UNC0642 for COVID-19

ABSTRACT Since the outbreak of SARS-CoV-2, viral mutations have posed significant challenges in identifying therapeutic targets and developing broad-spectrum antiviral drugs. Post-translational modifications of genes involved in interferon production and signaling pathways play a crucial role in regulating interferon responses. In this study, we employed CRISPR-Cas9 screening based on adenine base editors to investigate functional amino acids in 1,278 innate immune-related genes. This approach, which converts A-T base pairs into G-C base pairs to probe the functional importance of specific amino acids, allowed us to identify 17 vital factors involved in SARS-CoV-2 infection. Among the candidate genes, genetic knockdown of EHMT2 exhibited the strongest antiviral effect. Further analysis revealed that UNC0638, a selective inhibitor of EHMT2, significantly reduced the endosomal entry of SARS-CoV-2 in pseudovirus assays. The observed inhibitory effect was consistently observed across multiple SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Mechanistically, UNC0638 reduced mature cathepsin L (CTSL) levels, impairing the proteolytic cleavage of SARS-CoV-2 spike protein and subsequent membrane fusion, a critical step for viral entry. Our findings uncover EHMT2 as a host dependency factor and reveal the antiviral mechanism of EHMT2 inhibitors through CTSL maturation blockade. These results advance the understanding of host factors in SARS-CoV-2 infection and provide a strategic framework for developing host-targeted antiviral therapies. IMPORTANCE In this study, we demonstrated that knockdown or knockout of EHMT2 inhibited SARS-CoV-2 infection, and inhibitors of EHMT2, including UNC0638, UNC0642, and BIX01294 showed similar restrictive effects. Mechanistically, the EHMT2 inhibitor UNC0638 restricts spike-mediated cell entry by inhibiting the maturation of CTSL, a critical protease required for SARS-CoV-2 entry via the endosomal pathway. Importantly, CTSL is not only essential for SARS-CoV-2 but also plays a key role in the entry of other coronaviruses that utilize similar pathways. Therefore, EHMT2 inhibitors could have broader applications as pan-coronavirus therapeutic agents.

AbstractSince the emergence of SARS‐CoV‐2 at the end of 2019, the virus has caused significant global health and economic disruptions. Despite the rapid development of antiviral vaccines and some approved treatments such as remdesivir and paxlovid, effective antiviral pharmacological treatments for COVID‐19 patients remain limited. This study explores Nsp15, a 3′‐uridylate‐specific RNA endonuclease, which has a critical role in immune system evasion and hence in escaping the innate immune sensors. We conducted a comprehensive drug repurposing screen and identified 44 compounds that showed more than 55% inhibition of Nsp15 activity in a real‐time fluorescence assay. A validation pipeline was employed to exclude unspecific interactions, and dose–response assays confirmed 29 compounds with an IC50 below 10 μM. Structural studies, including molecular docking and x‐ray crystallography, revealed key interactions of identified inhibitors, such as TAS‐103 and YM‐155, with the Nsp15 active site and other critical regions. Our findings show that the identified compounds, particularly those retaining potency under different assay conditions, could serve as promising hits for developing Nsp15 inhibitors. Additionally, the study emphasizes the potential of combination therapies targeting multiple viral processes to enhance treatment efficacy and reduce the risk of drug resistance. This research contributes to the ongoing efforts to develop effective antiviral therapies for SARS‐CoV‐2 and possibly other coronaviruses.