Scientific paper
Biochemical Characterization of MurF from Streptococcus pneumoniae and the Identification of a New MurF Inhibitor Through Ligand-based Virtual Screening
Samo Turk,1 Martina Hrast,1 Izidor Sosi~,1 Hélène Barreteau,2 Dominique Mengin-Lecreulx,2 Didier Blanot2 and Stanislav Gobec1*
1 Faculty of Pharmacy, University of Ljubljana, A{ker~eva 7, 1000 Ljubljana, Slovenia.
2 University Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, 91405 Orsay, France.
Tel: +33 1 69 15 81 65; Fax: +33 1 69 85 37 15
* Corresponding author: E-mail: stanislav.gobec@ffa.uni-lj.si Tel: +386 1 47 69 500; Fax: +3861 42 58 031
Received: 18-10-2012
Abstract
MurF is an essential bacterial enzyme that is involved in the last intracellular stage of peptidoglycan biosynthesis, and therefore it has the potential to be exploited as a target for the development of new antibacterials. Here, we report on the expression, purification and biochemical characterization of MurF from an important pathogen, Streptococcus pneumoniae. Additionally, ligand-based virtual screening was successfully used and a new hit compound with micromolar inhibitory activities against MurF enzymes from S. pneumoniae and Escherichia coli was identified.
Keywords: MurF, peptidoglycan, Streptococcus pneumoniae, ligand-based virtual screening
1. Introduction
The bacterial cell wall is a vital and essential structure as it provides structural support and protection from the outside environment, and it functions as a semipermeable filter. Enzymes involved in cell-wall biosynthesis have long been successfully exploited as drug targets, and there are several classes of antibacterials that target these enzymes in clinical use. Unfortunately, resistance to those agents has become an ever-increasing problem.1,2 Broad awareness of the growing resistance has spurred several different strategies to fight the arms race against bacteria. One of those strategies is to exploit new targets, and this is where MurF comes in.3 MurF is an essential, widely conserved enzyme that is involved in the last intracellular stage of biosynthesis of bacterial peptidoglycan.4,5 MurF catalyzes the addition of D-Ala-D-Ala to a UDP-MurNAc-tri-peptide (UMtri), which is UDP-MurNAc-L-Ala-y-D-Glu-L-Lys (UMtri-L-Lys) or UDP-MurNAc-L-Ala-y-D-Glu-meso-DAP (UMtri-mDAP) in Gram-positive bacteria and Gram-negative bacteria, respectively.4,5 The MurF enzymes from several bacterial species have been isolated and characterized;6-8 moreover, several attempts to design inhibitors of these enzymes have been made.9-14 The best in-
hibitors of MurF were reported by Abbott (Compound 1, Figure 1) and were designed for the inhibition of MurF from Streptococcus pneumoniae (MurFSp). The published IC50 for compound 1 is 1 pM.10,11 While the authors reported the expression and purification of MurFSp, they neglected to report if any attempt of characterization of MurF from S. pneumoniae was carried out.10
Virtual screening has become a useful method for rapid identification of hit compounds,15-18 and it has had many successful applications in the discovery and design of antibacterials.19 Three-dimensional (3D) ligand-based virtual screening methods are one of the fastest methodologies here, and if the starting compound (the 'query') is carefully selected, this can provide surprisingly good re-sults.17 Combining fast ligand-based methods with a vast resource of commercially available compounds in the
Figure 1. Compound 1, a representative MurFSp inhibitor identified by Abbott.
form of the constantly updated ZINC database20 thus enables rapid exploration of the continuously expanding chemical space.
Herein, we report in detail the isolation and characterization of MurF from S. pneumoniae. Furthermore, we also report on a successfully used 3D ligand-based virtual screening campaign, which yielded a new micromolar inhibitor of two enzymes, MurF from S. pneumoniae and MurF from Escherichia coli (MurFEc).
2. Experimental
2. 1. Expression, Purification and
Characterization of S. pneumoniae MurF
Materials. DNA restriction enzymes and synthetic oligonucleotides were purchased from New England Biolabs and Eurofins-MWG, respectively.
