Pesticide Chemical Research in Toxicology: Lessons from Nature

Perspective pubs.acs.org/crt

Pesticide Chemical Research in Toxicology: Lessons from Nature John E. Casida*,† and Kathleen A. Durkin‡ †

Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley 94720, United States ‡ Molecular Graphics and Computational Facility, College of Chemistry, University of California, Berkeley 94720, United States ABSTRACT: Pesticide researchers are students of nature, and each new compound and mechanism turns a page in the ever-expanding encyclopedia of life. Pesticides are both probes to learn about life processes and tools for pest management to facilitate food production and enhance health. In contrast to some household and industrial chemicals, pesticides are assumed to be hazardous to health and the environment until proven otherwise. About a thousand current pesticides working by more than 100 different mechanisms have helped understand many processes and coupled events. Pesticide chemical research is a major source of toxicology information on new natural products, novel targets or modes of action, resistance mechanisms, xenobiotic metabolism, selective toxicity, safety evaluations, and recommendations for safe and effective pest management. Target binding site models help define the effect of substituent changes and predict modifications for enhanced potency and safety and circumvention of resistance. The contribution of pesticide chemical research in toxicology is illustrated here with two each of the newer or most important insecticides, herbicides, and fungicides. The insecticides are imidacloprid and chlorantraniliprole acting on the nicotinic acetylcholine receptor and the ryanodine receptor Ca2+ channel, respectively. The herbicides are glyphosate that inhibits aromatic amino acid biosynthesis and mesotrione that prevents plastoquinone and carotenoid formation. The fungicides are azoxystrobin inhibiting the Qo site of the cytochrome bc1 complex and prothioconazole inhibiting the 14α-demethylase in ergosterol biosynthesis. The two target sites involved for each type of pesticide account for 27−40% of worldwide sales for all insecticides, herbicides, and fungicides. In each case, selection for resistance involving a single amino acid change in the binding site or detoxifying enzyme circumvents the pesticide chemists’s structure optimization and guarantees survival of the pest and a continuing job for the design chemist. These lessons from nature are a continuing part of pest management and maintaining human and environmental health.

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CONTENTS

Introduction Pesticide Research Chemistry Toxicology Insecticides Beginnings Imidacloprid and nAChR (Figure 2) Chlorantraniliprole and RyR (Figure 3) Herbicides Beginnings Glyphosate and EPSPS (Figure 4) Mesotrione and HPPD (Figure 5) Fungicides Beginnings Azoxystrobin and Qo of bc1 (Figure 6) Prothioconazole and 14α-Demethylase (Figure 7) Lessons from Nature Author Information Corresponding Author Notes Biographies Acknowledgments © XXXX American Chemical Society

Abbreviations References

A B B B C C C D D D D E F F F

H H

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INTRODUCTION It is a pleasure to celebrate 30 years of Chemical Research in Toxicology with my 46th paper in this prestigious journal joined this time by Kathleen Durkin, my Berkeley colleague and frequent coauthor for the past 14 years. As a teenager, when DDT and 2,4-D first became available, I became fascinated with pesticides, particularly their mode of action and metabolism. The questions of what, why, and how have not changed, but the ability to research these areas has improved beyond belief but still with the knowledge that the answers are partial and that progress is led by curiosity and chance. Chemical research on the toxicology of six major pesticides and their target sites (Figure 1 and Table 1) is used here to illustrate the fascinating progress and challenges in learning and using the lessons of nature.

G G H H H H H

Special Issue: CRT30 Received: August 31, 2016

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DOI: 10.1021/acs.chemrestox.6b00303 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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A pesticide is applied to do a job and then disappear as if it was never there. Pesticide “ecokinetics” involves movement and degradation in crops, land, water, and air. It also involves stabilizers and other formulation ingredients that change the behavior. Fortunately, the factors and changes involved become more predictable with accumulating knowledge. Toxicology. Target identification involves a combination of chemistry, biochemistry, and physiology. The goal is to define the primary lesion, binding site, and interactions, and any secondary targets of concern using an increasing variety of analytical and omic technologies. Pesticides provide a way to dissect and manipulate life processes. A small change in chemical structure can make a big change in mode of action, target site, side effects, resistance, and metabolism. Current compounds work by about 100 different mechanisms and have helped us understand those processes and coupled events. Phylogenetic trees, available for each of the targets specifically considered in this perspective, establish the target site relationships between different types of organisms related to but not necessarily accounting for selective toxicity. A major goal of pesticide science is to avoid overusing and losing a sensitive target leading to selection of resistant strains. Cross-resistance between different types of chemicals implies a common target or detoxification mechanism. Another goal is to find a “new target” so the Insecticide, Herbicide or Fungicide Resistance Action Committee (IRAC, HRAC and FRAC) classification is unique and therefore not subject to use restrictions to minimize target site cross-resistance.5−7 Radioligand binding studies or equivalent target site assays allow characterization of common, modulator, or coupled sites. The likelihood of showing a common site or establishing a different target depends in part on the selection of species or assay conditions. Pesticide resistance and cross-resistance can also involve selection for enhanced detoxification, i.e., a mutation that gives increased metabolism in the resistant strain. This type of resistance can sometimes be overcome by changing molecular substituents, thereby altering the site of metabolic attack or adding a synergist that inhibits the detoxification. The importance of resistance and cross-resistance makes these target site and detoxification relationships and classifications more critical in pesticide research than other areas of chemical toxicology. More specifically in the case of pesticides, a minor and seemingly inconsequential change in one amino acid of a

Figure 1. Six major pesticides and their diverse targets.

