Rational Design And Screening Research Of Novel Lead Compound Based On Acetolactate Synthase Structure

Acetolactate synthase (ALS) is the first enzyme to play a catalyst role in the biosynthetic pathway of branched chain amino acid, and also the most widely used target of commercialized herbicides. This kind of inhibitors is known for its high efficiency, broad spectrum, and low toxicity for humans and animals, and also has the extremely wide range of applications in the world. In recent years, while protecting the crops from weeds, the problem of weed resistance is gradually appearing and getting worse. In addition, the period of generating resistance is shortened from10years to4～5years. Therefore, the development of novel herbicides is extremely urgent, and is also the most effective method to solve the problem of weed resistance. In our study, we had systematically researched the ALS crystal structure and the mechanism of action between the active site of enzyme and the ligand (inhibitors) by applying molecular modeling, biological test, organic synthesis and spectral characterization, and so on. Based on the above research results, the study on rational design and screening of the novel inhibitor lead compound to ALS had been performed. The specific research contents are as follows:Firstly, the nature, function, three-dimensional crystal structure, mechanism of action, inhibitors and research advances in ALS were introduced, and the advantage, classification, principle, research achievements and software of molecular modeling in computer-aided drug design were discussed in detail.Secondly, the theoretical screening study of lead compound:based on the interaction mechanism between active site of ALS and inhibitor, the specific screening process and the docking parameters of Surflex-Dock and FlexX-Pharm constraints were defined. The commercialized small molecule database was filtered by the receptor-ligand mechanism, binding modes. Finally,33compounds were screened out as the candidate compounds from a database of more than200,000compounds. Then the compounds were purchased from Specs and used to the further bioactive assay.Thirdly, the bioactive assay of candidate compounds:the bioassay method of enzyme in vitro was established according to the principle that activity of ALS was indicated by the change in absorbance of acetoin at525nm by ultraviolet absorption spectrophotometry. Using the established bioassay method, the bioactivity of33compounds was determined. The result showed that compound No.10had the highest value of IC50(47.41mg/L), and the docking conformation and binding force were similar to the reference structure. Therefore, compound No.10was treated as a lead compound used in the next rational molecular design. Fourthly, the further optimized design of lead compound:the structure of lead compound was analyzed and designed according to the interaction mechanism between the active site of ALS and inhibitor and the major binding conformation and binding force reported in the literatures. The optimized basic structures of target compounds were defined as (5-benzyl-1,3,4-thiadiazol-2-carbamic acid phenyl ester). The synthetic route of target compounds was established, and the reaction mechanism of these compounds was explored. The structure characterization of target compounds was made by IR,1HNMR. Using the established bioassay method, the bioactive test was carried out. The result showed that the compound2c had the highest value of IC50(23.54mg/L). Compared with the bioactivity of lead compound, the inhibition rate of2c had increased by50%. Then we performed the research of docking analysis, discussed the reason causing the change of bioactivity.Finally, the contents of virtual screening, structure design of lead compound, bioactive test, synthesis and structural characterization of the target compounds was summarized in detail. The structural design method of lead compounds was discussed, and some reasonable suggestions for the following research directions of the kind of compounds were proposed.