel therapeutic agonist, which possesses moderate acet-strategy to combat Alzheimer’s disease. This ylcholinesterase (AChE) inhibition properties. hypothesis is based on a series of findings This resulted in the delivery of the first example from cell and animal studies as well as in silico of a dual agent, named Donecopride. modelling showing that it is possible to tar- get both enzymes simultaneously. The pro- This lead compound has demonstrated both inflammatory microglial response triggered by precognitive and antiamnesic effects in sev-amyloid beta (A β) plaques leads to overactiv- eral animal models of AD. In the continuation ation of p38 α MAPK, which in turn enhances A β of the study, we will present an undisclosed synthesis, hyperphosphorylation of tau and al- modulation that led to Neocopride, our pre- teration of synaptic plasticity. Crucially, over- clinical candidate, which has been validated activation of microglia exacerbates neuroin- by a complete drug development plan. flammation, which worsens along with cholin- 54 OR-32 to cross the blood-brain-barrier, showing sat- Cholinesterase-based inhibitors as multit- isfactory in vitro pharmacological properties arget small molecules for the therapy of on selected biological targets (hChEs, hMAOs, Alzheimer’s disease hH3R, and hS1R) involved in the progress of José Marco Contelles 1, Francisco López- Alzheimer’s disease (AD), being able to restore Muñoz2 the cognitive impairment in appropriate in in 1Institute of General Organic Chemistry (CSIC), vivo AD animal models, comparing very favor-Spain ably with donepezil, a drug in the clinics for AD 2Faculty of Health, Camilo JoseĆela University of patients tretament. Thus, these data suggest Madrid (UCJC), 28692 Villafranca del Castillo (Mad-that Contilisant is a new “lead-compound” for rid), Spain AD therapy, ready to enter in the pre-clinical Introduction. Alzheimer’s disease (AD) is a phase. neurodegenerative disease characterized by OR-33 progressive memory decline, affecting vari- Discovery of novel BChE inhibitors for cog-ous cognitive domains such as attention, lan- nitive improvement guage comprehension, and problem-solving Baichen Xiong, Yuanyuan Wang, Weiting Zhang, skills. Despite dedicated efforts to understand Haopeng Sun the causes of AD, there is still an urgent need to School of Pharmacy, China Pharmaceutical Univer-uncover the origin of this disease in detail and sity, Nanjing, China, China to advance the development of new thera- peutic interventions. Butyrylcholinesterase (BChE) has been Methodology. Based on the therapeutic considered as a potential therapeutic tar-strategy of “one molecule-multiple targets” get for Alzheimer’s disease (AD) because for the development of drugs to treat mul-of its compensation capacity to hydrolyze tifactorial diseases such as AD, we have dis- acetylcholine and its close association with covered Contilisant as a multifunctional ligand A β deposit. In our study, hierarchical vir- that inhibits cholinesterases (ChEs) increas- tual screening protocols were applied, and ing the level of neurotransmitters such acet- several kinds of potential BChE inhibitors ylcholine, in clear deficit in the brain of AD pa-with different skeletons were selected as tients. a lead compounds. Based on the binding Results. Contilisant has been investigated in model of compounds and BChE protein pre- vitro and in vivo in several biological targets dicted by computer simulation, activity and involved in the progress and development of druggability optimization was conducted. the disease, and whose pharmacological in- Two classes of compounds, one contain- hibition and/or modulation it is expected to ing benzimidazole-aminofurazan structures produce satisfactory results in order to recover and the other N-benzyl benzamide struc-from the cognitive impairment, showing the tures, have been identified as candidate following results: compounds due to their promising inhib- hAChE (IC50 = 0.53 µ M); hBuChE (IC50 = 1.69 itory activity and cyto-safety. Candidate µ M); compounds (S11-1014, hBChE IC50 = 0.08 nM; hMAO-A (IC50 = 0.145 µ M); hMAO-B (IC50 = S11-1031, hBChE IC50 = 0.039 nM and S06-1064, 0.078 µ M); hBChE IC50 = 45.2 nM) possessed blood–brain hH3R (antagonist), Ki = 10.8 nM; barrier penetrating ability, a long T1/2, and low hS1R (agonist) Ki = 65.2 nM)] intrinsic clearance. BChE inhibitors exhibited Conclusions. Contilisant is a neuroprotective, neuroprotective effects and the ability to im-non-toxic, antioxidant, permeable ligand, able prove cognition in APP/PS1 transgenic mouse 55 model, by benefiting cholinergic system, redu- phore design with molecular probes. For both cing the total A β amount and increasing the enzymes, we performed classical molecular ghrelin content. Simultaneous modulation in dynamics simulations and simulations in a the center and periphery greatly improves the water-cosolvent system containing mixed efficiency of BChE inhibitors. Thus, candidate solvents. The use of other molecules than BChE inhibitors are promising compounds with water treated as specific molecular probes drug-like properties for improving cognitive allowed us to predict the preferred location dysfunction, providing a potential strategy for of functional groups with different physico-the treatment of AD. chemical properties. Defining the points of highest probe entry density allowed the cre- OR-34 ation of a map of potential inhibitor-protein Development of dual pharmacophore mod- interactions, which combined between en-els of acetylcholinesterase and human sol- zymes creates consistent pharmacophore uble epoxide hydrolase based on local co- model. solvent molecules distribution We gratefully acknowledge Poland’s high- Weronika Bagrowska, Artur Góra performance Infrastructure PLGrid (ACK Tunneling Group, Biotechnology Centre, Silesian Cyfronet AGH) for providing computer facilit- University of Technology, Poland ies and support within computational grants’ The complicated nature of Alzheimer’s dis- no. PLG/2023/016344, PLG/2023/016484, ease (AD) involves several pathways in which PLG/2024/017171 dysregulation can stimulate its progression. OR-35 Modulation of multiple targets may provide Pleiotropic prodrugs for both sympto-benefits during therapy. The main cognitive matic and disease-modifying treatment of impairment in AD patients results in a cholin-Alzheimer’s disease ergic deficit in the central nervous system. Anže Meden 1, Neža Žnidaršič2, Damijan Knez1, Acetylcholinesterase (AchE) is the main en-Yuanyuan Wang3, Ziwei Xu3, Huajing Yang3, Weiting zyme involved in cholinergic signalling through Zhang3, Anja Pišlar1, Andrej Perdih1, Simona Kranjc the hydrolysis of acetylcholine (Ach). Another Brezar4, Neža Grgurevič2, Stane Pajk1, Haopeng problem is neuroinflammation, caused by Sun3, Stanislav Gobec1 high concentrations of pro-inflammatory 1University of Ljubljana, Faculty of Pharmacy, Slov- cytokines, which gradually causes neuronal enia damage. This can be effectively reduced by 2University of Ljubljana, Faculty of Veterinary Medi-epoxyeicosatrienoic acids (EETs) metabolised cine, Slovenia by human soluble epoxide hydrolase (hsEH). 3China Pharmaceutical University, Nanjing, China Increased levels of ACh may promote the 4Department of Experimental Oncology, Institute metabolism of arachidonic acid to EETs, re- of Oncology, Slovenia inforcing anti-neuroinflammatory effect. For this reason, new drugs targeting both enzymes Alzheimer’s disease (AD) complexity and may prove more effective in treating the AD. failed clinical trials have spiked the interest The search for new drugs targeting a specific of the community in multifunctional ligands molecular target often begins with the design that target at least two key macromolecules of a pharmacophore model - matrix from in AD pathology. Here we report on a fo- which potential inhibitors are designed. Here, cused series of pleiotropic N-carbamoylazole we aim to propose new pharmacophores prodrugs with dual mechanism of action. models that are compatible for both enzymes. Pseudo-irreversible inhibition of the first thera- We used a novel technique using pharmaco- peutic target, human butyrylcholinesterase (hBChE), enhances cholinergic transmission, 56 and thereby provides symptomatic treatment. ergic traits are found in the epidermis con-Simultaneously, this represents metabolic ac- sisting of melanocytes and keratinocytes, and tivation that liberates a nanomolar select- acetylcholine (ACh) mediates signal transduc-ive α 2-adrenergic antagonist atipamezole, tion between the epidermal cells. Ultraviolet which blocks pathological amyloid β (A β)- B (UVB) serves as a “switch” controlling the induced and noradrenaline-dependent ac- release of ACh from epidermal keratinocytes tivation of GSK3 β that ultimately leads to to trigger the event. The expression of acet-hyperphosphorylation of tau, thus achiev- ylcholinesterase (AChE) in keratinocytes, both ing a disease-modifying effect. Lead comin vivo and in vitro models, was suppressed fol- pound 8 demonstrated long-term pseudo- lowing exposure to UVB irradiation: this reg- irreversible hBChE inhibition, metabolic ac- ulation was mediated by an upregulation of tivation in human plasma, blood-brain bar- miR-132 and miR-212. Under the low level rier permeability, and oral availability in mice. of AChE, i.e., AChE inhibition or AChE knock- Multi-day in vivo treatment with 8 in an A β- down, the overflow of ACh led by UVB irradi-induced AD murine model revealed a sig- ation caused a promoted pro-inflammatory nificant alleviation of cognitive deficit that response in the skin epidermis, demonstrated was better or comparable to rivastigmine, by increased secretion of cytokines IL-1 and the current drug of choice for AD therapy, IL-6, as well as COX-2. The activation of α 7 and also a decrease in tau phosphorylation. nicotinic ACh receptor (nAChR) in keratino- This surpasses the symptomatic-only treat- cytes is responsible for the ACh-induced in-ment with cholinesterase inhibitors, as it dir- flammatory response. In melanocytes, the ectly blocks an essential pathological cascade challenge of ACh regulates the production of in AD. Therefore, these multifunctional α 2- melanin, as well as the release of melanin syn-adrenergic antagonists–butyrylcholinesterase thesizing organelle melanosome, via M2/M4 inhibitors, exemplified by lead compound 8, muscarinic AChRs (mAChRs). In parallel, the present a potentially revolutionary small mo-activation of α 7 nAChR in keratinocytes plays lecule disease-modifying treatment for AD. a role in regulating the uptake of released melanosome through phagocytic activity. This Session VIII - Functions of phenomenon was markedly enhanced by ap- plying ACh, AChE inhibitor and α 7 nAChR ag- cholinesterases in different onist. Besides, the intracellular Ca2+ mobiliza- tissues tion in keratinocytes, triggered by UVB expos- ure, is attributed to initiation of phagocytosis, Wednesday - Afternoon while the blockage of Ca2+ influx with BAPTA- Lectures AM, or α 7 nAChR antagonist, terminates the Sep 18, 14:00 - 16:00 event completely. The findings provide insights into the development of novel therapeutic KN-08 strategies for photoprotection, tackling in- Acetylcholinesterase regulates inflammat- flammation and hyper-pigmentation caused ory responses and pigmentation in skin epi- by UVB irradiation. dermis Karl Wah-Keung Tsim The Hong Kong University of Science and Techno- logy, Hong Kong ***Keynote Lecture*** The cholinergic system presented in skin is proposed as “skin synapse”, where the cholin- 57 OR-36 BChE knockout mice, basal hemodynamic Characterizing Cardiac and Vascular parameters are preserved, but their response Cholinesterases: From Molecular Insights to to adrenergic stimulation is diminished. In Therapeutic Prospects mice, aortic BChE expression is negligible; Dominika Dingová, Kristína Szmicseková, Matej however, in rats, there is high BChE expression Kučera, Lenka Bies Pivačková, Tibor Hodbod, Parsa in rat aorta, primarily in the smooth muscle, Shafieikazerooni, Peter Křenek, Eric Krejci, Anna possibly compensating for very low BChE Hrabovska activity in serum. BChE inhibition enhances Faculty of Pharmacy, Comenius University Bratis- acetylcholine-induced relaxation of rat isol- lava, Slovakia ated aorta. In conclusion, AChE mediates Autonomic imbalance is a well- the bradycardic effects of cholinesterase documented feature of cardiovascular dis- inhibitors, while BChE plays a significant role eases. For the past six decades, the adrenergic in cholinergic signaling in the heart and blood system became a significant pharmacolo-vessels, potentially offering therapeutic bene- gical target. Recent studies in animal models fits in cardiovascular pathologies like chronic and human patients have highlighted the heart failure. Supported by APVV-22-0541 and beneficial effects of enhanced cholinergic VEGA 1/0283/22. signaling in the heart, prompting interest in OR-37 cholinesterase (ChE) inhibitors. Our research Mice with inactive cholinesterases: new aims to characterize ChE in the cardiovascu- tools to evaluate cholinergic and non-lar system. Through a series of biochemical, cholinergic functions of AChE microscopic, physiological, and pharma- Eric Krejci cological experiments in rats and mutant université Paris cité, CNRS centre Borelli, France mice lacking different ChE forms, we analyzed the molecular composition, localization, Organophosphate (OP) pesticides, nerve physiological functions, and pharmacological agents or drugs have dramatic immediate responses of ChE in the heart and blood and long-term effects on various physiolo- vessels. Both acetylcholinesterase (AChE) and gical functions (respiration, cardiovascular butyrylcholinesterase (BChE) are present in system, digestion, inflammation, brain func- the heart, with the highest activities in the tions (seizures)...). Inhibition of acetylcholin- sinoatrial and atrioventricular nodes. Their ex- esterase (AChE) is a key target of these highly pression levels are similar in the atria, but BChE reactive molecules. Since AChE terminates predominates in the ventricles. Only anchored synaptic transmission at the neuromuscular AChE is present in the heart where it colocal-junction (NMJ) as well as at other choliner- izes with neurons. AChE participates in heart gic synapses in the central and autonomic rate control and provides the bradycardic nervous systems, the canonical explanation for effect of ChE inhibitors. AChE expression in these effects is related to an excess of ACh at blood vessels is limited. BChE, detected as a the synapses. AChE KO mice survive because precursor monomer in the heart, is localized butyrylcholinesterase (BChE) compensates for intracellularly. BChE knockout mice exhibit AChE, presumably because BChE hydrolyses a more pronounced bradycardic response acetylcholine. However, BChE is not local- to ChE inhibitors, prolonged recovery after ized in the synaptic cleft. For example, BChE muscarinic blockade, and reduced sensitivity is abundant at the mouse NMJ, anchored by to acetylcholine under adrenergic stimula- PRiMA to the surface of the terminal Schwann tion, indicating adaptations in acetylcholine cells, where BChE limits the activation of an release and muscarinic receptor signaling. In α 7 receptor signaling pathway that reduces 58 ACh release. Thus, inhibition of BChE reduces tional and post-transcriptional regulation of ACh release while toxicity results from more AChE by TCDD. Whereas, in rodent-derived ACh or, if not, from unrelated effects of the models, AhR-independent mechanisms are inhibitors. To know whether the toxicity is re- involved in TCDD-induced AChE dysregula-lated to an excess of endogenous ACh (cholin- tion. ergic function) or to non-enzymatic functions Methodology: C2C12 is a well-established of the protein (non-cholinergic function), we mouse model for myogenic differentiation generated mice with a point mutation in the studies and is a sensitive system for assessing AChE or BChE gene. As a result, two mutants, the effects of dioxins on AChE. In light of the WAChE and SBChE mice, produce AChE and growing global concern about the health BChE proteins, respectively, that are correctly impacts of emerging pollutants, we employ localized but inactive. We used the growth of the C2C12 model to investigate the poten-the mice as a marker of toxicity. I will show tial interference effects of several emerging how different combinations of mutations af- dioxin-like pollutants on AChE. fect the growth and viability of the mice, sup- Results: We found that certain brominated porting a severe toxicity of ACh during post- dioxins and polyhalogenated carbazoles natal development. Unexpectedly, these ge- (PHCZs) were able to downregulate AChE netic approaches reveal that skeletal muscle is gene expression, that was in parallel with their a major source of toxic ACh. I will discuss how inhibitory effect on myogenous differentiation the hydrolysis of ACh by AChE and BChE may in C2C12 cells. Similar to classical dioxins, AChE explain the pleiotropic effects of ChE inhibit- dysregulations caused by these emerging ors. dioxin-like pollutants are mediated by AhR- independent mechanisms. OR-38 Conclusions: The emerging dioxin-like pollut- New perspectives on the effects of dioxin- ants are new AChE disruptors, which act by like pollutants on acetylcholinesterase interfering with the expression of the AChE Heidi QH Xie, Guanglei Yang, Ruihong Zhu, Jiahui genes. An, Yangsheng Chen, Li Xu, Bin Zhao Research Center for Eco-Environmental Sciences, OR-39 Chinese Academy of Sciences, China The cholinergic enigma of pigmented and Müller glial cells Introduction: Acetylcholinesterase (AChE, Paul G. Layer 1, Gesine Bachmann1, Alex EC3.1.1.7) plays an important role in choliner- Bausch1, Nicola Coronato1, Gopenath Thangaraj2 gic neurotransmission and has been widely 1Technischen Universität Darmstadt, Germany recognized as a biomarker for monitoring 2Science College of Musare, India organophosphate and carbamate pesticide contamination and poisoning. Dioxins are Cholinergic mechanisms are not restric- newly discovered AChE environmental dis- ted to neural functions. Using in vivo and ruptors. 