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PESTICIDE RESEARCH Chemistry. Pesticides are currently discovered by design or by chance on testing or screening more than 500,000 new compounds each year on one or more biological systems including insects, weeds, fungi, organs, cells, enzymes, and receptors.2−4 Some are natural products, and others are libraries of synthetic compounds at random or of selected types. The response may be death of an organism or altered growth or behavior. This search of nature for chemical−biological interactions from such a vast pool of compounds is more extensive than that achievable in any other way. Multiple organisms are involved in total metabolic degradation (mineralization) of pesticides by pathways that are increasingly predictable but still fascinating and often surprising in their balance. A pesticide may also undergo extensive photodegradation usually with initial reactions similar to those enzymatically catalyzed but sometimes generating totally different products with greater persistence and a change in toxic mechanism. Some pesticides have an unusual and often unique set of substituents to achieve maximum effect. The metabolic studies required for registration are thorough and involve a variety of systems.

Table 1. Six Major Pesticide Targets, Example Compounds, Sensitivity Comparisons, and Worldwide Sales percent worldwide salesa

target site type and target

example compound

species

sensitive (S)

resistant (R)

example

others

total

T81 E4946

9.1 7.6

15.9b 2.6c

25.0 10.2

d f

19.8 3.1

2.0e 1.7g

21.8 4.8

A143 F136

9.4 4.1

18.0h 8.8i

27.4 12.9

Insecticides nAChR RyR

IMI CHL

EPSPS HPPD

GLY MES

Qo of bc1 14α-demethylase

AZO PRO

Myzus persicae Plutella xylostella

R81 G4946

Herbicides Amaranthus palmeri d Arabidopsis thalliana f Fungicides Pyricularia grisea G143 Erysiphe necator Y136

a

Agranova 2015.1 Percentage relative to the total of the pesticide type as 100%. bNitroimines clothianidin and thiamethoxam: cyanoimines acetamiprid and thiacloprid. cFlubendiamide. dSensitive weed compared to resistant soybean from expressed CP4 with A100. eGlyphosate trimesium. fPlant target compared to mammalian pharmaceutical target. gTriketone tembotrione and isoxazole isoxaflutole which is bioactivated to a diketonitrile. hMethoxyacrylate picoxystrobin, oximinoacetates kresoxim-methyl and trifloxystrobin, dihydrodioxazine fluoxastrobin, and methoxycarbamate pyraclostrobin. iTriazoles difenoconazole, epoxiconazole, propiconazole, and tebuconazole. B



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single target or detoxifying protein can have worldwide ecological, social, economic, and political implications if a resistance-conferring site is involved. Pesticides are intended to control pests with minimal effects on other environmental organisms by design of the chemicals or defining the manner and place of their application. They are thoroughly studied and carefully classified as to their potential as carcinogens, mutagens, teratogens, delayed neurotoxicants, and other adverse effects. Representative organisms including birds, fish, honeybees, and Daphnia are used as ecotoxicology indicators to predict more broadly. Pharmaceuticals depend on organ and system selective toxicity. Pesticides require species selectivity with particular emphasis for insecticides on pollinators, pets, and people and for herbicides on crops versus weeds. If the selectivity is not there for a nonspecific herbicide such as glyphosate, it can be introduced by genetic modification of crops to reduce target site sensitivity and enhance detoxification. In the case of the herbicide mesotrione, a slight structural change makes it a pharmaceutical with adequate safety for human use.

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INSECTICIDES Beginnings. Before 1940, pest insects were controlled, although not very effectively, with inorganics and botanicals. The next three decades led to dozens of major effective compounds with diverse targets, for example, DDT and pyrethroids at the voltage-dependent sodium channel, organophosphorus compounds and methylcarbamates at acetylcholinesterse (AChE), and polychlorocycloalkanes, avermectins, and spinosyns at the ϒ-aminobutyric acid and glutamate-receptor chloride channels.5,8 Selection of resistant strains reduced their effectiveness, and toxicological concerns led to a need for alternatives. Two different chemical classes acting on new targets considered here helped fill the gap. Imidacloprid and nAChR (Figure 2). Screening new chemical types revealed weak insecticidal activity for 2(dibromonitromethy)-3-methylpyridine which was modified to the very effective but photolabile nitromethylene nithiazine9 and further to the photostabilized nitroimine imidacloprid (IMI)10−13 introduced in 1991, which was for many years the top selling insecticide. IMI (Figure 2) has chloropyridinyl, imidazolyl, and nitroimine moieties. The chloropyridinyl substituent (similar to the pyridinyl of nicotine) can be replaced with chlorothiazolyl or tetrahydrofuranyl, and the nitroimine moiety can be changed to a nitromethylene, nitroimine, sulfoximine, or butenolide substituent with an electronegative tip. These compounds are collectively designated the neonicotinoids (neonics).13−18 They are outstanding systemic insecticides used as seed, soil, and foliage treatments with contact and stomach action for aphids, plant hoppers, and many other pests. Four neonics (IMI, dinotefuran, thiamethoxam, and nitenpyram) are major flea control agents for dogs and cats by topical application (spot ons) and ingestion (pills) with adequate selective toxicity and rapid action.19 IMI and its neonic analogues (particularly thiamethoxam and clothianidin) currently dominate the insecticide market with 25% of the worldwide value. The neonics are nicotinic acetylcholine (ACh) receptor (nAChR) agonists which, in contrast to nicotine of similar overall action, are more toxic to insects than mammals.20−22 Mammals but not insects have a cholinergic neuromuscular junction. Insects have a protective barrier to entry of ionized compounds such as nicotine into their central nervous

