The Discovery Of A Irreversible Benzo Imidazole Thiazole Derivative Inhibitor For Protein Tyrosine Phosphatases

BackgroundThe phosphorylation of protein tyrosine is an important regulatory mechanism for regulating signal transduction in eukaryotic cells,reversible and dynamic protein tyrosine phosphorylation degree is governed by the mutually antagonistic activities of protein tyrosine kinases(PTK)and protein tyrosine phosphatases(PTP).PTP plays key roles in many physiological processes,it has a strict regulation on cell growth and development,cell metabolism,cell cycle,cell migration,immune responses and neural activities and so on.Inappropriate activity of the PTP could lead to human diseases,such as cancer or diabetes.PTP IB plays an important role of negative regulation in insulin signal pathways,the abnormal activities of PTP IB is related to diabetes.YopH is an important factor in the prevention of the host innate immune response for the Yersinia.Striatal tyrosine phosphatase STEP as a kind of central nervous system tyrosine phosphatase,plays an important role in synaptic plasticity and neuronal survival and development,dysfunction of STEP may lead to a variety of nervous system diseases including,Alzheimer’s disease.Currently,PTP has attracted increasing attention as the potential drug target.The small molecule compounds which specific regulate PTP is not only intended to be used in clinical pharmaceutical preparations,but also as probes to study and explain the important role of PTP in specific cell signaling pathways.Therefore,it has great significance to develop efficient chemical small molecule probe and in-depth study its mechanism of action.Objects:To obtain the pure STEP,LYP,PTP1B,YopH and MEG2 protein tyrosine phosphatases,then conduct related studies of enzyme Kinetics and inhibitor screening.Materials and MethodsSTEP,LYP,PTP1B,YopH and MEG2 wild type protein tyrosine phosphatases were sub-cloned into the pET-15b,pET-28a and pET-22b vectors and they were transformed to the E.coli BL21.The proteins were purified by affinity chromatography and ion exchange chromatography,then applied to determine the enzymatic activity and used the continuous reading method,IC50 and inhibitor enzyme kinetic assay to get the inhibitor screening.The specificity was further revealed by measuring the selectivity of the compound to other phosphatases and PPM families,and to determine the effect of the compound.ResultsThe results showed that STEP,YopH,MEG2 and other related wild type protein tyrosine phosphatases had been successfully expressed,and the enzymes with more than 90%purity could be used for enzymatic assays in vitro.The SDS-PAGE electrophoresis apparent molecular weight of purified STEP,YopH,MEG2 and other phosphatase were 35 kDa,33 kDa,38 kDa and so on.Subsequently,the basic enzyme activities of STEP and other phosphatases were determined,after that we acquired one kind of inhibitor from the library of compounds,which has significant inhibition with a novel structure.Compound E4 inactivate STEP in a time-and concentration-dependent fashion.Further study showed that compound E4 inhibits a series of PTP in a time dependent manner,whereas it shows little or no inhibition toward metal dependent protein phosphatases.ConclusionsHere,we characterize compound E4 as a new class of PTP activity probes.Functional studies of PTP can be greatly facilitated by chemical probes that covalently label the active site of a PTP through an activity-dependent chemical reaction.Collectively,this new identified covalent inhibitor of PTP has the potential to be developed to an active site Cys directed PTP probes to study the active properties of the PTP in cell signaling.