2,3,7,8-Tetrachlorodibenzo-p-dioxin in vitro models, the developmental appear- (TCDD) is representative of this group of ance of cholinergic components in verteb-compounds and inhibits AChE activity by dis- rate eye structures allows to analyse cholin-rupting its expression in cultured neurons and ergic functions both in a synaptic and non- muscles. The role of aryl hydrocarbon receptor synaptic context. For proper development (AhR)-dependent pathway has diverse and of the inner retina, a particular cholinergic species-specific roles in the suppression of amacrine cell type together with radial glial AChE expression by TCDD. In human-derived cells (Müller cells, MCs) is crucial. Less well neuroblastoma cells, AhR mediates transcrip- understood are possible cholinergic interac-59 tions of the outer retina, i.e., at the inter- responses. However, how AChE-regulated face of photoreceptors (PRs) and retinal pig- neuroinflammation affects the pathology of mented epithelium (RPE). Our retinal organoid Alzheimer’s disease (AD) is still unclear. Here, and explant work has established that retinal we applied two mouse models, conditional lamina and network formation depends both AChE knock-in mouse (cKI), in which AChE on RPE and on MCs (or, growth factors de- is specifically overexpressed in myeloid cell rived from them). But what does „choliner- linkage, and 5×FAD mouse. By using the cKI gic“ mean within the triad PRs, RPE and MCs? mice, the LPS-induced neuroinflammation, Literature data have shown that ACh is pro- including the expressions of proinflammat-duced in, and released from PRs, while func- ory cytokines and the activation of both tional α 7-nAChRs located on the apical sur- microglia and astrocytes, was aggravated face of RPE cells receive the ACh signal. Fur- in the brain of mice having overexpression thermore, MCs of adult mice retinae provide of AChE. Transcriptomics analysis confirm a source for adult mammalian retinal regen- the severer inflammation in the AChE over-eration (tradit. thought impossible). Not- expressing mice than the wildtypes after ably, these MCs are triggered by cholinergic LPS administration. Subsequently, the effect communication via α 7-nAChR onto RPE cells of AChE overexpression on AD pathology and from there back onto MCs. Thus sim- was explored by crossbreeding the AChE cKI ilar to cancer cells, MCs have preserved some mouse with 5×FAD mouse. Amyloid beta (A β) transitory state (as indicated by their BChE plaque is one of the hallmarks of AD, which is expression), which - via cholinergic actions - co-localized with activated glial cells in the can be (re)activated to turn into proliferat- brain. Surprisingly, the AChE overexpression ive progenitor cells. The cholinergic enigma at significantly relieved the A β burden in the the retinal-pigmented-glial triad bears emin- brain of AD mice. Microglia and astrocytes ent biomedical relevance, by far not restric- are primarily responsible for A β clearance in ted to eye structures (e.g., cancer biology, tis- the AD brain. The A β plaques were closely sue regeneration and engineering, etc.). How surrounded by activated glial cells and were cholinergic stimulation of MCs leads them to reduced significantly in the AChE cKI mice, as divide, is at the heart of stem cell biology. compared with wildtypes. Notably, there were more activated glial cells, i.e., astrocytes and OR-40 microglia, around A β aggregates in the AChE Acetylcholinesterase Reliefs Beta-Amyloid cKI mice. Thus, the high expression of AChE Plaque Burden via Enhancing Glial Activa- could relief A β plaque burden via stimulating tion in the Brain of 5×FAD Mouse the neuroinflammation and subsequently Yingjie Xia, Xiaoyang Wang, Maggie Suisui Guo, enhancing the glial activation in the brain of Ran Duan, Tingxia Dong, Karl Wah-Keung Tsim AD mice. The Hong Kong University of Science and Techno- logy, Hong Kong Acetylcholinesterase (AChE) has been reported to have additional functions in neuroinflammation, i.e., AChE regulates in- flammatory responses via being involved in cholinergic anti-inflammatory pathway (CAP). In our previous in vitro study, AChE was upregu- lated in LPS-induced microglia/macrophage, and contrarily potentiated the inflammatory 60 P O S T E R P R E S E N T A T I O N S ’ A B S T R A C T S Poster Session molecular dynamics. The best possible regres- sion models for BChE inhibition/theoretical Monday - Poster Session data based on various statistical paramet- Sep 16, 18:05 - 19:30 ers will be presented and utilized for future smart design of new inhibitors based only on PO-01 the in silico simulations. ACKNOWLEDGE- Target-guided synthesis of novel bu- MENTS: This work was financially supported tyrylcholinesterase inhibitors by the Croatian Science Foundation (Grant Ines Primožič, Alma Ramić, Toni Divjak, Tomica IP-2022-10-9525). Hrenar University of Zagreb, Faculty of Science, Croatia PO-02 Capitalizing on human BChE-ligand com- In this work, target-guided synthesis of plex structures for the design of BChE-compounds was combined with quantum specific reactivator against nerve agent in-chemical methods and extensive machine toxication learning protocol to find new inhibitors Damijan Knez1, Masa Zorman1, Anne- of cholinesterases for possible use in the Julie Gastellier2, Charlotte Courageux2, Janek treatment of Alzheimer’s disease. Com- Bzdrenga2, José Dias2, Xavier Brazzolotto 2 pounds with chemical scaffolds based on 1University of Ljubljana, Faculty of Pharmacy, Slov- α-acylamino amides were prepared by enia multicomponent reaction to obtain novel 2Institut de Recherche Biomédicale des Armées, inhibitors of butyrylcholinesterase (BChE). France Several different ketones (acetone, car- Among the potential treatment strategies bonyldiimidazole) and/or aldehydes (formal- studied against nerve agent poisoning, the dehyde, imidazole-2-carbaldehyde), amines use of a nerve agent bioscavenger in com- (benzamine, isobutylamine), isocyanides bination with a specific reactivator - i.e., (tert-butyl, para-toluenesulfonylmethyl) and the pseudo- catalytic bioscavenger strategy, carboxylic acids (acetic, benzoic) were chosen should provide the best of both worlds, by as components. Compounds were prepared reducing the quantity of the expensive bioin a conventional manner and reaction was scavenger. To this aim, determining multiple conduct in the presence of the enzyme for in structures of cholinesterases in complex with situ selection of reaction components (as a different ligands is a powerful tool to enrich proof-of-principle). The impact of substitu- the structural database required to ration-ents on the inhibition potency was analyzed ally design new reactivators of nerve-agents using the quantum chemical flexible simultan- poisoned cholinesterases. eous multiple ligands docking scheme. Using In the last few years, multiple crystal struc- the docking simulations, characterization of tures of human butyrylcholinesterase (BChE) optimal binding modes within the binding site were solved in complex with a family of BChE-and estimation of standard Gibbs binding specific ligands originally designed as poten- energies were determined for all synthes- tial anti- Alzheimer’s disease drugs. Among the ized compounds. The relationship between different structural motifs composing these measured binding affinities (IC50 values) and ligands, a remarkable cycloheptyl-based pat- theoretical data was established using ma- tern emerged due to its specific arrange- chine learning multivariate linear regression. ment into human BChE. After verification by Experimentally obtained inhibition data were molecular docking, such motif arrangement regressed on theoretically calculated poten- would be possible into OP-inhibited BChE and tial energy surfaces sampled from the ab initio 63 some molecules sharing both the cyclohep- ChEs compared to their bisquaternary oximes. tyl motif and the reference reactivator, pral- The reactivation ability of novel monoqua-idoxime, were synthesized. Despite the rel- ternary compounds for OP-inhibited ChEs ative structural simplicity of these molecules, was found to be similar or lower compared to a molecule presented noticeable reactiva- bisquaternary K-oximes. These results high-tion potency towards VX- and sarin-inhibited light the importance of a second charge for BChE compared to the reference oximes pral- binding into OP-inhibited cholinesterase and idoxime and HI-6, especially by increasing the for the reactivation. This work was suppor- affinity towards the enzyme. ted by the University of Hradec Kralove (no. SV2111-2024) and Czech Science Foundation PO-03 (no. GA21-03000S). Monoquaternary analogues of double charged K-oximes (K027, K048 and K203) PO-04 are less effective reactivators of cholin- Halogenated pralidoxime analogues are ef-esterases inhibited by organophosphates ficiently reactivating cholinesterases Zuzana Kohoutova, Rudolf Andrys, Kamil Musilek, Sara Rademacherova, Karolina Knittelova, Ad-David Malinak ela Fuchsova, Rudolf Andrys, Kamil Musilek, David University of Hradec Kralove, Department of Chem- Malinak istry, Hradec Kralove, Czech Republic, Czech Re- University of Hradec Kralove, Hradec Kralove, Czech public Republic Irreversible inhibition of acetylcholin- Organophosphorus compounds (OP) are esterase (AChE) by organophosphorus inhibiting cholinesterases, disrupt cholinergic compounds (OPs) such as nerve agents or functions and leading to the development pesticides can be life threatening. On the of an acute cholinergic crisis. Treatment other hand, inhibition of butyrylcholinesterase of OP poisoning involves symptomatic anti- (BChE) has no adverse effects and therefore dotes and causal oximes, such as pralidoxime, BChE can be used as bioscavenger of OPs. which act as reactivators of inhibited cholin- Reactivators of cholinesterases (ChEs) cleave esterases. However, the efficacy of charged the OP moiety from the active site of the oximes is limited due to their hydrophilic char- enzyme by making a covalent bond to form acter, resulting in low permeability through the a phosphyloxime and restore the activity of blood-brain barrier or an inability to reactiv-ChEs. There are five clinically used reactivators ate ”aged” acetylcholinesterase in vivo. Ad-of AChE (e.g. asoxime or pralidoxime), but they ditionally, none of the commercially available have certain drawbacks. The promising results oximes is universally effective against multiple were obtained for K-oximes K027, K048, K203 structurally variable OPs. and their halogenated analogues, especially This study proposes halogenated oxime react- oxime K868, which showed very promising ivators, when the various halogens were po-reactivation ability for both ChEs. The aim sitioned to pralidoxime scaffold. The pres- of this research was synthesis and in vitro ence of a halogen atom in a close proximity evaluation of monoquaternary analogues of of oxime group could lower its pKa value and K-oximes K027, K048 and K203 with or without increase their reactivation efficiency. The re- halogen substituents. Their stability and pKa activators were synthesized, their stability and values were determined and were found to be oximate forming properties were evaluated. analogous to bisquaternary oximes. Similarly, They were challenged to OP surrogate inhib-no significant change was observed in the ited acetylcholinesterase and butyrylcholin- inhibitory effect of novel compounds for both esterase with promising results. Overall, the C3 64 positioned halogens demonstrated a higher group can be valuable tool for design of reactivation ability compared to pralidoxime more potent reactivators. The most promising and confirmed the importance of higher oxim- brominated oxime was tested in vivo on sarin-ate formation for better reactivation.This work and VX-poisoned rats. This brominated oxime was supported by the University of Hradec showed interesting CNS distribution and signi-Kralove (no. SV2111-2024) and Czech Science ficant reactivation effectiveness in blood and Foundation (no. GA21-03000S). resulted with the best protective index for VX- poisoned rats. PO-05 This work was supported by the Univer- Brominated oxime nucleophiles are effi- sity of Hradec Kralove (Faculty of Science, no. ciently reactivating cholinesterases inhib- SV2111-2024) and Czech Science Foundation ited by nerve agents (no. GA21-03000S). Eliska Prchalova 1, Rudolf Andrys1, Jaroslav [1] PRCHALOVA, E. et al. Arch. Toxicol. 2023, Pejchal2, Zuzana Kohoutova1, Kamil Musilek1, Jana 97(11), 2839–2860. Zdarova Karasova2, David Malinak1 [2] PRCHALOVA, E. et al. Arch. Toxicol. 2024, 1University of Hradec Kralove, Faculty of Science, published. Czech Republic [3] ZORBAZ, T. et al. Eur. J. Med. Chem. 2022, 2University of Defence, Military Faculty of Medicine, 238, 114377. Hradec Kralove, Czech Republic Cholinesterases (ChEs) are essential for PO-06 maintaining function of nervous system. Un- Importance of the shape of the linker fortunately, they are targets of organophos- between two quaternary pyridinium rings phates (OPs) and can be inhibited which leads on reactivation process in oximes – in vitro to cholinergic crisis and in serious cases death. and in silico study The causal antidotes for OPs poisoning are re- Tanos Celmar Costa Franca 1, Fernanda Geor- gia Figueiredo Taborda Barbosa1, Joyce Sobreiro activators of ChEs. To date five commercially Francisco Diz de Almeida1, Eugenie Nepovimova2, available reactivators are used. However, they Rafael Dolezal2, Steven Laplate3, Kamil Kuca2 have several limitations; i.e. inability to re- 1Military Institute of Engineering, Rio de Janeiro, activate “aged” acetylcholinesterase (AChE) Brazil in vivo, low permeability through blood-brain 2Faculty of Science, University of Hradec Kralove, barrier (BBB) and none of the commercial Hradec Kralove, Czech Republic reactivators is universal for all OPs [1]. Six 3Institut National de la Recherche Scientifique novel brominated bis-pyridinium oximes were (INRS), Canada designed and synthesised with the aim to increase their nucleophilicity and reactiva- Acetylcholinesterase (AChE) reactivators tion ability of phosphorylated AChE and bu- are commonly employed as antidotes for the tyrylcholinesterase (BChE) [2]. Bis-brominated treatment of nerve agent intoxications. Ac-oximes showed decreased stability in a buf- cording to recent literature, oxime K203 is fer solution as well as their previously prepared among the drug candidates exhibiting prom-fluorinated and chlorinated analogues [2, 3]. ising reactivation efficacy both in vitro and in Their degradation proceeds through isoxazole vivo. In this study, we examined three oxime re-formation to the formation of nitriles. The activators in vitro, each differing slightly from brominated reactivators showed increased oxime K203. The sole structural distinction lies reactivation ability to non-halogenated ana-in the connecting chain between the two qua- logues, confirming that an electron withdraw- ternary nitrogen atoms in their molecules. Tra-ing group in a close proximity to the oxime ditionally, the connecting linker is believed to play a passive role in the reactivation process. 