Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.

• Keywords
• Förster resonance energy transfer (FRET), SARS-CoV-2 main protease, activation, autoprocessing, cell-based assay, fluorescence cross-correlation spectroscopy (FCCS), fluorescence life-time imaging, inhibitor, maturation, nsp5, precursor, protease, virus,
• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• COVID-19 virology   MeSH
• COVID-19 Drug Treatment * MeSH
• HEK293 Cells MeSH
• Protease Inhibitors * pharmacology   chemistry   MeSH
• Coronavirus 3C Proteases * metabolism   genetics   antagonists & inhibitors   chemistry   MeSH
• Humans MeSH
• Mutation MeSH
• Drug Discovery * MeSH
• SARS-CoV-2 * enzymology   drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3C-like proteinase, SARS-CoV-2 MeSH Browser
• Antiviral Agents * MeSH
• Protease Inhibitors * MeSH
• Coronavirus 3C Proteases * MeSH

The emergence of SARS-CoV-2, the causative agent of COVID-19, has highlighted the need for advanced antiviral strategies. Targeting the coronaviral methyltransferase nsp14, which is essential for RNA capping, offers a promising approach for the development of small-molecule inhibitors. We designed and synthesized a series of adenosine 5'-carboxamide derivatives as potential nsp14 inhibitors and identified coumarin analogs to be particularly effective. Structural modifications revealed the importance of the 5'-carboxyl moiety for the inhibitory activity, showing superior efficacy compared to other modifications. Notably, compound 18l (HK370) demonstrated high selectivity and favorable in vitro pharmacokinetic properties and exhibited moderate antiviral activity in cell-based assays. These findings provide a robust foundation for developing targeted nsp14 inhibitors as a potential treatment for COVID-19 and related diseases.

• Publication type
• Journal Article MeSH

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

• Keywords
• antiviral, coronavirus, covalent inhibitors, nsp16 methyltransferase, structural biology,
• MeSH
• COVID-19 * MeSH
• Rats MeSH
• Methyltransferases MeSH
• Mice MeSH
• S-Adenosylmethionine chemistry   metabolism   MeSH
• SARS-CoV-2 * metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Methyltransferases MeSH
• S-Adenosylmethionine MeSH

Monkeypox, or mpox, is a disease that has recently resurfaced and spread across the globe. Despite the availability of an FDA-approved vaccine (JYNNEOS) and an effective drug (tecovirimat), concerns remain over the possible recurrence of a viral pandemic. Like any other virus, mpox virus must overcome the immune system to replicate. Viruses have evolved various strategies to overcome both innate and adaptive immunity. Poxviruses possess an unusual nuclease, poxin, which cleaves 2'-3'-cGAMP, a cyclic dinucleotide, which is an important second messenger in the cGAS-STING signaling pathway. Here, we present the crystal structure of mpox poxin. The structure reveals a conserved, predominantly β-sheet fold and highlights the high conservation of the cGAMP binding site and of the catalytic residues His17, Tyr138, and Lys142. This research suggests that poxin inhibitors could be effective against multiple poxviruses.

• MeSH
• Humans MeSH
• Mpox, Monkeypox * MeSH
• Poxviridae * MeSH
• Drug Design MeSH
• Signal Transduction MeSH
• Monkeypox virus MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.

• MeSH
• COVID-19 * MeSH
• Zika Virus Infection * MeSH
• Humans MeSH
• Methyltransferases metabolism   MeSH
• RNA, Viral genetics   MeSH
• RNA MeSH
• SARS-CoV-2 genetics   MeSH
• Viral Nonstructural Proteins chemistry   MeSH
• Monkeypox virus genetics   metabolism   MeSH
• Zika Virus * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methyltransferases MeSH
• RNA, Viral MeSH
• RNA MeSH
• Viral Nonstructural Proteins MeSH