Radioligand Recognition of Insecticide Targets - American Chemical

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Radioligand Recognition of Insecticide Targets John E. Casida J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05984 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Radioligand Recognition of Insecticide Targets John E. Casida* * Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720, United States ABSTRACT: Insecticide radioligands allow the direct recognition and analysis of the targets and mechanisms of toxic action critical to effective and safe pest control. These radioligands are either the insecticides themselves or analogs that bind at the same or coupled sites. Preferred radioligands and their targets, often in both insects and mammals, are trioxabicyclooctanes for the GABA receptor, avermectin for the glutamate receptor, imidacloprid for the nicotinic receptor, ryanodine and chlorantraniliprole for the ryanodine receptor, and rotenone or pyridaben for NADH+ ubiquinone oxidoreductase. Pyrethroids and other Na+ channel modulator insecticides are generally poor radioligands due to lipophilicity and high nonspecific binding. For target site validation the structure-activity relationships competing with the radioligand in the binding assays should be the same as that for insecticidal activity or toxicity except for rapidly detoxified or proinsecticide analogs. Once the radioligand assay is validated for relevance it will often help define target site modifications on selection of resistant pest strains, selectivity between insects and mammals, and interaction with antidotes and other chemicals at modulator sites. Binding assays also serve for receptor isolation and photoaffinity labeling to characterize the interactions involved. KEYWORDS: GABA, glutamate, nicotinic, and ryanodine receptors

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CONTENTS INTRODUCTION Figs. 1 and 2 RADIOLIGANDS Table 1 GABA-R Fig. 3; Table 2 Receptors Radioligands Targets Glu-R Table 3 Receptors Radioligands Targets nAChR Fig. 4; Table 4 Receptors Radioligands Targets + Na CHANNEL Table 5 Channel Radioligands Targets RyR Table 6 Receptors Radioligands Targets VARIOUS TARGETS Tables 6 and 7 NADH: Ubiquinone Oxidoreductase Other Targets Modulators and Interacting Compounds CONCLUDING COMMENTS POSTSCRIPT AUTHOR INFORMATION ACKNOWLEDGEMENTS ABBREVIATIONS USED REFERENCES


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INTRODUCTION Insecticide mode of action research involves defining the receptor or enzyme target as a critical step in developing and insuring their safe and effective use.1, 2 Radioligand binding studies are often an essential step in recognizing the target which can be isolated, identified and verified by site-specific mutation (Figure 1)3-6 The six critical steps for insecticide radioligand binding studies are shown in Figure 2. The toxic mechanism can also be defined by physiological and proteomic investigations or by using a resistant pest strain for genomic studies to define the target site modification.

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RADIOLIGANDS The radioligand must allow the binding site to be analyzed at pM to nM levels which is the case for the specific activities of 3H, 18F, 32P, 35S, and 125I by liquid scintillation counting whereas 14 C specific activity may require the greatly enhanced sensitivity from analysis by accelerator mass spectrometry (Table 1). The examples that follow are almost entirely 3H-labeled compounds with the label introduced at or near a final step in synthesis as a tritiated methyl group or by adding tritium across an unsaturated substituent such as those in natural products including dehydroryanodine. The positions of labeling are indicated by asterisks in most cases.

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This review considers most but not all of the insecticide radioligands. They are grouped by their target sites or primary mode of action as classified by the Insecticide Resistance Action Committee (IRAC).7 Acetylcholinesterase (AChE) and other serine hydrolases inhibited by organophosphorus (OP) compounds and methylcarbamates (MCs) are not included here because the studies normally involve enzymatic assays instead of radioligand binding.8, 9 The radioligands and related compounds included are indicated in the text by number and in the tables by number, name and/or abbreviation, chemical type and IRAC resistance classification. Little or no attempt is made here to relate radioligand selectivity to receptor subunit or isoform composition either across or within a species.

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GABA-R Receptors. Chloride channels are modulated by γ-aminobutyric acid (GABA) as the opener and glutamate (Glu) as the closer both in a concentration-dependent manner. GABA is the major inhibitory neurotransmitter in both mammals and insects, acting to open the pentameric transmembrane chloride channel within the GABA receptor (GABA-R) which is a Cys loop ligand-gated ion channel. The vertebrate receptor has various combinations of β and γ subunits, typically α2β2γ. Insect GABA-Rs are expressed as three homopentamers, Rdl or LCCH1, LCCH2 and LCCH3.10, 11 Alternative splicing and RNA editing of insect GABA-Rs yield numerous receptor subtypes from a single gene. The Rdl-containing GABA-Rs are the site of insecticide action.11 Radioligands (Table 2). The classical toxicant acting at the GABA-R of both mammals and insects is picrotoxinin (PTX) (1A) which blocks the chloride channel. The first GABA-R radioligand was [3H]dihydropicrotoxinin (1B) with specific binding in rat brain membranes 3 ACS Paragon Plus Environment


