Unveiling the interdomain dynamics of type II DNA topoisomerase through all-atom simulations: Implications for understanding its catalytic cycle.

Type IIA DNA topoisomerases are complex molecular nanomachines that manage topological states of the DNA molecule in the cell and play a crucial role in cellular processes such as cell division and transcription. They are also established targets of cancer chemotherapy. Starting from the available crystal structure of a fully catalytic topoisomerase IIA homodimer from Saccharomyces cerevisiae, we constructed three states of this molecular motor primarily changing the configurations of the DNA segment bound in the DNA gate and performed µs-long all-atom molecular simulations. A comprehensive analysis revealed a sliding motion within the DNA gate and a teamwork between the N-gate and DNA gate that may be associated with the necessary molecular events that allow passage of the T-segment of DNA. The observed movement of the ATPase dimer relative to the DNA domain was reflected in different interaction patterns between the K-loops of the transducer domain and the B-A-B form of the bound DNA. Based on the obtained results, we mapped simulated configurations to the structures in the proposed catalytic cycle through which type IIA topoisomerases exert their function and discussed the possible transition events. The results extend our understanding of the mechanism of action of type IIA topoisomerases and provide an atomistic interpretation of some of the observed features of these molecular motors.

Catalytic Mechanism of ATP Hydrolysis in the ATPase Domain of Human DNA Topoisomerase IIα.

Human DNA topoisomerase IIα is a biological nanomachine that regulates the topological changes of the DNA molecule and is considered a prime target for anticancer drugs. Despite intensive research, many atomic details about its mechanism of action remain unknown. We investigated the ATPase domain, a segment of the human DNA topoisomerase IIα, using all-atom molecular simulations, multiscale quantum mechanics/molecular mechanics (QM/MM) calculations, and a point mutation study. The results suggested that the binding of ATP affects the overall dynamics of the ATPase dimer. Reaction modeling revealed that ATP hydrolysis favors the dissociative substrate-assisted reaction mechanism with the catalytic Glu87 serving to properly position and polarize the lytic water molecule. The point mutation study complemented our computational results, demonstrating that Lys378, part of the important QTK loop, acts as a stabilizing residue. The work aims to pave the way to a deeper understanding of these important molecular motors and to advance the development of new therapeutics.

Design, Synthesis and Evaluation of Fused Bicyclo[2.2.2]octene as a Potential Core Scaffold for the Non-Covalent Inhibitors of SARS-CoV-2 3CLpro Main Protease.

The emergence of SARS-CoV-2, responsible for the global COVID-19 pandemic, requires the rapid development of novel antiviral drugs that would contribute to an effective treatment alongside vaccines. Drug repurposing and development of new molecules targeting numerous viral targets have already led to promising drug candidates. To this end, versatile molecular scaffolds with high functionalization capabilities play a key role. Starting with the clinically used conformationally flexible HIV-1 protease inhibitors that inhibit replication of SARS-CoV-2 and bind major protease 3CLpro, we designed and synthesized a series of rigid bicyclo[2.2.2]octenes fused to N-substituted succinimides to test whether this core scaffold could support the development of non-covalent 3CLpro inhibitors. Inhibition assays confirmed that some compounds can inhibit the SARS-CoV-2 main protease; the most promising compound 11a inhibited 3CLpro in micromolar range (IC50 = 102.2 µM). Molecular simulations of the target-ligand complex in conjunction with dynophore analyses and endpoint free energy calculations provide additional insight and first recommendations for future optimization. The fused bicyclo[2.2.2]octenes can be used as a new potential starting point in the development of non-covalent SARS-CoV-2 3CLpro protease inhibitors and the study also substantiates the potential of this versatile scaffold for the development of biologically active molecules.

Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα.

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

Design and synthesis of 3,5-substituted 1,2,4-oxadiazoles as catalytic inhibitors of human DNA topoisomerase IIα.

Cancer constitutes a group of diseases linked to abnormal cell growth that can potentially spread to other parts of the body and is one of the most common causes of death. The molecular motors - DNA topoisomerases - that enable topological changes of the DNA molecule are one of the most established targets of cancer therapies. Due to known limitations of established topo II poisons such as cardiotoxicity, induction of secondary malignancies and recognized cancer cell resistance, an emerging group of catalytic topo II inhibitors attempts to circumvent these challenges. Currently, this approach comprises several subgroups of mechanistically diverse inhibitors, one of which are compounds that act by binding to their ATPase domain. In this study we have designed, synthesized and characterized a new series of 3,5-substituted 1,2,4-oxadiazoles that act as catalytic inhibitors of human topo IIα. The introduction of the substituted rigid substitutions on the oxadiazole backbone was intended to enhance the interactions with the ATP binding site. In the inhibition assays selected compounds revealed a new class of catalytic inhibitors targeting this molecular motor and showed binding to the isolated topo IIα ATPase domain. The predicted inhibitor binding geometries were evaluated in molecular dynamics simulations and subsequently dynophore models were derived, which provided a deeper insight into molecular recognition with its macromolecular target. Selected compounds also displayed in vitro cytotoxicity on the investigated MCF-7 cancer cell line and did not induce double-strand breaks (DSB), thus displaying a mechanism of action diverse from the topo II poisons also on the cellular level. The substituted oxadiazoles thus comprise a chemical class of interesting compounds that are synthetically fully amenable for further optimization to anticancer drugs.

Structure-guided optimization of 4,6-substituted-1,3,5-triazin-2(1H)-ones as catalytic inhibitors of human DNA topoisomerase IIα.

Human DNA topoisomerases represent one of the key targets of modern chemotherapy. An emerging group of catalytic inhibitors of human DNA topoisomerase IIα comprises a new paradigm directed to circumvent the known limitations of topoisomerase II poisons such as cardiotoxicity and induction of secondary tumors. In our previous studies, 4,6-substituted-1,3,5-triazin-2(1H)-ones were discovered as catalytic inhibitors of topo IIα. Here, we report the results of our efforts to optimize several properties of the initial chemical series that did not exhibit cytotoxicity on cancer cell lines. Using an optimized synthetic route, a focused chemical library was designed aimed at further functionalizing substituents at the position 4 of the 1,3,5-triazin-2(1H)-one scaffold to enable additional interactions with the topo IIα ATP binding site. After virtual screening, selected 36 analogues were synthesized and experimentally evaluated for human topo IIα inhibition. The optimized series displayed improved inhibition of topo IIα over the initial series and the catalytic mode of inhibition was confirmed for the selected active compounds. The optimized series also showed cytotoxicity against HepG2 and MCF-7 cell lines and did not induce double-strand breaks, thus displaying a mechanism of action that differs from the topo II poisons on the cellular level. The new series represents a new step in the development of the 4,6-substituted-1,3,5-triazin-2(1H)-one class towards novel efficient anticancer therapies utilizing the catalytic topo IIα inhibition paradigm.