Potent SARS-CoV-2 mRNA Cap Methyltransferase Inhibitors by Bioisosteric Replacement of Methionine in SAM Cosubstrate

Abstract

Viral mRNA cap methyltransferases (MTases) are emerging targets for the development of broad-spectrum antiviral agents. In this work, we designed potential SARS-CoV-2 MTase Nsp14 and Nsp16 inhibitors by using bioisosteric substitution of the sulfonium and amino acid substructures of the cosubstrate S-adenosylmethionine (SAM), which serves as the methyl donor in the enzymatic reaction. The synthetically accessible target structures were prioritized using molecular docking. Testing of the inhibitory activity of the synthesized compounds showed nanomolar to submicromolar IC 50 values for five compounds. To evaluate selectivity, enzymatic inhibition of the human glycine N -methyltransferase involved in cellular SAM/SAH ratio regulation was also determined, which indicated that the discovered compounds are nonselective inhibitors of the studied MTases with slight selectivity for Nsp16. No cytotoxic effects were observed; however, this is most likely a result of the poor cell permeability of all evaluated compounds.

Crystal structure of SARS‐CoV‐2 nsp10–nsp16 in complex with small molecule inhibitors, SS148 and WZ16

“…To our knowledge, only nonselective nanomolar inhibitors of nsp16 have been reported to‐date. 33 Although the MTase active site of nsp14 and nsp16, the two SARS‐CoV‐2 MTases, are quite different (Figure S1 ), our cross‐screening of inhibitors of nsp14 MTase activity against nsp10–nsp16 resulted in identifying SS148 and WZ16 as nsp16 MTase inhibitors. Our structural and biochemical assays elucidated a new mechanism of RNA‐dependent SAM‐competitive inhibition.…”

“…SARS‐CoV‐2 nsp10–nsp16 complex is an RNA MTase involved in RNA cap formation, enabling the virus to evade the immune system in humans. To our knowledge, only nonselective nanomolar inhibitors of nsp16 have been reported to‐date 33 . Although the MTase active site of nsp14 and nsp16, the two SARS‐CoV‐2 MTases, are quite different (Figure S1), our cross‐screening of inhibitors of nsp14 MTase activity against nsp10–nsp16 resulted in identifying SS148 and WZ16 as nsp16 MTase inhibitors.…”

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

“…Recently, several studies have reported high-throughput screening of existing libraries of small molecules against nsp14 activity. However, very few compounds have been identified as potential inhibitors of SARS-CoV-2. − 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. , However, both articles did not report studies on the inhibitory activity of these nsp14 inhibitors in SARS-CoV-2-infected cells. Before the emergence of SARS-CoV-2, our group had pioneered the synthesis of selective inhibitors targeting the SARS-CoV nsp14 N 7-MTase using dinucleosides as mimetics of the S -adenosyl- l -methionine (SAM) that is the methyl donor in the N 7-methylation of the cap (Figure ).…”

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

“… 17 − 19 There are potent S -adenosylhomocysteine (SAH)/SAM analogs that have been identified from a collection of other SAM-dependent MTase inhibitors 20 or rationally designed. 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.…”

“…17 or rationally designed. 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.…”