Research On Molecular Simulation Of Lymantria Dispar Methuselah-like And Targeting Small Molecular Inhibitors

Lymantria dispar is a worldwide leaf-eating pest with mixed feeding habits,strong vitality and a wide range of damage,which seriously endangers the growth of forest trees and causes significant losses to our economy and ecology.A protein inhibitor is an agent that can target the protein to reduce protein activity.The L.dispar Methuselah-like1 gene（Ldmthl1）belongs to a G protein-coupled receptor that regulates L.dispar resistance to heat,starvation,and paraquat oxidation.This paper presents a virtual screening of small molecule inhibitors of the receptor of the Ldmthl1 and analysis of the binding strength of six potential small molecule inhibitors to the Ldmthl1 receptor using molecular dynamics and MM-PBSA calculated binding free energy;the toxicity of the six potential small molecule inhibitors and their effects on the survival rate of the L.dispar under high temperature and starvation stress were analyzed by bioassay;the six potential small molecule inhibitors were also used in combination with deltamethrin to investigate whether the six potential compounds have synergistic effects.The main findings are as follows:1.Obtain a homology model of Ldmthl1,which contains seven transmembrane structures consistent with the structural features of G protein-coupled receptors and,upon evaluation,meet the criteria for protein model assessment;Discovery studio 2019 Client and Schr?dinger software was used to perform rapid and precise molecular docking of ZINC and SPECS databases,respectively.In order to obtain 20 candidate compounds,analyze the binding mode and binding energy,and finally identify six small molecule compounds as potential small molecule inhibitors.2.Molecular dynamics simulations were used to analyze the binding of six potential small molecule inhibitors to Ldmthl1.The results showed that the six potential small molecule inhibitors stably bound to Ldmthl1 through hydrogen bonding and hydrophobic forces;isoleucine（ILE）238,glycine（GLY）239,leucine（LEU）234,histidine（HIS）237 and cysteine（CYS）334 were the key amino acid residues in the binding process of Ldmthl1;the free energy of binding calculated by MM-PBSA revealed that the six potential small molecule inhibitors bound tightly to Ldmthl1,indicating that the binding mode of the six potential small molecule inhibitors to Ldmthl1 was stable and trustworthy.3.Previous research found that the silencing of Ldmthl1 gene reduced the resistance to deltamethrin,and the inhibition effect of the six inhibitors was analyzed by feeding the larvae of L.dispar at 1:1,1:2 and 2:1 ratios with the sublethal concentration of LC₃₀ and six inhibitors.At the three ratios,the larvae of L.dispar did not die at 48 h in the solvent（DMSO）control group,70.00%survived at 48 h in the deltamethrin-treated group at the 1:1 ratio,75.00%or more survived in all six inhibitor single treatment groups,and 6.67%～63.34%survived at 48 h in the six combined treatment groups;at the ratio of 1:2,L.dispar in the isoquinoline single treatment group died,while the survival rate of L.dispar in the other five inhibitor single treatment groups was above 60.00%.The survival rates of the six combined treatment groups ranged from 0%to60.00%and were significantly different from those of the deltamethrin and inhibitor single treatment groups;at the ratio of 2:1,L.dispar larvae died within 36 h.The survival rate of the combined treatment group was not significantly different from that of the deltamethrin and inhibitor single-treatment groups.6 inhibitors showed synergistic and additive effects in combination with deltamethrin,indicating that the combination with deltamethrin had a synergistic effect to enhance the toxicity of L.dispar.4 The 3rd instar 1st d L.dispar larvae were given six small molecule inhibitors LC₃₀.It was found that the average survival time of the six inhibitor treatment groups under high temperature stress was 37.63%～86.14%of the average survival time of the control group.The average survival time of the six inhibitor treatment groups under starvation stress was 78.63%～95.52%of the average survival time of the control group,indicating that the six inhibitors could affect the resistance of L.dispar.Using bromoxynil LC₃₀ concentrations in combination with six inhibitors at 1:1,1:2 and 2:1 ratios,the survival rate of the combined treatment group at 12 h～48 h was lower than the survival rate of the combined treatment group at room temperature under high temperature stress at all three ratios;under 1:1 ratio,the 48 h survival rate of the six combined treatment groups under high temperature stress was significantly lower than that of the deltamethrin and inhibitor single-dose treatment groups,and under starvation stress,the 156h survival rate of the control group was 6.67%and the 156 h survival rate of the six combined treatment groups was 0%,and the 84 h～156 h survival rate of the combined treatment groups was always lower than that of the deltamethrin treatment groups;survival rates at all time points under combined treatment group survival under 1:2 ratio of heat stress and starvation stress were lower than those under 1:1 ratio;the survival rate of L.dispar under high temperature stress in the 2:1 ratio was not significantly different from that of the deltamethrin single treatment group;the survival rate of L.dispar under starvation stress in the 2:1 ratio was similar to that of the 1:2ratio at 84 h～156 h.The above results indicated that the combination of inhibitor and deltamethrin had a synergistic effect and a greater effect on the resistance of L.dispar.The combination of 1:2 ratio achieved the best effect.In summary,we studied the Ldmthl1 function of the L.dispar by molecular dynamics simulation,MM-PBSA calculation of binding free energy,inhibitors and combination with deltamethrin.The results showed that six potential small molecule inhibitors inhibited the Ldmthl1 protein function and reduced the resistance to high temperature and starvation of L.dispar,laying the theoretical foundation for developing new Ldmthl1 targeted insecticides.