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

We have recently described 2 a strategy to design and synthesize viral polymerase inhibitors, by combining the ProTide Prodrug approach 17 used in the development of Sofosbuvir with the use of 3'-blocking groups that we have previously built into nucleotide analogues that function as terminators for DNA sequencing by synthesis. 29,30, 45 We reasoned that (i) the hydrophobic phosphate masking groups will allow entry of the compounds into the virus-infected cells, (ii) the 3'-blocking group on the 3'-OH with either free 2'-OH or modifications at the 2' position will encourage incorporation of the activated triphosphate analogue by viral polymerases but not host cell polymerases, thus reducing any side effects, and (iii) once incorporated, further extension will be prevented by virtue of the 3'-blocking group, thereby completely inhibiting viral replication and potentially overcoming the development of resistance due to the accumulation of new mutations in the RdRp. 46 Our design criterion is to identify groups for attachment to the 3'-OH with appropriate structural and chemical properties (e.g., size, shape, rigidity, flexibility, polarity, reactivity [e.g., stability to cellular enzymes]), 47, 48 along with appropriate 2'-substitutions, so that they will be incorporated by the viral RdRp, while minimizing incorporation by the host polymerases. We previously used this chemical and structural principle to select a variety of chemical moieties that block the 3'-OH of the nucleotide analogues as polymerase terminators. 31, 49, 50 In conclusion, we demonstrated the capability of more tolerant DNA polymerases, as well as SARS CoV RNA-dependent RNA polymerase, which is nearly identical to the SARS-CoV-2 RdRp responsible for COVID-19, to incorporate 2'-F,Me-UTP, the active form of Sofosbuvir, where it serves to terminate the polymerase reaction. We also showed two other nucleotide triphosphates, 3'-F-dTTP, the active form of Alovudine, and 3'-N 3 -dTTP, the active form of AZT, can also be incorporated and terminate further nucleotide extension by the RdRp in the polymerase reaction, potentially preventing further replication of the virus. Prodrug versions of these compounds and their derivatives therefore can be developed as potent broadspectrum therapeutics for coronavirus infectious diseases, including SARS, MERS and COVID-19. which is also less conservative in other coronaviruses. 5 Within the next region (the interface domain, aa ~250 . 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 through 400), the first 15 amino acid positions have multiple substitutions, but the rest of the interface domain is quite conservative. The region beyond the interface domain, corresponding to the nsp12 C-terminus, contains polymerase functional domains. These domains constitute the canonical fingers, palm, and thumb of the polymerase enzyme and contain several motifs that are conservative among coronaviruses (Motifs A through F). Out of the 34 amino acid substitutions in the nsp12 between SARS-CoV and SARS-CoV-2, only three substitutions are located within these motifs, and all three are between similar amino acids.