Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro

Haixia Su; Sheng Yao; Wenfeng Zhao; Minjun Li; Jia Liu; Weijuan Shang; Hang Xie; Changqiang Ke; Meina Gao; Kunqian Yu; Hong Liu; Jingshan Shen; Wei Tang; Leike Zhang; Jianping Zuo; Hualiang Jiang; Fang Bai; Yan Wu; Yang Ye; Yechun Xu

The mode of action of baicalein and the structural determinants associated with its binding with SARS-CoV-2 3CLpro were further explored using X-ray protein crystallography. The crystal structure of SARS-CoV-2 3CLpro in complex with baicalein was determined at a resolution of 2.2 Å ( Fig. 2 ; Table S2 ). The protease has a catalytic Cys145-His41 dyad and an extended binding site, features shared by SARS-CoV 3CLpro and MERS-CoV 3CLpro ( Fig. 2A ). The inhibitor binds in a core region of the substrate-binding site at the surface of the protease, between domains I and II (Fig. 2, A and B) . Examination of the active site of the complex revealed that three phenolic hydroxyl groups of baicalein made multiple hydrogen bonds with main chains of Leu141/Gly143 as well as side chains of Ser144/His163 with or without the aid of a buried water molecule (Fig. 2C ). The only carbonyl group established a hydrogen bond with the main chain of Glu166, while the free phenyl ring inserted into the S2 sub-pocket by making hydrophobic interactions with multiple residues Gln189/Arg188/Met49/Cys44/His41. Notably, apart from the hydrophobic interactions, the catalytic Cys145 and His41 also formed S- (a distance of 3.3 Å from the sulfur to the centroid of the phenyl ring) and  - interactions with the aromatic rings of baicalein, respectively. The side chain of Asn142 also established NH2- (a distance of 3.6 Å from the nitrogen to the centroid of the phenyl ring) interactions with baicalein, which together with the interactions contributed by Cys145 lead the phenyl ring with three OH groups to be sandwiched between Cys145 and Asn142. Moreover, Met165 contacted the middle ring of baicalein via hydrophobic interactions. Consequently, baicalein is perfectly ensconced in the core of the substrate-binding pocket and interacts with two catalytic residues, the oxyanion loop (residues 138-145), Glu166, and the S1/S2 subsites, which are the key elements for recognition of substrates as well as peptidomimetic inhibitors (17). Although baicalein did not move deeply author/funder. All rights reserved. No reuse allowed without permission.