Discovery of SARS-CoV-2 Nsp14 and Nsp16 Methyltransferase Inhibitors by High-Throughput Virtual Screening

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput virtual screening against these two enzymes. The docking of seven million commercially available drug-like compounds and S-adenosylmethionine (SAM) co-substrate analogues against both MTases resulted in 80 virtual screening hits (39 against nsp14 and 41 against nsp16), which were purchased and tested using an enzymatic homogeneous time-resolved fluorescent energy transfer (HTRF) assay. Nine compounds showed micromolar inhibition activity (IC50 < 200 μM). The selectivity of the identified inhibitors was evaluated by cross-checking their activity against human glycine N-methyltransferase. The majority of the compounds showed poor selectivity for a specific MTase, no cytotoxic effects, and rather poor cell permeability. Nevertheless, the identified compounds represent good starting points that have the potential to be developed into efficient viral MTase inhibitors.

Optimizing peptide inhibitors of SARS-Cov-2 nsp10/nsp16 methyltransferase predicted through molecular simulation and machine learning

“…Several studies have searched for small molecules that can inhibit MTase activity. Bobrovs et al computationally screened the docking of 7 million compounds with nsp14 and nsp16 and found 80 candidates, 39 for nsp14 and 41 for nsp16 [ 6 ]. They experimentally assayed them and found 9 that displayed MTase inhibition with an IC 50 < 200 μM.…”

“…There have been numerous efforts to find ways to disrupt SARS-CoV-2 MT activity using small molecules [ [6] , [7] , [8] , [9] ]. Other SARS-CoV studies have shown that synthetic peptides can interfere in the binding of nsp10 to nsp14 or to nsp16 and can diminish or modulate viral replication [ [10] , [11] , [12] , [13] , [14] ].…”

Potent Inhibition of SARS-CoV-2 nsp14 N7-Methyltransferase by Sulfonamide-Based Bisubstrate Analogues

“…However, very few compounds have been identified as potential inhibitors of SARS-CoV-2. 14 − 18 In addition to the drug-repurposing approach, the method of de novo drug discovery with structure-guided design of nsp14 substrates yielded potent inhibitors with IC 50 values in the nanomolar to submicromolar range. 19 , 20 However, both articles did not report studies on the inhibitory activity of these nsp14 inhibitors in SARS-CoV-2-infected cells.…”

Bisubstrate Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 Nsp14 Methyltransferase

“… 21 , 22 In addition, virtual screenings have been performed to identify potential Nsp14 MTase inhibitors. 23 However, most of these inhibitors are highly polar and no anti-SARS-CoV-2 activity has been reported. During the preparation of this manuscript, potent and selective bisubstrate inhibitors that contained an sulfonamide functionality were reported; however, no antiviral activity was disclosed.…”