65 However, our findings suggest that the con-to rescue the enzyme activity. Our experi- nection linker is a significant structural element mental data indicated that pralidoxime and influencing the efficacy of reactivators. These trimedoxime were the most efficient oximes for conclusions are bolstered by our in vitro find- reactivation of acetylcholinesterase inhibited ings and molecular modeling studies. by A-230 and A-242 surrogates, respectively. PO-07 PO-08 Synthesis and in vitro assessment of the re- Synthesis, modeling and in vitro assessment activation profile of clinical oximes on the of the reactivation profile of monocationic acetylcholinesterase model inhibited by A- isatin-oximes hibrids on the acetylcholin-230 and A-242 nerve agents’ surrogates esterase model inhibited by nerve agents’ Tanos Celmar Costa Franca 1, Samir surrogates Cavalcante2, Daniel Kitagawa2, Caio Borges2, Tanos Celmar Costa Franca 1, Amanda Moraes1, Marcelo Carneiro dos Santos1, Pedro Buitrago2, Samir Cavalcante2, Dipanjan Bhattacharyya3, Roberto Souza2, Antonio Luis Santos Lima1, Leandro Steven Laplate4, Joyce Sobreiro Francisco Diz de Bernardo2, Kamil Kuca3 Almeida1, Pat Forgione3 1Military Institute of Engineering, Rio de Janeiro, 1Military Institute of Engineering, Rio de Janeiro, Brazil Brazil 2Institute of Chemical, Biological, Radiological and 2Institute of Chemical, Biological, Radiological and Nuclear Defense (IDQBRN), Brazilian Army Techno-Nuclear Defense (IDQBRN), Brazil logical Center (CTEx), Rio de Janeiro, Brazil 3Department of Chemistry and Biochemistry, Con- 3University of Hradec Králové, Czech Republic cordia University, Montreal, Canada 4Institut National de la Recherche Scientifique The risk of use of toxic chemicals for unlaw- (INRS), Canada ful acts has been matter of concern for different governments and multilateral agencies. The risk of using toxic organophosphorus The Organisation for the Prohibition of Chem- (OP) compounds, potent cholinesterase inhib- ical Weapons (OPCW), which oversees the im- itors, as methods of warfare or for unlawful ac- plementation of the Chemical Weapons Con- tions is an ongoing concern. Despite the avail- vention (CWC), considering recent events in- ability of medical countermeasures, such as volving chemical warfare agents, has recently pyridinium oximes, these chemicals do not ex-expanded the CWC “Annex on Chemicals” hibit a broad spectrum and have poor phar-by including some organophosphorus com- macokinetic properties that prevent them pounds that are regarded as cholinesterase from crossing the blood-brain barrier, hinder-inhibitors, acting similarly to G- and V-series of ing the rescue of enzymatic activity in the nerve agents, the A-series. Hence, knowledge central nervous system. Therefore, research on on the activity of the pyridinium oximes, the novel and more effective reactivators is of ut- sole class of clinically available acetylcholin- most importance. Taking this into account, our esterase reactivators so far, is clearly war- Research Group at IDQBRN, the only OPCW ranted. Taking this into account, our Re- Designated Laboratory in the GRULAC Area search Group at IDQBRN, the only OPCW Des- (environmental samples, 2023-2024), in part-ignated Laboratory in the GRULAC Area (en- nership with the Military Institute of Engineer-vironmental samples, 2023-2024) synthesized ing (IME) – Rio de Janeiro/Brazil and Concor-surrogates for A-230 and A-242 nerve agents dia University – Montreal/Canada, synthes-and employed a modified Ellman’s assay in or- ized a series of monocationic isatin-oxime hyder to evaluate their ability to inhibit the acet- brids aiming to test their reactivation pro-ylcholinesterase from Electrophorus eel, and file towards acetylcholinesterase from Elec- if the clinically available antidotes are able trophorus eel (EeAChE) inhibited by surrog- 66 ates for nerve agents VX, A-230, and A-242 in Methodology comparison to pyridinium oximes already ap- Tridimensional structures of 12 proposed com- proved for clinical use, such as pralidoxime, pounds were constructed, had partial charges trimedoxime, and obidoxime. Simultaneously, and geometry optimization performed using molecular modeling studies will be performed software Spartan08. The systems of AChE on computational models of the AChE/OP inhibited by VX and A-242 were constructed complexes to investigate the reactivation per- from the crystallographic structure obtained formances of the hybrids. This comprehens- on Protein Data Bank, and validated with ive approach aims to develop more effect- Ramachandran graphics. Docking studies ive countermeasures against OP poisoning, of selected compounds on the enzyme were enhancing both safety and therapeutic out- performed using Molegro Virtual Docker and comes. The interdisciplinary nature of this results were evaluated by Near-Attack Con- research, combining chemical synthesis, bio- formation (NAC), total energy of interaction, chemical testing, and computational model- residues of interaction profiles criteria. ing, underscores its potential impact on public Results health and safety. Docking studies point to an neutral proposed compound 2 as better reactivator for AChE PO-09 inhibited by VX. These results suggest that Triazoles as potential reactivators of human the proposed modification in the structure acetylcholinesterase inhibited by the nerve can increase reactivation, considering that agents VX and Novichok A-242 penetration in BBB is better for uncharged Fernanda Pires1, Pedro Buitrago2, Tanos Celmar compounds. For A-242, charged compounds Costa Franca 1, Samir Cavalcante2, Joyce Sobreiro presented better results. All the ligands Francisco Diz de Almeida1 presented interaction with Ser203, a residue of 1Military Institute of Engineering, Rio de Janeiro, catalytic triad, and also with residues located Brazil on the peripheral anionic site (PAS). These 2Institute of Chemical, Biological, Radiological and results point that all the proposed compounds Nuclear Defense, Rio de Janeiro, Brazil present stabilization and affinity for the sites Introduction of the enzyme. Nerve agents are organophosphate com- pounds and irreversible inhibitors of the Conclusion enzyme acetylcholinesterase (AChE), con- For both systems, the most of the ligands sidered Chemical Warfare Agents. Traditional presented better results than reference oxime treatment includes quaternary pyridine oxime HI-6, according to all evaluation criteria. In compounds administration, but they are sequence, molecular dynamic calculations will not universal antidotes, because they are be performed to corroborate docking results. not efficient to all types of neurotoxicants2. Studies show that structural modifications PO-10 that increase lipophilicity can provide better Beyond carbamates: N-substituted permeation through the blood-brain barrier piperidine ureas as butyrylcholinesterase (BBB) and it may turn the compounds better inhibitors reactivators of AChE. In this work, 12 new Peter Mastnak-Sokolov 1, Urban Košak1, Dami- jan Knez1, Svit Ferjančič Benetik1, Anja Pišlar1, Xavier triazoles compounds that differ in lipophilicity Brazzolotto2, Stanislav Gobec1 were proposed and evaluated by molecular 1University of Ljubljana, Faculty of Pharmacy, Slov- docking studies on the enzyme inhibited by VX enia and Novichok A-242. 2Institut de Recherche Biomédicale des Armées, 67 France PO-11 Piperidine-carboxamides, -sulfonamides Alzheimer’s disease (AD) is the leading and -carbamates as selective bu- cause of dementia worldwide. The cognitive tyrylcholinesterase inhibitors decline in AD is associated with deficient Urban Košak 1, Damijan Knez1, Anže Meden1, Sicholinergic transmission in the central nervous mon Žakelj1, Jurij Trontelj1, Jure Stojan2, Maja Za-system (CNS). In healthy individuals, acet- košek Pipan4, Kinga Sałat3, Florian Nachon5, Xavier ylcholinesterase (AChE) is the primary enzyme Brazzolotto5, Gregor Majdič4, Stanislav Gobec1 that hydrolyzes acetylcholine (ACh), while 1University of Ljubljana, Faculty of Pharmacy, butyrylcholinesterase (BChE) plays a support- Slovenia ing role. However, as AD progresses, BChE 2Institute of Biochemistry, Faculty of Medicine, becomes the main enzyme deactivating ACh, University of Ljubljana, Ljubljana, Slovenia providing a rationale for BChE inhibitors to be 3Faculty of Pharmacy, Jagiellonian University Med- used as a symptomatic treatment of AD. ical College, Krakow, Poland Our group has developed several efficient 4Veterinary Faculty, University of Ljubljana, N-substituted piperidines as BChE inhibitors of Ljubljana, Slovenia different structural classes: from amides over 5Institut de Recherche Biomédicale des Armées, sulphonamides to carbamates. To further France investigate the structure-activity relationship (SAR) and expand the chemical space of our Introduction: Butyrylcholinesterase (BChE) CNS library, a series of N-alkyl piperidine ureas is a nonspecific cholinesterase involved in as inhibitors of BChE was designed, synthes- neurotransmission, (drug) metabolism, em-ized end evaluated. bryonic neural development, inflammation and cellular differentiation. This serine hy- A library of 95 compounds was prepared drolase is a potential drug target for treating and their activity against AChE and BChE Alzheimer’s disease (AD), multiple sclerosis and was assessed in vitro using Ellman’s test. Most substance abuse and selective BChE inhibitors compounds demonstrated good selectivity could thus be used to treat these diseases. for BChE over AChE. The most active com- Methodology: We first used virtual screen- pound showed an IC50 value of just under 100 ing to discover our hit compound, a novel nM, and its crystal structure was obtained. The 1,3-disubstituted piperidine-based nanomolar best compounds were tested in vitro for cyto- selective human (h)BChE inhibitor. To ex- toxicity and were found to be non-cytotoxic plore the chemical space around our hit com-on the BV-2 cell line. pound we utilized ligand-based and structure- based drug design for hit-to-lead optimiza- The SAR of N-substituted piperidine ureas tion and designed novel carboxamide, sulfon-was determined. Based on in vitro activity and amide and carbamate derivatives which we cytotoxicity profiles, a candidate for in vivo then synthesized and biologically evaluated. studies using the scopolamine model will be Results: We synthesized and evaluated close selected. Additionally, our in-house library of to 1000 compounds and obtained over 30 BChE inhibitors has been expanded, provid- crystal structures of inhibitor-hBChE coming a robust foundation for further activity plexes. The most potent piperidinecarbox- screening on various CNS targets in the quest amide (IC50 ≥ 1 nM) we developed is one of for multifunctional ligands as potential treat- the most potent BChE inhibitor ever reported. ments for neurodegenerative conditions like It improves memory, cognitive functions and AD. learning abilities of mice with scopolamine- induced AD-like symptoms. Our most prom- 68 ising piperidinesulfonamide (IC50 = 19 nM) a mild inhibitor of HssAChE. This compound also has procognitive effects in mice with could serve as a starting molecule for the scopolamine-induced symptoms of AD and design of new, more potent antidotes against improves cognitive functions and quality of A-agents. life of dogs suffering from canine cognitive dysfunction (CCD), which has many similar- PO-13 ities with AD in humans. We showed with Computational investigation of hardwickic our piperidinecarbamates that not all car- acid-derived amides using molecular dock-bamates are covalent BChE inhibitors and ing and prediction of ADME/Tox properties that we can produce multi-target-directed as potential inhibitors of cholinesterase en-ligands simply by placing the right group onto zymes the piperidine nitrogen. Rayssa Ribeiro1, Franco Leite2, Gessica Mendes1, Fernanda Georgia Figueiredo Taborda Barbosa 1, Conclusions: The piperidine-carboxamides, - Samir Cavalcante3, Marcelo Carneiro dos Santos1, sulfonamides and -carbamates we developed Tanos Celmar Costa França1, Valdir Veiga-Junior1 are some of the most potent and select- 1IME, Brazil ive BChE inhibitors ever reported and could 2UEFS, Brazil be used to treat and/or develop new drugs 3Institute of Chemical, Biological, Radiological and for treating CCD, Alzheimer’s disease, multiple Nuclear Defense (IDQBRN), Brazil sclerosis and substance abuse. Alzheimer’s disease (AD) is the leading PO-12 cause of dementia worldwide, characterized In vitro reactivation screening of A-234- by progressive cognitive decline. Pharmaco-inhibited human recombinant acetylcholin- logical treatment involves cholinesterase inesterase and butyrylcholinesterase hibitors targeting human acetylcholinesterase Martina Hrabinova (hAChE) and butyrylcholinesterase (hBuChE). University of Defence, Military Faculty of Medicine, Current drugs have low therapeutic efficacy, Czech Republic diverse side effects, and reduced patient ad- The A-series agent A-234 belongs to a new herence over time. Developing inhibitors that generation of nerve agents that directly inhibit target multiple targets could enhance thera- human acetylcholinesterase (HssAChE) and peutic efficacy at lower doses. Historically, butyrylcholinesterase (HssBChE). Although most marketed drugs were natural products A-agents were synthesized during the Cold or their analogs. Many drugs for various War, studies devoted to their therapeutic diseases feature amide bonds in their bio-countermeasures are nonexistent. Sympto- active compounds, a crucial group due to matic treatment is the mainstay of therapy; their role in forming peptide bonds in pro-however, there is no evidence causal anti- teins. Considering the importance of amide dotes are ineffective. Using a modified Ell- bonds in medicinal chemistry and the use of man’s method, we carried out extensive in natural products to synthesize new drugs, this vitro screening of oxime and non-oxime com- study investigated amides derived from hard- pounds to reactivate wickic acid amides as inhibitors of hAChE and A-234 inhibited by HssAChE and HssBChE hBuChE. Thirteen compounds were sketched in the search for potential antidotes. Only and optimized using GaussView 5.0.8 with the one compound showed limited recovery of Hs- DFT method, employing the B3LYP/6-31G basis sAChE activity, but none of them could react- set, to visualize molecular electrostatic poten-ivate HssBChE inhibited A-234. Subsequent ex- tial maps (MEP) and frontier orbitals (HOMO periments confirmed that compound is also and LUMO). In addition, pharmacokinetic and toxicological properties were studied using the 69 online servers PreADMET and SwissADME. Mo- A-234 was used in assassination attempts in lecular docking was performed against 3D England. These agents are considered more structures of acetylcholinesterase (AChE; PDB toxic than the nerve agents of the G and ID 4M0E) and butyrylcholinesterase (BuChE; V series, and as far as we know, there is no PDB ID 4BDS) prepared with the biopoly- reported antidote yet capable of reactivat-mer module in SYBYL-X 2.0. The results in- ing acetylcholinesterase (AChE) inhibited by dicated similar profiles in surface maps and them. Aiming to contribute to mitigating this molecular orbitals for the nitro substituent situation, our research group has developed group. Pharmacokinetic predictions demon-and used protocols to search for potential strated that all 13 hardwickic acid amide de- reactivators against AChE inhibited by sur- rivatives showed significant values for blood- rogates of the A-series nerve agents. Moving brain barrier (BBB) penetration, classifying forward in this direction, we investigated the them as active in the central nervous system reactivation profiles of oximes K027, K048, (CNS), a crucial pathway for AD treatment. In-K170, and K203 towards acetylcholinesterase termolecular interactions between the com- from Electrophorus eel (EeAChE) inhibited pounds and targets showed that benzyl amide by a surrogate of agente A-242. A modi- derivative J had the best binding energy at fied Ellman’s assay was employed to assess hAChE binding site (-10.1 kcal/mol) and hidroxy the reactivation rate in comparison to the amide derivative M for hBuChE binding site (- commercial oximes pralidoxime, trimedoxime, 9.7 kcal/mol). These findings can guide future and obidoxime. Simultaneously, molecular enzymatic assays of hardwickic acid amide modeling studies were performed on compu- derivatives as AChE and BuChE inhibitors. tational models of the AChE/A-242 complex to investigate the reactivation performances PO-14 of the oximes and correlate them to the Modeling studies and experimental evalu- experimental data. ation of the reactivation potential of oximes K027, K048, K170 and K203 against the nerve PO-15 agent A-242 Novichok A-232: basic knowledge of bio- Daniel de Jesus de Oliveira1, Fernanda Diniz chemical and toxicological properties Botelho1, Fernanda Georgia Figueiredo Taborda Daniel Jun, Martina Hrabinova, Lubica Muckova, Barbosa 1, Kamil Kuca2, Steven Laplate3, Samir Jakub Opravil, Dominik Krupka, Alzbeta Dlabkova Cavalcante4, Marcelo Carneiro dos Santos1, Tanos University of Defence, Military Faculty of Medicine, Celmar Costa França1 Department of Toxicology and Military Pharmacy, 1Military Institute of Engineering (IME), Brazil Czech Republic 2University of Hradec Králové (UHK), Czech Republic 3Institut National de la Recherche Scientifique In the last few years, CBRN threat research (INRS), Canada institutes have intensively studied the topic of 4Institute of Chemical, Biological, Radiological and fourth-generation nerve agents, code-named Nuclear Defense (IDQBRN), Brazil “A-agents”, called novichoks. There has never been an official unveiling of the chemical Despite being known since the 1970s, the structures of these extremely toxic organo- nerve agents belonging to the A-series re- phosphorus compounds. Vil Mirzayanov pub-mained out of the headlines and were not lished possible formulas of the substances in listed in Schedule 1 of the Annex on Chem-his book in 2009, but they do not correspond icals of the Chemical Weapons Convention to the structures published by Hoenig. (CWC) (https:/ /www.opcw.org/chemical- We evaluated novichok A-232 in our laboratory weapons-convention/annexes/annex-and compared its physical-chemical and bio- chemicals/schedule-1) until 2018, when agent 70 logical properties to well-known structures of between the oxygen atom of the oxime and nerve agents such as sarin or VX. Before start- the phosphorus atom of the nerve agent. Ading the experiments, we observed the stability ditionally, we computed the interaction ener-of the substance in an aqueous solution. In the gies, composed of short-range Coulombic en- next step, we determined the in vitro inhibition ergies and short-range Lennard-Jones ener- kinetics of human acetylcholinesterase and gies, between the oxime and the active site of butyrylcholinesterase. We also evaluated tox- the inhibited enzyme, between the oxime and icity in vivo on an animal model using WISTAR nerve agent, and between nerve agent and rats by estimating the LD50 value. Further, we the enzyme. The nerve agent-HssAChE com-assessed the ability of standard oxime nerve plex appears to be highly stable, forming sta-agent antidotes to reactivate both inhibited bilizing hydrophobic and electrostatic interac-cholinesterases. tions at the choline-binding site. Furthermore, These findings are preliminary data in our re- we calculated the partial charges on the oxy-search of fourth-generation nerve agents and gen atom of the oximate group to estimate should be useful for developing effective anti- the nucleophilicity strength of each oxime. dotes and possible subsequent therapy. This research has enhanced our under- standing of the behavior of individual react- PO-16 ivators in interaction with the inhibited en- Reactivation