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 cDNA of full length SARS-CoV-2 3CLpro or SARS-CoV 3CLpro was cloned into the PGEX6p-1 vector. To obtain SARS-CoV-2 3CLpro or SARS-CoV 3CLpro with authentic N and C terminals, four amino acids (AVLQ) were inserted between the GST tag and the full length SARS-CoV-2 3CLpro or SARS-CoV 3CLpro, while eight amino acid (GPHHHHHH) were added to the C-terminal of SARS-CoV-2-3CLpro. The plasmid was then transformed into BL21 (DE3) cells for protein expression. The N terminal GST tag and four amino acids (AVLQ) was self-cleavable. The expressed protein with authentic N terminal was purified by a Ni-NTA column (GE Healthcare) and transformed into the cleavage buffer (150 mM NaCl, 25 mM Tris, pH 7.5) containing human rhinovirus 3C protease for removing the additional residues. The resulting protein sample was further passed through a size exclusion chromatography (Superdex200, GE Healthcare). The eluted protein samples were stored in a solution (10 mM Tris, pH 7.5) for the enzymatic inhibition assay, native state mass spectrometry studies and protein crystallization.