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 performed polymerase extension assays with 2'-F,Me-UTP, 3'-F-dTTP, 3'-N 3 -dTTP or UTP following the addition of an pre-annealed RNA template and primer to a pre-assembled mixture of the RdRp (nsp12) and two cofactor proteins (nsp7 and nsp8). The extended primer products from the reaction were subjected to MALDI-TOF-MS analysis. The RNA template and primer, corresponding to the N1 epitope region of the N protein of the SARS-CoV-2 virus, were used for the polymerase assay, and their sequences are indicated at the top of Fig. 4 . Because there are two As in a row in the next available positions of the template for RNA polymerase extension downstream of the priming site, if the 2'-F,Me-UTP, 3'-F-dTTP or 3'-N 3 -dTTP are incorporated by the viral RdRp, a single nucleotide analogue will be added to the 3'-end of the primer strand. If they are indeed inhibitors of the polymerase, the extension should stop after this incorporation; further 3'extension should be prevented. In the case of the UTP control reaction, two UTP's should be incorporated. As shown in Fig. 4 and Extended Data Fig. 2 , this is exactly what we observed. In the MALDI-TOF MS trace in Fig. 4a, a peak In summary, these results demonstrate that the nucleotide analogues 2'-F,Me-UTP, 3'-F-dTTP and 3'-N 3 -dTTP, are permanent terminators for the SARS-CoV RdRp. Their prodrug versions (Sofosbuvir, 3'-F-5'-Ophosphoramidate dT nucleoside and 3'-N 3 -5'-O-phosphoramidate dT nucleoside) can be readily synthesized using the ProTide prodrug approach, as shown in Fig. 2a, c and d, and can be developed as therapeutics for both SARS and COVID-19.