Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

Abstract

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication–transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease

“…We next used this assay to determine the effect of substrate concentration on initial reaction velocities. We found that, in contrast with all of the other enzymes we have examined in this series of papers [31][32][33][34][35][36], reaction rates did not plateau at higher substrate concentrations but instead continued to increase non-linearly, indicating that nsp15 endoribonuclease behaves as an allosteric enzyme towards the 6 nt U substrate, with a K half of 2140 nM (Figure 3A and Supplementary Figure S2A).…”

“…Reaction slopes in the presence of each compound were calculated and normalised to the DMSO-containing positive controls (control reactions) on each plate (Figure 3C and Supplementary Figure S2B). We did not find any inhibitors that showed residual activities <0.7 in both concentrations (Figure 3D), which was surprising given the results of similar screens from our accompanying manuscripts [31][32][33][34][35][36][37]. Therefore, we considered compounds that had residual activities <0.9 and had a Z-score <−4 (Figure 3E and Supplementary Figure S2D).…”

“…However, this high enzyme concentration combined with high substrate competition (for potential competitive inhibitors) might explain why we did not detect many compounds able to inhibit nsp15 EndoU activity, and why our hit compounds only reduced nsp15 activity by 10-20% in the original conditions of the screen. We and our colleagues were able to detect hits for the other SARS-CoV-2 enzymes using the same drug library and general screening strategy (see accompanying manuscripts [31][32][33][34][35][36][37]) and, therefore, it would be advisable for future studies to use higher compound to enzyme or substrate ratios to improve detection of nsp15 inhibitors. We found that NSC95397 is an inhibitor of nsp15 but not a general nuclease inhibitor in vitro.…”

An all-out assault on SARS-CoV-2 replication

“…Compounds that inhibited viral replication without deleterious effects on uninfected cells were selected as candidate antivirals. The seven papers describing these results are published in this addition of the Biochemical Journal [1][2][3][4][5][6][7].…”

“…While all inhibited viral replication and had synergistic interactions with remdesivir the severe side effects associated with Trifluperidol may limit its use as a single agent. A series of compounds related to suramin were shown to inhibit the RdRp encoded by nsp12 in complex with its processivity factors nsps 7 and 8 [3]. Suramin was developed over 100 years ago by Bayer for the treatment of river blindness and sleeping sickness and is known to inhibit multiple viruses.…”