potency of GB, VX and A-234- zyme. Our research attempted to contrib-inhibited human recombinant acetylcholin- ute to explanation of right usage of in silico esterase in vitro and in silico methods in the discovery of new reactivators. Jakub Opravil, Jakub Fibigar, Zbyněk Večeřa Future studies combining molecular dynam- University of Defence, Military Faculty of Medicine, ics and quantum mechanics could provide a Department of Toxicology and Military Pharmacy, more comprehensive understanding of the re- Czech Republic activation mechanisms. The A-series agents, also known as PO-17 ’Novichoks’, are the latest generation of Exploring drug modality switch from in situ chemical warfare nerve agents (CWAs) that assembly to reversibility: reversible modu- directly inhibit human acetylcholinesterase lators of choline O-acetyltransferase activ- (HssAChE). Although these agents were syn- ity thesized during the Cold War, studies on Nina Forsgren, Frida Jonsson, Marcus Carls- their therapeutic countermeasures are rare. son, Robin Afshin Sander, Cecilia Springer Engdahl, Traditional cholinesterase reactivators have Daniel Wiktelius, Christopher Öberg, Fredrik Ekström proven ineffective against these agents. Swedish Defense Research Agency, Sweden In the search for potential antidotes, we conducted an in vitro reactivation assay and Research on the development of new in silico study to evaluate five commercially treatments of intoxications by organo- available oximes and a few compounds as phosphorus nerve agents (OPNAs) is largely new potential reactivators of recombinant Hs- focused on cholinergic receptors and acet-sAChE inhibited by nerve agents GB (sarin), VX ylcholinesterase, the enzyme that is the and A-234. We performed a reactivation as- primary target of OPNAs. Currently available say using modified Ellman’s protocol to ob-antidotes are limited in their clinical applicab- tain the percentage of reactivation potency. ility and new alternative targets are needed. Next, we utilized computational methods such Choline acetyltransferase (ChAT) catalyzes as molecular docking and molecular dynamics the synthesis of the neurotransmitter acet- simulations to calculate the average distance ylcholine in cholinergic neurons, and drugs 71 targeting ChAT could potentially be used for PO-18 broad-spectrum symptomatic treatment of Kinetic and structural evidence for specific intoxications caused by OPNAs. DMSO interference with reversible binding of uncharged bis-oximes to hAChE and their We have recently discovered that one of the reactivation kinetics of OP-hAChE. most studied classes of ChAT inhibitors, the Dora Kolić 1, Nichole Joiner2, Oksana Gerlits3, arylvinylpyridiniums (AVPs), act as substrate in Andrey Kovalevsky4, Zoran Radic2 1 an in situ synthesis in ChAT yielding an adduct Institute for Medical Research and Occupational that is the actual inhibitor of the enzyme. Health, Croatia 2 In this study, we used structure-based drug University of California San Diego, La Jolla, United design to change from the reactive inhibition States 3 mechanism of AVPs to compounds that act Department of Natural Sciences, Tennessee Wes- through a reversible inhibition mechanism. leyan University, Athens, United States 4 With the crystal structure of an in situ formed Neutron Scattering Division, Oak Ridge National inhibitor as a template, the objective was to Laboratory, Oak Ridge, United States design chemically stable, reversible inhibitors The structural basis of the inhibitory effect and investigate their potency, selectivity of the solvent dimethyl sulfoxide (DMSO) on and binding mode using a combination of the kinetics of reversible binding of novel biochemical, biophysical and structural tech- uncharged, heterocyclic bis-oximes to human niques. acetylcholinesterase (EC 3.1.1.7; hAChE) and on rates of reactivation of inactive organophos- By comparing the thermal stabilization phate (OP)-hAChE conjugates by bis-oximes provided by reactive inhibitors, we found a was studied. The reversible inhibition constant stabilization in ChAT that was not found in the of DMSO for hAChE in 0.1 M phosphate buffer related carnitine-O-acyltransferase (CrAT), pH 7.4 at 22 °C, was Ki= (0.257 ± 0.048) % (or 33 indicating that AVP-like scaffolds could po- ± 6 mM), competitive with acetylthiocholine tentially be used to gain selectivity for ChAT (K(S)=0.176 ± 0.062 mM), evaluated from the over CrAT. A set of reversible molecules was Hunter & Downs analysis. The Ki of the bis-synthesized and evaluated for inhibition of oxime LG-703 for hAChE was ˜ 4-fold larger in ChAT and CrAT activity. Furthermore, analysis 1% DMSO, consistent with direct competition of X-ray crystal structures of binary complexes between LG-703 and DMSO. The X-ray struc-with ChAT confirmed the expected binding ture of the LG-703*hAChE complex (PDB ID: site of the reversible inhibitors. 6U3P) shows both DMSO and LG-703 bound to individual hAChE monomers. In the chain To conclude, we have switched modality from A monomer, LG-703 extends along the gorge in situ assembly to reversibility and present axis from its opening to the base without insights to the selectivity and potency of the DMSO, and in the chain B monomer DMSO first class of ChAT inhibitors with a confirmed molecule binds at its base, without LG-703. reversible mechanism, offering valuable in- Since co-crystallization of the LG-703*hAChE sights for future efforts of developing ChAT was in ˜ 2% (or ˜0.28 M) DMSO (originating inhibitors. from the LG-703 stock) and with ˜1.5 mM LG- 703 final concentration, both small molecules were present at a similar excess over their corresponding Ki values for hAChE (7.8-fold for DMSO and 6.5-fold for LG-703). The formation of two different complexes (DMSO*hAChE 72 and LG-703*hAChE) of otherwise competing provements of the oxime and the design of ligands, in the same crystal, is thus consistent more potent reactivators. The liver is the ma-with competitive kinetics of their reversible jor organ responsible for detoxification, so we inhibition of hAChE. Furthermore, rates of evaluated the effect of the investigated or-reactivation of paraoxon-inhibited hAChE ganophosphates on cell death in a hepatic (POX-hAChE) were reduced 2 – 3-fold in 1% cell line. Some of the tested compounds ex- DMSO, consistent with observation of DMSO hibited toxic effects independently of their ef- molecules in POX-hAChE structures obstruct- fects on cholinergic transmission. Overall, this ing the access to the conjugated P atom. study contributed to a better understanding of these poorly characterized phosphoramid- This research was supported by the Counter- ates by determining their kinetic and cyto-ACT Program, NIH Office of the Director (OD), toxic properties. Although the A-series posed and the NINDS, [Grant Numbers 1U01NS083451, challenges for reactivation, important insights 1R21NS120839-01A2 and 1R21NS120884-01A2], were gained, and the reported results will con-and the UCSD AS award BG114128. tribute to and accelerate the progress in de- veloping therapy. PO-19 Acknowledgment: This work was supported Outlining the A-series of organophosphorus by the Croatian Science Foundation (IP-2022-compounds – cholinesterase inhibition, re- 10-6685) and the European Regional Develop-activation, cytotoxicity ment Fund (KK.01.1.1.02.0007). Nikolina Maček Hrvat, Dora Kolić, Tena Cadez, Goran Šinko, Zrinka Kovarik PO-20 Institute for Medical Research and Occupational Evaluation of resveratrol compounds as Health, Croatia therapeutics in organophosphorus poison- ing The A-series has been known for quite some Tena Cadez, Milena Mlakić, Nikolina Maček Hr- time now but gained widespread attention vat, Irena Škorić, Zrinka Kovarik after the UK incident in 2018. These phosphor- 1Institute for Medical Research and Occupational amidates inhibit cholinesterases (ChE), poten- Health, Croatia tially leading to death, but little else is known 2Faculty of Chemical Engineering and Technology, about them. This study aimed to investigate University of Zagreb, Croatia the rate of ChE inhibition by five OP compounds from the A-series and compare it to The dephosphorylation of acetylcholin- the G-series and VX. The most potent inhibit- esterase (AChE), a crucial enzyme in the hy-ors were A-230, A-232, and A-234, with a phos- drolysis of the neurotransmitter acetylcholine, phorylation rate comparable to cyclosarin remains insufficiently addressed by current and soman. We also tested standard oximes, medical countermeasures, especially when 2-PAM, HI-6, obidoxime, TMB-4, and MMB-4, inhibited by organophosphorus (OP) com-as reactivators of ChE inhibited by the A-series pounds such as A-series agents. The ability of compounds. Our results identified MMB-4 and butyrylcholinesterase (BChE) to bind OP com- HI-6 oxime as the most promising reactivat- pounds lowering their concentration in the cir-ors, especially of the A230-AChE conjugate, culation and preventing the inhibition of AChE restoring AChE activity in 2 and 4 hours, re- represents a basis for potential therapy for OP spectively. Furthermore, we modeled a near- poisoning. This approach calls for the devel-attack conformation of oxime MMB-4 and opment of effective oximes to address pos- HI-6 in phosphorylated acetylcholinesterase, itive outcomes in circulation for reactivating providing guidelines for further structural im-inhibited BChE. In our study, we investigated 73 heterostilbene derivatives as potential drugs Oxime antidotes are used in standard for OP poisoning focusing on their ability to medical treatment of organophosphate (OP) reactivate cholinesterases, mostly circulating nerve agent and pesticide poisoning, but the BChE in blood. These compounds were de-search for novel antidotes with the capacity to signed as analogs of resveratrol, a polyphenol cross the blood-brain barrier (BBB) is an on- known for its antioxidant, anti-inflammatory, going pursuit. The cationic pyridinium oximes and neuroprotective properties, which have do not cross the BBB in sufficient concen-shown promise in treating various neural dis- trations for reactivation of AChE in the brain orders. Oximes were screened for oxime- due to their permanent charge. Cholinergic dependent reactivation of sarin-, cyclosarin- receptor overstimulation can therefore trig-inhibited human BChE as well as BChE inhib- ger neuroinflammation and lead to perman- ited with A-series agents. We also estimated ent brain damage. In this work we analysed the AChE and BChE binding affinity for hetero- antidotal potency of a new class of uncharged stilbene oximes in terms of the oxime-enzyme but ionizable bis-oxime antidotes, which ex-dissociation constants. Among the tested hibited efficient in vitro reactivation of both compounds, 10 showed potential as selective AChE and BChE inhibited by sarin, cyclosarin, inhibitors of cholinesterase enzymes, with dis- VX, paraoxon, and phosphoroamidate insect-sociation constants (Ki) ranging from 7 to 68 icide fenamiphos. All three tested bis-oximes �M. A comprehensive in vitro analysis of enzyme were effectively comparable to 2-PAM, and kinetics, enabled detection of possible more the reversible inhibition constants (Ki) of bis-efficient reactivators of phosphorylated BChE oxime-ChE complexes were 150 to 800 �M. compared to standard oximes. Encouragingly, Cytotoxicity assay showed that bis-oximes did some compounds achieved up to 80% recov- not affect the viability of neural (SH-SY5Y) and ery of inhibited enzyme activity. Furthermore, hepatic (HepG2) cell lines up to 1.6 mM con-we evaluated the effects of these oximes on centration within 24 hours. Analysis of cholin- cell death in hepatic cell lines. Remarkably, esterase activity in blood and brain of mice oximes did not induce significant toxicity in exposed to sarin and bis-oxime demonstrated cells within 24 hours suggesting their potential 30-40% recovery of ChE activity within 30 for further investigation and development as minutes after poisoning. We further invest- therapeutic for OP neurotoxicity. igated bis-oxime neuroprotection capacity in Acknowledgments: This research was sup- the brain of mice exposed to a sublethal dose ported by the University of Zagreb, the Croa- of sarin w/o bis-oxime by assessing the change tian Science Foundation (IP-2022-10-6685), of expression in neuroinflammation markers the European Regional Development Fund GFAP (glial fibrillary acidic protein) and IBA1 (KK.01.1.1.02.0007), and the European Union – (ionized calcium-binding adapter molecule 1). Next Generation EU (BioMolTox project). The results showed that both GFAP and IBA1 levels were in the control range and signific- PO-21 antly lower in mice which received treatment Neuroprotective role of CNS-active un- compared to mice exposed to sarin only. The charged bis-oxime antidotes in mice ex- results of this study provide evidence for in vivo posed to organophosphate compounds effectiveness, low toxicity and good neuropro- Dora Kolić 1, Nikolina Maček Hrvat1, Zrinka tective efficacy in mice, and can serve as basis Kovarik1, Zoran Radic2 for further in vivo research. 1Institute for Medical Research and Occupational This research was supported by the Coun- Health, Croatia terACT Program, NIH OD, and the NINDS 2University of California San Diego, United States [Grant Number 1R21NS120884-01A2]. 74 PO-22 PO-23 Synthesis of broad-spectrum antidotes to Bis-pyridinium mono-aldoxime K203: a organophosphorus neurotoxins promising prophylactic cholinesterase re- Estelle Beaupparain 1, Karine Porte1, Pierre-Yves activator for organophosphate poisoning. Renard1, Ludovic Jean2 Syed Nurulain 1, Zarish Riaz2, Huba Kalasz3, Sajid 1Laboratoire COBRA, Université de Rouen, France Mehmood1, Kamil Kuca4 2Laboratoire CiTCoM, Université Paris Cité, France 1Department of Biosciences, Grand Asian University Sialkot, Pakistan Organophosphorous nerve agents (NOPs) 2COMSATS University Islamabad, Pakistan are phosphorus containing compounds that 3Semmelweis University, Budapest, Hungary can be found as pesticides or chemical war- 4University of Hradec Králové, Czech Republic fare agents. In case of intoxication, NOPs will phosphylate the catalytic serine of acet- Introduction ylcholinesterase (AChE), an enzyme that plays Oximes like acetylcholinesterase reactivators a major role during the nerve impulse transmis- (AChR) are used therapeutically to revive sion in the brain and at neuromuscular junc- inhibited acetylcholinesterase (AChE) by tions, which ends up with an irreversible in- organophosphorus compounds (OPs). OPs hibition of this enzyme. After AChE inhibi- are potent inhibitors of AChE, constituting tion, acetylcholine neurotransmitter (ACh) re- a wide variety of structurally different com-leased at cholinergic synapses are no longer pounds with lethality ranging from extremely eliminated, resulting in its accumulation in poisonous like warfare chemicals to extreme, the synaptic cleft, and thus an overstimula- moderate and mild poisonous OPs used as tion of post-synaptic receptors (muscarinic pesticides. With increasing threats of terror-and nicotinic ACh Receptors and a cholinergic ist activities and wartime scenarios, finding crisis, with deadly consequences. As medical an effective pretreatment compound for countermeasures, research is focusing on the organophosphorus poisoning is of utmost synthesis of new compounds called reactivat- importance. Currently, pyridostigmine is the ors of AChE as antidotes against organophos- only FDA-approved drug in the USA for pro-phorus nerve agents through dephosphylation phylactic treatment, but it has shown limited of the serine residue, associated with an anti- efficacy and significant side effects. convulsant and a muscarinic AChR antagonist. Methodology Along the years, research groups have tested This study aimed to identify a compound that a large variety of compounds with different could be used effectively and efficiently as a functions and structures. Our new strategy pretreatment for organophosphate poisoning is to obtain a broad-spectrum antidote with and tentative attack. An in vitro study on two different targets and roles within one mo- human blood was conducted using oximes K- lecule. On one hand, our antidote aims to 203, K-027, and pralidoxime, comparing them dephospylate AChE leading to elimination of with the pretreatment drugs pyridostigmine abnormal concentration of ACh in the syn- and eserine against the highly toxic organo-aptic gap. On the other hand, the molecule phosphorus compound paraoxon-ethyl (POX). should also act as antagonist of post-synaptic The oximes and drugs were administered at nicotinic AChR in order to limit the continuous 1/05, 1/10, and 1/20 of their IC50 values at 0, nerve impulse and limit the cholinergic crisis. 