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

One of the most important druggable targets for coronaviruses is the RNA-dependent RNA polymerase (RdRp). This polymerase is highly conserved at the protein level among different positive sense RNA viruses, to which coronaviruses and HCV belong, and shares common structural features in these viruses. 5 Like RdRps in other viruses, the coronavirus enzyme is highly error-prone, 6 which might increase its ability to accept modified nucleotide analogues. Nucleotide and nucleoside analogues that inhibit polymerases are an important group of anti-viral agents. [7] [8] [9] [10] Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved heptatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. 2 Sofosbuvir is a pyrimidine nucleotide analogue prodrug with a hydrophobic masked phosphate group enabling it to enter infected eukaryotic cells, and then converted into its active triphosphate form by cellular enzymes (Fig. 1 ). In this activated form, it inhibits the HCV RNA-dependent RNA polymerase NS5B. 11, 12 The activated drug (2'-F,Me-UTP) binds in the active site of the RdRp, where it is incorporated into RNA, and due to fluoro and methyl modifications at the 2' position, inhibits further RNA chain extension, thereby halting RNA replication and stopping viral growth. It acts as an RNA polymerase inhibitor by competing with natural ribonucleotides. Velpatasvir inhibits NS5A, a key protein required for HCV replication. NS5A enhances the function of RNA polymerase NS5B during viral RNA synthesis. 13, 14 Fig. 1 | Conversion of Sofosbuvir to active triphosphate (2'-F,Me-UTP) in vivo to inhibit viral polymerases. Adapted from 11 There are many other RNA polymerase inhibitors that have been evaluated as antiviral drugs. A related purine nucleotide prodrug, Remdesivir (Fig. 2b) , was developed by Gilead to treat Ebola virus infections, though not successfully, and is currently in clinical trials for treating COVID-19. 15, 16 In contrast to Sofosbuvir (Fig. 2a) , both the 2'-and 3'-OH groups in Remdesivir (Fig. 2b) are unmodified, but a cyano group at the 1' position serves to inhibit the RdRp in the active triphosphate form. In addition to the use of hydrophobic groups to mask the phosphate in the Protide-based prodrug strategy, 17 as with Sofosbuvir and Remdesivir, there are other classes of nucleoside prodrugs including those based on ester derivatives of the ribose hydroxyl groups to enhance cellular delivery. 18,19 The replication cycle of HCV 4 is very similar to that of the coronaviruses. 3 Analyzing the structure of the active triphosphate form of Sofosbuvir (Fig. 2a) compared to that of Remdesivir (Fig. 2b) , both of which have already been shown to inhibit the replication of specific RNA viruses (Sofosbuvir for HCV, Remdesivir for SARS-CoV-2), we noted in particular that the 2'-modifications in Sofosbuvir (a fluoro and a methyl group) are substantially smaller than the 1'-cyano group and the 2'-OH group in Remdesivir. The bulky cyano group in close proximity to the 2'-OH may lead to steric hindrance that will impact the incorporation efficiency of the activated form of Remdesivir. Interestingly, it was recently reported that, using the MERS-CoV polymerase, the triphosphate of Remdesivir was preferentially incorporated relative to ATP in solution assays. 20 Nevertheless, it has bee