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

拓扑异构酶是解决DNA新陈代谢中的拓扑问题所必需的，如复制和转录。我们团队的原创性发现是TOP3B是一种双活性拓扑异构酶，它不仅可以改变DNA的拓扑结构，而且可以改变RNA的拓扑结构(Xu等人，NA.Neurosci，2013)。这一发现使我们提出，RNA代谢，如mRNA翻译，可能类似于DNA代谢，产生需要RNA拓扑异构酶才能解决的拓扑问题。事实上，来自我们团队和其他人的越来越多的证据表明，TOP3B是动物中唯一的RNA拓扑异构酶，与TDRD3形成复合体，并与FMRP(脆性X智力低下蛋白)相互作用，调节神经发育过程中的mRNA翻译。在这种情况下，Prasanth的发现TOP3B和TDRD3是有效的病毒RNA复制所必需的，这进一步支持了我们的假设。 我们注意到Prasanth的研究缺乏关键证据表明TOP3B需要其催化活性来促进RNA病毒复制。如果没有这样的证据，TOP3B可能只在病毒复制过程中作为RNA结合蛋白发挥作用。如果是这样的话，TOP3B酶活性的抑制剂在治疗新冠肺炎和核糖核酸病毒诱导的大流行中将无用。相反，TOP3B RNA结合活性的抑制剂应该更合适。我们认为，这个问题是关键的，应该在我们开始漫长而昂贵的TOP3B抑制剂筛选之旅之前得到解决。我们提出的目标1就是解决这个问题。 虽然拓扑异构酶1和拓扑异构酶2的抑制剂已经被成功地开发并用于治疗不同的癌症，但还没有开发出针对TOP3B家族(IA型)的良好的拓扑异构酶抑制剂。新发现的TOP3B和SARS-CoV-2之间的联系需要开发一种TOP3B抑制剂。我们的小组先前已经证实，RNA拓扑异构酶活性普遍存在于细菌、古菌和真核生物的IA型拓扑异构酶家族中(Ahmad等人，NAR，2016)。我们参与了为这个家族开发一种抑制剂的合作，这导致了一系列小化学物质的发现，这些化学物质可以抑制TOP3B的大肠杆菌同系物(Ranjan等人)。在目标2中，我们建议重新努力开发TOP3B的抑制剂。 Prasanth发现，TOP3B和TDRD3-KO细胞减少了SARS-CoV-2和其他RNA病毒的生物生成，这可能是由于病毒RNA复制减少，或削弱了病毒RNA翻译。这是因为(+)病毒RNA在复制之前被翻译成病毒蛋白质。鉴于目前关于TOP3B在细胞mRNA翻译中的证据，我们提出目标3来研究TOP3B是否同样需要TOP3B来有效地翻译病毒RNA。 我们目前正在开发一种细胞系模型，以研究TOP3B-TDRD3复合体是否需要小鼠冠状病毒、小鼠肝炎病毒(MHV)的有效复制。我们的初步数据表明，从小鼠细胞系中去除TOP3B可以减少MHV的复制。我们正在重复这个实验，并计划建立TOP3B-TDRD3的基因敲除细胞系来验证这一发现。

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.