Overcoming insecticide resistance through computational inhibitor design

Significance Pesticides and insecticides are crucial for agricultural productivity, global food security, and control of disease vectors. However, resistance to insecticides is a widespread and urgent problem, which leads to increased insecticide usage with dire consequences to the environment. A common resistance mechanism against 2 of the most widely used insecticide classes, organophosphates and carbamates, involves carboxylesterases. We applied covalent virtual screening to discover potent inhibitors for a common, resistance-mediating carboxylesterase. The compounds reversed insecticide resistance in 2 different pest species, representing a sustainable strategy that allows significant reduction of insecticide use without compromising their efficacy. Such synergists could have major economic and environmental benefits, and the approach demonstrated in this work should be applicable to additional resistance mechanisms. 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.

[1]  A. Yudin,et al.  The versatility of boron in biological target engagement. , 2017, Nature chemistry.

[2]  R. Lehner,et al.  Carboxylesterases in lipid metabolism: from mouse to human , 2017, Protein & Cell.

[3]  Michael R. Taylor,et al.  Carboxylesterases: General detoxifying enzymes. , 2016, Chemico-biological interactions.

[4]  J. Bloomquist,et al.  An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria , 2016, Scientific Reports.

[5]  C. Jackson,et al.  Mapping the Accessible Conformational Landscape of an Insect Carboxylesterase Using Conformational Ensemble Analysis and Kinetic Crystallography. , 2016, Structure.

[6]  J. Oakeshott,et al.  Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability. , 2016, Journal of molecular biology.

[7]  C. Jackson,et al.  Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency. , 2016, Biochemistry.

[8]  D. Fairlie,et al.  Histone deacetylase enzymes as drug targets for the control of the sheep blowfly, Lucilia cuprina , 2015, International journal for parasitology. Drugs and drug resistance.

[9]  Ralf Nauen,et al.  IRAC: Mode of action classification and insecticide resistance management. , 2015, Pesticide biochemistry and physiology.

[10]  Nir London,et al.  Covalent Docking Predicts Substrates for Haloalkanoate Dehalogenase Superfamily Phosphatases , 2014, Biochemistry.

[11]  Nir London,et al.  Covalent Docking of Large Libraries for the Discovery of Chemical Probes , 2014, Nature chemical biology.

[12]  K. Diederichs,et al.  Better models by discarding data? , 2013, Acta crystallographica. Section D, Biological crystallography.

[13]  J. Oakeshott,et al.  Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance , 2013, Proceedings of the National Academy of Sciences.

[14]  P. Potter,et al.  Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors. , 2013, Chemico-biological interactions.

[15]  M. Rudolph,et al.  Structures of human acetylcholinesterase in complex with pharmacologically important ligands. , 2012, Journal of medicinal chemistry.

[16]  P. Andrew Karplus,et al.  Linking Crystallographic Model and Data Quality , 2012, Science.

[17]  P. Zwart,et al.  Towards automated crystallographic structure refinement with phenix.refine , 2012, Acta crystallographica. Section D, Biological crystallography.

[18]  A. Hermetter,et al.  Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7. , 2012, Insect biochemistry and molecular biology.

[19]  L. Field,et al.  Gene amplification and insecticide resistance. , 2011, Pest management science.

[20]  J. Oakeshott,et al.  The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects , 2011, Evolutionary applications.

[21]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[22]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[23]  Laura Robinson,et al.  The Material Safety Data Sheet , 2009 .

[24]  M. Whalon,et al.  Global Pesticide Resistance in Arthropods , 2008 .

[25]  Jia-Wei Wang,et al.  Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol , 2007, Nature Biotechnology.

[26]  J. Cooper,et al.  The benefits of pesticides to mankind and the environment , 2007 .

[27]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[28]  Peter Kuhn,et al.  Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1. , 2006, Journal of molecular biology.

[29]  T. Torres,et al.  A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles. , 2006, Veterinary parasitology.

[30]  G. Geiss,et al.  Role of paraoxonase (PON1) status in pesticide sensitivity: genetic and temporal determinants. , 2005, Neurotoxicology.

[31]  D. Hall.  Boronic acids : preparation and applications in organic synthesis and medicine , 2005 .

[32]  J. Oakeshott,et al.  Biochemical Genetics and Genomics of Insect Esterases , 2005, Reference Module in Life Sciences.

[33]  J. Oakeshott,et al.  Multiple Mutations and Gene Duplications Conferring Organophosphorus Insecticide Resistance Have Been Selected at the Rop-1 Locus of the Sheep Blowfly, Lucilia cuprina , 2005, Journal of Molecular Evolution.

[34]  Brian K. Shoichet,et al.  Virtual screening of chemical libraries , 2004, Nature.

[35]  J. Oakeshott,et al.  Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides , 2004 .

[36]  Dale L Boger,et al.  Discovering potent and selective reversible inhibitors of enzymes in complex proteomes , 2003, Nature Biotechnology.

