Eshelman School of Pharmacy, Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
Proc Natl Acad Sci U S A. 2024 Oct 15;121(42):e2409166121. doi: 10.1073/pnas.2409166121. Epub 2024 Oct 10.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV nonstructural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow-up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k /K of 6.4 × 10 Ms. LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity in proteomic experiments or against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the identification and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic.
基孔肯雅病毒（CHIKV）是一种蚊媒甲病毒，在过去二十年中已导致多次大规模爆发。目前，尚无针对任何甲病毒感染的 FDA 批准的治疗方法。CHIKV 非结构蛋白 2（nsP2）包含半胱氨酸蛋白酶结构域，对于病毒复制至关重要，因此是药物发现的有吸引力的靶标。在这里，我们优化了 CHIKV nsP2 蛋白酶（nsP2pro）生化测定法，以筛选 6,120 种化合物半胱氨酸定向共价片段文库。使用 50%抑制阈值，我们鉴定出 153 个命中物（2.5%的命中率）。在剂量反应后续研究中，包含乙烯砜弹头的共价片段 RA-0002034 对 CHIKV nsP2pro 的抑制作用的 IC 为 58±17 nM，进一步的时间依赖性抑制研究表明 k/K 为 6.4×10 Ms。LC-MS/MS 分析确定 RA-0002034 以位点特异性方式共价修饰催化半胱氨酸。此外，RA-0002034 在蛋白质组学实验或针对一组半胱氨酸蛋白酶中没有表现出明显的非靶标反应性。除了对 CHIKV nsP2pro 活性具有强大的生化抑制作用和卓越的选择性外，RA-0002034 还在甲型病毒感染的细胞模型中进行了测试，并有效抑制了 CHIKV 和相关甲病毒的病毒复制。这项研究突出了 RA-0002034 的鉴定和表征，作为基于共价片段筛选的有前途的命中化合物，用于开发针对 CHIKV 或泛甲病毒的治疗方法。
