The Identification Of A Non-Competitive Inhibitor NC1 Of LYP And Its Inhibition Mechanism Research

BackgroundThere are various regulatory mechanis of cell signal transduction,Reversible protein tyrosine phosphorylation is one of the important ways of adjustment.Under the action of protein tyrosine kinase PTK and protein tyrosine phosphatase,the protein tyrosine is phosphorylated and dephosphorylated to maintain the dynamic balance in its phosphorylation level.The process also affects many physiological activities,such as gene expression,cell growth,cell differentiation,cell communication,cell migration,blood glucose metabolic regulation,immune regulation,etc.The misfunction of protein tyrosine phosphatase can affect protein tyrosine phosphorylation level and lead to many diseases such as type I diabetes,systemic lupus erythematosus,rheumatoid arthritis.It becomes one of the important reasons for causing many diseases.So the protein tyrosine phosphatase has raised scientists great research interes,especially is lymphoid-specific protein tyrosine phosphatase(LYP)highly correlated with a spectrum of autoimmune diseases.LYP is encoded by PTPN22 and is exclusively expressed in hematopoietic immune cells.It plays the key role in the inhibition of T cell activation,by interacting with T cell receptors.Many studies have revealed that LYP mutation is closely related to many autoimmune diseases.Therefore,LYP has become the potential drug target on a variety of autoimmune diseases and identifying the LYP-specific inhibitor is of great significance.LYP is a member of the classic protein tyrosine family(PTP)in which,active site is highly conserved.So rare selective inhibitors have been developed.In the past,some kinds of typical protein tyrosine phosphatase,including selective inhibitors of PTP1B,LYP,played the roles by acting on the substrate-binding pocket,and its nearby area.Recently,as an additional kind of inhibition,some protein tyrosine phosphatase which is related with desease,including PTP1B,SHP2,the non-competitive inhibitors have been developed.But the non-competitive inhibitor of LYP has not yet been found.ObjectsTo identify the non-commpetitive inhibitor of LYP,and illuminate the allosteric inhiibition mechanism Materials and MethodsFirst of all,we construct a series of prokaryotic expression plasmids and transform them into the E.coli BL21 to get the proteins.By ion exchange chromatograph and Molecular Sieve,we have obtained high purity proteins.By testing the IC50 and Ki of inhibitor,we screened better inhibitors and found NCI fits a pattern of the noncompetitive inhibition.Then in PTP family range,with small molecular compounds of nitrobenzene disodium phosphate as the simulated substrate,we respectively tested the inhibition effect on phosphatase STEP,PTPN18,Glepp,slingshot2,PPM1A,PPM1G,PP1,measured inhibition constant to evaluate NCI specificity.Further,at the cellular level,using siRNA technology,in LYP regulation pathways,by Western blot we detect phosphorylation level of molucular LCK?ERK to test NCI specificity of inhibiting the action of the LYP in cells,and its inhibition effect.We find NCI can restrain activity of LYP specifically.By-site-directed mutagenesis,fragment-centric topographic mapping and molecular dynamics simulation,we studies the bind site of NC1.In addition,using a new technology,unnatural amino acid F2Y(2-fluorine-tyrosine)incorporation method and 19F-NMR spectroscopy,we provide direct evidence that NCI allostericallyregulates LYP activity.ResultsWe got the first LYP non-competitive inhibitors-NC1.The most active compound,NC1(Figure 1B),showed LYP inhibitory activity(Ki=4.66 ?M).Further enzyme inhibition tests indicated that NCI displayed at least two-fold higher selectivity against a panel of other protein phosphatases,including STEP,PTPN18,Glepp,and the Ser/Thr phosphatases PPMIA and PP1.It shows better inhibitory effect on LYP.At the T cell level,TCR activation in Jurkat T cells significantly increased the phosphorylation levels of ERK and LCK,which were substantially augmented by the application of 20 ?M NC1.Importantly,knockdown of LYP by siRNA increased both the phosphorylation of ERK and LCK to a similar extent to solely administration of NCI.Moreover,without endogenous LYP expression in T cells,the NCI shows no effect on CD3 induced LCK and ERK phoshphorylation in T cells.Mutatons?fragment-centric topographic mapping and molecular dynamics simulation results suggest that NC1,unlike other known LYP inhibitors,concurrently binds to a"WPD"pocket and a "second pocket" surrounded by LYP-specific insert,which contributes to its selectivity against other phosphatases.Moreover,using our newly developed unnatural amino acid F2Y(2-fluorine-tyrosine)incorporation method and 19F-NMR spectroscopy,we provide direct evidence that NC1 allosterically regulates LYP activity by restricting WPD-loop movement.ConclusionsUnlike other known LYP inhibitors,NC1 concurrently binds to a "WPD" pocket and a "second pocket" surrounded by a LYP-specific insert,which contributes to its selectivity against other phosphatases.Research significanceOur study not only identifies a new allosteric binding site of LYP for further selective LYP inhibitor development,but also provides a novel 19F-NMR probe that can be used to characterize and identify allosteric inhibitors of other tyrosine phosphatases.