| Magnaporthe grisea is one of the three major crop fungi.Rice infected with Magnaporthe grisea will lead to rice yield reduction.Studies have shown that the use of a large number of fungicides has caused fungal resistance.but the use of a large number of fungicides has caused fungal resistance.However,the use of a large number of fungicides has caused fungal resistance.Fructose 1,6-bisphosphate aldolase(FBA)is an important catalytic enzyme in the process of sugar metabolism and plays an important role in the growth of Magnaporthe grisea.A large number of literatures have shown that inhibition of FBA activity can inhibit the process of glucose metabolism in living organisms,thereby inhibiting the growth of Magnaporthe grisea.Based on this idea,this paper first established the activity test system of Magnaporthe grisea type II fructose 1,6-bisphosphate aldolase(Mg-FBA-Ⅱ),and expressed and purified 24 key amino acid residue mutants.In this paper,Mg-FBA-Ⅱ was used as the target,threel types of inhibitors were screened on Mg-FBA-Ⅱ,and the structure-activity relationship of several inhibitors was analyzed.The interactional mechanism of inhibitors and proteins was also studied.The details shown as following:(1)The inhibitory effect of N-substituted phenyl-1,2.3-triazoleacetamides(N series)compounds on Mg-FBA-Ⅱ was tested.It was found that N11 is the best inhibitors with IC50=4.8110.20 μM.These compounds were found to be non-competitive inhibitors by testing the relationship between inhibitors and FBP.Therefore,the binding model of these compounds were predicted for the first time.Secondly,the molecular docking technique was used to explore the binding mode of inhibitors and proteins.Finally,the site-directed mutagenesis technique and K1 determination were used to further verify.Whether the theoretical docking mode is correct,and the well consistence between predicted and experimental results.Therefore,a series of non-competitive inhibitors were found on Mg-FBA-Ⅱ,and the site of action was successfully found and the binding mode of compound to protein was explored,this provides direction for designing non-competitive compounds on Mg-FBA-Ⅱ.(2)The test evaluated the inhibitory activity of N-phenyl-N’-aryl formylthioureas(L series)on Mg-FBA-Ⅱ and Magnaporthe oryzae,and found such compounds in Mg-FBA-Ⅱ and Magnaporthe oryzae has good inhibitory activity.The molecular docking and site-directed mutagenesis techniques were used to explore the binding mode of inhibitors to target proteins.On this basis,the compound was further modified and finally obtained the most active compound L15(EC50=0.4±0.03 ppm),and its antibacterial activity was close to the activity of the commercial drug diniconazole(EC50=0.25±0.02 ppm),It is an order of magnitude higher than the antibacterial activity of another commercial drug,triadimefon(EC50=6.4±6.4±0.30 ppm).The inhibitory effect is higher about 5 times.Therefore,a class of inhibitors with Mg-FBA-Ⅱas a target and a similar inhibitory effect on the commercial drug diniconazole was found on the Magnaporthe oryzae.(3)Sothiocyanates are a natural class of products.According to reports,these compounds have particularly good effects on both anticancer and antifungal properties,so our team synthesized a series of such compounds(SF series).The biological activities of these compounds on Mg-FBA-Ⅱ,Magnaporthe oryzae and Synechocysis PCC 6803 were tested and found to be inhibitory at low concentrations in Mg-FBA-Ⅱ and Magnaporthe oryzae.Such compounds have no good inhibitory effect on Mg-FBA-Ⅱ and Magnaporthe oryzae,but have excellent anti-algae effects on Synechocystis PCC 6803.The best inhibitory effect was SF10,EC50=0.69±0.014 μM in 96-well plates,and EC50=0.34±0.054 pM in the amplification experiment.It is an order of magnitude higher than the inhibition effect of CuSO4(EC50=2.21±0.048 μM).In order to find the target of such compounds,the EC50 of SF10 on PSB Ⅰ(overexpression of FBP)and PSBⅡ(overexpression of FBA)was tested,The results showed that the EC50 on the two enzymes did not change much with the EC50 on the algae,indicating that these compounds do not act on these two targets. |