Bacterial strains and growth conditions. E. coli DH5a was used as the host for plasmids, and E. coli BL21(DE3)(pLysS) was used for the overproduction of the MurFSp enzyme. The construction of the pET2160 vector from pET21d (Novagen) was as described previously.21 The 2YT medium22 was used for growing the cells, and their growth was followed by monitoring the optical density of the cultures at 600 nm (OD600) using a Shimadzu UV-1601 spectrophotometer. Ampicillin and chloramphenicol were used at 100 pg ■ mL1 and 25 pg ■ mL1, respectively.
DNA manipulation. PCR amplification of the mur-FSp gene was performed in a Thermocycler 60 PCR machine (Bio-med) using Expand high-fidelity DNA poly-merase. DNA fragments were purified using Wizard PCR preps DNA purification kits (Promega), and standard procedures were used for DNA digestion, ligation and agarose gel electrophoresis.23 Plasmid isolation was carried out using kits purchased from Macherey-Nagel. E. coli cells were transformed with plasmid DNA by electroporation.
The MurF expression plasmid was constructed as follows. The murFSp gene was amplified from the chromosome of the S. pneumoniae R6 strain by PCR using the primers SpnO3 (5'-CGCGTCATGAAATTAACAATC-CATGAAATTGCC-3') and SpnO4 (5'-CGCGAGATCT-CTTGTCTTCATTTTCTAAACTTTCTACCAACTTG-GC-3'), which were designed to incorporate BspH1 and Bglll sites (in bold) at the 5' and 3' extremities of the gene (initiation codon underlined), respectively. The resulting fragment was digested with BspH1 and Bglll and ligated between the compatible Ncol and Bglll sites of the vector pET2160. The resulting plasmid, pET2160::murFSp, allowed expression of the corresponding protein with an Arg-Ser-His6 C-terminal extension. DNA sequencing was performed to confirm the sequence of the cloned fragment.
Overproduction and purification of MurFSp. E. coli BL21(DE3)(pLysS) cells carrying the pET2160::murFSp plasmid were grown at 37 °C in 2YT medium (1.0 L cultures) containing ampicillin and chloramphenicol. When the OD600 of the culture reached 0.8, isopropyl-P-D-thio-galactopyranoside was added at a final concentration of 1 mM and the incubation was continued overnight at 37 °C, with shaking. The cells were harvested at 4 °C and washed with cold 20 mM phosphate buffer, pH 7.2, containing 1 mM dithiothreitol (DTT) (buffer A). The cell pellet was resuspended in buffer A (12 mL), and the cells were disrupted by sonication in the cold (Bioblock Vibracell so-nicator; model 72412). The resulting suspension was cen-trifuged at 4 °C for 30 min at 200,000 x g in a Beckman TL100 centrifuge, and the pellet was discarded. The supernatant was stored at -20 °C.
MurFSp was purified on Ni2+-nitrilotriacetate (Ni-NTA)-agarose following the manufacturer recommendations (Qiagen). Crude protein extracts were mixed for 1 h at 4 °C with the polymer pre-equilibrated in buffer A containing 300 mM KCl and 1 mM DTT. The washing and elu-tion steps were performed with a discontinuous gradient of imidazole (20 mM to 200 mM) in buffer A containing 300 mM KCl and 1 mM DTT. MurFSp was eluted with 200 mM imidazole, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The relevant fractions were pooled, concentrated by ultrafiltration (30,000 Amicon Ultra-15 centrifugal filter; Millipore) and dialyzed overnight against 100 volumes of buffer consisting of 20 mM Hepes, pH 7.4, 200 mM NaCl, 5 mM DTT and 0.05% NaN3. The final preparations were stored at -20 °C after the addition of glycerol (10% final concentration).
SDS-PAGE analysis of the proteins was performed by the method of Laemmli and Favre.24 Protein concentrations were determined by the method of Bradford,25 with bovine serum albumin as the standard, or by amino-acid analysis with a Hitachi L8800 analyzer (ScienceTec), after hydrolysis of the samples in 6 M HCl at 105 °C for 24 h.