15, 20, and 30 minutes before the application of the IC70 concentration of POX. Results The results demonstrated that K-203 is effic- acious, and more effective than K-027 and 75 pralidoxime oxime, followed by the standard zymatic cleavage, regeneration with fluoride compounds pyridostigmine and physostig- and purification using SPE. The samples are mine (eserine) as prophylactic treatment. It then separated via high performance liquid is concluded that K-203 could be a promising chromatography and detected using mass pretreatment drug for organophosphate and spectrometry via total ion current and parallel organophosphonate poisoning. Further re- reaction monitoring using orbitrap mass spec-search on organophosphonates is suggested. trometer. Conclusion Up – to-date we were able to detect A230, K-203 may be a candidate drug for pre- A232 and A234 as tyrosine adducts broken treatment against organophosphorus/OP from albumin and their acidic remnants, which warfare chemicals. However, efficacy against could be found for example in urine. On the different OP warfare chemicals is suggested. other hand, when trying to regenerate com- pounds from plasmatic butyrylcholinesterase PO-24 with fluorine and derivatize them, mixed results A-agents: more resistant than expected? were obtained. Nevertheless, we were able to Biomarker detection in biological matrices detect original compounds. The preparation Lukáš Prchal 1, Alzbeta Dlabkova2 of samples and their regeneration afterwards 1Biomedical Research Centre, University Hospital showed distinct differences between A230 and Hradec Králové, Czech Republic A232 in reactivity and regenerability while A232 2University of Defence, Military Faculty of Medicine, regeneration went as expected, A230 binded Department of Toxicology and Military Pharmacy, to plasma rather slowly, however proved hard Czech Republic to regenerate. When almost all countries adhere to the This project is supported by Ministry of the In- Chemical weapons convention and have their terior (OPSEC VK01020171). stockpiles destroyed, the phantom of the Cold war started to emerge in what became to be Poster Session known as A-agents family or „Novichoks“, very potent, irreversible, acetylcholine esterase in- Tuesday - Poster Session hibitors. They came from dangerous yet al- Sep 17, 11:00 - 12:35 most forgotten chemical curiosity to the most watched theme in the late 20‘ of the 21st PO-25 century when several people were poisoned PON1 gene polymorphisms and inflammat- by compounds that probably came from this ory markers in organophosphate pesticides family. With this renewed interest the meth- cohorts from Cameroon and Pakistan ods of their detection as well as their biomark-Leonel Javeres Mbah Ntepe ers and their effects in the body and environ- Institute of Medical Research and Medicinal Plant, ment became interesting research topic be-Cameroon cause the determination of the correct sub- Background and objectives: The detri- stance can be vital in administering the right mental e�ects of organophosphates (OPs) on treatment. human health are thought to be of systemic, In our work we focus on the detection of i.e., irreversible inhibition of acetylcholin-residues, biomarkers and unreacted parent esterase (AChE) at nerve synapses. However, molecules of several A-agents after exposure several studies have shown that AChE inhibi-in rats as well as human plasma. To separ- tion alone cannot explain all the toxicological ate the compounds from the biological matrix manifestations in prolonged exposure to OPs. several methods are employed including en- Predisposition to population heterogeneity 76 and irregularities in various biochemicals like PO-26 paraoxonases and inflammatory biochemic- Screening and Characterization of Inhibit-als are the possible affects of OPs long term ors for the Recombinant Variant of Paraox-exposure that may lead to sequels of diseases onase 1 and are less addressed in literature. The study Neja Žnidaršič, Janez Smerkolj, Jure Stojan, Alwas aimed to assess the cholinergic enzymes joša Bavec, Marko Goličnik (AChE and BChE), PON1, and inflammatory University of Ljubljana, Faculty of Medicine, Institute markers (IL1 β, IL6, TNF α, CRP, Apo AI, Apo B) of Biochemistry and Molecular Genetics, Slovenia and determine the toxicogenetics association of PON1 gene to chronically OPs exposed Paraoxonase 1 (PON1) is a serum enzyme groups from Pakistan and Cameroon. primarily associated with high-density lipo- Materials and methods: AChE, BChE and proteins (HDL). While its physiological func- PON1 were measured by colorimetric method tion is not completely understood, PON1 dis-using spectrophotometry. Inflammatory plays great substrate promiscuity, acting on markers were determined by Elisa assay. PCR- lactones, aryl esters, and organophosphate restriction fragment length polymorphism paraoxon. Additionally, PON1 plays a signific- (PCR- RFLP) using salting out method was ant role in protecting against oxidative stress employed for SNP genotyping. by contributing to the antioxidant proper- Results: The results revealed the signific- ties of HDL and potentially preventing ath-ant (p ≤ 0.05) inhibition of cholinergic en- erosclerosis and several other diseases. One zymes PON 1 was found to be 6.91 ng/mL±1.03 of the best-known inhibitors of PON1 is 2-and 2.84 ng/mL±1.40 (mean ±SD) in Pakistan hydroxyquinoline (2HQ), but searching under-and Cameroon groups respectively. IL6, TNF α, standing of the inhibitory mechanisms of PON1 CRP were increased and Apo AI was less while is crucial to interpreting its physiological roles, Apo B was increased in OP exposed groups particularly its protective functions against ox-in both population groups. SNPs analysis of idative stress and its potential role in prevent-PON1 showed significant differences in allelic ing atherosclerosis. By identifying and charac- and genotype frequencies of OPs exposed terizing other inhibitory compounds, we could and non-exposed groups. better understand how PON1 activity is af- Conclusions: PON1 was noticeably less fected within the human body. in Cameroonian than Pakistani, albeit both This study aims to screen 20 organic mo- groups have significant decrease in PON1 lecules for their inhibitory effects on the re- actity. In addition, the study concludes that combinant G2E6 variant of paraoxonase 1 (re- OPs induce low grade inflammation, an ae- PON1). We aim to enhance the understand- tiology of many diseases. Selected PON1 ing of rePON1 activity and inhibition mechan- SNPs analysis showed a significant toxicogen- isms, providing further valuable insights into etics association with OPs exposure marker the PON1 physiological roles. enzymes. The results of this study may help in Time-courses of product formation (pro- regulation of usage of OPs anticholinesterases gress curves) were measured spectrophoto-in different populations. The study will further metrically at 270 nm for the enzyme-catalyzed open new avenues in toxicogenetic and ex- hydrolysis of dihydrocoumarin (DHC) by re-ploration of SNPs based strategies on organ- PON1, and kinetic models for inhibited enzyme ophosphate intoxication. reactions were fitted to the progress curves by using ENZO software. This method provided quantitative data on the inhibitory effects of the various molecules on rePON1, allowing us 77 to assess enzyme activity and inhibitor im- (rePON1). By comparing the binding affinit- pact. For inhibitors with high absorbance at ies, thermodynamic interactions, and kinetic 270 nm, we used thio-phenylacetate as sub- activities of rePON1 in the presence of these strate which coupled with Ellman’s reagent en- ions, we seek to gain deeper insights into the abled time-product measurements in visible specific contributions of calcium (II) ions to the spectrum at 412 nm. enzyme’s functionality. The screening of 20 various organic mo- We conducted a comprehensive kinetic lecules revealed several inhibitors of rePON1 analysis and Isothermal Titration Calorimetry activity. These findings can enhance our (ITC) to examine the interactions between re- understanding of the promiscuous nature of PON1 and various lanthanide (III) ions. The PON1. The kinetic analysis provided quantitat- kinetic analysis utilized dihydrocoumarin as a ive data on the inhibitory effects and helped substrate to assess the inhibitory effects of to determine the mechanisms of inhibition for lanthanide ions on rePON1 activity. The bind-the studied compounds. ing affinities and thermodynamic parameters of interactions between rePON1, calcium, and PO-27 the lanthanide ions were measured by using Exploring the Impact of Lanthanide (III) Ions ITC. on the Function of Paraoxonase 1 (PON1) Our study revealed distinct interactions Janez Smerkolj, Jure Stojan, Aljoša Bavec, Marko between rePON1 and each lanthanide ion. The Goličnik results indicated that all lanthanide ions can University of Ljubljana, Faculty of Medicine, Institute reversibly replace both calcium (II) ions from of Biochemistry and Molecular Genetics, Slovenia the protein backbone of rePON1 as the process Paraoxonase 1 (PON1) is a metallohydro- is driven spontaneously, and a reversible loss of lase that enables to protect the human enzymatic activity was observed. against oxidative stress and detoxifies harm- ful organophosphates, such as pesticides and PO-28 PON1 plasma activity in the aftermath of nerve agents. It is primarily associated with bariatric metabolic surgery: the benefits of high-density lipoprotein (HDL) in the blood- investigating more than one substrate stream, enhancing HDL’s antioxidant and Boštjan Petrič1, Aljoša Bavec1, Tadeja Pintar2, anti-atherosclerotic properties. PON1’s activ- Živa Mesarič 1 ity varies significantly among individuals due 1Institute of Biochemistry and Molecular Genetics, to genetic polymorphisms affecting its expres- Faculty of Medicine, University of Ljubljana, Slove-sion and functionality. The enzyme’s activ- nia, Slovenia ity relies on two calcium (II) ions for structural 2Department of Abdominal Surgery, University integrity and catalytic functions. Hence, the Medical Centre, Ljubljana, Slovenia, Slovenia specific roles of calcium (II) ions in PON1’s func- tion are essential for understanding the en- Previous research has indicated a connec- zyme’s biological mechanisms. In this con- tion between PON1 and both obesity and type text, the lanthanide (III) ions, which share sim- 2 diabetes, but most studies only investig- ilar ionic radii and coordination chemistry with ated PON1 with a single substrate, and no calcium (II) ions, offer a unique opportunity to study investigated kinetic parameters. Bari-probe these roles and explore potential mod- atric metabolic surgery (BMS) is used to treat ulation of PON1 activity. severe obesity and usually results in signific- This study aims to investigate the effects ant weight loss as a result of reduced calorie of the entire line of lanthanide (III) ions on the consumption and metabolic adaptation. We structure and function of recombinant PON1 wanted to investigate a BMS-related change 78 in patients’ weight would correlate with a are potent neurotoxicants, acting by rapid change in the patients’ PON1 activity. For a inhibition of the acetylcholinesterase (AChE) sample of 69 patients who underwent BMS, we pool of the intoxicated individual, ultimately acquired blood samples on the day of surgery leading to death. The threat posed by these and used three different substrates to meas- chemicals has recently attracted renewed ature three parameters of PON1 activity in blood tention after nearly two decades of silence. plasma: rate of hydrolysis (substrates DHC, PA, Not only sarin was used during the Syrian and PX), Km and Vmax (substrates DHC and conflict in 2013 and 2017, but a new genera- PA). We showed that the rate of hydrolysis for tion, a.k.a Novichok or A-series nerve agents, PX does not correlate with those for PA and emerged in 2018 and 2020, like a remnant of DHC, but it does correlate with Km for both, the past. which stresses the importance of measuring Even though medical countermeasures activity for at least two substrates and cal- have been a part of the scientific landscape culating kinetics in similar studies. For a sub- for several decades now, no technology has sample of 19 patients, we acquired follow-up emerged as a credible alternative to the blood samples and clinical data and com-historically available emergency treatment pared PON1 and clinical parameters between (i.e combination of atropine, an oxime, and an operation and follow-up. Of the 7 enzyme-anticonvulsant) for both military and civilians. activity parameters, all correlated significantly To prevent AChE inhibition in vivo, inject- between operation and follow-up; the correl- able enzymes were considered as CWNA bio-ation was especially strong for PX rate of hy- scavengers and constitute an already well-drolysis (Pearson CC = 0.846, p = 3.62×10-5). We studied field. Despite numerous engineering calculated the difference between the values efforts on several target candidates, catalytic at both time-points for each parameter and scavengers are not yet part of the therapeutic checked the correlations between these dif- arsenal, but their development is still ongoing, ferences: notably, the only significant correl- with promising candidates such as PTEs. ations were between all three rates of hydro- In this context, several mutants of hu- lysis, and there was no significant correlation man paraoxonase-1 (PON-1) were assessed as of any PON1 parameter with changes in BMI or catalytic bioscavenger candidates against A- weight. Our research thus suggests that PON1 230, A-232 & A-234. The enzymes consist of activity changes in the aftermath of BMS in a variants of the human PON-1, fused to a half- way that is not clearly connected to weight life extension partner (HLEP) to enhance their loss. in vivo pharmacokinetics, as previously repor- ted in a prophylaxis study against pesticides. PO-29 Here, we describe the evaluation of these re- Recombinant human paraoxonase-1 vari- combinant PON-1 to hydrolyze the Novichok ants depict hydrolyzing capabilities of A- agents in vitro, using butyrylcholinesterase inseries nerve agents in vitro hibition as a reporter in complement to LC-MS Janek Bzdrenga 1, Prakash Khandave2, Thomas analysis to directly assess compounds hydro- Soirot1, Nicolas Belverge1, Nicolas Taudon1, Florian lysis and their degradation products. Nachon1, Xavier Brazzolotto1, Abhay H. Pande2 1Institut de Recherche Biomédicale des armées, France 2National Institute of Pharmaceutical Education & Research, India Chemical warfare nerve agents (CWNA) 79 PO-30 sequence (DAEHK)) show hydrolysis levels (120 Copper-dependent stereoselective hydro- �M) of HDCP very similar to its respective wild lysis of O-hexyl O-2,5-dichlorophenyl phos- albumin. This same lack of activating effect phoramidate by recombinant serum albu- of Cu2+-dependent HDCPase activity was mins also observed between the rHSA (DAEHK) with Laura Ramirez Gonzalez glutamic acid inserted in position 3 and itś Universidad Autonoma del Estado de Morelos, Mex- corresponding HSA wild protein (DAHK). Both ico proteins showed 12-15% of HDCPase activity. These results suggest that the N-terminal A-esterases are enzymes that hydrolyze sequence of these animal albumins does not organophosphorus (OP) compounds. These participate in the A-estrasic catalytic center enzymes are metalloproteins that are iden- of albumin. tified in bacteria and mammalian tissues including human serum. In our laboratory, a PO-31 Cu2+-dependent hydrolyzing activity of chiral Update of ESTHER, the database and server OPs, such as O-hexyl O-2,5-dichlorophenyl dedicated to the analysis of protein and phosphoramidate (HDCP) and O-ethyl O-nucleic acid sequences within the super- 2,4,5-trichlorophenyl ethylphosphonothioate family of cholinesterase relative (trichloronate) has been identified in serum Arnaud Chatonnet 1, Zhou Yu2, Nicolas Roche2, albumin of birds and mammals. On the other Pascale Marchot3 hand, it is well known that albumin is the 1Université de Montpellier, Montpellier, France protein that transports 15% of the copper in 2Terre Nourricière, Montpellier, France the bloodstream to the tissues and organs in 3CNRS/Aix-Marseille Univ., Faculté des Sciences - vertebrate animals. Physicochemical studies Campus Luminy, Marseille, France have revealed that the N-terminal site of the serum albumin of these animals is one of For IT security reasons, the ESTHER data- the sites with the highest affinity for copper. base, which is dedicated to ESTerases and al-It is different with respect to its sequence pha/beta Hydrolase Enzymes and Relatives among mammals, birds and reptiles. There- headed by the cholinesterases, switched to a fore, has been suggested the participation new operating system in April 2024. After 30 of this sequence in the A-esterase catalytic years of using the ACEDB database system, site of this protein. In order to identify the we had to start again from scratch to warrant involvement of the N-terminal sequence the quality of the transition. Thanks to the ro- of albumin as the catalytic site responsible bustness of the model and data structure refor the Cu2+-dependent A-esterase activity covered as text files, the procedure was relat- (HDCPase and trichloronatase), an aliquot of ively smooth, and most of the data and tools 400 �M racemic HDCP was incubated with 200 are now operational again. �g of pentapeptide Ala-Glu-His-Lys (DAEHK) ESTHER is growing at fast pace: nowadays it and the tetrapeptide Asp-Ala-His-Lys (DAHK), contains ca. 70.000 gene-proteins, grouped peptides derived from the N-terminal site in 247 families. In 2024, the number of 3D of CSA and HSA, respectively, as well as re- structures available in the RCSB-PDB reached combinant albumins of CSA and HSA. The 3000, corresponding to 739 distinct proteins HDCP hydrolysis was quantified by UV-Vis validated as superfamily members. There are spectrophotometry and chiral chromato- 143 families in which at least one 3D struc-graphy. The results show a lack of hydrolysis of ture is known, and new families are regu-HDCP by the peptides, while the recombinant larly added to the superfamily. For the hu-chicken albumin (truncated in the N-terminal man genome, 120 genes belong to 59 fam-80 ilies described in the database. A 3D struc- molecular docking study. Triazole scores ture is available for only 51 proteins encoded were compared to the scores obtained for by these genes. Mutations associated with anticholinesterases. Additionally, the physico- a disease are found in 35 of human genes. chemical properties of the tested fungicides The 768 published mutations in the genes of were compared to the properties of CNS Block C proteins (Carboxylesterase, Cholester- active drugs to estimate the blood-brain olesterase, Cholinesterase, Neuroligin, Thyro- barrier permeability. As mefentrifluconazole globulin…) correspond to 341 residue positions is commercially available individually or in a in the protein sequence of Torpedo (Tetron- binary fungicidal mixture with the carbamate arce californica) acetylcholinesterase used as pyraclostrobin (Ravycare®), a kinetic study a reference. 