Application Of Binding Spectra In DMIs Fungicide Screening

The method of binding-spectra had been used to screening fungicides and to investigate the interaction of chemical molecules. CYP51 of fungus is membrane-binding protein. The purification of CYP51 protein is difficult so that it is not easy to obtain sufficient quantity of enzyme for fungicide screening. Those investigations of screening fungicides usually use the soluble protein from prokaryote as target enzyme, so the results inevitably generated inaccuracy. The establishment of a fast and accurate method for novel DMIs fungicide screening is important for large scale screening of novel fungicides, and offer evaluation tool for the designation and synthesis of highly active, harmfuless and highly selective fungicides.The lanosterol 14α-demethylase of Magnaporthe grisea expressed in E.coli was used as target enzyme and the DMI fungicides diniconazole, tebuconazole, triadimenol and triadimefon were used as representative fungicide, the effects of enzyme activity, enzyme purity and concentration on the binding spectra were investigated. The results showed that active enzyme, elimination of interference of other P-450s and proper enzyme concentration were necessary for obtaining accurate binding spectra. The Kd values of diniconazole, tebuconazole, triadimenol and triadimefon were 0.143μM, 0.24μM, 0.257μM and 0.307μM respectively, which significantly correlated to their 120h-EC₅₀ values on the growth of Magnaporthe grisea. The results indicated that the binding spectra of fungicide and lanosterol 14α-demethylase can serve as a reliable and fast method for novel fungicide screening.The binding of novel drugs with different structure to CYP51 of Magnaporthe grisea expressed in E.coli was investigated. The results showed that: EVE-2, EVE-3, LHEXP-10, LHEXP-3s, FBQbp-25, SYL-25, SYL-35, SYL-36, DM-2, SC-11could bind to CYP51, the Kd values are 73.039μM, 18.634μM, 2.18μM, 66.998μM, 1.607μM, 5.147μM, 8.653μM, 10.199μM, 3.603μM and 3.795μM, respectively. FBQbp-57, SYL-27, DM-5, SC-17, SYC-4 could not bind to CYP51. SYL-26, SYL-33, SYC-11 and ZD-6 could partly bind to CYP51. The bingding spectra of some drugs didn't match the results of growth inhibition test of Magnaporthe grisea, which may due to the different target protein in cell or the metabolism in cell. Though their basic structure were identical of the same type drug, their affinity to CYP51 were differrent due to the difference of number, variety and position of substituent groups. By screening 19 kinds of drugs, the results showed that EVE-3, LHEXP-10, FBQbp-25, SYL-25, SYL-35, SYL-36, DM-2, SC-11 had potential antifungal ablity.