Screen The Inhibitors Of Protein Tyrosine Phosphatase 1B

The insulin-impedance is a main reason for causing thetype II diabetes,and can cause the organism to be fat , discover the Protein tyrosinephospnatase 1B (PTP1B ) is closely related to this course, evidence suggests ,the signal that PTP1B blocks insulin through making the insulin receptor (IR )go for phosphorylation transfers to the route of leading . So any change ofexpress competence and vigor about PTP1B which is related to IR mayinfluence insulin signal transduction , and may cause the emergence of insulinimpedance . So, for preventing and treating II -type diabetes and havingpositive function fatly to effective , specific inhibitor of PTP1B.Mainly there are two kinds of effective , alternative PTP1B inhibitor selectedabroads: One substrate analog-phosphorus tyrosine polypeptide of PTP1B;Another kind is a phosphoric acid salt small molecule material ofsweet-smelling base , but these two kinds of inhibitors both have its one's ownlimitation, they can't be used in the clinical treatment of the II -type diabetes.Peroxovanadate , the effective inhibitor regarded as PTP, does not have thealternative , can only be used in the body to study. So the research ofnon-peptide and non-phosphoric acid saline PTP1B inhibitor become expectedwinner of this field.The most effective tactics to screen the inhibitor of PTP is regard activeposition of PTP as target order at present. So, this research uses the structuralland of catalysis of hPTP1B (â–³ PTP1B ) screening to carry on inhibitor, in thehope of , get , can used in clinical , effective , specific inhibitor.1.The expression and purification of â–³P TP1BTo suspend again the fungus body that is collected with buffer liquid andbreak it with supersonic wave. Centrifuging and adopting the supernatant ,then treat the supernatant with the anion exchange resin balanced by PH7.5bufferQ . Stage-elute with Buffer Q including 0.1M and 0.2M NaCl, collectsthe elution component of 0.2M NaCl, the purity of ? PTP1B collected can beup to 70%. After Elute group bing dialysed and the salt being removed , freezeand concentrate it, utilize S-100 chromatogram column to collect the activecomponent. It can be found by SDS-PAGE that the molecular weight ofpurified albumen is about 35kDa and its purity is more than 90%, Km is2.4mM .2. Screen the Inhibitors of â–³PTP1BTwo kinds of valid â–³PTP1B inhibitors , compound 11 and 12 has beenscreened by the experiment , whereafter, researches to compond 11and 12were done about enzyme kinetic and cell biology , found that compound 11and 12 display great inhibition .Enzyme kinetic study incubates that theinhibition styles of compound 11 and 12 are both competition inhibition ,and Ki value are 1.13mM and 0.48 mM respectively , Cell biology studyshows that compound 11 and 12 could go through cell membrane , inhibitthe endocellular PTP1B activation, enhance the phosphorilation of insulinreceotor(IR), so they can promote singnal transduction of insulin .From theresearch above , We could get another conclusion , that the inhibition ofcompound 12 is better than compound 11 . This result provide an possibilityof exploration a new specific, but the effect and safety of compound 12 needmore supports .