MurFSp characterization. For the determination of the kinetic constants of MurFSp, the Malachite green method was used,26 which detects the orthophosphate generated during the reaction. The mixture, with a final volume of 50 pL, contained 50 mM Hepes, pH 8.0, 50 mM Mg-Cl2, 0.005% Triton X-114, 600 pM D-Ala-D-Ala, 200 pM UMtri-L-Lys, 250 pM ATP and purified MurF from S. pneumoniae diluted in 50 mM Hepes and 5 mM DTT. The reaction mixture was incubated at 37 °C for 10 min and then quenched with 100 pL Biomol® reagent. The absor-bance was read at 650 nm after 5 min. All of the experiments were run in duplicate. Identical assay conditions were used when the nucleotide precursor was UMtri-m-DAP. All of the initial velocity experiments were performed under these assay conditions using different concentrations of one substrate and fixed concentrations of the others. The data were fitted to the Michaelis equation, v = V S/(K + S).
max v m '
Inhibition assay. The compounds were tested for inhibition of the addition of D-Ala-D-Ala to either UMtri-L-Lys or UMtri-mDAP catalyzed by MurFSp or MurFEc, respectively. The final volume of the mixture was 50 pL and it contained:
S. pneumoniae MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114, 100 pM D-Ala-D-Ala, 50 pM UMtri-L-Lys, 250 pM ATP, purified MurFSp and 5% of either DMSO (assay control) or compound dissolved in DMSO.
E. coli MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114, 600 pM D-Ala-D-Ala, 100 pM UMtri-mDAP, 500 pM ATP, purified MurFEc27 and 5% of either DMSO (assay control) or compound dissolved in DMSO.
The mixtures were incubated at 37 °C for 15 min and then quenched with 100 pL Biomol®reagent. After 5 min, the absorbance at 650 nm was measured. All of the experiments were run in duplicate. Residual activities (RAs) were calculated with respect to the assay control. The IC50 values were determined by measuring the residual activities at seven different concentrations.
2. 2. Computational
Virtual screening. Virtual screening was performed on a HP workstation with two quad core Intel Xeon 2.2 GHz processors, 8 GB of RAM, 320 GB and 1 TB hard drives, and a Nvidia Quadro FX 4800 graphic card, and it was running the current version of 64-bit Arch Linux.
The compound library for virtual screening was downloaded from the ZINC database.20 The ZINC druglike subset, which at the time contained 11 million compounds, was selected and downloaded in sdf form. The compound library was prepared with the Omega program (OpenEye Scientific Software Inc.)28 to yield on average 152 different conformations per compound.
Ligand-based virtual screening was performed with ROCS (OpenEye Scientific Software Inc.)29 using compound 1, identified by Abbott, as a query. The conformation of compound 1 was extracted from the co-crystal structure of MurFSp with compound 1 (PDB code 2AM1).10 The previously prepared compound library of 11 million compounds with 152 conformations on average per compound was screened and the compounds were ranked according to the TanimotoCombo score. Forty of the highest ranked and available compounds were obtained and evaluated in vitro.
Molecular docking. The three-dimensional structures of compound 2 used for the docking experiments was prepared with OMEGA (OpenEye Scientific Software, Inc.).28 Two hundred conformations of compound 2 were generated. FRED_receptor was used for the preparation of the MurF active site from the S. pneumoniae (PDB code 2AM1)10 active site. The active site was defined as a box around the co-crystallized ligand with a volume of
5652 À3. Molecular probe was used for site detection. The volumes for the inner and outer contours were 78 À3 and 1578 À3, respectively. Chemgauss3 scoring function was used for the exhaustive search and optimization. Validation was done by re-docking of the co-crystallized ligand.
3. Results and Discussion
3. 1. Expression, Purification and
Characterization of S. pneumoniae MurF
The murFSp gene was inserted into the vector pET2160. The resulting plasmid pET2160::murFSp enabled the expression of the corresponding protein with an Arg-Ser-His6 C-terminal extension. The E. coli BL21(DE3) (pLysS) strain carrying the pET2160::murFSp gene was used for the overproduction of the MurFSp protein. The protein was purified on Ni-NTA-agarose and the relevant fractions were pooled, as judged by SDS-PAGE. This resulted in 11.5 mg enzyme per litre of culture. MALDI-TOF mass spectrometry analysis revealed peaks at m/z 25,805 and 51,617 for the [M+2H]2+ and [M+H]+ ions, respectively, which is in agreement with the calculated value (Mr 51,569).
