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Inflammation Caspase (Cystine proteases with aspartic acid substrate specificity) is the Caspases family is important and the inflammatory response related protease, its main function is to produce mature proinflammatory factor IL-1beta and IL-18. The maturation of IL-1beta and IL-18 having a wide range of biological activity, and are associated with a variety of diseases, such as a variety of viruses, bacteria, fungi and microorganisms such as infection, trauma, myocardial infarction, hepatitis, pancreatitis, arthritis, neuroinflammation and neurodegenerative diseases. Inflammation Caspase Caspase-1, -4, -5, -11, -12. One of the most representative caspase-1, the efficient cutting IL-1beta and IL-18 specific protease. Vitro and in vivo experimental results show that inhibition of inflammation Caspase Caspase-1 activity, can alleviate excessive inflammation caused by various diseases, such as arthritis, psoriasis, nerve injury. Therefore, caspase-1 to become an important target for the treatment of excessive inflammatory diseases, looking for Caspase-1 efficient and highly selective inhibitor provides a new way for the treatment of these diseases. The purpose of this paper is that the expression and purification of Caspase-1 recombinant protein, to study the enzymatic properties, Caspase-1 inhibitors at the molecular level and on this basis to establish high-throughput screening model, and through national compound sample library screening, looking for high affinity Caspase-1 inhibitors, by their nature study, further compounds of structural transformation as well as the level of cell and animal pharmacology, pharmacodynamic studies lay the foundation. The content of this study include Caspase-1 full-length gene was cloned using RT-PCR method, and according to the literature will Caspase-1 of 381 amino acid mutation of the recombinant protein is more stable. Caspase-1 catalytic domain was cloned into the expression vector pET21b, and the N-terminus of the recombinant protein with (His) 6 tags. The recombinant plasmids were transformed into E. coli BL21-CodonPlus (DE3) and induced by IPTG. Ni 2 was purified by affinity chromatography to obtain the pure active form of Caspase-1 recombinant protein. Through a series of enzymatic characteristics for Caspase-1, including the effect of DTT, pH-dependent, kinetic analysis and positive inhibitor inhibition analysis, optimization Caspase-1 activity detection method, to create a Caspase-1 inhibitor highpass screening standard operating procedures. To filter the national compound sample library of 47,360 samples, 16 the IC 50 less than 10μM inhibit the activity of compounds. Wherein one active compound MXQ-1 the IC 50 163nM, new Caspase-1 inhibitor. Molecular level, enzyme kinetics method its inhibitory properties of the compound is reversible, slow combination of broad-spectrum caspase inhibitor, and Caspase-1 modes of interaction between the simple interaction, does not occur enzyme isomerization process. The discovery of this new Caspase-1 inhibitors and inhibiting properties of its further structural optimization and pharmacology, pharmacodynamic studies lay a solid foundation.
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