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inhibited by both PTX and dieldrin suggesting that this was the target of polychlorocycloalkane (PCCA) insecticide action.12, 13 A completely different toxicant chemotype, the trioxabicyclooctanes (TBOs), was noted to give poisoning signs in mammals very similar to those of PTX14 leading to the preparation of TBPS (2)15 as a 35S-labeled candidate radioligand which proved to be a potent GABA antagonist much more effective than PTX.16 The TBPS site in mammalian brain was also sensitive to benzodiazepines and other GABA-R agonists and antagonists. Importantly, the [35S]TBPS binding site proved to be predictive in inhibitor SAR studies of the toxicity not only of TBOs but also of the PCCA insecticides from studies both in vitro and in vivo in mice and rats.17, 18 In addition, [35S]TBPS was found effective for insect GABA-Rs although it had a lower affinity.19 This radioligand was improved with [3H]TBOB (3) which has a much longer half-life.20 Preparation of [3H]EBOB (4A) as a far more potent insecticide analog21 then allowed studies with house flies establishing by SAR (in vitro receptor inhibition versus organismal toxicity of synergized analogs) that this was an outstanding radioligand for the GABA-R toxicant site of both insects and mammals.22, 23 The propenyl analog of 4B was the critical intermediate to obtain [3H]EBOB.21 Activity was retained with a dithiane substituent replacing the TBO24, 25 and a related dithiane sulfone (5A) with a 18F label is a candidate imaging agent for PET.26 Other dithianes have been studied as GABA-R affinity probes (5B) with [3H]photoactivatable, fluorescent, biotin, agarose and protein substituents.27 The proposal that PCCA toxicants act at the GABA-R prompted the preparation of two candidate PCCA radioligands. [3H]BIDN (6) reproduces most of the binding properties of [3H]EBOB28, 29 verifying the hypothesis for PCCA toxicity. Even more direct, the major PCCA endosulfan was prepared as a radioligand ([3H]α-endosulfan) (7) and shown to give the expected binding properties after minimizing non-specific binding by albumin washes.30 The highly toxic rodenticide TETS (8) is proposed to be a GABA-R antagonist31 but [3H]TETS could not be prepared as a stable radioligand for binding assays. As a less favorable alternative [14C]TETS was synthesized and the appropriate sensitivity for binding assays achieved by accelerator mass spectrometry showing that it also acted at the GABA-R but at a different site.32 The current insecticidally most important chloride channel blocker is the phenylpyrazole fipronil (9A)33, 34 prepared as a potent 3H-labeled TFMD-fipronil analog (9B) photoaffinity probe.35,36 Two new insecticide chemotypes, the isoxazolines (10) and meta-diamides (11), have many of the toxicological properties of GABA-R antagonists and as such were prepared with tritium ([3H]fluralaner (10)37-40 and [3H]BPB 1 (11A)41) and also found to act at the GABA-R after Ndemethylation for bioactivation of brofanilide (11B).40-43 Targets. Binding assays with [3H]EBOB greatly facilitated the understanding of GABAergic insecticide toxicity.44 The [3H]EBOB assay results correlated with inhibition of chloride [35Cl-] flux in mouse brain membranes providing physiological basis for the approach.45, 46 Autoradiography studies then established the binding site localization in the cerebellum of mouse brain.47 Action in the cerebellum was verified by accumulation of cyclic GMP associated with inhibition of [3H]TBOB binding in rats poisoned with PTX and lindane.48 Using expressed GABA-R subunits from rat brain it was found that the β3 subunit was the principal insecticide4 ACS Paragon Plus Environment

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sensitive site and most interestingly that the β3 homopentamer duplicated the sensitivity and SAR of house fly brain in inhibition and poisoning.49, 50 Species specificity studies showed very similar sites for [3H]EBOB (4A) in mammalian and insect brain. In contrast, TMS-EBOB (4B) has high potency in insect but not mammalian brain suggesting a modified binding site or facile bioactivation in insects51. The use of four radioligands led to the proposal of four distinct binding sites in the GABA-R for non-competitive antagonists (NCAs) [3H]EBOB and [35S]TBPS (NCA IA), [14C]TETS (NCA IB), and [3H]fluralaner and [3H]BPB 1 (NCA II) and modulators [3H]AVE (avermectin site) with distinct physiological effects (Figure 3).52 They also confirmed that lindane, dieldrin, and toxaphene A2 (the most toxic component of toxaphene) act as GABAA receptor antagonists.53 Several botanical flavors or toxicants also act at the GABA-R, e.g. αthujone54, 55 and 3-pinanones.56 The complexities of GABA-R modulating drug action are considered in a proposed model for the binding sites of convulsants.57 Glu-R Receptors. The relative importance of the GABA-R and the Glu receptor (Glu-R) in modulating the chloride channel varies between nematodes, insects, and mammals allowing considerable selective toxicity.58 The principal agents of interest are the macrocyclic lactones (Table 3).59,60 The discovery and use of AVE to control river blindness (onchocerciasis) in Africa by controlling the filarial worm Onchocerca volvulus was the basis for the 2015 Nobel Prize in Physiology and Medicine awarded to William C. Campbell and Satoshi Omura.61 There is no target site cross-resistance in Drosophila between the cyclodienes and AVE,62 i.e. they act at different sites (IRAC 2A for the cyclodienes and IRAC 6 for the macrocyclic lactones).7 The sites are closely coupled in insects but are distinctly different in physiological profiles in mammalian brain.63 Cyclodiene resistance in Drosophila is due to the Rdl A301S or A301G mutation whereas AVE resistance is attributed to a P299S site mutation.64-66 AVEs affect invertebrates either by directly activating the Glu-R and GABA-R or by potentiating their actions, resulting in an influx of chloride ions into nerve cells and muscle. In nematodes the AVEs potentiate or directly open the Glu-gated chloride channel67,68 whereas in insects the GABA-R may be more important. Several macrocyclic lactones including AVE B1a (12A) increase ion conductance by disrupting inhibitory or excitatory action on binding to vertebrate and invertebrate GABA-gated chloride channels and invertebrate Glu-gated chloride channels. This means that GABA-R and Glu-R sometimes have similar properties in inhibitor binding studies although distinct sites are involved. Radioligands (Table 3). [3H]AVE (12A) was originally shown to undergo relevant specific binding to membranes of the nematode Caenorhabditis elegans.69 The same radioligand undergoes high affinity specific binding in insect nerve and with house fly brain the SAR for AVE analog inhibition of [3H]AVE B1a binding correlates with the synergized toxicity.63 Interestingly, the same AVE analog SAR for [3H]AVE binding also holds for [3H]EBOB binding. Thus, [3H]AVE (a channel opener) binds to a house fly chloride channel site coupled to the site for [3H]EBOB (a channel blocker).63 Ivermectin (IVM) (12B) noncompetitively inhibits 5 ACS Paragon Plus Environment