Figure 2. Imidacloprid (IMI) and insect nicotinic acetylcholine receptor (nAChR). (A) Structure and some sites of metabolic attack. (B) Agonist site for ACh, IMI, and neonics. (C) Effect of R81T resistance mutation in Myzus persicae (major aphid pest).

system.13,20 The selective toxicity of neonics is conferred largely by differences in nAChR binding site amino acids and conformations in insects and mammals.17,22 The neonics as a group are low in acute and chronic toxicity for mammals, birds, and environment organisms except bees.9,15,16 Although the cyanoimine neonics are of reduced toxicity to bees, the high toxicity of IMI and nitroimine and nitromethylene neonics has led to restrictions on their use. Neonics are readily metabolized23 and photodecomposed so that prolonged persistence is not a problem except in groundwater. IMI metabolism involves CYP 450 mediated hydroxylation of the imidazolidine ring, reduction (by aldehyde oxidase) and loss of the nitro group, and cleavage to chloronicotinic acid.23 The selection of resistant strains has curtailed the use of IMI and other neonics for several major pests attributed in some cases to resistance mutations in the binding site and in others to CYP 450 mutations conferring enhanced detoxification. IMI is much more potent on insect than mammalian nAChRs consistent with major differences in their binding site amino acids and conformations defined in part by ACh binding protein (AChBP) surrogates from snail hemolymph, one mimicking the insect and another the mammalian site.17,22 Aplysia californica AChBP has been a model for the ligand binding domain of insect nAChRs, whereas Lymnaea stagnalis AChBP has been the corresponding mammalian model, with high selectivity of IMI for Aplysia between the two. Crystal structures are available in both cases for AChBP with bound neonics.22,24 The insect nAChR specific binding site for IMI is illustrated here with Myzus persicae in which resistance is conferred by both enhanced detoxification and the R81T mutation shown with a homology model of the α2β1 dimer.25 The IMI pyridine N hydrogen bonds to a water molecule which itself is hydrogen bonded to backbone atoms in I143 of the (−)-face of the β1 subunit in Myzus. The imidazoline ring pi stacks with the W174 and Y224, and perhaps most importantly, the electronegative tip of the nitro group interacts with the Cysloop C226 and Y224 on the (+)-face.26 Additionally, there is a C



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key interaction on the (−)-face of the binding site between the negatively charged ligand tip of IMI and a positively charged R81, which is lost in the R81T mutant. Changing both the length and chemical nature of the side chain is observed to confer resistance. For neonics other than IMI, cation−pi or related interactions may exist with R81 but are lost in the mutant. A different IMI resistance mechanism is observed with the brown planthopper Nilaparvata lugens27 in which the Y151S mutation (numbering per ref 27) is not directly in the IMI binding site but instead is in loop B immediately adjacent to residues lining the agonist binding pocket. Chlorantraniliprole and RyR (Figure 3). A pyrazine dicarboxamide herbicide, observed to have weak insecticidal activity, was the lead followed independently in two companies leading to flubendiamide (FLU) reported in 1995 and chlorantraniliprole and cyantraniliprole (CHL and CYAN) announced in 2006 and shortly thereafter.28−30 CHL (Figure 3)

