Muthu Ramalingam Bose, Oh Hans J, Sears John D, Chen Chun-Hsing, Vala Anand, Liu Shubin, Talbot Kacey M, Hossain Mohammed Anwar, Brown Peter J, Houliston Scott, Garcia Perez Julia, Li Fengling, Amare Meareg G, Halfmann Peter, Smith Jessica L, Hirsch Alec J, Arrowsmith Cheryl H, Halabelian Levon, Vargason Ava, Couñago Rafael M, Arnold Jamie J, Cameron Craig E, Moorman Nathaniel J, Heise Mark T, Willson Timothy M
Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
READDI AViDD Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
ACS Infect Dis. 2025 Aug 8;11(8):2331-2341. doi: 10.1021/acsinfecdis.5c00351. Epub 2025 Jul 18.
Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine , a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer ()- exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the ()- enantiomer was >100-fold less active. The ()- enantiomer also demonstrated a high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of ()- to the nsP2hel protein was confirmed by F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of ()-, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate ()- (RA-NSP2-) as a high-quality chemical probe and ()- (RA-NSP2-N) as a negative control for probing the biology of alphavirus RNA helicases.
基孔肯雅病毒（CHIKV）的复制依赖于多功能非结构蛋白2（nsP2），这使其成为抗病毒药物研发的一个有吸引力的靶点。在此，我们报告了氧杂螺哌啶（一种CHIKV nsP2 RNA解旋酶（nsP2hel）的一流抑制剂）拆分为其组成对映体的过程及其抗病毒活性的表征。对映体（）-表现出对病毒复制、nsP2hel ATP酶活性和双链RNA解旋的有效抑制，而（）-对映体的活性则低100倍以上。（）-对映体还显示出对CHIKV相对于其他RNA病毒以及对nsP2hel相对于其他RNA解旋酶具有高选择性。通过F NMR证实了（）-与nsP2hel蛋白的直接结合。生物物理和结构研究揭示了（）-的螺环支架中的构象多态性，表明配体的热运动性在nsP2hel变构抑制中的潜在作用。总体而言，这些发现将（）-（RA-NSP2-）指定为用于探究甲病毒RNA解旋酶生物学的高质量化学探针，而将（）-（RA-NSP2-N）指定为阴性对照。
