Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors

Abstract

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure–activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

“…However, these two domains share an extensive interface ( 19 ). It has been shown that NSP14 C-terminal MTase activity does not require NSP10 and is, in fact, robust in the absence of NSP10 consistent with our observation that NSP14 is stabilized by SAH above the IC 50 for inhibition of the C-terminal activity ( 16 , 27 ). The C-terminal MTase activity of NSP14 has been targeted for small molecule inhibitor design ( 17 , 27 ).…”

“…It is well documented that the methyltransferase reaction product S-adenosyl-homocysteine (SAH) is a non-specific inhibitor of methyltransferases ( 26 ). Consistent with this, it has been shown that SAH inhibits the methyltransferase activity of NSP14 ( 27 ), yet it is unclear the impact SAH may have on the nuclease activity of NSP14. To address this issue, we first used DSF to determine the impact that SAH imparts on the thermal stability of NSP14 and NSP10/14 complex.…”

Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role inBetacoronavirusreplication

“…To enhance our overall understanding of nsp14 N7-MTase structure and function, also in the light of its emergence as an important drug target in the battle against SARS-CoV-2 ( 50 , 67 – 69 ), we now revisited the SARS-CoV nsp14 X-ray structure to define the most likely residues involved in N7-MTase substrate binding and catalysis. Instead of a βαβ architecture (a seven-stranded β-sheet surrounded by six α-helices) and the canonical MTase motifs, the CoV N7-MTase incorporates 12 β-strands and five α-helices that form a five-stranded β-sheet core ( 36 , 45 ).…”

Structural biology of SARS-CoV-2: open the door for novel therapies

“… 281 , 282 (3) SAM competitive inhibitors and SAM analogs, such as S-adenosyl-homocysteine (SAH), sinefungin (SFG), and aurintricarboxylic acid (ATA) 273 , 283 interfere with N7-Mase activity and subsequently impede 5′-end cap formation. 284 In addition to these inhibitors, mutations in Nsp14 can lead to virus attenuation and induction of higher interferon response. Live attenuated virus vaccine development is, therefore, an option for this target in addition to the development of antibodies.…”