CHL, CYAN, and FLU activate insect RyRs leading to upregulated loss of internal Ca2+ stores and flaccid paralysis.35,36 CHL, FLU, and Ry act at independent but coupled sites in the insect Ca2+-activated Ca2+ channel,37,38 whereas specific binding of [3H]CHL was not observed in rabbit and lobster muscle preparations.38 Metabolism of CHL involves oxidation at each methyl group and cyclization to a quinazolinone derivative.39 The anthranilic diamides are readily photodecomposed by intramolecular rearrangements and molecular cleavage. CHL is quite persistent on crops and in soil with limited mobility.4 Fortunately, the diamides are of very low toxicity to honeybees, birds, fish, and most nontarget organisms.40 Resistance is a rapidly increasing problem with the diamides.41−43 The precise location of the diamide binding site in the insect RyR remains to be defined. However, a tentative model can be prepared based on three recent observations: determination of the structure of a mammalian RyR;44,45 defining the partial sequence of the Plutella xylostella RyR;46 and assignment of a resistance-associated G4649E mutation in the highly conserved C-terminal membranespanning domain of the Plutella RyR.47 The exploratory homology model shown here of the membrane spanning region of the RyR for P. xylostella (Genbank AFW97408) used PDB 3J8H48 as a template. This model is similar to that reported by Steinbach43 and confirms that the G4946E mutation associated with resistance to diamides is at the join position between the S4−S5 linker and the S4 transmembrane helix. This is at the boundary of the cytoplasm and reticulum membrane and may play a role in the dynamics of the channel pore. The I4790M mutation also associated with resistance in this species41,43 is 15−20 Å away in the S2 helix as shown here and by Steinbach43 or S3 according to Guo41 but is also at the membrane boundary and could have a related chemical impact. While this model is preliminary, there is evidence of potential binding pockets suitable for CHL insertion in this region with pi stacking, cation pi, and H-bonding networks possible at this hydrophilic−hydrophobic interface. The observed mutations associated with resistance could disrupt both the shape and dynamic character of this interface. Herbicides. Beginnings. Weed control was mostly mechanical until the 1930s and 1940s when auxins and other plant growth regulators (2,4-D and related chlorophenoxyaliphatic acids) and many photosynthesis inhibitors (ureas and triazines) and carotenoid biosynthesis inhibitors were introduced with little or no problems for many years from selection of resistant strains of weeds.6 Inhibitors of amino acid biosynthesis (sulfonylureas and imidazolinones) followed with excellent selectivity for plants versus mammals. There were also protein and lipid biosynthesis inhibitors (chloroacetanilides), antitubulins (trifluralin), and membrane disruptors (paraquat), protox inhibitors, and bleachers. Major changes came about with the discovery of glyphosate (GLY) and mesotrione (MES). Glyphosate and EPSPS (Figure 4). GLY is phosphonylmethylglycine used primarily as the isopropylamine or trimesium salt.49−52 The herbicidal activity was reported in 1971 with market introduction in 1974. GLY (Figure 4) controls both annual and perennial weeds with postemergence applications and remarkable systemic action. GLY currently accounts for 20% of the worldwide herbicide market value. The herbicidal action of GLY is due to inhibiting enolpyruvylshikimate phosphate (EPSP) synthase

Figure 3. Chlorantraniliprole (CHL) and insect ryanodine receptor (RyR). (A) Structure and major sites of metabolic attack. (B) CHL in a possible binding pocket of Plutella xylostella (major lepidoteran pest) indicating the position of the G and I resistance mutations to E and M, respectively. (C) Speculative zoomed out version of panel B showing the entire transmembrane region.

and CYAN are anthranilic diamides, while FLU is a phthalic diamide. They all have bromo or iodo substituents in the central aromatic or heterocyclic rings, and FLU has a perfluoroalkyl side chain. CHL and FLU have exceptional potency on lepidopterous larvae. CYAN with cyanophenyl replacing chlorophenyl is more polar and more systemic in plants with outstanding activity on aphids and other sucking insects. CHL is currently the major non-neonic insecticide with 7.6% of the insecticide worldwide market value. Progress on diamide insecticide action was greatly facilitated by early studies on ryanodine (Ry) chemistry. Ry is the ester alkaloid toxicant for insects and mammals in the botanical insecticide Ryania (no longer used). Attempts to simplify the structure and improve the selective toxicity of Ry and its derivatives were not successful by reconstructive chemistry of the natural material31,32 and synthesis of the very complex molecule (finally successful in 42 steps).33 [3H]Ry was the critical radioligand in characterizing the Ry receptor (RyR) relative to Ca2+ functions.34 Ryanodol, the hydrolysis product of Ry, is also a natural product insecticide possibly acting at a channel-related site.31 D



