Nucleotide Analogues as Inhibitors of SARS-CoV Polymerase

Jingyue Ju; Xiaoxu Li; Shiv Kumar; Steffen Jockusch; Minchen Chien; Chuanjuan Tao; Irina Morozova; Sergey Kalachikov; Robert N. Kirchdoerfer; James J. Russo

n shown that the active triphosphate form of Remdesivir does not cause complete polymerase reaction termination and actually delays polymerase termination in Ebola virus and respiratory syncytial virus, likely due to its 1'-cyano group and the free 2'-OH and 3'-OH groups. 20, 21 Compared to the active form of Sofosbuvir (2'-fluoro-2'-methyl-UTP), two other nucleotide inhibitors with related structures were reviewed: 2'-fluoro-UTP is incorporated by polymerase, but RNA synthesis may continue past the incorporated nucleotide analogue; 22 2'-C-methyl-UTP has been shown to terminate the reaction catalyzed by HCV RdRp, 22 but proofreading mechanisms can revert this inhibition in mitochondrial DNA-dependent RNA polymerases. 23 Additionally, HCV develops resistance to 2'-C-methyl-UTP due to mutations of the RdRp. 24 A computational study published in 2017 25 considered the ability of various anti-HCV drugs to dock in the active site of SARS and MERS coronavirus RdRps as potential inhibitors. Recently, Elfiky used a computational approach to predict that Sofosbuvir, IDX-184, Ribavirin, and Remidisvir might be potent drugs against . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.12.989186 doi: bioRxiv preprint Thus, based on our analysis of the biological pathways of hepatitis C and coronaviruses, the molecular structures and activities of viral inhibitors, model polymerase and SARS-CoV RdRp extension experiments described below, and the efficacy of Sofosbuvir in inhibiting the HCV RdRp, we expect that Sofosbuvir or its modified forms should also inhibit the SARS-CoV-2 polymerase. 2 The active triphosphate form of Sofosbuvir (2'-F,Me-UTP) was shown to be incorporated by HCV RdRp and prevent any further incorporation by this polymerase. 22, 27 Other viral polymerases have also been shown to incorporate active forms of various anti-viral prodrugs to inhibit replication. 28 Since, at the time of the preparation of this manuscript, we did not have access to the RdRp from SARS-CoV-2, we first selected two groups of polymerases to test the termination efficiency of the active form of Sofosbuvir, one group with high fidelity behavior with regard to incorporation of modified nucleotide analogues, which one would expect for host cell polymerases, the other group with low fidelity mimicking viral polymerases, as well as the RdRp from SARS-CoV, the virus causing the 2003 and subsequent outbreaks of SARS. Our rationale is that the low fidelity viral-like enzymes would incorporate 2'-F,Me-UTP and stop further replication, while the high fidelity polymerases, typical of host cell polymerases, would not. Experimental proof for termination of the SARS-CoV polymerase catalyzed RNA replication would provide further support for this rationale, indicating that Sofosbuvir or its modified forms will inhibit SARS-CoV-2.