Overcoming insecticide resistance through computational inhibitor design

Abstract

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

Keywords: Lucilia cuprina; carboxylesterase; covalent docking; insecticide resistance; organophosphates.

Fig. 1.

Overview of synergists for OP insecticides. (A) OP insecticides inhibit AChE and prevent the hydrolysis of acetylcholine. (B) CEs, like αE7, rescue AChE activity by binding and hydrolyzing OP insecticides. (C) An inhibitor that outcompetes OPs for binding to CEs could act as a synergist to restore insecticide activity.

Fig. 2.

Covalent docking predicts potent inhibitors of LcαE7. (A) Chemical structures of predicted LcαE7 inhibitors. Compounds 1 to 5 were ranked 8th, 169th, 202nd, 210th, and 478th in the DOCKovalent screen. (B) In vitro K_i inhibition constants for WT and Gly137Asp LcαE7 with PBA and compounds 1 to 5. Data are presented as ±95% confidence intervals for 3 repeat measurements of enzyme activity at each concentration of compound. (C) Compounds 1 to 5 (purple sticks) form covalent adducts with the catalytic serine of LcαE7 (Ser218). The omit mF_O-DF_C difference electron density is shown (green mesh contoured at 3 σ). The docking predictions (yellow sticks) are overlaid onto the corresponding cocrystal structures. Active site residues are shown as white sticks. (D) Surface view of LcαE7 active site, with compound 3 shown as spheres.

Fig. 3.

Second generation boronic acids are potent inhibitors of Gly137Asp LcαE7. (A) Chemical structures of compound 3 analogs. Compounds 3.9 and 3.10, which combine the disubstitution pattern of compound 3 and the relatively large flexible substituent of compound 2, are highlighted with a dashed box. (B) In vitro K_i inhibition constants for WT and Gly137Asp LcαE7 with compounds 3.1 to 3.12. Data are presented as ±95% confidence intervals for 3 repeat measurements of enzyme activity at each concentration of compound. (C) Surface view of the cocrystal structure of compound 3 with WT LcαE7 (white surface and sticks) overlaid with the apo Gly137Asp LcαE7 structure (yellow sticks). (D) The active site is rearranged in the cocrystal structure of compound 3.10 with Gly137Asp LcαE7 (white sticks and surface). The boronic acid compounds are shown as purple spheres.

Fig. 4.

Boronic acid compounds are highly selective against L. cuprina, mouse, and human enzymes. (A) In-gel fluorescence ABPP of serine hydrolases in L. cuprina using an FP-RH probe. (B) Heat map showing compound selectivity against human AChE, BChE, CES1, and CES2. Selectivity was calculated as the ratio of K_i inhibition constants between WT LcαE7 and each of the human enzymes. (C) Heat map showing inhibition of 26 human serine and threonine proteases by compounds 3, 3.9, and 3.10. Percentage inhibition was determined at a single compound concentration (100 μM) in duplicate. SI Appendix, Table S3 shows assay conditions. (D) In-gel ABPP of serine hydrolases in mouse liver using an FP-RH probe.

Fig. 5.

Boronic acid compounds synergize with OP insecticides. (A) Chemical structures of the active forms of the insecticides diazinon and malathion chlorpyrifos. (B) Compounds 3, 3.9, and 3.10 synergized diazinon and malathion against the susceptible LS blowfly strain, while all compounds tested synergized diazinon against the resistant Tara strain. EC₅₀ values were calculated from 3 (diazinon) or 2 (malathion) repeat measurements of pupation rate, with 50 larvae at each diazinon/malathion concentration. EC₅₀ values are presented as ±95% confidence intervals with the diazinon-only control compared with treatment with diazinon and the boronic acid compounds (1-way ANOVA followed by Dunnett’s multiple comparison test). ns, not significant. ****P < 0.0001. (C) Compounds 3.7, 3.10, and 5 synergized chlorpyrifos against the peach–potato aphid. After initial infestation with 9 adult aphids, the numbers of alive adults and new larvae were counted after 1, 3, and 7 d. Data are mean ± SEM for 4 replicate experiments with the chlorpyrifos-only control compared with treatment with chlorpyrifos and the boronic acid compounds (1-way ANOVA followed by Dunnett’s multiple comparison test). ****P < 0.0001.