The Discovery Of Novel Caspse-1 Inhibitors

Inflammatory Caspase (Cystine proteases with aspartic acid substrate specificity) belong to Caspase family, is a kind of proteases related to inflammatory responses. Their main function is processing pro-inflammatory cytokines IL-16 and IL-18 into their mature form. Mature IL-16 and IL-18 have many activities and related to many diseases, such as infection by virus, bacterial and fungi, trauma, miocardial infarction, hepatitis, acute pancreatitis, osteoarthritis(OA), neuron inflammation and neurodegenerative diseases. Inflammatory Caspases include Caspase-1, -4, -5, -11, -12. Caspase-1 is the best characterized one in inflammatory Caspase subfamily because it is the best efficient and specific in processing IL-16 and IL-18. Researches in vitro and in vivo revealed that inhibition the activities of inflammatory caspsaes, especially Caspase-1, can alleviate many diseases such as OA, psoriasis, neuron damage etc. Therefore, Caspase-1 became an important drug target in treatment of diseases with excess inflammation. Efficient and potent inhibitor toward Caspase-1 has become a new way to inflammatory diseases therapy. Therefore, the aim of this work is to express Caspase-1 in E.Coli, investigate the characterization of Caspase-1 and establish a molecular model of high-throughput screening for Caspase-1 inhibitors. By screening the national compounds library, we found some compounds that can inhibit Caspase-1 with high affinity. Based on the result of screening, we investigated the inhibition mechanism of these Caspase-1 inhibitors, which will help for compound modification, cell-based and animal-based pharmacological and pharmic effects researches in the future.Caspase-1 gene was amplified by RT-PCR. According to the reported method, Caspase-1 381st A.A was mutant for the purpose of high stability. Caspase-1 catalyze domain with a (His)6 tag at 5' sequence was amplified was cloned into vector pET21b. The recombinant plasmid was transformed into E.coli BL21-CodonPlus(DE3), and expression Caspse-1 after IPTG induction. Active Caspse-1 was purified by Hitrap chealting column. The characteristics of active Caspase-1 were investigated, including DTT influence, pH dependence, the kinetics of enzyme reaction and inhibition by positive inhibitor. By optimizing the reaction condition and screening system, we established the standard operation process of high-throughput screening for Caspase-1 inhibitors. 47360 samples of national compound library were screened. We found 16 potent compounds with IC50 below 10 μM. Among them, a compound denominated...