112 distinct mutations in acet- was performed on both compounds and ylcholinesterase or carboxylesterase genes showed that mefentrifluconazole reversibly where described in insecticide-resistant pop- inhibited human acetylcholinesterase (AChE) ulations of 43 arthropod species. and butyrylcholinesterase (BChE) with a 7-fold The new version of the web server with im- higher potency toward AChE (Ki = 101 ± 19 µ M), proved layout and new tools is available at while pyraclostrobin inhibited AChE and BChE http:/ /bioweb.supagro.inrae.fr/ESTHER. Feed- progressively with a rate constant of ki = 6.6 backs from the users on this rejuvenated ES- · 103 M-1 min-1 and ki = 9.2 · 103 M-1 min-1, THER will be most welcome. Acknowledge- respectively. The approach in which in silico ment: INRAE Phase department. activity/property evaluation and a kinetic study are combined may help in the future de- PO-32 velopment of biologically active compounds In silico evaluation of the anticholinesterase where in silico methods can be used to test the activity of triazole fungicides possible unwanted biological activity toward Goran Šinko non-primary biological targets, already char- Institute for Medical Research and Occupational acterized by drug design and QSAR regression Health, Zagreb, Croatia models including ChEs. Triazoles are compounds with various Acknowledgment: This work was supported biological activities, including fungicidal ac- by the European Regional Development Fund tion. Triazoles are currently the most used (KK.01.1.1.02.0007), Next Generation EU (Bio- class of antifungals in agriculture and medi- MolTox project) cine. Unwanted cross-reactivity with human PO-33 cytochrome P450s enzymes forced the devel- Cholinesterase monitoring for nerve agent opment of safer azoles and it was shown that exposure voriconazole inhibited fewer human P450s. Nick Coe1, Jennifer Dawson 1, Georgia Smith2, Mefentrifluconazole (Ravystar®) is the first Sarah Goodchild1 isopropanol triazole fungicide, developed 1Dstl, United Kingdom to overcome fungus resistance in plant dis- 2Imperial College London, United Kingdom ease management, which like voriconazole inhibits about 1 800-fold fewer human P450s. In our organisation, research scientists work Evaluation of the anticholinesterase effect with organophosphorus (OP) compounds such of the triazole fungicide mefentrifluconazol,e as nerve agents for defensive purposes, for together with structurally related fungi- example for the development of medical cides was performed using in silico methods countermeasures. Blood cholinesterase (ChE) including CHARMm-based scoring func- activity measurements have been monitored tion Cdocker interaction energy used in a in numerous individuals for over 40 years. This 81 method is particularly suited for routine mon-sue distribution, or inhibition sensitivity, itoring in laboratory situations because col- two human cholinesterases can be distin- lection of an effective baseline measurement guished: acetylcholinesterase (AChE) and is possible. In a military setting where ex- butyrylcholinesterase (BChE). Many com-posure risks are greater, continuous wear- pounds interact with human cholinesterases. able monitoring could be of significant bene- Organophosphorus nerve agents (e.g. VX, fit. This would enable pre-symptomatic expos- sarin, or tabun) and pesticides (e.g. car- ure warning and subsequently early adminis- bofuran or bendiocarb) present significant tration of medical countermeasures in addi-threats to civilian and military populations tion to supporting the medical management [1]. On the other hand, some compounds are of prognosis by monitoring recovery of ChE irreplaceable helpers in alleviating the symp- and treatment effectiveness. These are either toms of neurodegenerative disorders such as not possible or are difficult and labour intens- Alzheimer’s disease. Precise determination ive with the current available in vitro methods. of the mechanism of interaction between Our expertise in measuring ChE activity is be- these substances and cholinesterases is thus ing used to help develop a real-time mi- of fundamental importance for civilian and crofluidic assay with collaborators at Imper- military use [2]. ial College London, UK. This system uses mi- The main challenge when working with en- crodialysis sampling to measure choline res- zymes is their cost, limited stability, possible ulting from acetylcholine hydrolysis, detected interference with analytical methods, and inusing microelectrode biosensors housed in a ability to reuse. In this project, human recom- 3D-printed chip. Although still in proof-of- binant AChE and BChE were non-covalently concept phase, we have shown that changes immobilized on the surface of porous and in ChE activity can be continuously detec- non-porous magnetic microparticles to over-ted and quantified following OP exposure by come these limitations. By attaching ChEs measuring the pick-up of choline via microdia- to magnetic solid support, a powerful, cost-lysis. effective, stable, recyclable, and easy-to-use These assays and technologies provide op- tool for studying enzyme kinetics, and inhibi- tions to those at risk of exposure to OPs in tion mechanisms has emerged [3]. Moreover, the military setting which can be utilised at the resulting immobilized biocatalysts were various points in the medical management used to investigate the reactivation potential chain, from mobile units with limited options of a series of novel oximes. By utilizing immob-for equipment, to field hospitals back at base. ilized enzymes, we were able to observe pure reactivation potential without the confound- PO-34 ing effect of oxime inhibition. Additionally, Immobilization of cholinesterases on mag- the immobilized ChEs facilitated the study of netic microparticles for enhanced stability phosphorylated-oxime intermediates, which and biosensing applications are crucial byproducts of the reactivation Rudolf Andrys 1, Charline Monnier1, Veronika reaction. This work was supported by the Mickova1, Louise Nemery1, Evica Antonijevic2, Kamil Czech Science Foundation (no. GA22-14568S), Musilek1, Lucie Zemanova1 International mobility for research activities 1University of Hradec Kralove, Department of of the University of Hradec Kralove II (no. Chemistry, Hradec Kralove, Czech Republic CZ.02.2.69/0.0/0.0/18_053/0017841), Univer- 2University of Belgrade, Department of Toxicology , sity of Hradec Kralove (Faculty of Science, Serbia no. SV2106-2023, Excellence project PrF UHK According to substrate specificity, tis- 2201/2024-2025. 82 PO-35 above 1000-fold selectivity was designed and Highly potent and selective butyrylcholin- synthesized. BChE inhibitors exhibited neuro-esterase inhibitors for cognitive improve- protective effects and the ability to improve ment and neuroprotection cognition in several AD mouse models. BChE Baichen Xiong, Zuoaoyun Song, Haopeng Sun inhibitors were proved to up-regulate ghrelin School of Pharmacy, China Pharmaceutical Univer- levels. Simultaneous modulation in the center sity, Nanjing, China and periphery improves the efficiency of drug. Butyrylcholinesterase (BChE) has been PO-36 found to regulate the cholinergic system, Detection of butyrylcholinesterase signal neuroinflammation and energy metabolism peptide in human brains in the brain of advanced Alzheimer’s disease Jacek Jasiecki 1, Andrew Reid2, Meghan Cash2, (AD) patients. Therefore, BChE has been more Monika Targońska1, Bartosz Wasąg1, Sultan and more attractive for treating neurodegen- Darvesh2 erative diseases. However, there is still a lack of 1Medical University of Gdańsk, Gdańsk, Poland BChE inhibitors with high selectivity and activ- 2Department of Medical Neuroscience, Dalhousie ity, and the biological function of BChE in AD University, Halifax, Canada has not been clarified. In this study, a hierarch- The pathogenesis of Alzheimer’s disease ical virtual screening protocol was applied, (AD) is attributed to extracellular aggreg- and a potential BChE inhibitor 8012-9656 ates of amyloid- β (A β) plaques and intracel-was selected as a lead compound. We have lular neurofibrillary tangles (NFTs) in the hu-conducted three rounds of structure-activity man brain. Butyrylcholinesterase (BChE) has relationship studies. Based on the results of also been reported to be associated with A β inhibiting activity and cyto-safety evaluations, plaques and NFTs in the brains of AD patients. compound S06-1064 (hBChE IC50 = 45.2 nM) We previously found that a substitution in the has been selected as candidate. Candidate 5’ untranslated region (5’UTR) of BChE resul-compound S06-1064 possessed outstand- ted in an in-frame N-terminal 41-amino acid ing safety and pharmacokinetic profiles. As extension of the BChE signal peptide. The the results of pharmacological evaluations, resulting variant with a 69-amino acid sig-candidate compound exhibited potent anti- nal peptide, designated N-BChE, may play a oxidant activity and neuroprotective effects role in AD development. We recently repor-against A β-, glutamate- and H2O2-induced ted that the BChE signal sequence, when pro-cytotoxicity at very low concentrations (1, 2, 5 duced in an extended version of 69 amino µ M). They could also decline the production acids, can self-aggregate and form seeds that of ROS caused by LPS and A β, exerting an enhance amyloid fibril formation in vitro in a anti-inflammation profile. Furthermore, the dose-dependent manner and generate larger analogs S06-1064 were able to improve the co-aggregates. Based on these observations, cognition and learning in APP/PS1 transgenic we hypothesized that a similar phenomenon mouse model, without leading to body weight of an extended version of 69 amino acids ex-loss, verifying the efficacy and safety of BChE pression could be observed in the brain. Our inhibition. Additionally, we demonstrated that recent experiments confirm the presence of BChE inhibitors can up-regulate ghrelin levels the BChE signal peptide in human brains. We in vivo and in vitro. Simultaneously enhan- have detected this signal peptide using spe- cing ghrelin level in the center and periphery cific antibodies by western blotting and ob- greatly improves the application efficiency of served different aggregation forms of it in the BChE inhibitors. In conclusion, a series of BChE brain samples as well as in the purified protein inhibitors with nanomolar IC50 values and 83 in vitro. The exact role of this signal peptide in there was an increase in both BChE expression AD requires further investigation. and activity. Interestingly, increased ALT and AST enzyme activities, clinically used markers PO-37 of liver injury, were also detected. Others col- Impact of Type 1 Diabetes Mellitus on Bu- lected tissues showed no changes in relative tyrylcholinesterase Expression and Activity expression or activity of BChE. In conclusion, in Rats the development of STZ-induced type 1 DM Tibor Hodbod, Lukas Nemet, Katarina Hadova, led to an increase of BChE activity in plasma Anna Hrabovska and an increase in BChE expression and activ- Faculty of Pharmacy, Comenius University Bratis- ity in liver but not in other studied organs. lava, Slovakia Importantly, increased BChE levels were ob- Serum butyrylcholinesterase (BChE) activ- served despite increased markers of liver dam-ity is altered in pathologies associated with age; thus, plasma BChE should not be used in impaired metabolism, such as diabetes mel- clinical practice to diagnose liver injury. litus (DM). Increased activity has been ob- This project was supported by APVV-22- served in both animal models and human pa- 0541 and VEGA 1/0283/22. tients. Although BChE is an abundant en- zyme throughout the body, information about PO-38 Expression of cholinesterases in rats its expression and activity in tissues other than Lukas Nemet, Tibor Hodbod, Monika Zelinová, blood is sparse. The main aim of the pro- Anna Hrabovska ject was therefore to investigate changes in Comenius University Bratislava, Slovakia BChE expression and activities in major tissues in an animal model of type 1 DM. Twelve-week- Introduction: Wistar rats are suitable model old male Wistar rats were used in the exper- for studying various pathological states in iment. DM was induced by administering a pharmacological research. Cholinergic sys- single-dose intraperitoneal injection of strep- tem plays an important role in many patholo-tozotocin (STZ, 55 mg/kg) dissolved in 0.1M cit- gies, but information about cholinergic system rate buffer. Hyperglycemia was confirmed across individual tissues in rats is sparse. There-three days after STZ administration by meas- fore the goal of this research was to study the uring glucose concentration in blood collec- expression of cholinesterases in major organs ted from tail vein. Four weeks after DM induc- of rats. tion, the animals were sacrificed in CO2 cham- Methodology: Nineteen-week-old male ber, and blood, liver, lungs, spleen, pancreas, Wistar rats were used. After euthanasia with hypothalamus, and heart (dissected into its CO2, brain, liver, lungs, spleen and heart (dis- functional compartments) were collected and sected to its functional compartments) were flash-frozen in liquid nitrogen. The relative ex- swiftly removed, flash frozen in liquid nitrogen, pression of BChE mRNA was determined by and stored at -80°C until further analysis. Total RT-qPCR. BChE activity was measured using a RNA was isolated using TRI-Reagent, quanti- Ellman method. Plasma lipid profile and ALT fied and checked for quality and reverse tran-and AST activities were assessed in a certi- scribed. RT-qPCR was conducted with SYBR fied laboratory. Successful induction of DM Select Master Mix. Gene expression was nor- was confirmed by increased fasting glycemia, malized to ActB, and β 2 µ G. Results were ana-elevated levels of VLDL and triglycerides in lyzed relative to a pool of liver cDNA from plasma. The development of DM was accom- Wistar rats. panied by increased plasma BChE activity. In Results: Rat mRNA relative expression the liver, the main synthetic organ of BChE, patterns, considering liver cDNA pool as a 84 baseline, differed for AChE and BChE. For The animals were weighted weekly. Vital func-AChE, the highest expression was observed in tions (heart rate, hemoglobin oxygen satur- brain and the lowest in the lungs: brain (88 ation, and breathing frequency) were recor-x) > right atrium (8x) > left atrium (4x) > left ded by non-invasive pulse oximetry four weeks ventricle and septum (3x) > right ventricle (2x) after injection. Animals were euthanized 24 > liver (baseline) > spleen (1 x) > lungs (0.1x). For h later, and plasma, lungs, pulmonary artery, BChE, the highest expression was observed and right and left heart ventricles were colin right atrium and the lowest in spleen: right lected, flash frozen and store at -80°C until atrium (9x) > left ventricle and septum (7x) > further analysis. Cholinesterase mRNA relative right ventricle (6x) > left atrium (5x) > brain (2x) expression was determined by RT-qPCR, and > lungs (0.2x) > spleen (0.1). activities were measured by Ellman’s activity Conclusion: In rat, the expression of AChE assay. in the brain is dramatically higher than in other Results: We observed a progressive de- studied tissues, while the expression of BChE crease in body weight and an increase in heart was highest in heart compartments. The ex- rate and breathing frequency following MCT pression patterns of AChE and BChE in rats dif- application. Post-mortem gravimetry con- fers from the ones reported for mice or hu- firmed hypertrophy of the right ventricle. There mans. was a decrease in plasma BChE activity in This research was funded by APVV-22-0541 MCT- treated rats as compared to controls. and VEGA 1/0283/22 grants. BChE expression and activity were also de- creased in pulmonary artery and left and right PO-39 ventricles. BChE activity and mRNA expres- Investigating the Link between Bu- sion in the lungs was, however, comparable tyrylcholinesterase and Pulmonary Vas- between the two experimental groups. cular Disease in Rats Conclusion: Plasma BChE activity is de- Parsa Shafieikazerooni, Monika Zelinová, Jana creased in MCT-induced pulmonary vascular Veteskova, Eva Velasova, Tibor Hodbod, Peter syndrome but due to high variability in the val- Krenek, Anna Hrabovska ues, it is not a reliable biomarker. Decreased Comenius University Bratislava, Slovakia BChE levels in pulmonary artery may suggest Introduction: Recent research suggests a endothelial dysfunction, and decreased BChE link between butyrylcholinesterase (BChE) and levels in ventricles are consistent findings from pulmonary vascular disease, underscored by heart failure research. inflammation, metabolic dysregulation, vas- This project was supported by APVV-22- cular dysfunction, and oxidative stress. The 0541, APVV-19-0458 and VEGA 1/0283/22. aim of this project was to study this link in monocrotaline(MCT)-induced pulmonary PO-40 vascular syndrome in rat, which is charac- Acetylcholinesterase and muscarinic re-terized by proliferative pulmonary vasculitis, ceptors control the ultraviolet-mediated pulmonary hypertension, and cor pulmonale release of melanosomes in cultured melan- (i.e., structural and functional pathological oma changes in right ventricle). Maggie Suisui Guo, Yingjie Xia, Jiahui Wu, Xiaoyang Wang, Gary Ka Wing