Inhibitor Screening And Crystallography Research Of CVB3-3C

Currently viral myocarditis cause serious disease to human.Antiviral myocarditis drug design and screening for treatment is particularly important. Coxsackievirus group B (CVB) is the main pathogens of human viral heart diseases. CVB3 has a single open reading frame (ORF), encoding form 4 viral capsid proteins (VP1-VP4) and 7 structural protein (2A-2C、 3A-3D). The 3C protein is responsible for cleavage polyprotein into mature protein.It is important to replication of viral RNA.The central roles played by CVB3-3C, make it an appealing target for anti-CVB3 drug development.Prokaryotic expression system expressed CVB3-3C protease in vitro. CVB3-3C protease was purified by affinity chromatography and ion exchange chromatography and gel filtration chromatography. To achieve drugs screening of CVB3-3C, we made optimization experiments to identifie the buffer type, salt concentration and pH, the concentration of protease and substrate, and DMSO leve of the screening system. Analyzed and evaluated the stability and reliability of the system.Finally, established a drug screening system and screened 2000 compounds.12 compounds with inhibition ratio more than 80% were selected,6 compounds(MDCE-10a, MDCE-10c, MDCE-10e, MDCE-A008, MDCE-A022, MDCE-A043)of them with lower IC50 alue,36.70 μmol/L,33.25 μmol/L, 21.07 μmol/L,60.60 μmol/L,23.55 μmol/L and105.70 μmol/L, respectively.Through protein crystallization technology to obtain compound crystal of CVB3-3C with inhibitors.Finally, we get CVB3-3C and MDCE-A008 compound crystal, and used X-ray crystal diffraction method to obtained the crystal data with 2.4 A resolution.This study established a drug screening system of CVB3-3C at the cellular level, which used to screen anti-CVB3 lead compounds.Meanwhile we get compound crystal of CVB3-3C with inhibitor. Which is provide support for optimizing lead compounds of anti-CVB3.