Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase

Minchen Chien; Thomas K. Anderson; Steffen Jockusch; Chuanjuan Tao; Shiv Kumar; Xiaoxu Li; James J. Russo; Robert Kirchdoerfer; Jingyue Ju

Potential inhibitors have been designed to target nearly every stage of the viral replication cycle. 2 However, despite decades of research, no effective drug is currently approved to treat serious coronavirus infections such as SARS, MERS, and now COVID-19. One of the most important druggable targets for coronaviruses is the RdRp. This polymerase displays similar catalytic mechanisms and some key conserved amino acids in the active site among different positive sense RNA viruses, to which coronaviruses and HCV belong. 4 Like RdRps in other viruses, the coronavirus enzyme is highly error-prone, 5 which might increase its ability to accept modified nucleotide analogues as substrates. Nucleotide and nucleoside analogues that inhibit polymerases are an important group of anti-viral agents. [6] [7] [8] [9] Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). 10 Using polymerase extension experiments, we demonstrated that the activated triphosphate form of Sofosbuvir was incorporated by SARS-CoV RdRp, and blocked further incorporation. Using the same molecular insight, we selected two other anti-viral agents, Alovudine and AZT (the first FDA approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. Alovudine and AZT share a similar backbone structure (base and ribose) to Sofosbuvir, but have fewer modification sites and less steric hindrance. Furthermore, because these modifications on Alovudine and AZT are on the 3' carbon in place of the OH group, they directly prevent further incorporation of nucleotides leading to permanent termination of RNA synthesis and replication of the virus. We also demonstrated the ability of two HIV reverse transcriptase inhibitors, 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate (the active triphosphate forms of Alovudine and AZT), to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. 10 In this paper, we first constructed SARS-CoV-2 RdRp based on a similar procedure to that of SARS-CoV, 11, 12 and then demonstrated that the above three nucleotide analogues ( Fig. 1 a, b , d) are inhibitors of SARS-CoV-2 RdRp. Using structure-activity based molecular insight, we selected the active triphosphate form of Tenofovir alafenamide (TAF, Vemlidy, an acyclic adenosine nucleotide) ( Fig. 1 c) , which is an FDA approved drug for HIV and hepatitis B virus (HBV) infection, for evaluation as a SARS-CoV-2 RdRp inhibitor. The results indicated that the active triphosphate form of this molecule, tenofovir diphosphate (TFV-DP), also inhibited this polymerase. TAF, a prodrug form of the nucleotide analogue viral polymerase inhibitor, shows potent activity for HIV and HBV, but only limited inhibition of host nuclear and mitochondrial polymerases. 13, 14 It is activated by a series of hydrolases to the deprotected monophosphate form, TFV, and then by two consecutive kinase reactions to TFV-DP. 15 TFV-DP is an acyclic nucleotide and does not have a 3'-OH group. Remarkably, this molecule is incorporated by both HIV and HBV polymerases, terminating nucleic acid elongation and viral replication. 13, 15 In addition, resistance mutations were rarely seen in patients treated with regimens including TAF. 16 In view of the fact that the active triphosphate