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EBOB binding in the insect GABA-gated chloride channel.63 Rat brain membranes bind [3H]IVM but with low affinity.70, 71 High potency photoaffinity probes for mouse, house fly, and fruit fly brain are obtained on replacing the AVE B1a dioleandrosyl substituent with suitable esters and ethers.72 AVE B1a is just one of the macrocyclic lactones used in defining the chloride channel binding sites. Others are emamectin (12B), moxidectin (13), and selamectin (14).73-76 Several of the macrocyclic lactones studied with both [3H]- and [35S]radioligands act as Pglycoprotein modulators (12C, 13, and 14).73 Studies with [3H]12C and [3H]14 showed that they are very effective substrates for ATP-binding cassette transporter P-glycoprotein.73 Targets. AVE B1a (12A) binds to two different sites in the GABA-gated chloride channel of cultured cerebellum granule neurons with dual effects, i.e. activating the channel on binding to the high affinity site and blocking it on further binding to the low affinity site.77,78 Nodulisporic acid (NA) (15A), a potent insecticide from an endophytic fungus79 studied as 3H and 35S derivatives (15B and 15C), 65 appears to act at the same site as IVE on GluClα channels from NA-resistant Drosophila.64 Drosophila have a distinct class of NA and IVE receptors that contain both GluClα and GABA-Rdl subunits based on binding studies with NA and IVM radioligands.66

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nAChR Receptors. The nicotinic acetylcholine (ACh) receptor (nAChR) is the target for a wide variety of toxicants in insects and mammals. The discovery of highly effective nicotinic agonists as insecticides started a renaissance in studies on nAChR structure and function.80-87 As with the GABA-R, the nAChR is a Cys loop ligand-gated ion channel. It is the principal excitatory receptor of the insect central nervous system. The majority of ACh-triggered nAChR activation results in cationic current conducted inward by Na+ and Ca2+ (depolarizing) with the cation selectivity depending on the subunit composition of the pentameric ACh channel. Insect receptors consist of α and β subunits examined as brain membrane preparations or recombinant hybrids of insect α and vertebrate β subunits. Mammalian preparations are normally brain membranes or α4β2 recombinant nAChRs. The insect and mammalian nAChRs have a very important difference in their agonist binding sites determining toxicity.81, 88-91 Nicotine (16) and epibatidine (17A) referred to as nicotinoids are ionized at physiological pH and more potent on mammalian than insect nAChRs and more toxic to mammals than to insects.90-93 In contrast, the neonicotinoids (nics) are not ionized and are more potent on insects and are very important insecticides. Radioligands (Table 4). The initial nicotinoid radioligand was [3H]nicotine (16)87, 89 with the same binding site SAR as desnitro-imidacloprid (DN-IMI) (18), an imidacloprid (IMI) metabolite.91 The highly poisonous dart frog toxicant epibatidine (17A)92 as a radioligand93 and azidoepibatidine as a photoaffinity ligand (17B) (considered later) have greatly improved the understanding of the nAChR nicotinoid binding site.92, 93 The impetus to prepare nAChR radioligands greatly increased with the discovery of the insecticidal activity of nithiazine (19) and other nitromethylenes (20-22) and particularly IMI (23) and the realization that their outstanding effectiveness and selective toxicity indicated a similar yet remarkably different 6 ACS Paragon Plus Environment

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activity from that of nicotine.94-97 Soon IMI became the number one insecticide in sales. Nitromethylene-IMI (20) and IMI (23) were prepared and assayed as candidate radioligands.97-99 Their binding site(s) in house fly brain was inhibited by carbachol and (with AChE inhibition) by ACh and other choline esters and it had the same neonic SAR as the insecticidal activity [with a cytochrome P450 (CYP) inhibitor to minimize detoxification].98,99 Other neonic radioligands followed with studies on clothianidin (24) as a potential metabolite of [3H]thiamethoxam (25),100 [3H]dinotefuran (26),101-103 and [3H]acetamiprid (27)104 in house fly head or aphid membranes. These nAChR radioligands are only a portion of those used in binding studies and photoaffinity labeling. Targets. Radioligand binding assays were major contributors in defining the selectivity mechanisms of neonics and nicotinoids and ultimately the binding site amino acids and configuration making this critical difference (Figure 4).81, 104 The essential feature for insect nAChR binding appears to be the negatively-charged tip or region of the nitro or cyano or equivalent group. In contrast the mammalian nAChR sensitivity to nicotinoids, epibatidine and DN-IMI is due to the positive charge of their ammonium nitrogen. Binding site models are based on or supported by photoaffinity labeling with azidonics (e.g. 17B)104-106 or azidoneonics107-111 and sequencing. They are confirmed by crystal structure determinations of ACh binding proteins (AChBPs) with various bound neonics (Figure 4).112, 113 The specificity of neonics for the insect versus mammalian nAChR is generally retained by all the compounds illustrated here. The nitroand cyanoimine (23-27) neonics appear to act at the same or similar sites (with the possible exception of compounds 24-26 in some insect species) but the trifluoroacetamide (28)114, the sulfoximines (29A, 29B),115-117 and butenolides (30),118, 119 bind differently and fall in different IRAC classes. The mesoionic triflumezopyrim (31) acts at an orthosteric nAChR site.120 Binding site assays reveal neonic nAChR target site mutations for several insect pests121,122 but CYPs also play a role in neonic resistance123-125 and possibly aldehyde oxidase reduction of the nitro substituent as well.126 Three natural product insecticides act at different sites than the neonics on insect nAChRs. The spinosads fall in a different IRAC class127-130 and [3H]5,6-dihydrospinosyn A (31) was partially successful as a Dα6-nAChR radioligand.127 Methyllycaconitine (32) and its [3H]radioligand bind to an α7-type neuronal nAChR as antagonists in mammalian brain and insect preparations.131-134 Cartap via its naturally-occurring cleavage product nereistoxin (33) is a nAChR channel blocker.135 Na+ CHANNEL Channel. The voltage-gated Na+ channel (VGSC) is the primary target for DDT (35) and the pyrethroids (36A-41B), two of the most important insecticide classes of the last 75 years.136-138 The impact of DDT was recognized by awarding the 1948 Nobel Prize in Physiology and Medicine to Paul Müller.139 Pyrethrum flowers have been used for centuries to control insect pests.140, 141 It was very surprising and important to discover that resistance to DDT conferred cross-resistance to pyrethrum.10, 142 Knockdown resistance (kdr) is attributed to kdr-like mutations at two sites (PγR1 and PγR2) associated with para homologous Na+ channel genes. 7 ACS Paragon Plus Environment