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issue,61 which could diminish or curtail GLY use with no replacement of similar properties. The EPSPS binding site positions the native substrates shikimate-3-phosphate (S3P) and phosphoenol pyruvate (PEP) in a complex electrostatic amino acid environment, wherein the ligands are embedded in a web of potential H-bonding, water bridging, and counterion stabilization interactions. More remote amino acids influence the shape and mobility of this site such that minor changes in the amino acid composition within and close to the active site impact binding and catalytic activity. GLY inhibition involves displacement of PEP in the active site. The homology model shown here for EPSPS of the sensitive weed Amaranthus palmeri with G175 (GenBank: ACV53022.1) is for GLY in a low energy extended active form in the PEP site, preventing subsequent catalytic action. The GLY-resistant CP4 from Agrobacterium with alanine as residue 100 (PDB code 2GGA) has been engineered into soybean as a Roundup Ready crop.55 In this, CP4 EPSPS bound with both S3P and GLY, the GLY (in the PEP site) only fits in a high energy conformational state due to crowding from A100 and is not active as an inhibitor. The A100G mutation restores GLY sensitivity in CP4. GLY resistance in Amaranthus has been reported, mostly due to gene amplification,62 although single mutations are observed with reduced catalytic activity.63 Mutations leading to GLY tolerance are often associated with decreased PEP affinity and catalytic activity. An engineered GLY tolerant maize includes a T102I/P106S double mutant (residue numbering from CP4, T97I/P101S in E. coli, aka TIPS).64 This double mutant is insensitive to GLY, yet retains high catalytic activity. This and other resistance mechanisms are available to organisms in the wild and in bioengineering. However, double mutations like TIPS are currently rare in the wild. Gene amplification is a more common resistance strategy.57,58 Mesotrione and HPPD (Figure 5). The discovery of MES is an outstanding example of natural product research leading to a major commercial pesticide.65,66 Brilliant red blossoms falling from the bottlebrush plant (Callistemon citrinus) were observed to produce unique bleaching symptoms on susceptible weeds in the surrounding ground. The possibility of a natural product herbicide led to isolation and identification of the triketone leptospermone as the herbicidal component. Structure optimization of benzoyl cyclohexanediones for potency and selectivity gave MES (Figure 5), introduced in 2001, which is a methylsulfonylnitrobenzoylcyclohexanedione triketone.65,66 The 2- and 4-substituents of the phenyl ring are electron-withdrawing groups, and the herbicidal activity correlates with the acidity of the benzoylcyclohexanedione. The isoxazole isoxaflutole of similar action is bioactivated to a diketonitrile.67 MES controls a large range of broad-leaf and grass weeds in maize. It is rapidly taken up by weeds and distributed by both acropetal and basipetal movement. Maize picks up MES more slowly and metabolizes it more rapidly than weeds. MES is the second most important herbicide with 3.1% of worldwide sales. MES blocks carotenoid biosynthesis in plants and bacteria by inhibiting 4-hydroxyphenylpyruvate dioxygenase (HPPD) in the tyrosine to plastoquinone and α-tocophenol pathway with action at extremely low levels.68−70 Metabolism of MES involves cleavage of the cyclohexanedione to the nitrobenzoic acid and reduction to the amino derivative.71 MES has very low acute and chronic mammalian toxicity to all species examined and little toxicity to honeybees and environmental nontarget

Figure 4. Glyphosate (GLY) and EPSP synthase. (A) Structure and major site of metabolic attack. (B) EPSPS reaction inhibited by the GLY transition state analog of PEP. (C) Comparison of Amaranthus palmeri (sensitive weed) with resistant soybean from expressed CP4 with A100.

(EPSPS).49−54 This blocks aromatic amino acid biosynthesis by a system important in plants but not mammals. A high structural specificity is involved with only N-hydroxy and Namino GLY approaching the potency of the parent compound.53 GLY is a chelator of copper, manganese, and zinc. The principal advantage and disadvantage of GLY is its lack of specificity controlling almost all weeds but damaging the crop as well as killing the weeds. This was successfully overcome by Monsanto scientists on introducing into the crops two new genetically engineered defense mechanisms of bacterial origin to decrease the target site sensitivity and speed up the detoxification. GLY-tolerant crops have an introduced epsps gene from Agrobacterium strain CP4 that gives a less sensitive EPSPS target and a modified glyphosate oxidase (GOX) gene to speed detoxification. These transgenic GLY-resistant crops became available in 1996. As applied to corn, soybeans, cotton, and many other crops, this is the most important of all applications of genetically modified organisms (GMOs) in crop production and the life sciences. More generally, GMOs provide the potential option of changing the organism or altering the chemical. The extensive use of GLY for many years has led to selection of resistant weeds55−58 as a major problem temporarily overcome by higher doses but with increased soil and water contamination and environmental impact. The overall amount of GLY used will probably decrease as the GMO-crops are no longer protected from the higher treatment levels required for resistant weeds.52,57,58 GLY metabolism primarily involves oxidative cleavage by GOX to aminomethylphosphonic and glyoxylic acids. GLY binds quickly and strongly in soil, so leaching is not a problem. It has a very favorable ecotoxicological profile.4 Mammalian toxicity was not considered to be a problem59 until a recent reevaluation classified it as a possible human carcinogen.60 This is a strongly debated E



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FUNGICIDES Beginnings. Sulfur was the first and is still a very major fungicide. Synthetic organic fungicides of nonspecific action started with the dimethyldithiocarbamates and ethylenebis (dithiocarbamates) as salts or disulfides in the 1940s controlling a great variety of plant diseases. These multivalent fungicides have remained effective for many years without selection of resistant strains, and mancozeb introduced in 1961 was still in 2015 the third most important fungicide in sales. Captan, a thiophosgene progenitor, also acted on multiple targets without selection for resistance. The first systemic fungicide was oxathiin followed by the benzimidazoles (benomyl, carbendazim, and thiophanate), the acylalanines, and many others.4,7 Current major classes are the strobilurins and triazoles considered here. Azoxystrobin and Qo of bc1 (Figure 6). The mushroom Strobilurus tenacellus has very potent antifungal agents designated as strobilurins reported in 197780 and structurally modified to give the strobilurin type of fungicides.81,82 Azoxystrobin (AZO) (Figure 6) introduced in 1996 is a

Figure 5. Mesotrione (MES) and 4-hydroxyphenylpyruvate dioxygenase (HPPD). (A) Structures of MES and isoxaflutole, and major sites of metabolic attack on MES. (B) HPPD reaction inhibited by MES. (C) Comparison of MES in Arabidopsis thaliana with nitisinone in mammals.

