Notes
DNA topoisomerases are a diverse family of enzymes, that work as molecular motors and enable topological changes of the DNA molecule. While there are several subgroups of topoisomerases, the subject of our research was the human DNA topoisomerase IIalpha, which is a validated and established anticancer target, as its concentration is higher in rapidly dividing cells compared to normal cells. Type II topoisomerases act via a complex catalytic cycle that offers multiple points for its modulation and consequently drug design. Clinically used inhibitors of this enzyme, topoisomerase II poisons, act by stabilizing a short-lived complex between the DNA and the enzyme turning it into a cellular toxin. However, they at the same time also damage the DNA molecule, and this can lead to serious side effects, such as cardiotoxicity and the onset of secondary forms of cancer. Therefore, in recent years, research has focused on the development of catalytic topo II inhibitors that do not cause DNA damage. In our research outlined by four research hypotheses, we investigated the identification, optimization, and evaluation of new catalytic inhibitors of human topoisomerase IIalpha that bind to its ATP binding site. In our first study substituted 4,5%-bitiazoles, known inhibitors of the bacterial topoisomerase type II DNA gyrase were used as a design starting point. By comparing the ATP binding sites of both enzymes, we pinpointed their key differences and similarities that were used in a virtual screening of a targeted chemical library to identify inhibitors that would better bind to the topoisomerase IIalpha ATP binding site. For selected compounds, we demonstrated at the in vitro level that they act as catalytic ATP-competitive inhibitors of topoisomerase IIalpha and bind to its isolated ATPase domain. The compounds were also effective at the cellular level, as some of them exhibited cytotoxicity on the HepG2 and MFC-7 cell lines in the same range as clinically used anticancer drug etoposide. The substituted 4,5%-bitiazoles stopped the cell cycle in the G1 phase, affected the cell proliferation, and did not cause the occurrence of DNA double-strand breaks, a further evidence that these compounds exhibit a different mode of action at the cellular level compared to topoisomerase II poisons. Our second study dealt with the optimization of position 4 of the 4,6-disubstituted 1,3,5 triazin-2(1H)-ones, for which previous studies have shown they act as catalytic topoisomerase IIalpha inhibitors and bind to the ATPase domain. Using a developed docking binding model, we screened a focused virtual library of possible analogues with different substituents at the position 4 of the 1,3,5-triazine ring that could form additional interactions with the ATP binding site. After screening, selected in silico optimized analogues were synthesized and were shown to be more potent catalytic inhibitors than the parent compounds. 4 Moreover, two of them also exhibited promising cytotoxicity on the HepG2 cancer cell line. In addition, this chemical class did not induce the formation of DNA double-strand breaks at the cellular level. In our final study, we started from the inactive compounds from the class of 3,5-disubstituted 1,2,4-oxadiazoles, which were originally designed as DNA gyrase inhibitors. We added more rigid moieties to the core structure along with functional groups, which could enable stronger interactions with the ATP binding pocket. In addition to the synthesized compounds, several commercially available substituted oxadiazoles were included in the evaluation of topo II% inhibition. The assays identified several 3,5-disubstituted 1,2,4-oxadiazoles with introduced rigid structures which possessed human topoisomerase IIalpha inhibition activity. These compounds acted via a catalytic inhibition mechanism and were able to bind to the isolated ATPase domain. They further displayed a cytotoxic potential on the MCF-7 cancer cell line and did not lead to DNA double-strand breaks. During our research we were able to confirm all four research hypotheses. In addition, the discovered lead compounds could via proper optimization lead to preclinical candidates with comparable efficacy as clinically used topoisomerase II poisons, and decreased incidence of serious side effects associated with this group of anticancer agents.