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Selective toxicity is attributed in part to target site sensitivity. The insect para channel is far more sensitive to pyrethroids than rat brain type IIA Na+ channels.143 Radioligands. (Table 5). [14C]DDT (35) and [14C]cis-permethrin (38A) studies with house fly head membranes revealed binding with many of the characteristics expected of the relevant VGSC target.144 The dibromo analog (38B) of [14C]1R,cis-permethrin (38A) undergoes a minor degree of stereospecific binding in mouse brain membranes.145 Azido-RU58487 (39) as a photosensitive radioligand bound specifically to a receptor site on the α subunit of rat brain membrane Na+ channels.146 Two 3H-labeled candidate pyrethroid radioligands [the most potent stereoisomers of cyhalothrin (40A) and 4’-fluorocyhalothrin (40B)]147 did not yield useful levels of specific binding in house fly head or mouse brain membranes. The insecticide fenvalerate (41A) served as a model for a similarly potent photoreactive analog decyanoazidofenvalerate (41B)148 used as a radioligand showing its photoreaction with a 36 kDa guanosine 5’triphosphate binding protein of rat brain membranes and the possibility that pyrethroid toxicity involves modification of G-proteins.149 Some Type II pyrethroids with the α-cyano group inhibit, albeit at micromolar not nanomolar levels, the binding of [35S]TBPS in mammalian brain and insect nerve membranes whereas the Type I pyrethroids without the α-cyano group do not.150-152 Although the VGSC is the primary target of pyrethroid action there may be secondary targets at higher insecticide levels.153, 154 Targets. Pyrethroid binding-site models are based on mutations conferring resistance rather than on direct binding interactions of pyrethroids at the target. The VGSC has 6 insecticide domains identified with specific radioligands in addition to the DDT-pyrethroid site which results in prolonged inactivation.151-154 Pyrethroids, batrachotoxin, and N-alkylamide BTG 502 (42) show overlapping binding sites on insect Na+ channels.154,155 The potent insecticide veratridine (43)156, 157 binds to intramembrane receptor site 2 and to a proteoglycolipid from rat gastrocnemius tissue possibly similar to a macromolecular complex in the axonal Na+-action potential ionophore.158 The bioactivated oxadiazine metabolite (44B) of indoxacarb (44A) binds selectively to and inhibits slow-inactivated (non-conducting) Na+ channel states.159, 160 Metaflumizone (45) acts at the indoxacarb site.160 There is no cross-resistance between the pyrethroid site and these other binding domains. SAR studies are available for each of these series but radioligands have not been successfully applied in optimizations.

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RyR Receptors. The ryanodine (46A) (Ry) receptor (RyR) is a Ca2+- activated Ca2+ release channel essential for muscle excitation-contraction coupling.161-163 Modulation of muscle contraction involves voltage-gated channels which regulate external Ca2+ entry and RyRs which regulate release of internal Ca2+ stores. The receptor can also be recognized and assayed by electrophysiological and Ca2+ release assays.161-164 The channel’s conductance state and muscle contractions are altered by the botanical insecticide ryania with 46A and dehydroryanodine (46B) as the active ingredients.165-170 Attempts to improve on the insecticidal activity of ryanodine by structural modifications were only marginally successful in part because of the complex structure.171, 172 8 ACS Paragon Plus Environment

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Radioligands. (Table 6). Properties of the RyR were compared for mammals170 and insects173 with [3H]46A providing a binding assay for in vitro studies. The insect and mammalian RyRs are similar in their Ca2+ requirement and enhanced binding with ATP.169-174 Mode of action research on two new chemotypes of diamide insecticides revealed that they were RyR modulators with very high potency on insects but very low on mammals.165, 175 The first compound commercialized was phthalic diamide flubendiamide (Flu) (47) and soon thereafter, anthranilic diamides chlorantraniliprole (Chlo) (48A) and cyantraniliprole (48B) with DP-O1O (49) as a model.175-181 Targets. Three radioligands were compared for species differences in binding of [3H]46A, [3H]47, and [3H]48A noting major differences with muscles of lobster, rabbit and four insect species.182, 183 Clearly this is an excellent target for insect/mammal specificity but the basis for this favorable balance remains to be defined. Rapid selection for 48A resistance in the moth Plutella xylostella has been associated with identified microRNAs and specific amino acid polymorphisms.184-186 VARIOUS TARGETS NADH: Ubiquinone Oxidoreductase (Table 6). Several natural and synthetic insecticides (50-53) inhibit mitochondrial respiration at Complex I or NADH: ubiquinone oxidoreductase (NADH oxidase).187 The natural products are rotenone (50A) and piericidin A (51) and the synthetics include pyridaben (52A) and fenazaquin (53A). In perhaps the first radioligand binding study with an insecticide, [14C]50A was shown to undergo specific and saturable binding in electron transport particles with an SAR consistent with its respiratory and toxic effects.188-190 The assay was improved with [3H]dihydrorotenone (50B)191 in studies of Parkinson’s disease and the binding site localized in the substantia nigra.192 The binding site within Complex I shared by [14C]50A, [14C]51, [3H]52A and [3H](52B),193 [3H]53A, and [3H]53B was further focused in a photoaffinity study with [3H]53B showing its localization at the interface of the 40 kDa and the PSST subunits of Complex I coupled to the quinone site.194 This allowed the binding site to be further defined relative to that for 1-methyl-4-phenylpyridinium and paraquat as known and suspected inducers of Parkinson’s disease.195-197 18F-Labeled Complex I inhibitors based on 50A, 52A, and 53A have been developed as radioligands for myocardial perfusion imaging with PET.198 Other Targets. (Table 6). Many ATPase inhibitors are potent insecticides and acaricides. Diafenthiuron (54A) is particularly effective with action involving bioactivation via the sulfoxide which undergoes spontaneous conversion to the reactive and toxic carbodiimide (54B) as established with the 3H-radioligand.199-201 Cantharidin (55A) is a cytotoxic blistering agent and purported aphrodisiac in humans and feeding deterrent in insects.202, 203 Isolation of the cantharidin-binding protein monitored with [3H]cantharidic acid (55B)203 allowed its identification as protein phosphatase 2A.204 Radioligand binding approaches have also been used in mode of action studies on formamidine insecticides,205-208 chitin synthesis inhibitors,209 insect