species.4,72,73 Dietary feeding of MES in rats inhibits HPPD and increases plasma tyrosine levels.72,73 In one of the few cases of a candidate pesticide used directly to treat a human disease, the herbicide and HPPD inhibitor with the SO2CH3 of MES replaced by CF3 and designated nitisinone as a pharmaceutical is successful in slowing the effects of hereditary tyrosinemia Type I.74 The HPPD triketone binding site has been defined by X-ray crystallography as involving iron bound to H308, H226, and E394 (numbering from Arabidopsis, PDB 5CTO) forming one side of the binding pocket.75 4-Hydroxyphenylpyruvate is the native substrate, and its diketone moiety is mimicked by the herbicide, serving to fill out 2 more positions of the Fe coordination sphere. The triketone of the herbicide is presumably active in an enol form, which allows the oxygens to be in plane and optimize orbital overlap in a delocalized bonding arrangement. The sixth Fe ligand is water, although this changes with oxidation state of the metal.76 There is pistacking with residues F381 and F424. Interactions with Q379, S267, and N282, among others, are believed to play a role in the reactivity with the native substrate.77 The triketone binding site in mammalian HPPD is essentially the same as that in plants (see figure): we note Arabidopsis K421 corresponds to mammalian A361.74 While this residue has not been implicated in catalysis, the plant lysine forms a stabilizing salt bridge (not shown in the figure) absent in the mammalian HPPD, impacting the dynamic nature of the active site region. Evolution of weed resistance thus far to the herbicide involves overexpression of HPPD, P450 mediated detoxification, and bypassing this pathway through semi synonymous alternate metabolic routes.78 Thus, far it does not include HPPD target mutations in the wild. However, the development of HPPD engineered crops, tolerant to HPPD inhibitor herbicides, is being investigated.79

Figure 6. Azoxystrobin (AZO) and the Qo site of cytochrome bc1 complex. (A) Structures of AZO and strobilurin A and some sites of metabolic attack on AZO. (B) Quinone/quinol (Qo) site of electron transfer and AZO inhibition. (C) Effect of G143A resistance mutation in Pyricularia grisea (rice blast fungus).

methyl cyanophenoxypyrimidinyloxyphenylmethoxyacrylate. The toxophore of AZO and the strobilurins is the methoxyacrylate substituent which is replaced by methoxyacetamide, methoxycarbamate, oximinoacetate, oximinoacetamide, oxazolidinedione, dihydrodioxazine, imidazolinone, or benzylcarbamate in other analogues. AZO is a very broad spectrum fungicide protecting against ascomycetes, deuteromycetes, basidomycetes, and oomycetes with protectant, curative, eradicant, translaminar, and systemic properties. It is widely used on cereals, vines, rice, citrus, potatoes, and tomatoes. The strobilurins collectively accounted for 27% of the 2015 fungicide worldwide sales. AZO inhibits mitochondrial respiration and energy production by blocking electron transfer at the quinone “outside” site of the cytochrome bc1 complex between cytochrome b and cytochrome c1 referred to as the ubiquinol oxidizing or Qo site F



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ultimately preventing the generation of ATP.80−82 AZO metabolism involves but is not limited to hydrolysis of the carbomethoxy group, cleavage of each aryl ether linkage, hydroxylation of each phenyl substituent,83 and glutathione conjugation on the cyanophenyl substituent. It has very low toxicity to mammals, birds, and bees but is toxic to algae, Daphnia, fish, and other aquatic organisms.4,84,85 Resistance develops quickly in many fungi and oomycetes and is due primarily to the G143A mutation, although F129L and G137R confer partial resistance.86 Homology models of cytochrome b of the rice blast fungus Pyricularia grisea (aka Magnaporthe grisea, Genbank Q85KP6) of both the G143A mutant and nonmutant G143 were built with A20 in the Qo site, using PDB 3CX5 (Saccharomyces) and 1SQB (Bos) as templates. It is proposed that G143A resistance is due to steric hindrance between the fungicide and cytochrome bc1 complex87 as shown here, but possibly the mutation also affects the flexibility of this dynamic reactive complex.88−94 Prothioconazole and 14α-Demethylase (Figure 7). Triazoles with suitable substituents provide highly effective, systemic, and broad spectrum control of major plant pathogens. They were introduced in 1973 with triadimeform, triadimenol, and propiconazole as early compounds followed by epoxiconazole, tebuconazole, and many others with increased potency to keep up with the selection for resistance. PRO (Figure 7) developed in 2002−2004 is the newest and currently

