Study On Screening Novel Selective Inhibitors Of Tumor Necrosis Factor-α Converting Enzyme By HPLC

Tumor necrosis factor-αconverting enzyme (TACE or ADAM17) is a zinc metalloproteinase. At present, a large number of experimental study found TACE has an important role in the physiology and pathology, its functions are involved in the regulation of the immune system, the induction of inflammatory bowel disease and nerve disease. The membrane-bound tumor necrosis factor-α(pro-TNF-α) is cleaved by TACE to release the soluble sTNF-α, inducing inflammatory diseases. Therefore, TACE becomes a new target of treating inflammatory diseases.When human cause inflammatory diseases, an alternative paradigm for affecting TNF-αlevels is via the inhibition of TACE by some small molecule TACE inhibitors. Therefore, it is quite important to screen the small molecule TACE inhibitors. Yet, it is the key point that established the method of assaying the activity of TACE inhibitors. So, the thesis studied on the determination method. The main content of this study are described as following:1. The method of assaying the activity of TACE inhibitors by HPLC was established.According to the structure of substrate(12 peptides), a new compound, DL-α-aminopropionic acid-Dpa was synthesized, as an internal standard substance of quantitative analysis. After that, some experimental conditions of TACE enzymatic reaction were optimized, such as enzyme concentration, time of enzymatic reaction, the mobile phase and so on. After obtaining the optimized parameters, a HPLC method was established for the determination of the TACE activity depending on the internal standard method. The linear range is from 10μmol/L to 400μmol/L, and the limit of detection (LOD) is 0.1μmol/L. Then, under the optimal experimental and determination conditions, 50% inhibitory concentration (IC50) value of GM6001 inhibiting TACE was determined by this newly proposed method. Ultimately, the evaluation of the activity of TACE inhibitor was accomplished. Finally, this newly proposed method was used to determine standard samples, and the and the results were showed as following: coefficients of variability of in-day and daytime is less than 5% and 10% respectively, and the recovery of standard samples is between85% and 115%. 2. The method of assaying the activity of MMP-9 inhibitors by HPLC was established.In order to meet the need of the effective screening of the selective TACE inhibitors, a method of assaying the activity of MMPs inhibitors by HPLC was established. The experimental conditions of MMP-9 enzymatic reaction were optimized using MMP-9 and seven peptides linked a UV group as substrate. After obtaining the optimized parameters, a HPLC method was established for the determination of the MMP-9 activity depending on the internal standard method. The calibration curve is linear between 10μmol/L and 360μmol/L, and the detection limit (LOD) is 0.1μmol/L. Then, this newly proposed method was used to evaluate the capability of GM6001 inhibiting TACE, by expression of IC50. The results were in good agreement with those obtained by literatures reported.