Abstract 1370 Revealing the nucleic acid interactions of human topoisomerase 3 beta, a unique dual-acting topoisomerase, and discovering its inhibitors

Human topoisomerase III beta (hTOP3B) is the only type IA topoisomerase in the human cell that works on DNA and RNA substrates. Studies show that lack of hTOP3B leads to increased R-loops and genome instability; irreversible TOP3B cleavage complexes (TOP3Bccs) lead to DNA damage and reduce cell survival; tumors lacking TOP3B grow more slowly, and positive-sense single-stranded RNA viruses utilize hTOP3B to replicate efficiently, making it a potential anticancer and antiviral drug target. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not elucidated. In this study, we highlight the interactions between the residues of hTOP3B and nucleic acids using molecular dynamics (MD) simulation and find inhibitors of the enzyme through in-silico and in-vitro approaches. MD simulation is a well-established tool for studying protein-substrate interaction, and we utilized this method to study the interactions between hTOP3B and nucleic acids. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure (PDB: 5GVC) and 8-mer single-stranded DNA and RNA sequences. These modeled complexes include both covalently and non-covalently complexed structures. We then performed MD simulations of all the generated models. From the simulations, we can highlight the stability of the complexes, conformational changes, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our binding affinity results also provide insight into the substrate preference of hTOP3B. Additionally, developing inhibitors of hTOP3B could serve not only as potential therapeutics but also as biomolecular probes to understand its mechanism of action and biology better. To this end, we conducted an in-silico screening of a library of potential inhibitors targeting the active site cavity of hTOP3B. We then evaluated the candidates with the best docking scores for their inhibition of hTOP3B relaxation activity and quantitatively analyzed the compounds that exhibited inhibition to determine their MIC. Our results show that in-silico screening combined with inhibition assay can help us find potential inhibitors of hTOP3B. Our work demonstrates that hTOP3B forms stable complexes with both RNA and DNA and highlights the suitability of the complexes for inhibitor discovery and binding study. It also provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.