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attractants, repellants, and pheromones and on the molecular targets of insect growth regulators which are beyond the scope of this review. Modulators and Interacting Compounds (Table 7). Coupled sites recognized by activation or inhibition of insecticide radioligand binding allow a rapid screen for in vitro interactions of possible relevance in vivo expressed as increased or decreased toxic effects. For example, barbiturates, benzodiazepines and neurosteroids selected for optimal in vitro potency may serve as antidotes for insecticides acting as GABA-A receptor chloride channel blockers (Figure 3).


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CONCLUDING COMMENTS An insecticide target site study has defined steps according to current knowledge but is never complete with the expanding variety of techniques, species, and strains. An important step is the radioligand assay yielding unique data sets not attainable by other methods. Many of the radioligands mentioned are not new and were not ever available on a commercial basis and must therefore be reprepared for use again. A recent approach uses photoredox-mediated hydrogenatom transfer to replace the hydrogens at C-H bonds adjacent to amines with tritium from T2O.210 Advances in fluorescence probes and their detection has started a movement towards fluorescent ligands rather than radioligands.27, 211-213 Radioligand design and synthesis has largely moved from academic to contract industrial laboratories. Radioligands have a role (for now) in studying selective toxicity, e.g. insects vs. mammals, brain vs. muscle. This approach works regardless of species and tissue if there is a specific binding site. Radioligands allow binding site assays when the mode of action (enzyme or receptor assay) is unknown. Much of the literature on insect toxicology involves cross-resistance mechanisms to each group of new chemicals as they are introduced. One approach is to establish the SAR for inhibition of a relevant radioligand compared with the cross-resistance spectrum, i.e. binding assays versus toxicity determinations. This can be done even without knowing the resistance mechanisms(s). The current alternative and more definitive approach is to select or obtain the resistant (R) and susceptible (S) strains and define the mutation(s) involved then to express the mutations to confirm that the same SAR is obtained on toxicity testing. The methods are combined by binding assays on the R and S strains. POSTSCRIPT This review gives the author the opportunity to place in perspective his personal involvement in radioligand research. His laboratory routinely made and used 32P-labeled insecticides in metabolism studies starting in the mid 1950’s, supported by the U.S. Atomic Energy Commission and the U.S. National Institutes of Health. Studies emphasizing insect toxicology led to [14C]rotenone and [14C]pyrethrin I. The laboratory moved in 1964 from Madison to Berkeley leading to radioligand binding studies on [14C]rotenone, [35S]TBPS, and [3H]dihydroryanodine. Our finding that TBOs were more effective radioligands than TBPS led to the preparation of [3H]TBOB in the National Tritium Labeling Facility of the Lawrence Berkeley National Laboratory of the University of California, followed soon thereafter by [3H]EBOB. For the next 17 years we labeled almost every new type of insecticide for metabolism and binding studies. Detoxification of esters by esterases was examined by two-phase scintillation counting in which the unhydrolyzed ester extracted into the organic phase containing the scintillation fluor was counted whereas the hydrolysis products in the aqueous phase were not. This was applied to carboxyl-radiolabeled endogenous esters in the cannabinoid system and neuropathy target esterase as well as labeled pesticides such as pyrethroid esters. The Berkeley lab was “downsized” in 2015 and the tritium-labeled compounds deaccessioned, regretfully discarding more than 1000 mCi of our tritiated candidate radioligands. Some of the compounds discussed are 11 ACS Paragon Plus Environment


J. Agric. Food Chem. 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422

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commercially available—[3H]BIDN, [3H]EBOB, [3H]TBOB, [35S]TBPS, [3H]ivermectin, [3H]IMI, and [3H]ryanodine. Newer compounds are now usually prepared in contract radiosynthesis laboratories. This takes away some of the flexibility and excitement of following through on a natural product or toxicant study by preparing the labeled compound in a few weeks in the “tritium lab” with the help and guidance of Hiromi Morimoto and Philip G. Williams so there was no delay or waning of the enthusiasm moving forward. This bye-gone era has shifted mostly now from academic curiosity to contract laboratories dedicated to pesticide and pharmaceutical development. Nevertheless, radioligands continue to play a major role in bioactives mode of action and development research. AUTHOR INFORMATION Corresponding Author Phone: (510) 642-5424. E-mail: [email protected]. ORCID* John E. Casida: 0000-0002-3181-7561 Notes The author declares no competing financial interest. ACKNOWLEDGMENTS I give special thanks to Minhchau (MC) Le Nguyen (B.S. 2017, Department of Nutritional Sciences and Toxicology) and Thomas Zy Lin (B.A. 2019, Department of Molecular and Cell Biology), University of California, Berkeley for outstanding contributions in searching, compiling, and presenting the information in this Review. Most importantly, I acknowledge my laboratory colleagues, many of whom are cited in the references, for sharing on a daily basis the excitement of their discoveries in insecticide chemistry and toxicology. ABBREVIATIONS USED* *Compound numbers with some abbreviations are given in Tables 2-6. ACh, acetylcholine; AChBP, ACh binding protein; AChE, acetylcholinesterase; AChR, ACh receptor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AVE, avermectin; Chlo, chlorantraniliprole; CYP, cytochrome P450; DN-IMI, desnitro-IMI; Flu, flubendiamide; GABA, γ-aminobutyric acid; GABA-R, GABA receptor; Glu, glutamate; Glu-R, glutamate receptor; IMI, imidacloprid; IRAC, Insecticide Resistance Action Committee; IVM, ivermectin; kdr, knockdown resistance; MCs, methylcarbamates; NA, nodulisporic acid; nAChR, nicotinic ACh receptor; NCA, non-competitive antagonist; NMDA, N-methyl-D-aspartic acid; OP, organophosphorus; PCCA, polychlorocycloalkane; PET, positive emission tomography; PTX, picrotoxinin; R, resistant strain; Ry, ryanodine; RyR, Ry receptor; S, susceptible strain; SAR, structure-activity relationship; TBOs, trioxabicyclooctanes; TFMD, trifluoromethyldiazirinyl; TMS, trimethylsilyl; VGSC, voltage-gated sodium channel


