The Inhibitor Of STAT3 Signaling Pathway And The Activation Mechanism Of GK

Signal transducer and activator of transcription 3(STAT3)signaling pathway is constitutively activated in human cancer cells.It is now established that the specificity in signaling are mediated by STAT3 Coiled-coil domain(CCD)and SH2 domain,which regulate and mediate the interaction with the phosphopeptide docking sites displayed by receptors,dimerization and subsequent DNA binding.Here we identify K116 as a novel allosteric inhibitor targeted to Coiled-coil domain of STAT3 and a new small molecule B9 binding for STAT3 SH2 domain.After structure-based screening we detected the activity of these compounds by fluorescence polarization(FP).We also identified that both of K116 and B9 inhibite the growth of tumor cells harboring abnormal activation of STAT3,such as,MDA-MB-468,MDA-MB-231 and DU145.Howerver,there is litte inhibition of MCF-7 cells by these two STAT3inhibitors.The binding affinity of K116 to CCD and WT of STAT3 are 3.2?M and2.397?M,almost as the same as mutation of STAT3,such as K591A,R595G,C712G,K591A/S611G,I634S/S635G.The K_d of B9 to STAT3(I634S/Q635G)is 22.75?M compared to 4.59?M for WT.As a result,the phosphorylation of 705 Tyr on STAT3 in MDA-MB-468 cells was obviously decreased after treament of K116 or B9 while has no effect of p-Src.The downstream targets of STAT3,such as Cyclin D1,C-myc and Bcl-xl were also downregulated in protein level.B9 displayed a remarkable dose-denpendent inhibitory effect on STAT3-induced luciferase activity in Hela cells transfected with STAT3-Luc plasmid.K116 and B9 potentially suppress the migration of human breast cell MDA-MB-468.Moreover,K116 decreases pY705STAT3 nuclear accumulation and induces MDA-MB-468 cell apoptosis.Our studies indicate two novel small molecules,K116 and B9,binding to STAT3 Coiled-coil domain and SH2domain,which decreases STAT3 activation and the function of STAT3 signal pathway,and thereby induces antitumor response in human cancer harboring constitutively active STAT3.Glucokinase(GK)is a glycolytic enzyme that plays an important role in regulating blood glucose level,thus acting as a potentially attractive target for drug discovery in the treatment of diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy.To characterize the activation mechanism of GK from the super-open state(inactive state)to the closed state(active state),a series of conventional molecular dynamics(MD)and targeted MD(TMD)simulations were performed on this enzyme.In combination with the inactivation process,our findings showed that the whole conformational pathway for the activation-inactivation-activation of GK is a one-direction circulation,and the active state is less stable than the inactive state in the circulation.Our observations reveal a complicated conformational process in the allosteric protein,resulting in new knowledge about the delicate mechanisms for allosteric biological macromolecules that will be useful in drug design for targeting allosteric proteins.