[37]  B. Wang,et al.  Boronic acid compounds as potential pharmaceutical agents , 2003, Medicinal research reviews.

[38]  Nick Buckley,et al.  Pesticide poisoning in the developing world—a minimum pesticides list , 2002, The Lancet.

[39]  J. Thorson.  Faculty Opinions recommendation of Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. , 2002 .

[40]  P. Taylor,et al.  Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. , 2002, Angewandte Chemie.

[41]  R. Edwards,et al.  Diazinon is activated by CYP2C19 in human liver. , 2001, Toxicology and applied pharmacology.

[42]  P. Batterham,et al.  The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina. , 2001, Insect biochemistry and molecular biology.

[43]  K. Lorentz,et al.  Continuous monitoring of arylesterase in human serum. , 2001, Clinica chimica acta; international journal of clinical chemistry.

[44]  J. Oakeshott,et al.  The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly. , 1999, Insect biochemistry and molecular biology.

[45]  J. Oakeshott,et al.  A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[46]  WHO Regional Office for the Eastern Mediterranean , 1995 .

[47]  G. Levot.  Resistance and the control of sheep ectoparasites. , 1995, International journal for parasitology.

[48]  R. Russell,et al.  Insecticide resistance and malathion carboxylesterase in the sheep blowfly,Lucilia cuprina , 1994, Biochemical Genetics.

[49]  P Taylor,et al.  Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. , 1993, Biochemistry.

[50]  Fukuto Tr.  Mechanism of action of organophosphorus and carbamate insecticides. , 1990 .

[51]  T. R. Fukuto,et al.  Mechanism of action of organophosphorus and carbamate insecticides. , 1990, Environmental health perspectives.

[52]  D. Agard,et al.  Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids. , 1988, Biochemistry.

[53]  P. Spencer,et al.  Phenyl-n-butylborinic acid is a potent transition state analog inhibitor of lipolytic enzymes. , 1986, Biochemical and biophysical research communications.

[54]  M. J. Whitten,et al.  The genetic basis for organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera, Calliphoridae) , 1976, Bulletin of Entomological Research.

[55]  Y. Cheng,et al.  Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. , 1973, Biochemical pharmacology.

[56]  G. Lienhard,et al.  2-phenylethaneboronic acid, a possible transition-state analog for chymotrypsin. , 1971, Biochemistry.

[57]  J. Casida,et al.  Properties of housefly microsomal cytochromes in relation to sex, strain, substrate specificity, and apparent inhibition and induction by synergist and insecticide chemicals. , 1970, Life sciences. Pt. 1: Physiology and pharmacology.

[58]  J. Casida.  Mixed-function oxidase involvement in the biochemistry of insecticide synergists. , 1970, Journal of agricultural and food chemistry.

[59]  G. Shanahan.  Development of a changed response in Lucilia cuprina (Wied.) to organophosphorus insecticides in New South Wales , 1966 .

[60]  K. Courtney,et al.  A new and rapid colorimetric determination of acetylcholinesterase activity. , 1961, Biochemical pharmacology.

[61]  Probing questions. , 2015, Nature chemical biology.

[62]  P. Potter,et al.  Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon. , 2012, Toxicology and applied pharmacology.

[63]  C. Walker,et al.  “A” esterases and their role in regulating the toxicity of organophosphates , 2004, Archives of Toxicology.

[64]  S. Bryant,et al.  Organophosphate poisoning. , 1968, Lancet.

[65]  N. Sales,et al.  New high level resistance to diflubenzuron detected in the Australian sheep blowfly, 'Lucilia cuprina' (Wiedemann) (Diptera: Calliphorddae) , 2002 .

[66]  J. Oakeshott,et al.  MCE activities and malathion resistances in field populations of the Australian sheep blowfly (Lucilia cuprina) , 2000, Heredity.

[67]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[68]  I. Barchia,et al.  In vitro larvicidal efficacy of flystrike dressings against the Australian sheep blowfly , 1999 .

[69]  Caixian Tang,et al.  The influence of alkalinity and water stress on the stomatal conductance, photosynthetic rate and growth of Lupinus angustifolius L. and Lupinus pilosus Murr. , 1999 .

[70]  J. Oakeshott,et al.  cDNA cloning, baculovirus-expression and kinetic properties of the esterase, E3, involved in organophosphorus resistance in Lucilia cuprina. , 1997, Insect biochemistry and molecular biology.

[71]  A. Devonshire,et al.  Gene amplification and insecticide resistance. , 1991, Annual review of entomology.

[72]  A. Devonshire.  Insecticide resistance in Myzus persicae : From field to gene and back again , 1989 .

[73]  J. Mckenzie.  Dieldrin and diazinon resistance in populations of the Australian sheep blowfly, Lucilia cuprina, from sheep-grazing areas and rubbish tips. , 1984, Australian journal of biological sciences.

[74]  R. Metcalf.  Mode of action of insecticide synergists. , 1967, Annual review of entomology.