Yuen, Tingxia Dong, Karl Methods: Male Wistar rats aged 10-12 Wah-Keung Tsim weeks were used in experiments. Pulmonary The Hong Kong University of Science and Technno- vascular syndrome was induced by MCT injec- logy, Hong Kong tion at dose of 60 mg/kg, s.c. (n=11). Controls were injected with 0.9% NaCl, s.c. (n=10). Melanosome is an intracellular organelle 85 responsible for melanin synthesis and stor- PO-41 age, functioning in determining the skin color. Trehalose restores the tacrine-induced en-In the epidermis, mature pigmented melano- doplasmic reticulum stress in cultured neur- somes are released by melanocytes, and sub- onal cells sequently internalized by surrounding kerat- Xiaoyang Wang, Yingjie Xia, Maggie Suisui Guo, inocytes via phagocytosis, leading to skin pig-Jiahui Wu, Tingxia Dong, Karl Wah-Keung Tsim mentation. This serves as a protective mech- The Hong Kong University of Science and Technno- anism for the epidermis, forming a nuclear logy, Hong Kong melanin shield and preventing the cells from Alzheimer’s disease (AD) is known as the ultraviolet (UV) radiation. The idea of “skin syn- most common dementia with progressive loss apse” describes the interaction between epi-of cognitive functions. Acetylcholinesterase dermal keratinocytes and melanocytes, medi- (AChE) inhibitors have been approved as con- ated by acetylcholine (ACh). Here, we aimed ventional pharmacotherapies for AD. Tacrine to investigate how the release of melanano- was the first AChE inhibitor introduced into somes could be regulated in the “skin syn- clinical use as a therapy for AD; however, it apse”. Cultured mouse melanoma B16F10 were was withdrawn from the usage in 2013 due to deployed as the cell model. Narrow-band safety concerns. In the brain, AChE is asso-UVB light, acetylcholinesterase (AChE) inhib- ciated with a proline-rich membrane anchor itor, agonists and antagonists targeting mus- (PRiMA), producing the tetrameric globular carinic ACh receptors (mAChRs) were applied (G4) form and anchoring to the membrane. In as the drug treatments. The extracellular cultured neurons, tacrine was found to induce melanin present in culture medium was col-endoplasmic reticulum (ER) stress and finally lected and quantified. Calcium indicator Fluo lead to apoptosis. This mechanism potentially 4-AM was used to measure the change of in- explains part of the adverse effects associ- tracellular Ca2+ level following the treatments. ated with tacrine. The phosphorylation of protein kinase C (PKC) In PRiMA-linked G4 AChE overexpressed was assessed by western blotting; while the NG108-15 cells, C/EBP homologous protein expression of tethering complex genes (i.e. (CHOP) was used to measure the level of ER synaptotagmin, Sec8, Exo70 and Rab11b) re- stress. The immunofluorescence staining of lated to melanosome exocytosis was studied AChE was employed to visualize the effect by western blotting and RT-qPCR. The applic- of tacrine on AChE protein expression and ation of ACh and AChE inhibitor was able to in-localization. The ER fraction was isolated to crease the amount of released melanosomes, analyze the distribution of different forms of as well as the intracellular Ca2+ level. The ant- AChE within ER by non-reducing gel electro-agonists of M1 and M3 could reverse the ef- phoresis. Tacrine could significantly increase fects, showing the role of M1 and/or M3 mA- the expression level of CHOP, which could ChRs in mediating the UVB-induced melano-be decreased by the co-treatment of tre- some release. Moreover, the expressions of halose, a known ER stress reducer. The result tethering complex for exocytosis were also of immunofluorescence staining showed that controlled in a similar manner. In conclusion, trehalose could relieve the tacrine-reduced M1/M3 mAChRs could be involved in regulat- G4 AChE level on the cell surface. In non-ing melanosome release following the UVB ra- reducing gel electrophoresis analysis of the ER diation. fraction, the tacrine-exposed group revealed a considerable G1/G2 forms of AChE accu- mulation, while the treatment of trehalose decreased the G1/G2 AChE accumulation. 86 Thus, tacrine could affect the assembly of G4 ous molecules for Wnt/ β-catenin signalling by AChE in the ER and finally lead to ER stress. real-time PCR. Activation of Wnt/ β-catenin Trehalose possesses the ability to relieve ER signalling was mediated by PI3K/AKT and stress by promoting the proper assembly of ERK signalling upon the stimulation of beth-G4 AChE. anechol. In addition, an increase in hair shaft elongation in mouse vibrissae and accelerate PO-42 re-entry of anagen in the in vivo hair growth The Muscarinic Acetylcholine Receptor in test was observed the upon the treatment of Dermal Papilla Cells Regulates Hair Growth bethanechol. These findings shed light on the Gary Ka Wing Yuen, Tingxia Dong, Daniel Ye, Aji-role of cholinergic system in hair growth. aikebaier Dilidaer, Karl Wah Keung Tsim The Hong Kong University of Science and Techno- PO-43 logy, Hong Kong The Regulatory Role of Gut Microbiota in Ex- pression of AChE in Epithelial Cells: a Regu- The role of the cholinergic system in hair lator of Inflammatory Bowel Disease biology remains an interesting topic with lim- Ajiaikebaier Dilidaer, Yingjie Xia, Jiahui Wu, ited research, having only a few reports ex- Tingxia Dong, Karl Wah-Keung Tsim ploring the relationship of the complex pro- The Hong Kong University of Science and Techno-cess. Several lines of evidence support the logy, Hong Kong notion of acetylcholine (ACh) playing role in hair biology. Alzheimer’s patients tak- ing oral acetylcholinesterase (AChE) and bu- Inflammatory bowel disease (IBD), including tyrylcholinesterase (BChE) inhibitor, rivastig- Crohn’s disease (CD) and ulcerative colitis mine, exhibited symptoms of hypertrichosis (UC), is a group of chronic inflammation in and hair re-pigmentation. In dermal papilla gastrointestinal tract, which is affecting mil- cells (DPCs) treated with AChE inhibitor, nor- lions of people all over the world and has a galantamine, a stimulation of anagen’s activ- significant impact on the quality of life, as ating signalling in the growth of hair follicles it is often accompanied by comorbid condi-has been identified. In addition, the knockout tions, such as cardiovascular disease, neuro-mice of M4 muscarinic receptor (M4 mAChR) psychological disorders, and metabolic syn-showed a defect in hair growth and which drome. Recent studies have focused on de- failed to produce pigmented hair shafts. Here, veloping effective treatments and potentially we reported that hair growth was regulated finding cures. Several potential drug targets by cholinergic signaling via mAChRs. DPCs ex- have been identified for IBD therapy, includ-pressed different cholinergic biomarkers. In ing gut bacteria and gut barrier. Studies il- addition, the release of ACh, induced by solar lustrate that gut bacteria produce different light, from DPC was determined by ELISA kit kinds of neurotransmitters, e.g., acetylcholine and LC–MS/MS, which thereafter activated (ACh). ACh can bind to α 7 nicotinic acet- the AChRs localized on DPCs. Inhibiting AChE ylcholine receptor ( α 7 nAChR) on the intest-or stimulating M4 mAChR in DPCs, culture vi- inal macrophages, reducing the production brissae and skin epidermis promoted the ac- of inflammatory cytokines, thereby inhibiting tivated Wnt/ β-catenin signalling was inhib-the intestinal inflammatory response. Besides, ited by tropicamide (M4 mAChR antagonist). the gut microbiome activates the cholinergic These results were supported by various in-anti-inflammatory pathway (CAP) via activ- dicative biomarkers, including pTOPflash lu- ating α 7 nAChR in intestinal epithelial cells, as ciferase assy, phosphorylation of GSK-3 β by well as altering the expression level of acet-western blot and mRNA expression of vari- ylcholinesterase (AChE). Here, IEC-6 cell line 87 was employed as the intestinal epithelial cell in a variety of cancers were extracted from model. The expression profiles of choliner- online websites and databases such as HPA, gic molecules, like AChE, AChR, and choline TIMER2, GEPIA2, cBioPortal, UALCAN, STRING, acetyltransferase were determined in cultured and Sangerbox. Pan-cancer patient data- IEC-6 cells. LPS/TNF- α was applied to in- sets were analyzed to reveal AChE expression duce inflammatory responses in the cultures. signatures, map protein-protein interactions, The expression and enzymatic activity of AChE and co-expression networks in cancers. Fur-were altered in LPS/TNF- α-induced cultured thermore, correlation between AChE expres-IEC-6 cells. The related inflammatory sig- sion and cancer prognoses was also predicted naling pathways, e.g., NF- κ B/CREB pathways, by survival analysis. were involved in this process. ACh, secreted Results: The pan-cancer analysis showed by the gut microbiome, could regulate the that AChE mRNA expression was significantly LPS-induced inflammatory responses, includ- downregulated in 9 cancers including cervical ing the production of inflammatory cytokines cancer, acute myeloid leukemia lung squam-via activating α 7 nAChR. The result suggested ous cell carcinoma, and ovarian cancer; while that ACh, released by gut bacteria, could in-upregulated in 6 cancers including stom- hibit intestinal inflammation and subsequently ach cancer, pheochromocytoma & paragan-alleviate the symptoms of IBD by activating glioma, kidney papillary cell carcinoma, and CAP and/or regulating AChE expression in in-thymoma. By gene set enrichment analysis, testinal cells. The study provides potential pre- co-expressing genes of AChE can be identi-ventive and therapeutic targets for IBD treat- fied. Taking breast cancer as an example, the ments. co-expression genes were found to be mainly involved in extracellular structure organization PO-44 and positive regulation of cell activation and Studying the Expression and Regulation of adhesion. Survival analysis showed that relat-AChE in Multiple Cancers Using Data-Driven ively higher AChE expression was significantly Approach associated with better overall survival (OS) in Jiahui An, Heidi QH Xie, Ruihong Zhu, Guanglei patients with melanoma, ovarian cancer, and Yang, Yangsheng Chen, Li Xu, Bin Zhao ocular melanoma, whereas with a poor OS in Research Center for Eco-Environmental Sciences, endometrioid cancer, adrenocortical cancer, Chinese Academy of Sciences, China and mesothelioma. Introduction: A growing body of in vitro Conclusions: Our study highlights the po- and in vivo studies has shown that the ex- tential of AChE as a novel biomarker for pre-pression levels of acetylcholinesterase (AChE) dicting prognosis across different human can-are associated with the development of a cers. variety of cancers. However, the association between AChE expression and cancer devel- PO-45 Advances in the development of new drugs opment and prognosis has not been revealed against Alzheimer’s disease based on tac-from a pan-cancer perspective. Therefore, rine scaffold in order to clarify the expression of AChE in Martin Novák 1, Vajrychova Marie1, Volker M. cancer tissues to find clues for its atypical Lauschke2, Ondrej Soukup1 functions in cancers, it is necessary to con- 1Biomedical Research Centre, University Hospital in duct a comprehensive bioinformatics analysis Hradec Kralove, Czech Republic of pan-cancer datasets based on a data- 2Department of Physiology and Pharmacology, Ka-driven approach. rolinska Institutet, Sweden Methods: The expression data of AChE 88 Current symptomatic pharmacotherapy tacrine-based compounds. of Alzheimer’s disease is primarily focused on acetylcholinesterase inhibitors and NMDA This work was supported by the Grant Agency (N-methyl-D-aspartate) receptor blocking. of the Czech Republic [No. 23-07570S], and by Tacrine, a molecule with both of the above Charles University [Nr. SVV 260 547]. mechanism of action was withdrawn from clinical use in 2013, mainly due to drug-induced PO-46 liver injury. The culprit of tacrine-associated Synthesis of a multifunctional compound hepatotoxicity is believed to be 7-OH-tacrine targeting neuroinflammation and choliner-metabolite, a possible precursor of quinone gic deficit in Alzheimer’s disease methide, which binds to intracellular -SH David Malinak 1, Zuzana Kohoutova1, Miroslav Psotka1, Karolina Knittelova1, Rudolf Andrys1, Kamil proteins. With regard to this toxicity pathway, Musilek1, Stanislav Gobec2 a new selective HPLC-MS/MS method for 1University of Hradec Kralove, Faculty of Science, monitoring of metabolites (especially for 7- Hradec Kralove, Czech Republic OH-tacrine) formed from tacrine derivatives 2University of Ljubljana, Faculty of Pharmacy, Slov-was developed. Further, we would like to enia find out if rational structure modifications of tacrine derivatives are less prone to quinone Neurodegenerative diseases, includ- methide formation. For this purpose the ing Alzheimerś disease (AD), pose a serious introduction of methoxy- or phenoxy- group healthcare threat with the staggering number to position 7 or chlorine into position 6 on the of patients diagnosed with these disorders. tacrine moiety was done to form 7-methoxy-, The therapy is currently limited to palliative 7-phenoxy-, and 6-chloro-tacrine. These care and relief of the symptoms with nu- substitutions may potentially hinder the form- merous novel drugs in the pipeline. Among ation of toxic species. these, multifunctional ligands are focused on Using our newly developed HPLC-MS/MS targeting multiple pathologies involved in the method we proved that tacrine and 7- AD progression. The main aim of this study is to methoxytacrine were primarily hydroxylated design and synthesized novel anti-AD agent to 7-OH-tacrine, whereas 7-phenoxytacrine able to simultaneously target neuroinflam-formed only trace amounts. Surprisingly, our mation by inhibiting p38 mitogen-activated study showed that 7-OH-tacrine which was protein kinase α (p38 MAPK α or p38 α) and predicted the most toxic according to the cholinergic deficit by inhibiting butyrylcholin- quinone methide hypothesis, was experi- esterase (BChE). That means this compound mentally determined as the least hepatotoxic should belong to a multi-target-directed (7-OH-tacrine < tacrine < 7-methoxytacrine ligands (MTDLs) group. Two compounds were < 7-phenoxytacrine) after incubation with developed for structure optimization towards primary human hepatocytes. These results designed lead molecule. First is selective were corroborated by mass spectrometry, p38 α inhibitor (Ki = 101 nM) and second com-which did not identify traces of quinone pound is selective BChE inhibitor (Ki = 11.1 pM). methide or -SH adducts formation by cysteine The newly designed compound has better quantification. Based on our results we sug-physical-chemical and biological properties. gests that 7-OH-tacrine and quinone methide For its in vivo evaluation, we have optimized formation is not the mechanism causing synthetic routes of the lead compound by tacrine toxicity as previously hypothesized. reducing the number of synthetic steps and Finally, we suggested via primary human hep-increasing yields of target molecule. This work atocytes, a new toxicity pathway, which could was supported by Czech Science Foundation bring a new insight to further development of 89 (No. GF23-42701L). matory pathway and the traditional choliner- gic hypothesis. The latter states that forebrain PO-47 cholinergic neuron loss is characteristic of AD. Dual inhibitors targeting BChE and p38 α By inhibiting the hydrolytic action of cholin-MAPK: a novel strategy for Alzheimer’s dis- esterases, in particular butyrylcholinesterase ease therapy (BChE) we may augment the activity of sur- Svit Ferjančič Benetik, Aleš Obreza, Urban Košak, viving cholinergic neurons. This multifaceted Damijan Knez, Stanislav Gobec approach seeks to address the complex in- University of Ljubljana, Faculty of Pharmacy, Slove- terplay of neuroinflammation and cholinergic nia dysfunction, offering a promising strategy for INTRODUCTION: AD intervention. Alzheimer’s disease (AD) is a progressive neuro- RESULTS AND DISCUSSION: First, a library degenerative disorder and the main cause of of small molecules with confirmed activity dementia. Currently, available therapeutic against p38 α MAPK was generated. The com-options for AD remain scarce with six drugs on pounds were categorised into 30 clusters ac- the market, three of which are small-molecule cording to their molecular fingerprint and cholinesterase inhibitors. As the efficacy of docked into the BChE active site gorge. Of the these drugs is limited to mild-to-moderate de-best docked ligands ARRY-371797, a p38 α MAPK mentia and adverse effects such as vasogenic inhibitor from Pfizer Inc. showed very prom-edema, hinder the progress of novel biolo- ising activity against BchE. This molecule was gical treatments against amyloid beta (A β) in then subjected to further optimization leading clinical trials, the development of innovative to two different series of compounds. small-molecule drugs targeting key proteins involved in the early pathophysiology of AD PO-48 Berberine in comparison to 7-MEOTA for presents a daunting task for medicinal chem- Alzheimer’s treatment: a polypharmacolo- ists. Although numerous hypotheses attempt gical approach to elucidate the intricate mechanisms under-Syed Nurulain 1, Areeba Kiran2, Sidra Batool3, lying pathophysiology of AD, they invariably Sosan Khan2, Huba Kalasz4, Sajid Mehmood1, Kamil converge on the extensively studied neuroin- Kuca5 flammation hypothesis. According to this hy- 1Department of Biosciences, Grand Asian University pothesis, the presence of A β plaques alongside Sialkot, Pakistan phosphorylated tau proteins leads to hyper- 2Department of Biosciences, COMSATS