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

National Institute On Aging

Investigators

Abstract

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. Aim 1. Progress 1. Top3b-KO may promote but not inhibit MHV replication in mouse cell lines. We examined MHV proliferation in Top3b-KO 17Cl-1 cells generated by CRISPR-Cas9. We made a total of 5 groups of KO cells by 5 different guide RNAs (Fig. A). In KO cells generated by 2 guide RNAs targeting the catalytic domain, we observed a 20-90 fold increase of MHV replication (Fig. B, C)). In KO cells generated by 3 guide RNAs targeting the N-terminal domain of Top3b, MHV replication shows much smaller difference (less than 5-fold increase or decrease). One explanation for the difference is that targeting catalytic domain can completely inactivate Top3b (sgRNA 2 and 3), whereas targeting the N-terminal domain (sgRNA1, 4, 5) may result in a partially active protein. These data imply that complete inactivation of Top3b may promote rather than inhibit MHV replication, so that they do not support the proposition by Mariano and colleagues. We have communicated our data to Marianos group, and their latest results are largely consistent with our data but inconsistent with their earlier reports. We have published one paper indicating that Top3b is dispensable for replication of MHV using cell line and mouse models (Zhang et al., Antiviral Res. 2022). Aim 1. Progress 2. Identify potent coronavirus inhibitors by drug screens We have established a collaboration with Mariano and 3 other labs to screen inhibitors of MHV and SARS-Cov-2 replication. Although Top3b protein inactivation may promote rather than inhibit MHV replication, an inhibitor of Top3b may still prevent viral RNA replication by forming an RNA cleavage complex containing Top3b protein covalently linked to the cleaved RNA. This is the same way by which a DNA topoisomerase inhibitor works to block DNA replicationformation of a Topoisomerase-containing DNA cleavage complex. We have successfully established MHV-EGFP and MHV-luciferase assays that allow us to screen inhibitors of MHV in 17Cl-1 host cells. We have so far screened about 80 compounds obtained from our collaborators. The compound 6002 from Arya group inhibits MHV with IC50 0.25 M at high MOI and short time infection conditions (Fig. E). 6002 is a derivative of Bisbenzimidazole family molecules which also bind DNA and RNA; and inhibit Top3b activity at about 10-50 M concentrations (Ranjan et al., 2016 Bioorg Med Chem Lett.). The combination of 6002 with low concentration Remdesivir shows some additive effects, although there is no strong synergistic effect (Fig. F). Compounds 4487-G08 from Pommiers group can trap Top3b on DNA and RNA to form a covalently linked protein-nucleic acid complex in vitro; and can also cause death in cells expressing Top3b. Importantly, 4487-G08 shows strong MHV inhibition with IC50 0.03 M (Fig. G) at low MOI and longtime infection conditions; and it is more potent than Remdesivir (IC50 0.1 M in our hands) (Fig. H), the FDA-approved drug for COVID patients. Furthermore, combination of 4487-G08 and Remdesivir shows synergistic effects on MHV replication (Fig. H). Consistent with our findings, Marianos group found that 4487-G08 shows significant SARS-Covid2 inhibition at 1 M concentration. Together, our data suggest that 4487-G08 is a strong drug candidate that can be developed to treat SARS-Covid2 patients. We recently identified a structural homolog of G08, G09, is more potent in inhibiting MHV replication in cell lines (IC506 nM). We are now testing this drug in the mouse model, and found that it has a strong trend to inhibit MHV replication in the lungs. We are expanding the mouse experiment, to find the optimal condition to use this compound to inhibit MHV replication in mouse models. Our most recent studies with collaborators show that G09 compound can inhibit RNA-dependent RNA polymerase (RdRP) in vitro. It can also inhibit retrovirus encoded reverse transcriptase in vitro. The data suggest that the compound may work against other viruses. Aim 2. Progress. Top3b-KO mice may be more vulnerable to MHV infection. With the supports of NIA and Bethesda animal facilities, we tested MHV replication in WT and Top3b-KO mouse tissues. MHV was injected into mouse brain, and brains and livers were dissected three days after injection to detect MHV RNA levels by qRT-PCR. MHV RNA levels are not significantly different in WT and Top3b-KO brains. However, MHV RNA levels in Top3b-KO livers are about 10-fold higher than those of WT (Fig. D). The results from mouse livers are similar to the increased MHV replication in Top3b-KO2 and -KO3 cell lines. The Top3b-KO mice were generated by gene targeting and the catalytic domain with Y336 of Top3b was replaced by Neo gene (Kwan & Wang, 2001, PNAS). The mouse data also support our cell line data that complete inactivation of Top3b may promote rather than inhibit MHV replication.