potency helps prolong its use as resistance appears. PRO represents 4.1% and four other triazoles account for an additional 8.8% of fungicide sales. The triazole fungicides for agriculture and medicine are ergosterol biosynthesis inhibitors; they block the CYP51 sterol 14α-demethylase acting on lanosterol, 24,25-dihydrolanosterol, and eburicol resulting in the depletion of ergosterol and concomitant increase of 14α-methylated sterols.100−104 They are sometimes referred to as demethylation inhibitors or DMIs. Humans have CYP51A1 involved in cholesterol synthesis and other CYPs in detoxification reactions so discrimination between the human and microbial enzymes is important. This has obviously been achieved with PRO and PRO-desthio based on all required mammalian toxicology, ecotoxicology, and environmental testing. Metabolism leads to the activated PROdesthio plus PRO-S-methyl and a large series of other detoxified metabolites. The 14α-demethylase like all other P450s positions the substrate in the active site with the FeO3+ entity as the oxidative power.103−105 The nucleophilic nitrogen of the azole fungicide heterocyclic ring directly coordinates as the sixth ligand of the heme ferric ion, and the azole side chain interacts with the CYP51 polypeptide structure.106,107 In the plant pathogen Erysiphe necator, which causes grapevine powdery mildew, Y136F is associated with DMI resistance (same as Y140F in Saccharomyces).108−111 We built a homology model of Erysiphe 14α-demethylase (Genbank AMM72596.1) using PDB 4UYL and 5EAD as templates, and observe that Y136 is embedded in a web of H-bonding interactions, possibly including PRO or PRO-desthio OH, a set of interactions that is lost in the Y136F mutant. The wild type tyrosine forms a water bridged hydrogen bonding interaction with PRO-desthio, fluconazole, and related DMI species.112 The mutant form lacks this stabilizing interaction. Given the position of the wild type tyrosine, it is likely critical for mediating proton transfer in the reaction of the native substrate. Thus, the resistance mutation results in decreased function and is reported to be coupled with increased gene expression to compensate.110 Cryptococcus neoformans is a serious human pathogen causing life threatening disease, mainly in immunocompromised individuals, and it is currently treated with triazole fungicides.113,114 Serotype A (var. grubeii), found in >90% of clinical isolates and >99% of isolates from AIDS patients, shows resistance to fluconazole and other triazoles.115 A likely cause of this resistance is the G484S point mutation in the lanosterol 14α-demethylase gene (AKJ77995.1). This mutation affects the shape and flexibility of the base of the binding pocket below the heme cofactor. A crystal structure is available for Saccharomyces cerevisiae CYP51 complexed with PRO-desthio.116

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LESSONS FROM NATURE Pesticide science has provided the chemical tools to produce food and fiber and protect the health of an expanding human population. This has been achieved by the collaboration of chemists and biologists and of industry and academia. The industrial groups continue to consolidate, so fewer and fewer companies will determine future directions. Pesticide potency has increased with the newer compounds and more knowledge of resistance mechanisms. Emerging technologies will reveal new approaches and targets and redirect the emphasis and judgments on regulations for enhanced safety and resistance management. Chemists will continue to design compounds to fit the targets, while the organisms under pesticide selection

Figure 7. Prothioconazole (PRO), PRO-desthio, and sterol 14αdemethylase. (A) Structures and some sites of metabolic attack. (B) Partial sterol pathway. (C) Effect of Y136F resistance mutation in Erysiphe necator (grapevine powdery mildew).

the most important modification of the triazoles.95−99 The 2hydroxypropyl moierty has chlorophenyl, chlorocyclopropyl, and dihydrothiazolethione substituents as a racemate (S is the more active enantiomer). It is a profungicide undergoing oxidative bioactivation to PRO-desthio. PRO is systemic, protective, curative, and eradicative and has long lasting activity against many diseases of wheat, barley, and other crops but does not stop spore germination. The exceptional fungicidal G



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pressure will evolve the targets so they no longer fit. Each new compound and mechanism turns a page in the ever-expanding encyclopedia of life. We are the students of nature, and the world is our classroom to keep clean and green.

