High Throughput Screening For Sterol Carrier Protein-2 Inhibitors Of Helicovera Armigera

The continued increase in pesticide resistance to agricultural pests is a serious threat to agricultural sustainable development and human survival.Looking for new targets to develop high efficiency,low toxicity,environment friendly new pesticides is an important urgent task of pest control.Cholesterol is the component of cell membrane and the precursor of molting hormone,but the insect can not synthesize cholesterol de novo themselves.To fulfill the needs of growth,development and reproduction,insects must obtain cholesterol from food or symbiotic microbes.So the absorption and transport of cholesterol is essential for the survival of insects.Sterol carrier protein 2(SCP-2)is a non-specific lipid transporter that participates in cholesterol absorption and transport in insects.Experiments show that the treatment of Diptera,Lepidoptera,Coleoptera agricultural pests larvae with mosquito SCP-2 inhibitors can significantly affect theirs growth and development,while causing high adult mortality and low egg hatching rate.Therefore,SCP-2 protein can be used as a potential target for the development of new insecticides.The main results are as follows:(1)The recombinant expression plasmid pET-22b/HaSCP2 was transformed into E.coli BL21(DE3)for the inducible expression of HaSCP-2.The optimal expression conditions were at the temperature of 25 ? and at the final concentration of 0.2 mmol/L IPTG for 10 h.HaSCP-2 protein and substrate binding activity analysis showed that 1,8-ANS bound to HaSCP-2 with binding constant Kd of 1.973 ?mol/L and 1,8-ANS was replaced by cholesterol bound to HaSCP-2 with a half effective concentration(EC50)of 29.03 ?mol/L.(2)Forty-five potential SCP-2 inhibitors were identified by high-throughput virtual screening(HTVS)based on the structure of Helicoverpa armigera SCP-2 from an HTS library of 218,780 compounds.According to the classification of chemical structure diversity,nine different structural scaffolds compounds were selected for bioassay experiments screening,and five HaSCP-2 inhibitors(H1,H7,H14,H19 and H29)with strong insecticidal effect and growth inhibition were identified.The binding pattern of HaSCP-2 inhibitor and HaSCP-2 revealed that small molecules were horizontally conformation through hydrophobic interactions.In addition,the benzene groups of inhibitors form a ?-? interaction with residues Tyr51,Phe53 or Phe110,respectively.The formation of weak H-bonds with residues Leu127,Thr128 and Gln131 might stabilize the complex.(3)Bioassay indicated that the order of mortality of the larvae was 54%,50%,48%,33% and 17%,respectively;With the increase of the concentration of the inhibitor,the weight of the larvae decreased by 14% to 46%,with the following order of inhibitory effect: H1> H14> H19> H29> H7;Pupation time of larvae treated with HaSCP-2 inhibitors delayed 3 to 5 days.In addition,the oviposition rate of adults decreased by 12.5% to 88.3%.Environmental toxicity predictions indicate that the five inhibitors are mild or low acute oral toxicity and are less toxic to beneficial insects.