Novel Multi-Target-Directed Inhibitors Discovered By Rational Design And Screening Research

As is known to all, in the Calvin cycle, glycolysis and gluconeogenesis, fructose-1,6-bisphosphate aldolase (Fba, EC4.1.2.13) catalyzes the reversible aldol condensation of dihydroxyacetonephosphate (DHAP) and glyceraldehyde3-phosphate (GAP) to fructose-1,6-biphosphate (FBP), while Fructose-1,6-bisphosphatase (FBPase, EC3.1.3.11) catalyzes the reversible hydrolysis of FBP into fructose-6-phosphate and inorganic phosphate. Because they both are essential to the energy metabolism of all organisms, they have important medical and agricultural significance. From the outset, the class Ⅱ Fba was viewed as a particularly attractive antibacterial and antifungal target and a lot of the attention is focused on the drug design of the enzyme. Human FBPase has attracted considerable attention and represents a highly attractive drug target for type Ⅱ diabetes. A unique FBPase from cyanobacterial not only can exhibit FBPase activity but also can act as a sedoheptulose-1,7-bisphosphatase (SBPase)(named Cy-FBPase),which was categorized into type Ⅱ FBPase together with GlpX from E.coli. Based on their crucial role and function in cyanobacteria, Cy-FBPase and class Ⅱ Fba from cyanobacterial (named Cy-FBA-Ⅱ) are good candidate targets for the development of specific algicides.Rice blast disease, caused by the fungus Pyricularia oryzae, is one of the most serious and damaging diseases in rice production worldwide. Sterol14a demethylase (CYP51, EC1.14.13.70) that catalyzes the removal of the14a-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, and a primary target for clinical and agricultural antifungal azoles. Trihydroxynaphthalene reductase (3HNR, EC1.1.1.252) and scytalone dehydratase (SD, EC4.2.1.94) in the melanin biosynthetic pathway have proven to be valuable targets for development of rice blast fungicides. In particular, inhibitors of3HNR and SD have yielded commercially useful rice fungicides. Because the biosynthetic pathway does not exist in off-target organisms and, consequently, low levels of off-target toxicity and few resistance development are reported for the commercial blasticides targeted to3HNR and SD.This thesis carried out the research by use of multidisciplinary approaches, such as molecular simulation, cloning and expression, site-specific mutagenesis, enzymatic analysis, protein crystallography and so on. Under the guidance of multi-target inhibitor design, the research combinated theory and experiment, and intended to in-depth systematically study the active site structure of these series of key regulatory enzymes in cyanobacteria and Magnaporthe grisea and their mechanism of interaction with substrates or inhibitors. On these basis points, multi-targeted directed inhibitors were design and screened for Cy-FBPase and Cy-FBA-Ⅱ from cyanobacteria, and CYP51and3HNR (or SD) from Magnaporthe grisea. The main study and the novelty described in the present thesis are about the following five areas:First, we constructed the expression plasmids of Cy-FBPase, Cy-FBA-Ⅱ, Magnaporthe grisea3HNR and SD, established the expression and purification systems of all these enzymes respectively.Second, based on homology models of Cy-FBPase and Cy-FBA-Ⅱ, key amino acid residues in the active site of Cy-FBPase and Cy-FBA-Ⅱ were predicted, and the site-directed mutagenesis were done. In computer theory term, molecular dynamic simulation and fragment molecular orbital (FMO) were performed to reveal that three metal ions and some of the impotant residues were involved in the mechanism of Cy-FBPase catalysis. Furhermore, we first crystallized the two crystal structures of Cy-FBPase complex with allosteric inhibitor AMP+/-substrate FBP. The crystal structures of Cy-FBPase proved the validity of the theoretical calculation results and showed Cy-FBPase was a novel tetramer which has three domains including substrate binding domain and AMP binding domain and disulfide linkages domain. Through complex collaborative regulatory mechanism these domains regulate the enzymes activies.Third, On the basis of the understanding of the target enzymes’hydrophilic active site structures which were rich in hydrogen bond donors and acceptors, we designed and screened a number of compounds targeted for both Cy-FBPase and Cy-FBA-Ⅱ. And then target enzyme inhibition test and algae inhibition experiments were done. Among these compounds, CFL20and CFL23showed very good inhibitory activities According to the binding model of CFL23in both Cy-FBPase and Cy-FBA-II, we guided the structure optimization and directly synthetized two series of compounds a and b. Generally, both series of compounds could well inhibit Cy-FBPase and Cy-FBA-Ⅱ activities and the growth of cyanobacterial. Compared to CFL23, their inhibitory activities were almost improved by an order of magnitude. The structure-activity relationship of these compounds was then analyzed.Fourth, a new theory3D-structure of CYP51form Magnaporthe grisea was obtained by homology modeling based on five homologous crystal structures. According to the characteristics of both active sites of CYP51and3HNR, the receptor pharmacophore was set and through the pre-screening strategy,44candidate compounds were screened out for further biological test. The inhibition experiment results shown that among these compounds, ZY9has a binding constant Kd of29.78nM for CYP51and inhibition constant IC50of19.7μM for3HNR. In vivo, ZY9could inhibit the growth of Magnaporthe grisea and the the secretion of melanin. ZY9was a good hit compound which proved the rationality of our virtual screening strategy and the feasibility of the design muti-target inhibitors against the Magnaporthe grisea CYP51and3HNR.Fifth, According to the characteristics of both active sites of CYP51and SD and their potent inhibitors, the receptor and ligand pharmacophore were set and through the pre-screening strategy,50candidate compounds were screened out for further biological test. The binding model of these compounds in the active sites of CYP51and SD were analyzed and they may exhibit good potential inhibit function on the two enzymes and thalli level.In surmmary, several important regulatory enzymes in the metabolic processes of cyanobacteria and Magnaporthe grisea were studied by using multidisciplinary approaches and the combination of theory and experiment. The active site structure of these series of key regulatory enzymes and their mechanism of interaction with substrates or inhibitors were understood. The two crystal structures of Cy-FBPase complex were obtained. Under the guidance of multi-target inhibitor design, multi-target inhibitors against Cy-FBPase and Cy-FBA-II and against CYP51and3HNR were successfully screened out and synthetized. All these results would shed light on the research of inhibitor exploitation and design for novel algicide pesticides and fungicides, and provide new ideas.