The Rational Design,Synthesis,Structure-Activity Relationship Of New Inhibitors Based On Cy-FBP/SBPase Structure

In recent years,the degree of eutrophication in China has deepened,resulting in the frequent occurrence of "cyanobacteria bloom",which has caused serious impact on the production and life of human beings.It is imperative to find a new type of high efficient,highly selective and environmentally friendly alga to effectively control "cyanobacteria bloom".In our previous research,we took cyanobacteria fructose-1,6-biphosphatase/sedoheptulose-1,7-bisphosphatase(Cy-FBP/SBPase)as the target,and obtained a HQ7(IC50=1.1±0.2 pM)which had a strong inhibitory effect on Cy-FBP/SBPase,then we obtained HQ series compounds by modification.Based on this theory,this paper established the binding model of compound HQ7 and Cy-FBP/SBPase preliminarily by using DOX method and analyzed the interaction between HQ series compounds with Cy-FBP/SBPase,established the interaction model between ligand HQ series compounds with receptor Cy-FBP/SBPase.Based on this,a new pharmacophore screening model based on the receptor structure is established,a work on screening,modification and synthesis of the new inhibitor that targeted on Cy-FBP/SBPase has carried out.The main works of this paper is as follows:1.Established the interaction model between ligand HQ series compounds with receptor Cy-FBP/SBPase:Based on the crystal structure of Cy-FBP/SBPase(PDB code:3RPL),the binding model of inhibitor HQ7 and Cy-FBP/SBPase was preliminarily established by DOX method.It shows that amino acid residues ARG176,ARG178,TYR131,THR102 in the substrate cavity can format hydrogen bonds with inhibitor HQ7.This is consistent with the results of protease kinetic analysis of amino acids site directed mutagenesis.which validate the reasonableness of the binding model of HQ7 and Cy-FBP/SBPase.According to the binding model,the interaction between HQ series compounds and Cy-FBP/SBPase was analysed detailedly.This binding model can basi-cally explain the structure-activity relationship between the structure and inhibitory activity of HQ series compounds,and further demonstrate the accuracy of the binding model of HQ7 and Cy-FBP/SBPase.Based on above all,we established the interaction model between Cy-FBP/SBPase receptor and its ligand compounds.This model consists of three parts:(1)the binding region,composed of hydrophilic amino acids(ARG176,ARG178,TYR131);(2)the catalytic region,composed of magnesium ions and amino acid residues THR102 in the catalytic site;(3)the hydrophobic region,composed of hy-drophobic amino acid residues(VAL201,ALA222).According to the study of inhibitors and enzyme in this chapter,we determined the pharmacophore of the next virtual screen-ing is the amino acid residues ARG176,ARG178,TYR131 and THR102 that play an important role in the substrate cavity.2.Virtual screening,acquisition and characterization of the hit compound(Z18):Based on the new pharmacophore screening model,using Flex-Pharm restrictive docking(restricted the interactions between small molecules with pharmacophore ARG176,ARG178,TYR131,THR102)and Surflex-Dock unrestrictive docking procedure of docking software SYBYL-X 2.1.1,two rounds of virtual screening from Maybridge compounds library were performed.20 candidate compounds were obtained.These compounds were purchased and tested the inhibitory effects on Cy-FBP/SBPase at final concentration 100 μM.The results showed that there were 4 compounds had inhibitory effects on Cy-FBP/SBPase,and compound Z18 showed the best inhibitory activity on Cy-FBP/SBPase,the inhibition rate on Cy-FBP/SBPase was over 80%at 100 μM.3.The acquisition and characterization of the new A series inhibitors:Compound Z18 was used as a pilot compound for further modification,and was renamed as compound A1.Combined with the binding model of compound A1 in the Cy-FBP/SBPase substrate cavity,we modify and transform the compound A1.First,we transform the tertiary butyl part into benzene ring and get compound A2,so that the compound molecule can occupy the cavity more comprehensively.The results showed that the inhibitory ac-tivity on Cy-FBP/SBPase was increased from IC50=14.6±0.2 μM to IC50=8.9±0.3μM,and the inhibitory activity was increased by 1.6 times.Then,the substituents on the benzene ring of A2 were transformed,a compound A16 with better inhibitory activity on Cy-FBP/SBPase was obtained.Its IC50=0.3±0.1 μM,the inhibitory activity is 30 times that of A2 and 49 times that of the pilot compound A1.4.Study on the binding model of new inhibitor A16 with Cy-FBP/SBPase:In theory,using docking method predict the binding model of the inhibitor A16 with Cy-FBP/SBPase.It can be seen from the binding model that the inhibitor A16 has formed hydrogen bonds with the amino acid residues ARG176,ARG178,TYR131 and THR102.Through site directed mutagenesis and enzyme inhibition kinetics experiments,we analysed the inhibitory effect of A16,a new inhibitor,on the important amino acid mutants of Cy-FBP/SBPase substrate cavity.The results showed that the inhibition effect of inhibitor A16 on the ARG176,ARG178,TYR131,THR102,ASP200 mutants is diminished or lost.The binding mode of inhibitor A16 to Cy-FBP/SBPase was further determined.