The Cloning And Expression Of Human PTP1B Catalytic Domain And The Screening Of Its Inhibitors

Type II diabetes is characterized by a resistance of insulin-sensitivetissues, such as muscle, liver, and fat, to insulin action. PTP1B is a negativeregulator of insulin signaling pathway, suggesting that the inhibitors of thisenzyme maybe enhance insulin sensitivity and cause resistance to obesity. Weneed specific, effective non-peptidic inhibitors. PTP1B is intracellular PTPthat is a potential target for the treatment of type II diabetes and obesity. As aconsequence, the potent and selective inhibitors play an active role in treatingtype II diabetes and obesity.Candidate PTP1B inhibitors in foreign countries fall in two major types:substrate analogues of PTP1B, phosphotyrosine containing peptides and aryIphosphates. However, both of them are still being experimented and cannot beapplied to clinic therapy. As a potent PTP inhibitor, vanadate is non-selective,which can be only used in vitro studies. Therefore, the study of these PTP1Binhibitors such as non-peptide and non-phosphonates become the bestcandidates in this field.At present, one of the most effective ways to screen PTP inhibitors is toemploy the catalytic domain of the PTP as a target. In our study, the catalyticdomain of the PTP1B (ΔPTP1B) has been employed in order to obtain potentand selective inhibitors that can be applied in clinics.1. Expression and purification of ΔPTP1BCollect and sonicate E.coli cells in extraction buffer. Clear the extract bycentrifugation at 15000 rpm. Then purify ΔPTP1B by Q-Sepharose that wasequilibrated with buffer Q, eluted Q-Sepharose column with 0.1M NaCL and0.2M NaCl in buffer Q. Collect the fraction eluted with 0.2M NaCl in buffer Q.For further purification, load the concentrated sample on a SephacryI S-100column running with buffer Q and collect active fraction. The purity of thesample is more than 95% by HPLC identification if p-NPP serves as thesubstrate.2. Screening the inhibitors of ΔPTP1B I choose ΔPTP1B as a target and select 48 samples of monomers ofChinese herbs to screen inhibitors of ΔPTP1B. Samples 1~15 were dissolvedin DMF, while 16~49 were dissolved in Methanol. The concentration ofsamples 1~48 is about 5mg/ml. First add pNPP(1mM)2.8ml to the cell,mixwith 100μl solution of monomer of Chinese herbs, monitor the absorbance at405nm within 3 minutes at RT after the addition of 100μl ΔPTP1B, andreplace 100μl solution of monomer of Chinese herbs with 100μl DMF orMethanol to measure ΔPTP1B activity with control. After many experiments,five effective inhibitors of ΔPTP1B have been chosen out. As preliminaryidentification turns out it has been considered as the selective inhibitors ofΔPTP1B. However, the mechanism of inhibition still needs further study. It is of great importance to find out selective herbal components that areable to inhibit PTP1B, since they can provide a novel specific drug to treatdiabetes and accelerate the modernization of Chinese herbs.3. The assay of enzyme kinetics and cell culture for inhibitors I have screened five effective inhibitors from traditional Chinesemedicines, and have assayed the IC50, Ki value for these inhibitors. We need...