DNA topoisomerases comprise an important family of enzymes that catalyse the induction of topological changes (e.g. relaxation/supercoiling, catenation/decatenation and knotting/unknotting) in the DNA molecule. Due to their ability to modulate the topology of the DNA molecule, DNA topoisomerases play a vital role in replication, transcription, chromosome separation and segregation, and thus represent an important collection of validated targets for novel anticancer drugs. These enzymes perform their functions by creating transient either single-stranded or double-stranded breaks in the DNA molecule. DNA topoisomerase IIα is already one of the established targets for cancer chemotherapy. Topoisomerase IIα targeting agents are classified into two groups that differ in their mechanism of action: poisons and catalytic inhibitors. Poisons stabilize the covalent cleavage complex and convert this enzyme into a cellular toxin which is lethal to normal cells. Several poisons have entered in clinical use. The other novel group of catalytic inhibitors interfere in other steps of the topoisomerase IIα catalytic cycle. Due to frequently experienced occurrence of serious side effects of these molecules during therapy, especially cardiotoxicity issues, further drug design efforts were initiated already yielding novel promising compounds than have overcome this issue and already entered into clinical studies.
In this doctoral thesis we designed and experimentally evaluated new catalytic inhibitors from different chemical classes that bind to ATPase domain in human topoisomerase IIα. Design of novel topoisomerase IIα inhibitors comprised from structure-based and ligand-based pharmacophore modelling. In structure-based pharmacophore modelling we used structural information about binding of AMP-PNP molecule in the active site of enzyme, whereas in the ligand-based approach we used informations of already known active inhibitors. Both models were used to screen commercial available compounds and hits were further experimentally evaluated. Subsequently, active compounds were found to be active against human topoisomerase IIα, were evaluated for their in vitro cytotoxicity. In first research project we started from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site. We designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines as monocyclic htIIα inhibitors targeting the ATP binding site. To provide insight into the dynamic behavior of 4-amino-6-(phenylamino)-1,3,5-triazine, molecular dynamics (MD) simulations were initiated for the selected docked conformation of active inhibitor in the solvated human DNA topoisomerase IIα ATP active site. The most potent compound from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma HepG2 cell lines.
Next, in the second research project, based on the structural data about the binding mode of AMP-PNP molecule to human topo IIα, we designed a two stage virtual screening campaign combining structure-based pharmacophores and molecular docking, identifying in the first stage several mono-substituted 9H-purine compounds and a novel class of 1H-pyrazolo[3,4]pyrimidine inhibitors of the htIIα. In the second stage, bi-substituted analogues with improved properties for cell activity were discovered. Compounds from both classes were shown to inhibit htIIα-mediated decatenation and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction pattern between compounds and htIIα were further analyzed in MD simulation studies. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma HepG2 and breast cancer MCF-7 cell lines. These discovered compounds represent a starting point for further hit to lead development in the anticancer drug discovery.
Finally, starting from our discovered 4-amino-1,3,5-triazine inhibitors of human topoisomerase IIα we initiated a complex in silico design study of a focused library of 2,4,6-trisubstituted-1,3,5-triazines to optimize the series. 6-substituted-4-(benzylthio)-1,3,5-triazin-2(1H)-ones which were identified as novel topoisomerase IIα inhibitors. The most active compound was able to inhibit the human DNA topoisomerase IIα-mediated DNA decatenation and did not induce double-stranded breaks. Furthermore it inhibited the cleavage reaction induced by etoposide showing its inhibition step is in the early stages of topoII catalytic cycle. Binding studies using the novel microscale thermophoresis (MST) method established binding of 1,3,5-triazin-2(1H)-one compound to the htIIα ATPase domain, confirming experimentally for the first time the binding of a monocyclic catalytic inhibitors to the htIIα ATPase domain. The performed molecular dynamics (MD) simulation provided further insights into proposed molecular recognition. The discovered 6-substituted-4-(benzylthio)-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the ATP binding site and have the potential for hit to lead development of a monocyclic series of catalytic inhibitors of human DNA topoisomerase IIα. All discovered compounds represent promising starting points for further development and optimization paving the way to novel, much needed, antitumor agents.
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