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(15) Milbrath, D.S.; Engel, J.L.; Verkade, J.G.; Casida, J.E. Structure-toxicity relationships of 1substituted-4-alkyl-2,6,7-trioxabicyclo[2.2.2]octanes. Toxicol. Appl. Pharmacol. 1979, 47, 287293.

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(16) Squires, R.F.; Casida, J.E.; Richardson, M.; Saederup, E. [35S]t-Butylbicyclophosphorothionate binds with high affinity to brain specific sites coupled to γ-aminobutyric acid-A and ion recognition sites. Mol. Pharmacol. 1983, 23, 326-336.

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(17) Lawrence, L.J.; Casida, J.E. Interactions of lindane, toxaphene and cyclodienes with brainspecific t-butylbicyclophosphorothionate receptor. Life Sci. 1984, 35, 171-178.

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(18) Cole, L.M.; J.E. Casida. Polychlorocycloalkane insecticide-induced convulsions in mice in relation to disruption of the GABA-regulated chloride ionophore. Life Sci. 1986, 39, 1855-1862.

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(19) Cohen, E.; J.E. Casida. Effects of insecticides and GABAergic agents on a housefly [35S]tbutylbicyclophosphorothionate binding site. Pestic. Biochem. Physiol. 1986, 25, 63-73.

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(20) Lawrence, L.J.; Palmer, C.J.; Gee, K.W.; Wang, X.; Yamamura, H.I.; Casida, J.E. t[3H]Butylbicycloorthobenzoate: new radioligand probe for the γ-aminobutyric acid-regulated chloride ionophore. J. Neurochem. 1985, 45, 798-804.

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(21) Palmer, C.J.; Casida, J.E. Novel selective catalytic reduction with tritium: Synthesis of the GABAA radioligand 1-(4-ethynylphenyl)-4-[2,3-3H2]propyl-2,6,7-trioxabicyclo[2.2.2]octane. J. Label. Compd. Radiopharm. 1991, 29, 829-839.

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(22) Deng, Y.; Palmer, C.J.; Casida, J.E. Housefly brain γ-aminobutyric acid-gated chloride channel: target for multiple classes of insecticides. Pestic. Biochem. Physiol. 1991, 41, 60-65.

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(23) Cole, L.M.; Casida, J.E. GABA-gated chloride channel: binding site for 4'-ethynyl-4-n-[2,3H2]propylbicycloorthobenzoate ([3H]EBOB) in vertebrate brain and insect head. Pestic. Biochem. Physiol. 1992, 44, 1-8.

479 480

(24) Elliott, M.; Pulman, D.A.; Larkin, J.P.; Casida, J.E. Insecticidal 1,3-dithianes. J. Agric. Food Chem. 1992, 40, 147-151.

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(25) Wacher, V.J.; Toia, R.F.; Casida, J.E. 2-Aryl-5-tert-butyl-1,3-dithianes and their S-oxidation products: structure-activity relationships of potent insecticides acting at the GABA-gated chloride channel. J. Agric. Food Chem. 1992, 40, 497-505.

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(26) Li, X.; Jung, Y.-W.; Snyder, S.E.; Blair, J.; Sherman, P.S.; Desomnd, T.; Frey, K.A.; Kilbourn, M.A. 5-tert-Butyl-2-(4’-[18F]fluoropropynylphenyl)-1,3-dithiane oxides: potential new GABAA receptor radioligands. Nucl. Med. Biol. 2008, 35, 549-559.

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(27) Li, Q.X.; Casida, J.E. Affinity probes for the GABA-gated chloride channel: 5e-tert-butyl2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes with photoactivatable, fluorescent, biotin, agarose and protein substituents. Bioorg. Med. Chem. 1995, 3, 1675-1684.

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(28) Rauh, J.J.; Benner, E; Shnee, M.E; Cordova, D; Holyoke, C.W.; Howard, M.H.; Bai, D.; Buckingham, S.D.; Hutton, M.L.; Hamon, A.; Roush, R.T.; Sattelle, D.B. Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates. Br. J. Pharmacol. 1997, 121, 1496-1505

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(29) Chen, L.; Durkin, K.A.; Casida, J.E. Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action. J. Biol. Chem. 2007, 281, 3887138878.

497 498 499

(30) Cole, L.M.; Saleh, M.A.; Casida, J.E. House fly head GABA-gated chloride channel: [3H]αendosulfan binding in relation to polychlorocycloalkane insecticide action. Pestic. Sci. 1994, 42, 59-63.

500 501 502

(31) Esser, T.; Karu, A.E.; Toia, R.F.; Casida, J.E. Recognition of tetramethylenedisulfotetramine and related sulfamides by the brain GABA-gated chloride channel and a cyclodiene-sensitive monoclonal antibody. Chem. Res. Toxicol. 1991, 4, 162-167.