A detailed investigation of the MurFSp kinetic parameters was performed. The pH in all of the assays was set at 8, given the optimal pH range for the Mur ligases has been reported to be from 8 to 9.2.30 The enzyme activity was measured indirectly by detecting orthophosphate generated during the reaction with Malachite green.26 Initial velocity measurements were performed while keeping two of the substrates at constant saturating concentrations and varying the concentration of the third. Based on these experiments, the Km values for UMtri-L-Lys, D-Ala-D-Ala and ATP were determined as 40 pM, 86 pM and 69 pM, respectively. The Vmax of purified MurFSp was 38 pmol min1 mg1.
The maximum velocity for MurFSp is between that of MurF from Staphylococcus aureus (MurFSa) (71 pmol min1 mg1)8 and that of MurFEc (16 pmol min1 mg1).6 The Km values for ATP and their respective UDP-MurNAc-tripeptide were similar among all of these MurF orthologs, although the Km for D-Ala-D-Ala was almost three-fold lower for MurFSp. As in the case of MurFSa,8 but contrary to MurFEc,31 no substrate inhibition by UDP-MurNAc-tripeptide was observed. Interestingly, there was a perceivable difference in the Km values for both forms of the nucleotide precursor (L-Lys and meso-DAP); the Km for UMtri-mDAP was estimated to be 240 pM, thereby showing that MurFSp discriminates between these substrates, which was not observed for MurFEc.31 Discrimination between these two forms of the UDP-MurNAc-tripeptide has previously been reported for MurF from Chlamydia trachomatis, with the preference being for UMtri-mDAP over UMtri-L-Lys.7
3. 2. Virtual Screening and Biochemical Evaluation of Hits
Three-dimensional, ligand-based, virtual screening was used to screen commercially available compounds for hit compounds with similar shapes, volumes and distributions of atom types (dubbed color in the OpenEye programs) to compound 1. A drug-like subset that contained 11 million compounds at the time, was downloaded from the ZINC library.20 To cover the conforma-tional space of the compounds, the virtual library was pre-processed using the Omega program (OpenEye Scientific Software Inc.),28 which yielded an average of 152 conformations per compound. Compound 1 was used as a query. The selection of the conformation of query compound is of great importance in 3D ligand-ba-sed virtual screening, and ideally this should be an active conformation.17 Fortunately, compound 1 was co-crystallized with MurFSp and so we were able to extract the bioactive conformation from the co-crystal structure (PDB code 2AM1)10 and use it as a query. The ROCS program (OpenEye Scientific Software Inc.)29 was used for the ligand-based virtual screening. Briefly, ROCS overlays screened compounds to the query structure and calculates shape similarities and the similarity of distribution of the atom types (color). Both similarities are calculated as Tanimoto indices. TanimotoCombo, as used here, is simply a sum of both the shape and color Tanimoto indices. Compounds from the virtual library were ranked according to the TanimotoCombo index, and subsequently 40 of highest ranked and available compounds were purchased and biochemically evaluated (Supplementary Information, Table 1) for inhibition of MurFSp and MurFEc.
The results of the MurFSp characterization were considered when setting up the assay, and the following concentrations of substrates were chosen: 100 pM D-Ala-D-Ala, 50 pM UMtri-L-Lys, and 250 pM ATP. The MurFEc purification as well as assay were described previ-ously.12,27 To reduce the chance of false positive results due to compound aggregation, a surfactant was used in all of the assays (0.005% Triton X-114).32 All 40 compounds were assayed for enzyme inhibition of both MurFSp and MurFEc, although only compound 2 (3-((4-chloro-1H-pyrazol-1-yl)methoxy)-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)benzamide) showed activity on both. The IC50 values were 126 pM and 56 pM for MurFSp and MurFEc, respectively.