University activated microglia, triggering the expression Islamabad, Pakistan of various enzymes that exacerbate the im-3School of Chemistry and Molecular Biosciences, balance between anti- and proinflammatory The University of Queensland, Australia cytokines. Among the many enzymes that are 4Semmelweis University, Budapest, Hungary overactivated p38 α MAP kinase (p38 α MAPK) 5University of Hradec Králové, Czech Republic has recently gained more attention. This ubiquitously expressed enzyme enhances the Introduction production of proinflammatory cytokines such Alzheimer’s disease (AD) is characterized by as TNF- α and IL-1 β, facilitates A β accumu- multiple pathophysiological mechanisms lation, and catalyzes the hyperphosphoryla- like cholinergic neuronal damage, abnor- tion of neurotoxic tau proteins (4). This in turn mal accumulation of β-amyloid forming makes it an interesting pharmacological tar- senile plaques, tau hyperphosphorylation, get to combat AD. Our aim is to develop a dual neuroinflammation, and oxidative stress. The inhibitor that targets both the neuroinflam- complexity of these mechanisms has hindered 90 the discovery of an effective treatment for PO-49 AD. Phytochemicals are bioactive compounds Novel amiridine-based multi-target dir- found in fruits, vegetables, and grains, and are ected ligands for the Alzheimer’s disease known to reduce the risk of major diseases. treatment Berberine, a plant alkaloid, has been reported Jan Konecny 1, Eva Mezeiova1, Martin Horak2, to be beneficial in various pathological condi- Galina Makhaeva3, Jan Korabecny1 1 tions. Faculty Hospital of Hradec Kralove, Biomedical Re- Methodology search Center, Czech Republic 2 This study aimed to evaluate the cholinergic Institute of Experimental Medicine of the Czech inhibitory activity of berberine against human Academy of Sciences, Department of Neurochem- RBC-AChE and human plasma BChE. To assess istry, Czech Republic 3 the polypharmacological effects of berberine Russian Academy of Sciences, Institute of Physiolo- and 7-MEOTA, thirteen different receptors gically Active Compounds at Federal Research like AChE, BChE, MAO-A, MAO-B, Amyloid β, Center of Problems of Chemical Physics and Medi-Apo-E, α 7, α 4 β 2, Catalase, Glutathione Re- cinal Chemistry, Russian Federation ductase, β-Secretase, Superoxide Dismutase, Alzheimerś disease (AD) is a complex and P38 α MAPK were selected for molecular disorder with significant economic impact. docking using standard softwares. Results Current treatment offers only temporary were compared with 7-Methoxytacrine- delay in disease progression. The complexity adamantylamine (7-MEOTA). of AD and its unknown etiology are the major Results obstacles for development of new thera- The results showed that berberine inhibited peutic treatment. As single-target therapies RBC-AChE and BChE at millimolar concen-have proven not effective, rational specific- trations, with IC50 values of 0.20 mM for targeted combination into a single molecule RBC-AChE and 7.24 mM for BChE, com-represent more promising approach for treat- pared to 0.50 mM and 1.08 mM for 7-MEOTA, ment of AD. Consequently, a new class of respectively. Computational polypharmaco-potential drugs called multi-target directed logy offers the ability to predict the activity ligands (MTDLs) has been introduced as an al- profile of a ligand against a set of targets ternative option to counter the AD. Within our using various computational approaches. contribution, we will present new concept of Molecular docking indicated that berberine MTDLs pursued by a rationally designed series had low binding energies of -8.9 kcal/mol and of small molecules based on amiridine linked -8.3kcal/mol, demonstrating higher binding with memantine or benzothiazole scaffolds, affinities for AChE and BChE compared to the simultaneously targeting impaired cholinergic 7-MEOTA. Additionally, berberine exhibited and glutamatergic neurotransmission, along good binding interactions with Apo-E, the with antioxidant properties. The putative α 7-nAChR receptor, glutathione reductase, targets of these molecules are cholinesterases β-secretase, P38 α MAP kinase, and superoxide and NMDA receptors. dismutase, outperforming 7-MEOTA in these interactions. This study was supported by the Czech Conclusions Science Foundation grant no. 22–24384S. Berberine is found to be better than 7-MEOTA and may be used as an adjunct in the treat- ment of AD. Further in silico studies with analogues of berberine and 7-MEOTA is sug- gested followed by experimental studies with potentially good analogues. 91 PO-50 that team sports such as basketball and vol- Investigating the effects of basketball and leyball altered acetylcholine (and thus cholin-volleyball as team sports on social cognit- esterase) and BDNF levels, which are associ-ive function and BDNF and cholinesterase ated with cognitive function, and that this ef-levels in young men and women fect was particularly pronounced in men. Muslum Gok, Halil Ates, Ergul Cansu incegil, Serkan Aksu PO-51 Faculty of Medicine, Mugla Sitki Kocman University, Design of AChE reactivators using versatile Turkey molecular platforms and nanodiamonds Yevgen Karpichev 1, Illia Kapitanov1, Denys Neurogenesis is the production of new Bondar1, Vadym Mochalin2 neurons in areas of the brain such as the 1Tallinn University of Technology (TalTech), Estonia hippocampus. In recent years, studies have 2Missouri University of Science and Technology, shown that exercise is one of the strongest United States promoters of neurogenesis in the human brain. Brain-derived neurotrophic factor (BDNF) and The currently available antidotes against acetylcholine (ACh) are two regulators of syn- toxic organophosphorus compounds suffer aptic plasticity, neuronal survival and differ- from poor permeability across the blood-entiation. The relationship between phys- brain barrier (BBB) and are limited in their ical activity and fitness, cognitive function ability to restore the activity of the inhibited and academic performance is of great in- acetylcholinesterase (AChE) in the central terest but remains unclear. Therefore, this nervous system (CNS). Another requirement study further examined the effect of two of nowadays is to target a synergy between the world’s most popular physical activities principles of green chemistry and chemical on cognitive development in sedentary men disarmament and nonproliferation. and women from the medical school basket- We report two novel approaches to address-ball (n=16) and volleyball (n=12) teams who ing this requirement. participated in a tournament for 6 months. The L-Phe-derived versatile molecular plat-Social cognitive function tests (emotion re- form was chosen as a prospective scaffold for cognition test) were performed and blood design of sustainable products with different samples were taken before and after the tour- functionality. Their oxime derivatives are the nament. BDNF level and cholinesterase level sustainable AChE reactivators demonstrating were measured in blood by ELISA and Ell-remarkable activity against the AChE inhib- man assay, respectively. Our results showed ited by VX, compared with, or exceeding those that the BDNF level increased in male bas- for2-PAM and obidoxime. The regularities ketball players (p=0.1611) and volleyball play- on antidotal activity, cell viability, plasma ers (p=0.0547), while it decreased significantly stability, biodegradability as well as molecular in female basketball players (p=0.0313). Total docking study of the newly synthesized oximes cholinesterase and acetylcholinesterase levels are being analyzed for further improvement increased only in basketball players (p=0.1613), of their structures. especially in male participants (p=0.0967), We designed functionalized detonation nan-while no changes were observed in volleyball odiamond (ND) nanocarrier platforms to players. The results of the emotion recogni- transport quaternary oxime moiety bound to tion tests showed that there was a time ef- a biocompatible linker covalently attached fect in the recognition of sadness (F=5.004; to the nanodiamonds. The ND-based AChE p=0.031) and an increase in both the basket- reactivators crossed Madin-Darby Canine ball and volleyball groups. This study showed Kidney (MDCK) cells and demonstrated a 92 dose-independent in vitro reactivation capacity towards human AChE inhibited by GB, VX, and paraoxon. Visualization of tight junc- tions and actin cytoskeleton in the MDCK and HUVEC assays points to a cell type-dependent internalization pathway of ND-based react- ivators. The results reveal the potential of detonation nanodiamonds as a promising delivery platform for charged therapeutic agents to CNS, aimed to restore the activity of the inhibited AChE and enhance treatment outcomes in organophosphorus poisoning. 93 Authors’ Index A Brazzolotto, Xavier 35, 49, 79, Dawson, Jennifer 81, Achanta, Nagendra sai kumar 36, 68, 63, 42, 67, De Sousa, Julien 35, 48, Brea, José 51, Decker, Michael 53, Afshin Sander, Robin 71, Brink, Christiaan 50, Dehouck, Marie-Pierre 35, Aksu, Serkan 92, Buitrago, Pedro 67, Denic, Milica 36, Alle, Thibault 34, 33, Buitrago, Pedro 66, Dias, José 63, 36, 35, Almenares-Lopez, Damianys Bzdrenga, Janek 63, 79, 49, Dilidaer, Ajiaikebaier 87, 87, 46, Dingová, Dominika 58, C An, Jiahui 48, 59, 88, Diniz Botelho, Fernanda 70, Cadez, Tena 48, 73, 73, 34, Andersson, David 32, Divjak, Toni 63, Calas, André-Guilhem 35, 39, Andrys, Rudolf 64, 64, 65, 89, Dlabkova, Alzbeta 70, 76, Capasso, Giovambattista 52, 82, 38, Doisne, Nicolas 49, Carles, Allison 53, Antonijevic, Evica 82, Dolezal, Rafael 65, Carlsson, Marcus 71, Araoz, Romulo 35, Dong, Tingxia 85, 87, 60, 87, 86, Carneiro dos Santos, Marcelo Ates, Halil 92, Dorandeu, Frédéric 49, 66, 70, B Duan, Ran 60, Carneiro dos Santos, Marcelo Baati, Rachid 35, d 69, Bachmann, Gesine 59, Cash, Meghan 83, de Jesus de Oliveira, Daniel 70, Bagrowska, Weronika 56, Catara, Giuliana 48, E Ballatore, Carlo 33, 34, Cavalcante, Samir 69, 66, 66, Ekström, Fredrik 44, 71, 32, Bar, Adi 27, 70, 67, Ekström, Fredrik 39, Bartolić, Marija 42, Celmar Costa França, Tanos Engdahl, Cecilia 39, Bartolini, Manuela 51, 70, 69, Estevan, Cermen 45, Bartra, Clara 51, Cervellati, Carlo 27, 47, Estévez, Jorge 45, 47, Batool, Sidra 90, Champault, Alexandre 39, 40, F Bausch, Alex 59, Chantegreil, Fabien 49, Bavec, Aljoša 78, 77, 78, 45, Feng, Feng ?? , Chatonnet, Arnaud 53, 80, Beaupparain, Estelle 75, Ferjančič Benetik, Svit 54, 90, Chen, Yangsheng 48, 88, 59, Bellver, Aina 51, 67, Coe, Nick 81, Belverge, Nicolas 49, 79, Fibigar, Jakub 71, Coisne, Caroline 35, Bennett, Estelle 27, Figueiredo Taborda Barbosa, Coronato, Nicola 59, Fernanda Georgia 70, 69, 65, Bergström, Tomas 39, Costa Franca, Tanos Celmar Forgione, Pat 66, Bernardo, Leandro 66, 66, 65, 67, 66, Forsgren, Nina 32, 71, 44, Bertrand-Gaday, Christelle Courageux, Charlotte 36, 35, Forsgren, Nina 39, 53, 63, Francisco Diz de Almeida, Bhattacharyya, Dipanjan 66, Crouzier, Lucie 53, Joyce Sobreiro 67, 65, 66, Bies Pivačková, Lenka 58, Fuchsova, Adela 64, Bondar, Denys 92, D Borges, Caio 66, Da Silva, Ophélie 35, G Bosak, Anita 42, Dal Bo, Grégory 40, 39, 40, Gasnot, Julien 35, Bouix, Julien 49, Dallemagne, Patrick 54, Gastellier, Anne-Julie 35, 36, Braiki, Anissa 35, Darvesh, Sultan 83, 63, 95 Gerlits, Oksana 33, 72, 34, Jourdain, Laurène 40, Lépinard, Lucie 40, Gobec, Stanislav 67, 54, 89, 56, Juárez-Jiménez, Jordi 51, Leterrier, Sarah 40, 68, 50, 90, Jun, Daniel 37, 70, Lindgren, Cecilia 44, 32, Gok, Muslum 92, K Linusson Jonsson, Anna 44, 32, Goličnik, Marko 77, López-Muñoz, Francisco 55, Kalanj Bognar, Svjetlana 43, Goličnik, Marko 78, 45, Kalasz, Huba 75, 90, Loza, María Isabel 51, Goodchild, Sarah 81, Kapitanov, Illia 92, M Góra, Artur 56, Karpichev, Yevgen 92, Maček Hrvat, Nikolina 43, 34, Gorecki, Lukas 43, Kenawi, Moussa 49, 73, 74, 34, Gosselet, Fabien 35, Khan, Sosan 90, Maček Hrvat, Nikolina 73, Greenberg, David 27, Khandave, Prakash 79, Madi, Méliati 39, 49, Grgurevič, Neža 56, Kiran, Areeba 90, Madrer, Nimrod 27, Griñán-Ferré, Christian 51, Kitagawa, Daniel 66, Magisson, Fanny 49, Guo, Maggie Suisui 60, 85, 86, Knez, Damijan 67, 68, 54, 63, Majdič, Gregor 68, H 90, 56, Makhaeva, Galina 91, Haberek, Wawrzyniec 51, Knittelova, Karolina 89, 64, Malinak, David 64, 65, 64, 38, Hadova, Katarina 84, Knoertzer, Julie 40, 89, Hammock, Bruce D. 51, Kohoutova, Zuzana 64, 65, 89, Manco, Giuseppe 52, 48, Hanak, Anne-Sophie 35, 39, Kolić, Dora 74, 72, 43, 73, 34, Maraković, Nikola 42, Harvey, Brian 50, Konecny, Jan 91, Marchot, Pascale 80, Haus, Rachel 49, Korabecny, Jan 91, Marco Contelles, José 55, Heger, Zbynek 38, Korabecny, Jan 43, Marie, Vajrychova 88, Hepnarova, Vendula 43, Košak, Urban 68, 50, 67, 90, 54, Marone, Maria 48, 52, Ho, Kwok-Yiu 41, Kovalevsky, Andrey 34, 33, 72, Mastnak-Sokolov, Peter 67, Hodbod, Tibor 84, 84, Kovarik, Zrinka 73, 73, 48, 43, 34, Matošević, Ana 42, Hodbod, Tibor 58, 85, 74, 34, Maurice, Tangui 53, Hoffmann, Matthias 53, Kranjc Brezar, Simona 56, Mbah Ntepe, Leonel Javeres Horak, Martin 91, Krejci, Eric 58, 76, Horn, Gabriele 37, Krejci, Eric 58, Meden, Anže 68, 56, Hoster, Norman 44, Křenek, Peter 58, Meden, Anže 32, Hrabinova, Martina 69, 70, 43, Krenek, Peter 85, Meesemaecker, Gwladys 39, Hrabovska, Anna 85, 58, 84, 84, Krupka, Dominik 70, Mehmood, Sajid 90, 75, Hrenar, Tomica 63, Kuca, Kamil 65, 66, 70, 90, 75, Mendes, Gessica 69, Kučera, Matej 58, I Mendes, Anne Christine 49, Kumari, Rajendra 32, Ilić, Katarina 43, Mesarič, Živa 78, Kumari, Rashmi 32, Mezeiova, Eva 91, i L Mickova, Veronika 82, incegil, Ergul Cansu 92, Lacerra, Giuseppina 48, Mlakić, Milena 73, J Lamassiaude, Nicolas 35, Mlinac Jerković, Kristina 43, Jaffré, Nina 49, Lamba, Doriano 32, Mochalin, Vadym 92, Jagadeesh, Yerri 35, Lampitella, Eros A. 48, 52, Modeste, Fabrice 36, Jasiecki, Jacek 83, Landry, Christophe 35, Mola, Gianmarco 27, 47, Jean, Ludovic 75, 35, Laplate, Steven 70, 66, 65, Monnier, Charline 82, Joiner, Nichole 72, Lauschke, Volker M. 88, Monroy-Noyola, Antonio 46, Jonsson, Frida 71, 44, Layer, Paul G. 59, 45, Jonsson, Frida 39, Leite, Franco 69, Moraes, Amanda 66, 96 Morisseau, Christophe 51, Puljko, Borna 43, Soreq, Hermona 27, Muckova, Lubica 70, p Soukup, Ondrej 88, Muñoz-Torrero, Diego 51, Soukup, Ondrej 37, porzio, Elena 48, Musilek, Kamil 89, 38, 82, 64, 65, Souza, Roberto 66, 64, R Springer Engdahl, Cecilia 71, Rademacherova, Sara 64, N 44, Radic, Zoran 42, 33, 34, 72, Starcuk, Zenon 38, Nachon, Florian 35, 79, 68, 36, Radic, Zoran 41, 74, Stojan, Jure 32, 68, 36, 49, 42, Ramić, Alma 42, Stojan, Jure 78, 77, Naldi, Marina 51, Ramić, Alma 63, Sullivan, Dennis 42, Nemery, Louise 82, Ramirez Gonzalez, Laura 46, Sun, Haopeng 83, 55, ?? , Nemet, Lukas 84, 84, 80, Sun, Haopeng 56, Nepovimova, Eugenie 65, Rancillac, Armelle 40, Sussman, Joel L. 31, Nervo, Aurélie 49, Raushel, Frank 31, Suzumoto, Yoko 52, Novák, Martin 88, Razafindrainibe, Franck 35, Szmicseková, Kristína 58, Novell, Anthony 40, Reid, Andrew 83, Nurulain, Syed 90, 75, Š Renard, Pierre-Yves 35, 75, Ö Šinko, Goran 73, 34, 81, Reymond, Chloé 49, Škorić, Irena 73, Öberg, Christopher 71, Riaz, Zarish 75, O Ribeiro, Rayssa 69, T Targońska, Monika 83, Obreza, Aleš 90, 54, Riccetti, Raffealla 27, Taudon, Nicolas 36, 49, 79, Olivier, Nadia 50, Riccetti, Raffaella 47, Taylor, Palmer 43, 41, 42, Opravil, Jakub 71, 70, Rochais, Christophe 54, 52, Thangaraj, Gopenath 59, Ozaydin, Beste 51, Roche, Nicolas 80, Rosenberg, Yvonne 42, Thibault, Karine 39, 40, 40, P Rosta, Valentina 27, 47, Trancart, Marilène 35, Pajk, Stane 56, Trancart, Marilène 39, Pallàs, Mercè 51, S Travers–Lesage, Valentin 52, Palomo, Valle 51, Sabate, Raimon 51, Trentini, Alessandro 47, 27, Pande, Abhay H. 79, Sampietro, Anna 51, Trepiccione, Francesco 52, Pejchal, Jaroslav 65, Sanfeliu, Coral 51, Trontelj, Jurij 68, Perdih, Andrej 56, Santos Lima, Antonio Luis 66, Tsim, Karl Wah-Keung 85, 57, Pérez, Belén 51, Sałat, Kinga 68, 60, 87, 86, Pérez de la Lastra Aranda, Scheiner, Matthias 53, Tsim, Karl Wah Keung 87, Carmen 51, Servent, Denis 35, Tzur, Yonat 27, Pesaresi, Alessandro 32, Servonnet, Aurelie 49, U Petrič, Boštjan 45, 78, Shafieikazerooni, Parsa 85, 58, Undiano, Elizabeth 46, Pintar, Tadeja 78, Shahid, Mohammed 50, Pires, Fernanda 67, Silman, Israel 28, V Pišlar, Anja 67, 56, Simchovitz, Alon 27, Vázquez, Santiago 51, Porte, Karine 75, Since, Marc 52, Večeřa, Zbyněk 71, Prchal, Lukáš 76, Skarka, Adam 38, Veiga-Junior, Valdir 69, Prchalova, Eliska 65, 38, Smerkolj, Janez 78, 77, Velasova, Eva 85, Primožič, Ines 42, Smith, Georgia 81, Verret, Catherine 49, Primožič, Ines 63, Sogorb, Miguel A 45, Veteskova, Jana 85, Probst, Nicolas 35, Soirot, Thomas 49, 79, Vilanova, Eugenio 45, 28, 46, Psotka, Miroslav 89, Song, Zuoaoyun 83, Voros, Camille 35, 97 W Xiong, Baichen 83, 55, Žnidaršič, Neža 56, Wang, Yuanyuan 56, ?? , 55, Xu, Li 59, 88, 48, Z Wang, Xiaoyang 60, 86, 85, Xu, Ziwei 56, Zakošek Pipan, Maja 68, Wang, Alice 52, Y Zandona, Antonio 48, Warnault, Pierre 35, Yang, Huajing 56, Zdarova Karasova, Jana 65, Wasąg, Bartosz 83, 43, Wiktelius, Daniel 71, Yang, Guanglei 88, 59, Zelinová, Monika 84, 85, Wille, Timo 44, Ye, Daniel 87, Zemanova, Lucie 82, Worek, Franz 37, 44, Yu, Zhou 80, Zhang, Weiting 56, Wu, Jiahui 87, 85, 86, Yuen, Gary Ka Wing 85, 87, Zhang, Weiting ?? , 55, X Ž Zhao, Bin 59, 48, 88, Xia, Yingjie 60, 86, 85, 87, Žakelj, Simon 50, 68, Zhu, Ruihong 48, 88, 59, Xie, Heidi QH 48, 88, 59, Žnidaršič, Neja 77, Zorman, Masa 63, 98 Sponsors / Partners 99 Sponsors / Partners 100 101 Document Outline cover_PBoA CHEPON2024_BoA P R O G R A M M E P L E N A R Y L E C T U R E S ' A B S T R A C T S O R A L P R E S E N T A T I O N S ' A B S T R A C T S P O S T E R P R E S E N T A T I O N S ' A B S T R A C T S Authors' Index