503 504 505

(32) Zhao, C.; Hwang, S.H.; Buchholz, B.; Lightstone, F.C.; Carpenter, T.S.; Yang, J.; Hammock, B.D.; Casida, J.E. The GABAA receptor target of tetramethylenedisulfotetramine. Proc. Natl. Acad. Sci. USA 2014, 111, 8607-8612.

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(33) Cole, L.M.; Nicholson, R.A.; Casida, J.E. Action of phenylpyrazole insecticides at the GABA-gated chloride channel. Pestic. Biochem. Physiol. 1993, 46, 47-54.

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(34) Hainzl, D.; Cole, L.M.; Casida, J.E. Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem. Res. Toxicol. 1998, 11, 15291535.

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(35) Sirisoma, N.S., Ratra, G.S., Tomizawa, M., Casida, J.E. Fipronil-based photoaffinity probe for Drosophila and human β3 GABA receptors. Bioorg. Med. Chem. Lett. 2001, 11, 2979-2981.

513 514

(36) Sammelson, R.E.; Casida, J.E. Synthesis of a tritium-labeled, fipronil-based, highly potent, photoaffinity probe for the GABA receptor. J. Org. Chem. 2003, 68, 8075-8079.

515 516

(37) García-Reynaga, P.; Zhao, C.; Sarpong; R., Casida, J.E. New GABA/glutamate receptor target for [3H]isoxazoline insecticide. Chem. Res. Tox. 2013, 26, 514-516.

517 518 519 520

(38) Zhao, C.; Casida, J.E. (2014) Insect γ-aminobutyric acid receptors and isoxazoline insecticides: toxicological profiles relative to the binding sites of [3H]fluralaner, [3H]-4′-ethynyl4-n-propylbicycloorthobenzoate, and [3H]avermectin. J. Agric. Food Chem. 2014, 62, 10191024.

521 522 523

(39) Gassel, M.; Wolf, C.; Noack, S.; Williams, H.; Ilg, T. The novel isoxazoline ectoparasiticide fluralaner: selective inhibition of arthropod γ-aminobutyric acid- and L-glutamate-gated chloride channels and insecticidal/acaricidal activity. Insect Biochem. Mol. Biol. 2014, 45, 111-124.




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(40) Casida, J.E. Golden age of RyR and GABA-R diamide and isoxazoline insecticides: common genesis, serendipity, surprises, selectivity, and safety. Chem. Res. Toxicol. 2015, 28, 560-566.

527 528 529

(41) Ozoe, Y.; Kita, T.; Ozoe, F.; Nakao, T.; Sato, K.; Hirase, K. Insecticidal 3-benzamido-Nphenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors. Pestic. Biochem. Physiol. 2013, 107, 285-292.

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(42) Nakao, T.; Banba, S. Minireview: mode of action of meta-diamide insecticides. Pestic. Biochem. Physiol. 2015, 121, 39-46.

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(43) Nakao, T.; Banba, S. Broflanilide: a meta-diamide insecticide with a novel mode of action. Bioorg. Med. Chem. 2016, 24, 372-377.

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538 539 540

(45) Huang, J.; Casida, J.E. Characterization of [3H]ethynylbicycloorthobenzoate ([3H]EBOB) binding and the action of insecticides on the γ-aminobutyric acid-gated chloride channel of cultured cerebellar granule neurons. J. Pharmacol. Exp. Ther. 1996, 279, 1191-1196.

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(47) Kamijima, M.; Casida, J.E. Regional modification of [3H]ethynyl-bicycloorthobenzoate binding in mouse brain GABAA receptor by endosulfan, fipronil and avermectin B1a. Toxicol. Appl. Pharmacol. 2000, 163, 188-194.

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(50) Ratra, G.S.; Casida, J.E. GABA receptor subunit composition relative to insecticide potency and selectivity. Toxicol. Lett. 2001, 122, 215-222.

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(82) Millar, N.; Denholm, I. Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invert. Neurosci. 2007, 7, 53-66.

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(186) Zhu, B.; Xu, M.; Shi, H.; Gao, X.; and Liang, P. Genome-wide identification of lncRNAs associated with chlorantraniliprole resistance in diamondback moth Plutella xylostella (L.). BMC Genomics. 2017, 18, 380.

932 933 934

(187) Miyoshi, H. Current topics of the inhibitors of Complex I. In A Structural Perspective on Respiratory Complex I: Structure and Function of NADH: Ubiquinone Oxidoreductase. Sazanov, L. Ed.; Springer Netherlands, 2012. pp 81-96.

935 936 937

(188) Horgan, D.J.; Singer, T.P.; Casida, J.E. Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase. XIII. Binding sites of rotenone, piericidin A, and amytal in the respiratory chain. J. Biol. Chem. 1968, 243, 834-843.

938 939 940

(189) Horgan, D.J.; Casida, J.E. Specific binding of [14C]piericidin A in the reduced nicotinamide-adenine dinucleotide dehydrogenase segment of the mitochondrial respiratory chain. Biochem. J. 1968, 108 153-154.

941 942 943

(190) Gutman, M.; Singer, T.P.; Casida, J.E. Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase. XVII. Reaction sites of piericidin A and rotenone. J. Biol. Chem. 1970, 245, 1992-1997.




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944 945

(191) Blandini, F.; Greenamyre, J.T. Assay of [3H]dihydrorotenone binding to Complex I in intact human platelets. Anal. Biochem. 1995, 230, 16-19.

946 947 948

(192) Higgins, Jr., D.S.; Greenamyre, J.T. [3H]Dihydrorotenone binding to NADH; ubiquinone reductase (Complex I) of the electron transport chain: an autoradiographic study. J. Neurosci. 1996, 16, 3807-3816.

949 950 951

(193) Latli, B.; Morimoto, H.; Williams, P.G.; Casida, J.E. Photoaffinity radioligand for NADH:ubiquinone oxidoreductase: [S-C3H2](trifluoromethyl)diazirinyl-pyridaben. J. Label. Compd. Radiopharm. 1998, 41, 191-199.

