Metal Ion Catalytic Regulation Of FBA-? And Screening And Inhibition Mechanism Of Novel Inhibitors Against FBA-?

The major energy for living organisms is provided by the carbohydrate metabolism.The carbohydrate metabolism pathway mainly includes the glycolytic pathway and the gluconeogenesis pathway.Fructose 1,6-bisphosphate aldolase is an essential regulatory enzyme for the glycolytic pathway and gluconeogenesis pathway.Extensive researches shows that it's likely that blocking the activity of fructose 1,6-bisphosphate aldolase(FBA)or the gene expressed FBA may affect the survival of organisms.The knockout experiments of fba-? also confirmed that FBA-? is a potential drug target.The development of a novel inhibitor with FBA-? as a target will help to address the resistance problems caused by the long-term use of commercial M.grisea fungicides,as well as environmental pollution caused by the chemical algaecide used to solve the cyanobacterial blooms.Compared with FBA-?,FBA-? is a kind of metal-dependent enzyme,and the catalytic regulation mechanism of metal ions has a great influence on the design of inhibitors.The study on the metal catalysis mechanism of Mg-FBA-? and Cy-FBA-? was first performed.The catalysis of metal ions on Cy-FBA-? and Mg-FBA-? is not completely the same.Monovalent metal ions have an activation effect on Mg-FBA-?,but no activation on Cy-FBA-?.The main reason may be that the monovalent metal ion binding site in Cy-FBA-? is replaced by K201,which does not require monovalent metal ions to participate in the catalysis.While Co2+can catalyzes Cy-FBA-?,and in Mg-FBA-?,Zn2+,Mn2+ and Ca2+ act as catalysts.In addition,studies have shown that K+and Zn2+synergistically catalyze in Mg-FBA-?,and a monovalent metal ion binding site(K2)and two divalent metal ion binding sites(Zn1 and Zn2)in Mg-FBA-? were discovered.And E174 was the important amino acid found interacting with Zn1.The importance of Zn2+ for the catalytic reaction of Mg-FBA-? was further confirmed,and the possible catalytic mechanism of metal ions in Mg-FBA-?was proposed.The research of the new sites provides a new idea of designing inhibitors of Mg-FBA-?,and these inhibitors may show high selectivity as there are no metal ions required for catalysis in FBA-?.The study on the catalytic mechanism of Mg-FBA-? provides a new idea and research basis for the study of inhibitors targeting Mg-FBA-?.Since Zn2+is so important for the catalytic reaction of Mg-FBA-?,it may be possible to achieve better inhibition by designing inhibitors for metal ions.Some compounds were screened for the metal ion binding site of Mg-FBA-?,and the inhibitory activity against M.grisea was tested.The inhibition mechanism was studied by the combination of theoretical prediction and experiment.The results show that most of the phenylhydrazone derivatives have higher inhibitory activity than thioureas derivatives.Inhibition mechanism studies show that since the benzoquinone compounds form a coordination with Zn2+,and the thiourea compounds do not form a coordination with Zn2+,which is the main reason that most of phenylhydrazone compounds have higher inhibitory activity than the the thiourea compounds,so that it can be seen that coordination with Zn2+is critical to the increase of inhibitory activity of the compounds.And R332 may be a key amino acid to increase the inhibitory activity of the compound.The structure-activity relationship analysis found that the introduction of OH and para-substituents on the benzene ring was beneficial to the improvement of the inhibitory activity of the compounds on Mg-FBA-?.With reference to the structure-activity relationship,binding mode and the importance of coordination of Zn2+,the two compounds represented by b1 and d2 were designed and synthesized.Among them,the phenylhydrazone derivative represented by b1(Ki=0.46 ?M)has the best inhibitory activity against Mg-FBA-?,mainly because of its enhanced coordination with Zn2+.The compounds d show good inhibitory activity against Mg-FBA-?.Studies have shown that it is a kind of non-competitive inhibitor,which binds to Znl binding cavity and forms coordination with metal Zn2+,and E174 is an important combination site for Znl binding cavity.Studies have shown that compounds d exhibit high selectivity for Mg-FBA-?.Among these compounds,only thiourea compounds have antifungal activity against M.grisea.Taking the Zn2+ion of the Mg-FBA-? in the substrate cavity as an important concern the compound e4 and 5a with a new skeleton was obtained through virtual screening,which provides a certain research basis for the design and modification of subsequent inhibitors.In this paper;through the screening and modification,two Mg-FBA-? inhibitors acting on different cavities were discovered,which confirmed the importance of forming coordination with Zn2+for inhibitors,and inhibitors combined with the Znl cavity design may have high selectivity,which provides a new research idea and direction for rational design of new inhibitors with high selectivity and high activity.Cyanobacteria is a large single-cell prokaryote with simple structure,and is an ideal model for prokaryotes.A new de novo computational strategy,FBVS,was developed by integrating Surflex-Dock,X-score and Percepta.Using this FBVS protocol,compound L1 was de novo design for the novel Cy-FBA-? inhibitors.Based on the structural skeleton of Ll,a class of thiourea compounds represented by Lll was designed and synthesized,and its inhibitory activity against Cy-FBA-? was evaluated.The inhibition mechanism of these compounds was studied.According to the structure-activity relationship of the compounds and their binding mode analysis with Cy-FBA-?,a series of thiourea derivatives represented by L14 were designed and synthesized.The inhibition activity showed better inhibitory effect on Cy-FBA-?.When the OH was introduced on the benzoylhydrazine moiety of the benzamide derivatives exhibit high Cy-FBA-? inhibitory activities.Furthermore,the algicide activities were determined for Synechocystis sp.PCC 6803.The results suggest that most of the compounds with high Cy-FBA-? inhibition might exhibit potential in vivo activities.The results have shown that L14 has a high inhibitory effect on Cy-FBA-?(Ki=0.65 ?M),and L14 also exhibits highest algicide activities(EC50=0.09 ppm),which is 7-fold potent than our previous inhibitor(EC50=0.6 ppm).The possible interactions of representative compound L14 and surrounding key residues around the active cavity of Cy-FBA-? were explained through combining DOX,MD simulations,MM-PBSA and site-directed mutagenesis assays.The results show that the ideas of the design and modification of compounds are reasonable and feasible,and R281 is confirmed to an important action site.The experiments of L14 against Synechocystis sp.PCC 6803 and fba-? overexpressing Synechocystis sp.PCC 6803(PSB?)demonstrated that the thiourea compounds inhibit the growth of Synechocystis sp.PCC 6803 by inhibiting the activity of Cy-FBA-?.This indicates that it is feasible to design synthetic algicides with Cy-FBA-? as a target.Thiourea compounds can be used as the lead compound for the development of specific and green ideal algicides,thus providing a basis for solving the problem of cyanobacteria blooms.In conclusion,FBA-? which plays an important role in the glycolysis and gluconeogenesis had been studied by combining theoretical calculations and experimental methods.With a combination of multidisciplinary methods,the catalysis mechanism of Mg-FBA-? was performed.And series of novel inhibitors against Mg-FBA-? and Cy-FBA-? were designed reasonably and optimized with high inhibition activities,and the studies on the inhibition mechanism of compounds were performed.These results shed light on the development of high-efficiency,high-selectivity new green fungicides and algaecides,and provide a certain research basis and new ideas.