Molecular choreography: Unveiling the dynamic landscape of type IIA DNA topoisomerases before T-segment passage through all-atom simulations.

Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations. The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery.

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

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.

Phenotypic Discovery of Thiocarbohydrazone with Anticancer Properties and Catalytic Inhibition of Human DNA Topoisomerase IIα.

Phenotypic screening of α-substituted thiocarbohydrazones revealed promising activity of 1,5-bis(salicylidene)thiocarbohydrazide against leukemia and breast cancer cells. Supplementary cell-based studies indicated an impairment of DNA replication via the ROS-independent pathway. The structural similarity of α-substituted thiocarbohydrazone to previously published thiosemicarbazone catalytic inhibitors targeting the ATP-binding site of human DNA topoisomerase IIα prompted us to investigate the inhibition activity on this target. Thiocarbohydrazone acted as a catalytic inhibitor and did not intercalate the DNA molecule, which validated their engagement with this cancer target. A comprehensive computational assessment of molecular recognition for a selected thiosemicarbazone and thiocarbohydrazone provided useful information for further optimization of this discovered lead compound for chemotherapeutic anticancer drug discovery.

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.

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids.

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.