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

The RdRp of SARS-CoV-2, referred to as nsp12, and its two protein cofactors, nsp7 and nsp8, whose homologs were shown to be required for the processive polymerase activity of nsp12 in SARS-CoV, 11, 12 were cloned and purified as described in the Methods. These three viral gene products in SARS-CoV-2 have high homology (e.g., 96% identity and 98% similarity for nsp12, with similar homology levels at the amino acid level for nsp7 and nsp8) to the equivalent gene products from SARS-CoV, the causative agent of SARS. 10 We performed polymerase extension assays with 2'-F,Me-UTP, 3'-F-dTTP, 3'-N 3 -dTTP or TFV-DP + UTP, following the addition of a pre-annealed RNA template and primer to a pre-assembled mixture of the SARS-CoV-2 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 3' end of the SARS-CoV-2 genome, were used for the polymerase assay, and their sequences are indicated at the top of Fig. 2 . Because there are two A's 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. Because the two A's in the template are followed by 4 U's, in the case of the TFV-DP/UTP mixture, two UTP's should be incorporated prior to the incorporation and termination by TFV-DP, which has an adenosine base. As shown in Fig. 2 , this is exactly what we observed. In the MALDI-TOF MS trace in Fig. 2a , a peak indicative of the molecular weight of a single base primer extension product with one 2'-F,Me-UTP was obtained (6644 Da observed, 6634 Da expected). Similarly, in the trace in Fig. 2b , a single extension peak indicative of a single base extension by 3'-F-dTTP is revealed (6623 Da observed, 6618 Da expected), with no further incorporation. In both of the above cases, the primer was nearly completely extended. In the trace in Fig. 2d , a single extension peak indicative of a single-base extension by 3'-N 3 -dTTP is seen (6633 Da observed, 6641 Da expected), with no evidence of further incorporation, though the incorporation efficiency was lower than for 2'-F,Me-UTP and 3'-F-dTTP, which may require further optimization. Finally, in the trace in Fig. 2c , a peak indicative of the molecular weight of a primer extension product formed by incorporating 2 U's and 1 TFV is found (7198 Da observed, 7193 Da expected), in addition to other peaks representing partial incorporation (1 U, 6623 Da observed, 6618 Da expected) or misincorporation (3 U's, 7235 Da observed, 7230 Da expected). Importantly, once the TFV-DP was incorporated, there was no further extension, indicating it was a permanent terminator.