Investigation of whether Topoisomerase 3b can be a drug target for SARS-CoV-2 and other positive-strand RNA virus; and development of such a drug

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Topoisomerases are necessary to solve topological problems during DNA metabolism, such as replication and transcription. Our group has made the original discovery that Top3b is a dual-activity topoisomerase that can change topology for not only DNA, but also RNA (Xu et al., Nat. Neurosci., 2013). This discovery has led us to propose that RNA metabolism, such as mRNA translation, may resemble DNA metabolism in producing topological problems that require an RNA topoisomerase to solve. Indeed, increasing evidence from our group and others has shown that Top3b is the only RNA topoisomerase in animals, forms a complex with TDRD3, and interacts with FMRP (Fragile X mental retardation protein) to regulate mRNA translation during neurodevelopment. In this context, Prasanths findings that Top3b and TDRD3 are needed for efficient viral RNA replication provides further support for our hypothesis. We noted that Prasanths study lacks key evidence to show that Top3b requires its catalytic activity to promote RNA virus replication. Without such evidence, it is possible that Top3b only acts as an RNA-binding protein during viral replication. If that is the case, an inhibitor of the Top3b enzymatic activity will not be useful in treatment of patients of COVID-19 and RNA virus-induced pandemics. Rather, an inhibitor of Top3b RNA-binding activity should be more appropriate. We believe that this issue is critical and should be resolved before we start a long and expensive journey to screen for a Top3b inhibitor. Our proposed Aim 1 is to address this issue. Although inhibitors of Topoisomerase 1 and 2 have been successfully developed and used to treat different cancers, no good inhibitors for the Top3b family (Type IA) of topoisomerases have been developed yet. The newly discovered connection between Top3b and SARS-CoV-2 necessitates development of a Top3b inhibitor. Our group has previously established that RNA topoisomerase activity is prevalent in Type IA family of topoisomerase from bacteria, archaea, and eukaryotes (Ahmad et al., NAR, 2016). We have participated in a collaboration to develop an inhibitor for this family, which resulted in discovery of a family of small chemicals that can inhibit an E.coli homolog of Top3b (Ranjan et al.). In Aim 2, we propose to renew our efforts to develop an inhibitor for Top3b. Prasanths findings that Top3b and TDRD3-KO cells have reduced biogenesis of SARS-Cov-2 and other RNA viruses could be due to either reduced viral RNA replication, or impaired viral RNA translation. This is because translation of (+) virus RNA into viral proteins precedes its replication. Given the current evidence on Top3b in cellular mRNA translation, we propose Aim 3 to investigate if Top3b is similarly needed for efficient viral RNA translation. We are currently developing a cell line model to study whether Top3b-TDRD3 complex is needed for efficient replication of mouse coronavirus, murine hepatitis virus (MHV). Our preliminary data show that depletion of Top3b from a mouse cell line can reduce replication of MHV. We are repeating this experiment, and also plan to generate Knockout cell lines of Top3b-TDRD3 to verify this finding.