Not surprisingly, compound 2 shares certain structural elements with query compound 1. Both compounds have a three-ring system that is connected with short linkers. The 3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene from compound 1 is replaced with 3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophene in compound 2. In both compounds, the amide bond connects the thiophene with the phenyl moiety, which is dichloro-substituted in the case of compound 1. The phenyl moiety is further connected via sulfonamide to morpholine in compound 1, or via ether to chloropyrazole in compound 2. Nonetheless, in spite of some structural similarity between these compounds, compound 2 expands the potentially useful chemical space of the cyanothiophene-type of MurF inhibitors.
To improve our understanding of the binding mode of compound 2, it was docked into the MurFSp active site (PDB code 2AM1; Figure 3), using the FRED program (OpenEye Scientific Software, Inc).33 The active site was defined as a box around the co-crystallized compound 1, with a volume of 5652 A3. The docking protocol was validated with successful re-docking of the co-crystallized compound 1. The predicted binding pose was similar to that of the co-crystallized inhibitor 1, as expected. For compound 2, it was predicted to occupy the same part of the active site as compound 1, with the three-ring systems of both compounds overlapping. In both cases, the nitrile moiety formed two H-bonds with the backbone nitrogens of Ala48 and Arg49. The amide nitrogen, which is also common to both structures, formed an H-bond with Thr330. The last shared structural element, the phenyl moiety, formed n-stacking interactions with Phe31. For the rest of compound 2, no other interactions were predicted, except weak Van der Waals bonds. In contrast, one of the sulfonamide oxygens of compound 1 forms two H-bonds, one with Asn326 and the other with Asn328. Moreover, the morpholine oxygen of compound 1 forms a weak H-bond with the backbone
Figure 2. Structure of compound 2
Figure 3. The predicted binding pose of compound 2 (dark grey). The co-crystallized compound 1 is shown as grey sticks, the relevant residues of MurFSp active site are shown as lines.
nitrogen of Gly140.10 The loss of three H-bonds might explain the somewhat higher IC50 of compound 2 compared to compound 1.
4. Conclusions
MurF from S. pneumoniae was successfully expressed, purified and subsequently characterized. The kinetic parameters were determined: the Km values for UMtri-L-Lys, D-Ala-D-Ala and ATP were 40 pM, 86 pM and 69 pM, respectively. These data extend our understanding of the MurF enzymes, and together with previous knowledge, this should facilitate the design of MurF inhibitors with potential broad-spectrum antibacterial activity. Additionally, it enabled us to set up an assay for inhibitor screening, with 40 compounds resulting from the ligand-based virtual screening campaign. These were assayed in vitro, and out of the 40 assayed compounds, compound 2 was identified as a hit compound. It showed micromolar inhibitory activities against MurFSp and MurFEc, with IC50 values of 126 pM and 56 pM, respectively. Similar to the query compound 1, compound 2 is a cyanothiophene-type MurF inhibitor, although it has different substituents, and so it further extends the potentially useful chemical space of this type of MurF inhibitors. Compound 2 thus represents a promising starting point for further development of new broad-spectrum antibacterials.
5. Acknowledgements
We thank OpenEye Scientific Software, Inc. for free academic licenses of their software, the Ministry of Higher Education, Science and Technology of the Republic of Slovenia for financial support and Dr. Chris Berrie for critical reading of the manuscript. This study was partially supported by a Young Researcher grant to MH and L1-4039 grant to SG, both from the Slovenian Research Agency.
6. Supporting Information
Complete results of biochemical evaluation of virtual hits and 1H NMR spectrum of active compound.
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Povzetek
MurF je esencialen bakterijski encim, ki sodeluje pri zadnji znotrajcelični stopnji biosinteze peptidoglikana in je kot tak obetavna tarča za razvoj novih protibakterijskih učinkovin. V tem prispevku poročamo o uspešni ekspresiji, izolaciji in biokemijski karakterizaciji encima MurF iz pomembnega patogenega seva Streptococcus pneumoniae. Poleg tega smo tudi uspešno uporabili virtualno rešetanje na osnovi liganda in odkrili novo spojino zadetek z zaviralno aktivnostjo v mi-kromolarnem območju na encima MurF iz S. pneumoniae in Escherichia coli.