Rational Design And Biological Evaluation Of Novel Inhibitors Against FBPase And FBA-? In Glycometabolism

Energy is the basis for the normal life activities of the organism,and carbohydrate is the main source of energy that life activities should need to.So theoretically lowing or blocking glycometabolism of a species should treat some diseases related to glycometabolism or kill it.Glycolysis is a near-universal pathway by which a glucose molecule is oxidized to two moleculesof pyruvate,with energy conserved as ATP and NADH.Gluconeogenesis is a predominantly hepatic pathway by which three-carbon precursors are converted enzymatically to glucose.In type II diabetes,increased gluconeogenesis is primarily responsible for increased endogenous glucose production(EGP)during fasting.Fructose-1,6-bisphosphatase(FBPase),the gluconeogenic enzyme in the middle substrate cycle,has provided new insights into the therapeutic utility of gluconeogenesis inhibitors and the potential of FBPase inhibitors as a new class of antidiabetic drugs.Human liver FBPase is a cytosolic enzyme that consists of four subunits each containing a substrate site and an allosteric site.Both the substrate site and the AMP site of FBPase have been the target of drug discovery efforts.But most efforts were made to the allosteric site.Adenosine 5'-monophosphate(AMP)is increasingly recognized as important physiological sensors of cellular energy status that modulate many targets in cell.So a strutcture-guided approach using AMP as a starting point yielded an inhibitor,which maybe has low selectivity.In the paper,a set of virtual screening strategies were performed to obtain inhibitors targeted by the substrate site of Hu-FBPase,some novel inhibitors have found,e.g.XY5(IC50=17.1 ?M),XY8(IC50=15.8 ?M),XY23(IC50= 8.3?M).In the other hand,a series of b-nitrostyrene compounds were rationally designed and synthesized using FBP and AMP as starting points.HS39,the most potent inhibitor,can inhibit Hu-FBPase with IC50 of 0.13?M.And HS14 not only inhibit Hu-FBPase with an IC50 value of 0.89 ?M but also inhibit the glucose production in primary rat hepatocytes at 100 ?M as well as Metformin at 2 mM.The study on the binding mode of inhibitor and its target is the basis of explanation and rational inhibitor design.In this study,the TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor(the active site or the allosteric site)using FBPase saturated by AMP and FBP,respectively.Using the TNP-AMP fluorescent probe we exam that HS49 maybe both bind to the substrate site and the allosteric site of Hu-FBPase.Identifying the binding models of the substrate site and the allosteric site of Hu-FBPase and HS12 was proformed by docking and site-directed mutagenesis.The results suggested that K274,Y264,N212,Y244,R243,S124 in the substrate site and T27 in the allosteric site were important for HS12 to bind into Hu-FBPase.Finding new targets and designing novle inhibitor is always a focus in the research of antifungal drugs.The inhibitor study on type II fructose-1,6-bisphosphate aldolase(FBA-?)in the glycometabolism maybe can provide an idea to solve the above problems.FBA-II is an essential regulatory enzyme in glycolysis and gluconeogenesis,which catalyzes the reversible cleavage of fructose-1,6-biphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.A set of virtual screening strategies were performed to obtain inhibitors targeted by Cy-FBA-II,H1 was found with a value of 17.8 ?M.Compound 1 and analogues were designed according to the binding mode of H1 and Cy-FBA-II,and their corresponding inhibitory activities in vitro and in vivo were evaluated Especially,compound 10 not only shows high Cy-FBA-? activities(IC50 =1.7 ?M)but also have highest in vivo activities against Synechocystis sp.PCC 6803(EC50 = 0.6 ppm).Thus,the compound 10 was selected as representative molecule,and its probable interactions with the surrounding important residues in the active site of Cy-FBA-II were elucidated by jointly using molecular docking,molecular dynamics(MD)simulations,ONIOM and enzymatic assays,to provide a new insight into the binding-mode of the inhibitors and Cy-FBA-?.The enzymatic and algal inhibitions assays suggest that the Cy-FBA-? is very likely to be a promising target for the design,synthesis and development of novel specific algicides to solve CHABs.In the papare,Ca-FBA-? was expressed in E.coli BL21(DE3)cells and purified.The kinetic parameters Km,Vmax,were determined,with Vmax.=70 U/mg,Km=61?M.Using the crystal structure of E.coli FBA-?(PDB ID:1B57)as a template,a plausible homology model of Ca-FBA-? was generated with the SWISSMODEL server.The quality of the homology model of Ca-FBA-? was assessed with PROCHECK,ProQ,ProSA-web,QMEAN.A set of virtual screening strategies were performed to obtain inhibitors targeted by Ca-FBA-?,FBA1 was found with a value of 16.5?M.Compound ZY1 and ZY15 were further designed according to the binding mode of FBA1 and Ca-FBA-?,which had IC50 values of 2.7 and 0.96?M.And compound ZY2 had in vitro antifungal activity against C.albicans(MIC80= 16 ?g/mL),C.glabrata(MIC80= 2 ?g/mL),C.tropicalis(MIC80=32 ?g/mL).In summary,FBPase and FBA-? in the glycolysis and gluconeogenesis had been studied using multidisciplinary approaches and combining theory and experiment.Series of novel Inhibitors against Hu-FBPase,Ca-FBA-?,and Cy-FBA-? were rationally designed.The above researchs for the treatment of type ? diabetes,the development of new fungicides and algaecides provide new ideas and necessary research base.