The main protease of SARS-CoV-2 (M pro ) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in M pro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 M pro mutants provided structural insight into their altered functionality. Additionally, we show M pro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting M pro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOC) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and subsequent significant decrease in cytokine secretion, providing evidence for alterations in escape of host-antiviral mechanisms. Accordingly, mutations associated with the highly virulent Delta VOC resulted in significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, Mpro and PLpro, that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 Mpro and PLpro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 Mpro and PLpro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.

PCLX-001 is a first-in-kind drug that targets a protein modification essential for membrane anchorage and signalling, and has been under human clinical trial evaluation for the treatment of lymphoma and solid malignancies for over a year. In human, over 600 proteoforms are modified with the fatty acid myristate by two N-myristoyltransferases: NMT1 and NMT2. These include many proto-oncogenic proteins (e.g. Src-family kinases and c-Abl) and metabolic regulators (e.g. AMPK β subunit). Thus, we validated the potential of NMTs as oncology targets on 300 cancer cell lines and uncovered its potential in hematological cancers. The multiple substrates of NMTs complicated the elucidation of a clear mechanism of action of the pan-NMT inhibitor PCLX-001. To circumvent this, we evaluated the individual roles of NMT1 and NMT2 in tumours as well as their relative contributions, including those of their substrates, to PCLX-001 sensitivity. Analysis of TCGA and CCLE databases revealed that NMT2 expression levels vary drastically (7 log2(TPM+1)) while those of NMT1 vary slightly (3 log2(TPM+1)). Hematological cancers comprised the vast majority of the lowest NMT2 expressing cells but lower NMT2 levels were also observed in solid tumour cell lines (e.g. breast & ovary). NMT2 levels were also lower in cancer versus normal corresponding tissues. We confirmed that NMT2 expression is epigenetically repressed at a CpG island found in the 5´end of the NMT2 gene thereby explaining the reduction in NMT2 expression. Since most hematological cancer cells are NMT2-deficient, we think that by targeting the remaining NMT1, PCLX-001 selectively kills these cells in a manner reminiscent of synthetic lethality, thereby sparing normal human cells with two NMTs. Therefore, identifying substrates specific to NMT1 in NMT2-deficient cancer cells became of the utmost importance to understand which NMT1 protein substrates were critical for survival. Using differential proteomics in the CRISPR/Cas9 NMT1 KO HAP1 near-haploid cell line derived from the chronic myelogenous leukemia versus WT HAP1 cells, we surprisingly found that few signaling proteins were downregulated, rather, 36 mitochondrial protein levels were decreased and 18 of these belonged to respiratory complex I. Myristoylated mitochondrial protein NDUFAF4, a major complex I assembly factor, was the most downregulated and PCLX-001 reduced its levels leading to decreased complex I activity. This seemingly ordinary observation could have transformational implications for cancer treatment since oxidative phosphorylation is essential for both cancer stem cell survival and metastasis. In addition, gene set enrichment analyses performed on RNASeq data obtained from 1200 cell lines treated with myristoylation inhibitors allowed us to derive an NMT inhibition sensitivity gene set that could be used to identify patients which would most benefit from NMT inhibition therapy. Citation Format: Erwan Beauchamp, Chistopher Cromwell, Eman Moussa, Aishwarya Iyer, Megan Yap, Rony Pain, Jay Gamma, Olivier Julien, Basil Hubbard, Luc Berthiaume. Understanding the sensitivity of cancer cells to myristoylation inhibitors for oncology applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1662.