title: Biochemical characterization of recombinant Enterovirus 71 3C protease with fluorogenic model peptide substrates and development of a biochemical assay.
abstract: Enterovirus 71 (EV71), a primary pathogen of hand, foot, and mouth disease (HFMD), affects primarily infants and children. Currently, there are no effective drugs against HFMD. EV71 3C protease performs multiple tasks in the viral replication, which makes it an ideal antiviral target. We synthesized a small set of fluorogenic model peptides derived from cleavage sites of EV71 polyprotein and examined their efficiencies of cleavage by EV71 3C protease. The novel peptide P08 [(2-(N-methylamino)benzoyl) (NMA)-IEALFQGPPK(DNP)FR] was determined to be the most efficiently cleaved by EV71 3C protease, with a kinetic constant kcat/Km of 11.8 ± 0.82 mM(-1) min(-1). Compared with literature reports, P08 gave significant improvement in the signal/background ratio, which makes it an attractive substrate for assay development. A Molecular dynamics simulation study elaborated the interactions between substrate P08 and EV71 3C protease. Arg39, which is located at the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a Z' factor of 0.87 ± 0.05 was developed. A novel compound (compound 3) (50% inhibitory concentration [IC50] = 1.89 ± 0.25 μM) was discovered using this assay, which effectively suppressed the proliferation of EV 71 (strain Fuyang) in rhabdomyosarcoma (RD) cells with a highly selective index (50% effective concentration [EC50] = 4.54 ± 0.51 μM; 50% cytotoxic concentration [CC50] > 100 μM). This fast and efficient assay for lead discovery and optimization provides an ideal platform for anti-EV71 drug development targeting 3C protease.
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People's Republic of China.
authors.forename: Luqing
authors.initials: L
authors.lastname: Shang
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People's Republic of China.
authors.forename: Shumei
authors.initials: S
authors.lastname: Zhang
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People's Republic of China.
authors.forename: Xi
authors.initials: X
authors.lastname: Yang
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China.
authors.forename: Jixue
authors.initials: J
authors.lastname: Sun
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People's Republic of China.
authors.forename: Linfeng
authors.initials: L
authors.lastname: Li
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People's Republic of China.
authors.forename: Zhengjie
authors.initials: Z
authors.lastname: Cui
authors.affiliation: High-Throughput Molecular Drug Discovery Center, Tianjin International Joint Academy of Biotechnology & Medicine, Tianjin, People's Republic of China.
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: He
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Guo
authors.affiliation: National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, People's Republic of China sunyn@moon.ibp.ac.cn zheng_yin@nankai.edu.cn.
authors.forename: Yuna
authors.initials: Y
authors.lastname: Sun
authors.affiliation: College of Pharmacy & State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, People's Republic of China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People's Republic of China sunyn@moon.ibp.ac.cn zheng_yin@nankai.edu.cn.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yin
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000138:chemical synthesis* / Q000494:pharmacology*; D001681:Biological Assay; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000096:biosynthesis; D004770:Enterovirus / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D029544:Foot-and-Mouth Disease Virus / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis
chemical_list: D000998:Antiviral Agents; D005456:Fluorescent Dyes; D010455:Peptides; D011480:Protease Inhibitors; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases