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

Based on our similar insight related to their molecular structures and previous antiviral activity studies, in comparison with Sofosbuvir, we selected the triphosphate forms of Alovudine (3'-deoxy-3'-fluorothymidine) and azidothymidine (AZT, the first FDA approved drug for HIV/AIDS) for evaluation as inhibitors of the SARS-CoV RdRp. These two molecules 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. The copyright holder for this preprint (which was not peer-reviewed) is the .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 Alovudine is one of the most potent inhibitors of HIV reverse transcriptase and HIV-1 replication. This promising drug was discontinued after a Phase II trial due to its hematological toxicity. 33 However, subsequent in vitro studies showed Alovudine was very effective at suppressing several nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-resistant HIV-1 mutants. 34 New clinical studies were then carried out in which low doses of Alovudine were given as supplements to patients showing evidence of infection by NRTI resistant HIV strains and not responding well to their current drug regimen. A 4-week course of 2 mg/day Alovudine reduced viral load significantly, and was relatively well tolerated with no unexpected adverse events. 35 AZT is another antiretroviral medication which has long been used to prevent and treat HIV/AIDS. [36] [37] [38] Upon entry into the infected cells, similar to Alovudine, cellular enzymes convert AZT into the effective 5'triphosphate form (3'-N 3 -dTTP, structure shown in Fig. 2d ), which competes with dTTP for incorporation into DNA by HIV-reverse transcriptase resulting in termination of HIV's DNA synthesis. 39 Since the side effects and toxicity of AZT are well understood, novel methodologies have been directed at enhancing AZT plasma levels and its bioavailability in all human organs in order to improve its therapeutic efficacy. Among these possibilities, an AZT prodrug strategy was proposed. 40 We thus assessed the ability of 3'-N 3 -dTTP and 3'-F-dTTP, the active triphosphate forms of AZT and Alovudine, along with 2'-F,Me-UTP, to be incorporated by SARS-CoV RdRp into an RNA primer and terminate the polymerase reaction.