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Mechanistic studies of metal-dependent DNA cleavage in Type II topoisomerase toward the rational design of novel anticancer drugs

Periodic Reporting for period 1 - Targeting TopoII (Mechanistic studies of metal-dependent DNA cleavage in Type II topoisomerase toward therational design of novel anticancer drugs)

Reporting period: 2017-11-06 to 2019-11-05

Type II topoisomerase (TopoII) metalloenzymes play a crucial role in regulating DNA topology in replication, transcription, recombination, and repair processes. TopoII is thus a validated target for clinical antibiotics and anticancer drugs, such as etoposide (ETO). ETO is an anticancer drug that acts by inducing TopoII-mediated DNA cleavage. The drug has been reported to target both TopoII isoforms, TopoIIα and TopoIIβ. However, the contributions of the two enzyme isoforms to the therapeutic and leukemogenic properties of the drug are unclear. Further, despite the wide use of ETO as a therapeutic agent for the treatment of cancer, the drug is associated with severe side effects, such as secondary leukemias. Understanding the mode of action of such TopoII inhibitors will assist the fight against the observed severe side effects and drug resistance. Moreover, it will open a path for the synthesis of new TopoII targeting drugs. To this end, docking and MD simulations can be a vital tool to facilitate the discovery of potent TopoII inhibitors as the first step toward more effective anticancer drugs. The cancer burden in the European Union is huge. Cancer is the second most common cause of death in the E.U. with more than 3.9 million new cases and 1.9 million deaths each year.(Ferlay et al. Eur. J. Cancer, 2018, 103, 356.) Sharing 9% of the world population, Europe shares 25% of the global cancer burden. Unfortunately, the current anticancer drugs often induce harmful side effects (such as treatment-related acute myeloid leukemia) that diminish their efficacy. Additionally, the frequent development of drug resistance hampers the drug action against cancer and bacterial infections, further enhancing the socio-economic burden. Considering the rising menace of cancer, the overall objective was to employ computational methods to determine the mechanisms underlying human TopoII catalysis and deciphering the key interactions of known/potential TopoII inhibitors with TopoII. In turn, the knowledge gained will assist in the design of novel TopoII inhibitors.
1. Dynamic Docking Studies of Human TopoII Bound to Potential TopoII Inhibitors. To develop an ETO-based drug with specificity for cancer cells the sugar moiety of the drug has been previously replaced with a polyamine tail. Such inhibitors would be preferentially taken up by cancer cells thereby lessening off-target effects in noncancerous cells. We analyzed the activity of a series of ETO-polyamine hybrids toward human TopoIIα and IIβ. All of these compounds induce higher/comparable enzyme-mediated DNA cleavage than induced by ETO. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward TopoIIβ than IIα. In collaboration with Prof. Neil Osheroff, Vanderbilt University, we provided the basis for the enhanced specificity of polyamine derivatives of ETO to TopoIIβ. Our enzymology & modeling studies form the basis of a testable hypothesis for the design of future drugs with enhanced specificity toward a single isoform of human TopoII.
2. Classical and Smoothed Potential MD Simulations to Unravel Isoform Specificity in Targeting Human TopoII. Current clinical TopoII poisons are nonspecific with regard to TopoIIα and TopoIIβ. However, strong support has been provided by both cellular and in vivo studies that TopoIIβ is primarily responsible for processes that initiate the secondary leukemias associated with ETO treatment. Hence, TopoIIα-specific poisoning might help mitigate the side effects observed with nonspecific TopoII drugs. However, the rational design of TopoIIα selective inhibitors is difficult as the two isoforms share ~78% identity at the catalytic site. We reported that TopoIIα exhibits ~3-fold more drug-target residence time with ETO as compared to TopoIIβ. This may reduce the adverse effects of specific TopoIIα poisons. To clarify how to design TopoIIα specific drugs, we employed classical molecular dynamics (MD) simulations to comparatively analyze the interactions formed within the TopoII/DNA/ETO complex in both isoforms. We also used smoothed potential MD to estimate ETO dissociation kinetics from the two isoform complexes. These extensive simulations revealed stabilizing interactions of ETO with amino acid residues in TopoIIα, which are missing in TopoIIβ. This may explain the greater persistence of ETO-stabilized cleavage complexes formed with TopoIIα. The study provides a structural and kinetic rationale for the design of novel TopoIIα-specific drugs, which can stably engage these amino acid residues.
3. Dynamic Docking to Elucidate the Interactions of TopoII and a New Class of TopoII Inhibitors. Based on the previously reported hybrid TopoII inhibitors that merge key pharmacophoric elements of ETO and merbarone, the molecular entity was expanded by synthesizing sixteen additional hybrid derivatives. Dynamic docking studies were performed to identify the vital interactions stabilizing the hybrid TopoII inhibitors. Most of these new compounds exhibited good inhibitory activity and cytotoxicity in three cancer cell lines with an IC50 in the low μM range. Hence, understanding the key molecular features needed for the drug-like properties in this novel chemical series of TopoII targeting compounds will aid in developing the new structural motif as a potential TopoII poison.

Dissemination
Publications
1. Oviatt, A. A.; Jissy, A. K. et al, N. Biorg. Med. Chem. Lett. 2018.
2. Jissy, A. K. et al. J. Chem. Inf. Model 2019 Just Accepted.
3. Arencibia, J. M.; Brindani, N.; Jissy A. K. et al. J. Med. Chem., 2019, Submitted.

Scientific Talks
2019
Aug: Recipient of Promise in COMP Award: ACS Fall Meeting, San Diego, CA.
July: GRS CADD, Mount Snow, USA.
June: MSCA IF event on Antimicrobial Resistance, Barcelona, Spain.
May: 12th European Workshop in Drug Design, Siena, Italy.
Mar: 6th CDDD Meeting, Rome, Italy.

Poster Presentations
2019
July: GRC on Computer Aided Drug Design, USA.
March: 6th CDDD Meeting, Rome, Italy.
2018
September: 22nd EuroQSAR, Thessaloniki, Greece.
July: GRC, Computational Chemistry, VT, USA.; GRS & GRC, Topoisomerases, Mt. Holyoke, MA, USA.; CECAM Workshop on modeling metal ions, 2018, Paris, France.
June: BioExcel Summer School, Sardinia, Italy.
TopoII is a central and validated target for anticancer research. Recent groundbreaking structures of TopoII – reported by Wang, et al (NAR, 2017, 45, 10861.) of TopoIIα, and Schmidt et al.(Nature 2010, 465, 641.) and Wu et al. (Science 2011, 333, 459) of TopoIIβ, respectively – were employed as novel starting points to initiate molecular simulations on TopoII enzymatic function and guide new informative SBDD efforts, based on TopoII in complex with DNA and the anticancer drug ETO. The targeting of TopoIIβ isoform has been correlated with the undesirable side effects such as secondary leukemia. Irrespective of having this knowledge, advancement in the field of developing alpha specific drugs has been slow. We employed the first reported crystal structure of TopoIIα and previously reported TopoIIβ structures bound to ETO to understand the key differences between the interactions of the drug molecule with the two isoforms. Also, a new enhanced sampling method, smoothed potential MD, was used to differentiate between the disassociation kinetics of the drug from the two TopoII isoform complexes. The knowledge gained provided a novel rationale for the design of TopoIIα poisons.