Mengxin Luo, Jun Mo, Ziqiao Wang, Huimin Wei, Kexin Chen, Liteng Shen, Ying Wang, Linjie Li, Yongkang Chen, Weihao Chen, Xue Li, Hui Feng, Xinyu Wang, Huan Zhou, Bizhi Li, Feng Xu, Qingwei Zhao, Yichen Xu, Jinxin Che, Peng Zou, Rong Zhang, Xiaowu Dong, Wei Xie
SARS-CoV-2 NSP14, an N7-guanosine methyltransferase, plays a critical role in viral RNA capping, enabling viral replication and immune evasion. While NSP14 has emerged as a promising drug target, its role in host-virus crosstalk and the cellular consequences of NSP14 inhibition remain poorly understood. Here, we present the identification and characterization of C10, a highly potent and selective non-nucleoside inhibitor of the NSP14 S-adenosylmethionine (SAM)-binding pocket. C10 demonstrates robust antiviral activity against SARS-CoV-2, including its variants, with EC50 values ranging from 64.03 to 301.9 nM, comparable to the FDA-approved drug remdesivir in our cell-based assays. C10 also exhibits broad-spectrum activity against other betacoronaviruses and inhibits SARS-CoV-2 at the replication stage. C10 suppresses viral translation and exhibits immunostimulatory effect. Additionally, C10 specifically reverses NSP14-mediated alterations in host transcriptome. The antiviral efficacy of C10 is further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings highlight C10 as a promising candidate for the development of effective treatments against SARS-CoV-2 and its emerging variants. This study also uncovers a novel mechanism of NSP14 in SARS-CoV-2 pathogenesis and its therapeutic potential, providing insights that may extend to other viral capping methyltransferases. C10, a potent non-nucleoside inhibitor targeting NSP14’s SAM-binding pocket, suppresses viral translation, exhibits immunostimulatory effects, and reverses NSP14-induced transcriptome alterations, offering promise for broader coronavirus treatment.