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(5) Insecticide Resistance Action Committee (2016) IRAC Mode of Action Classification Scheme, CropLife International, Brussels, Belgium. (6) Herbicide Resistance Action Committee (2016) HRAC Classification of Herbicides According to Site of Action,. (7) Fungicide Resistance Action Committee (2016) FRAC Code List©: Fungicides Sorted by Mode of Action (Including FRAC Code Numbering), CropsLife International, Brussels, Belgium. (8) Casida, J. E., and Quistad, G. B. (1998) Golden age of insecticide research: past, present, or future. Annu. Rev. Entomol. 43, 1−16. (9) Soloway, S. B., Henry, A. C., Kollmeyer, W. D., Padgett, W. M., Powell, J. E., Roman, S. A., Tieman, C. H., Corey, R. A., and Horne, C. A. (1978). Nitromethylene Heterocycles as Insecticides, in Pesticide and Venom Neurotoxicity (Shankland, D. L., Hollingworth, R. M., and Smyth, J., Jr., Eds.) pp 153−158, Plenum, New York. (10) Moriya, K., Shibuya, K., Hattori, Y., Tsuboi, S., Shiokawa, K., and Kagabu, S. (1992) 1-(6-Chloronicotinyl)-2-nitroimino-imidazolidines and related compounds as potential new insecticides. Biosci., Biotechnol., Biochem. 56, 364−365. (11) Kagabu, S. (1997) Chloronicotinyl insecticides: discovery, application and future perspective. Rev. Toxicol. 1, 75−129. (12) Ishaaya, I., and Degheele, D., Eds. (1998) Insecticides with Novel Modes of Action: Mechanisms and Application, p 289, Springer-Verlag, Berlin. (13) Yamamoto, I., and Casida, J. E., Eds. (1999) Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor, p 300, SpringerVerlag, Tokyo, Japan. (14) Nauen, R., Ebbinghaus-Kintscher, U., Elbert, A., Jeschke, P., and Tietjen, K. (2001) Acetylcholine receptors as sites for developing neonicotinoid insecticides. Biochemical Sites of Insecticide Action and Resistance, 77−105. (15) Tomizawa, M., and Casida, J. E. (2003) Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annu. Rev. Entomol. 48, 339−364. (16) Tomizawa, M., and Casida, J. E. (2005) Neonicotinoid insecticide toxicology: mechanisms of selective action. Annu. Rev. Pharmacol. Toxicol. 45, 247−268. (17) Tomizawa, M., and Casida, J. E. (2009) Molecular recognition of neonicotinoid insecticides: The determinants of life or death. Acc. Chem. Res. 42, 260−269. (18) Jeschke, P., Nauen, R., Schindler, M., and Elbert, A. (2011) Overview of the status and global strategy for neonicotinoids. J. Agric. Food Chem. 59, 2897−2908. (19) (2015) Neonicotinoids for Veterinary Use on Dogs, Cats and Livestock against External Parasites, retrieved August 01, 2016 from http://parasitipedia.net/index.php?option=com_content. (20) Sattelle, D. B., Buckingham, S. D., Wafford, K. A., Sherby, S. M., Bakry, N. M., Eldefrawi, A. T., Eldefrawi, M. E., and May, T.E. (1989) Actions of insecticide 2-(nitromethylene)-tetrahydro-1,3-thiazine on insect and vertebrate nicotinic acetylcholine receptors. Proc. R. Soc. London, Ser. B 237, 501−14. (21) Shao, X., Xia, S., Durkin, K. A., and Casida, J. E. (2013) Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist. Proc. Natl. Acad. Sci. U. S. A. 110, 17273−17277. (22) Talley, T. T., Harel, M., Hibbs, R. E., Radic, Z., Tomizawa, M., Casida, J. E., and Taylor, P. (2008) Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore. Proc. Natl. Acad. Sci. U. S. A. 105, 7606−7611. (23) Casida, J. E. (2011) Neonicotinoid metabolism: compounds, substituents, pathways, enzymes, organisms, and relevance. J. Agric. Food Chem. 59, 2923−2931. (24) Ihara, M., Okajima, T., Yamashita, A., Oda, T., Asano, T., Matsui, M., et al. (2014) Studies on an acetylcholine binding protein identify a basic residue in loop G on the β1 strand as a new structural determinant of neonicotinoid actions. Mol. Pharmacol. 86, 736−746. (25) Bass, C., Puinean, A. M., Zimmer, C. T., Denholm, I., Field, L. M., Foster, S. P., et al. (2014) The evolution of insecticide resistance in the peach potato aphid. Insect Biochem. Mol. Biol. 51, 41−51.

AUTHOR INFORMATION

Corresponding Author

*Tel: +1-510-642-5424. E-mail: [email protected]. Notes

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. The authors declare no competing financial interest. Biographies Dr. John E. Casida obtained his Ph.D. degree in entomology, biochemistry, and plant physiology at the University of Wisconsin, Madison (1954). He was Professor of Entomology at Madison from 1954 to 1963 and Professor of Entomology and Toxicology at Berkeley starting in 1964. He is currently Professor of the Graduate School and holds the Edward A. Dickson Emeriti Professorship. His research emphasizes pesticide chemistry, molecular toxicology, and comparative biochemistry. He is a member of the U.S. National Academy of Sciences, the U.K. Royal Society, and the European Academy of Sciences and a recipient of the Wolf Prize in Agriculture. Dr. Kathleen A. Durkin obtained her Ph.D. degree in Chemistry at Emory University (1992) in Atlanta, specializing in molecular modeling as applied to Organic and Bioorganic systems. She is Director of the Molecular Graphics and Computational Facility of the College of Chemistry, University of California, Berkeley.

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ACKNOWLEDGMENTS We give special thanks to Rencong (Dian) Jiang (BS 2017 Department of Nutritional Sciences-Physiology and Metabolism at the University of California, Berkeley) who assisted with devotion and distinction in searching, compiling, and presenting the information in this Perspective.

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ABBREVIATIONS ACh, acetylcholine; AChBP, ACh binding protein; AChR, ACh receptor; AZO, azoxystrobin; CHL, chlorantraniliprole; CYAN, cyantraniliprole; CYP51, sterol 14α-demethylase; DMI, demethylation inhibitor; EPSP, enolpyruvylshikimate phosphate; EPSPS and epsps, EPSP synthase; FLU, flubendiamide; FRAC, fungicide resistance action committee; GLY, glyphosate; GMO, genetically modified organism; GOX, glyphosate oxidase; HRAC, herbicide resistance action committee; IMI, imidacloprid; IRAC, insecticide resistance action committee; MES, mesotrione; nAChR, nicotinic AChR; neonic, neonicotinoid; PEP, phosphoenol pyruvate; PRO, prothioconazole; Qo, ubiquinol oxidizing site; RyR, ryanodine receptor; S3P, shikimate-3-phosphate; TIPS, T97I/P101S

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