SARS-CoV-2 nsp14-IN-10 is a highly effective and selective inhibitor of the NSP14 (IC50 = 0.34 µM) S-adenosylmethionine (SAM) binding pocket. This compound exhibits significant antiviral activity against SARS-CoV-2 and has broad-spectrum efficacy against other β-coronaviruses. It inhibits the replication phase of SARS-CoV-2 and also suppresses viral translation while stimulating immune responses. Additionally, SARS-CoV-2 nsp14-IN-10 can specifically reverse NSP14-mediated alterations in the host transcriptome. It is a valuable compound for research on SARS-CoV-2.

SARS-CoV-2 nsp14-IN-10 (Compound C10) specifically binds to the SAM recognition pocket of NSP14 in a competitive manner with a dissociation constant (K D) of 187 nM. At concentrations between 5-25 μM over 8 hours, it increases the thermal stability of NSP14 protein in HEK293-NSP14 cells, indicating target binding in live cells. After 24-hour exposure, it significantly reduces infection rates of original SARS-CoV-2, Delta, and Omicron variants in A549-ACE2 cells, with EC 50 values of 0.31 μM, 0.50 μM, and 0.06 μM, respectively. At 10 µM over 24 hours, it effectively hinders SARS-CoV-2 replication in HeLa cells. When used at 3-20 μM for 0-24 hours, it shows strong inhibitory activity against SARS-CoV-2 trVLP in A549-ACE2 cells, regardless of full-course or post-entry treatment, though no significant inhibition is observed in uninfected cell entry treatment. Concentrations of 20-40 μM over 8 hours inhibit translation initiation of SARS-CoV-2 mRNA in trVLP-infected A549-ACE2 cells, specifically obstructing viral cap maturation. It selectively inhibits viral translation under the same conditions and substantially reduces Spike mRNA and protein expression levels after 24-hour exposure at 20 μM. Additionally, it upregulates interferon-stimulated genes (ISG) like RSAD2, IFI6, and IRF7, and reverses host transcriptome changes induced by NSP14 expression in HEK293-NSP14 cells at 5-20 μM over 48 hours. Western Blot analysis indicates enhanced thermal stability of NSP14 protein at concentrations between 5 and 25 μM after an 8-hour incubation in HEK293-NSP14 cells.

SARS-CoV-2 nsp14-IN-10 (Compound C10) administered intraperitoneally at doses of 60-180 mg/kg twice daily for three days significantly reduces SARS-CoV-2 replication and pathological damage in the lungs of infected K18-hACE2 transgenic mice, while maintaining a broad safety margin.