952 953 954

(194) Schuler, F.; Yano, T.; Di Bernardo, S.; Yagi, T.; Yankovskaya, V.; Singer, T.P.; Casida, J.E. NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone. Proc. Natl. Acad. Sci. USA 1999, 96, 4149-4153.

955 956 957

(195) Wood, E.; Latli, B.; Casida, J.E. Fenazaquin acaricide specific binding sites in NADH: ubiquinone oxidoreductase and apparently the ATP synthase stalk. Pestic. Biochem. Physiol. 1996, 54, 135-145.

958 959

(196) Tocilescu, M.A.; Zickermann, V.; Zwicker, K.; Brandt, U. Quinone binding and reduction by respiratory complex I. Biochim. Biophys. Acta. 2010, 1797, 1883-1890.

960 961 962

(197) Giordano, S.; Lee, J.; Darley-Usmar, V.M.; Zhang, J. Distinct effects of rotenone, 1methyl-4-phenylpyridinium and 6-hydroxydopamine on cellular bioenergetics and cell death. PLOS ONE. 2012, 7, 44610.

963 964 965

(198) Mou, T.; Zhao, Z.; Fang, W.; Peng, C.; Guo, F.; Liu, B.; Ma, Y.; Zhang, X. Synthesis and preliminary evaluation of 18F-labeled pyridaben analogues for myocardial perfusion imaging with PET. J. Nucl. Med. 2012, 53, 1-8.

966 967 968

(199) Knox, J.R.; Toia, R.F.; Casida, J.E. Insecticidal thioureas: preparation of [phenoxy-43 H]diafenthiuron, the corresponding carbodiimide and related compounds. J. Agric. Food Chem. 1992, 40, 909-913.

969 970

(200) Petroske, E.; Casida, J.E. Diafenthiuron action: carbodiimide formation and ATPase inhibition. Pestic. Biochem. Physiol. 1995, 53, 60-74.

971 972 973

(201) Kayser, H.; Eilinger, P. Metabolism of diafenthiuron by microsomal oxidation: procide activation and inactivation as mechanisms contributing to selectivity. Pest Manag. Sci. 2001, 57, 975-980.

974 975

(202) Carrel, J.E.; Eisner, T. Cantharidin: potent feeding deterrent to insects. Science. 1974, 183, 755-757.

976 977

(203) Graziano, M.J.; Pessah, I.N.; Matsuzawa, M.; Casida, J.E. Partial characterization of specific cantharidin binding sites in mouse tissues. Mol. Pharmacol. 1988, 33, 706-712.


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978 979

(204) Li, Y.M.; Casida, J.E. Cantharidin-binding protein: identification as protein phosphatase 2A. Proc. Natl. Acad. Sci. USA. 1992, 89, 11867-11870.

980 981

(205) Hashemzadeh, H.; Hollingworth, R.; Voliva, A. Receptors for 3H-octopamine in the adult firefly light organ. Life Sci. 1985, 37, 433-440.

982 983

(206) Costa, L.G.; Murphy, S.D. Interaction of the pesticide chlordimeform with adrenergic receptors in mouse brain: an in vitro study. Arch. Toxicol. 1987, 59, 323-327.

984 985

(207) Nathanson, J.A. Development of a photoaffinity ligand for octopamine receptors. Mol. Pharmacol. 1989, 36, 34-43.

986 987 988

(208) Ohta, H.; Ozoe, Y. Molecular signaling, pharmacology, and physiology of octopamine and tyramine receptors as potential insect pest control targets. In Target Receptors in the Control of Insect Pests: Part II, Volume 46, 1st Ed.; Cohen, E. Ed.; Academic Press, 2014. pp. 73-166.

989 990 991

(209) Ando, T.; Tecle, B.; Toia, R.F.; Casida, J.E. Tritio-nikkomycin Z,[uracil-5-3H, pyridinyl2,4-3H]: radiolabeling of a potent inhibitor of fungi and insect chitin synthetase. J. Agric. Food Chem. 1990, 38, 1712-1715.

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(210) Loh, Y.Y.; Nagao, K.; Hoover, A.J.; Hesk, D.; Rivera, N.R.; Colletti, S.L.; Davies, I, W.; MacMillan, D.W.C. Photoredox-catalyzed deuteration and tritiation of pharmaceutical compounds. Science. 2017, 358, 1182-1187.

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(211) Yakimchuk, K. Protein receptor-ligand interaction/binding assays. Mater. Methods. 2011, 1, 199.

997 998 999

(212) Wang, Y.; Guo, L.; Qi, S.; Zhang, H.; Liu, K.; Liu, R.; Liang, P.; Casida, J.E.; Liu, S. Fluorescent probes for insect ryanodine receptors: candidate anthranilic diamides. Molecules 2014, 19, 4105-4114.

1000 1001 1002

(213) Liu, K.; Li, Q.; Wang, Y.; Liu, R.; Li, Q.; Liu, S. Affinity-based fluorescence polarization assay for screening molecules acting on insect ryanodine receptors. RSC Adv. 2016, 6, 3903939043.



J. Agric. Food Chem. 1003

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Graphic Abstract

1004 1005


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March 9, 2018

1006 1007 1008 1009 1010 1011

Figure 1. Radioligand binding site recognition and identification versus other methods for determining mode of action.

1012 1013

Figure 2B

Figure 2A

1014 1015




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1016

Figure 2C

1017 1018

Figure 2D

1019 1020

Figure 2E

1021 1022

Figure 2F

1023 1024 1025 1026 1027

Figure 2. Six steps of receptor binding assays with suitable radioligands. A. Radioligand is incubated with receptor or enzyme source followed by filtration or other method to separate free and bound ligand. B. Radioligand concentration is increased to determine total, nonspecific, and 32 ACS Paragon Plus Environment

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J. Agric. Food Chem. 1028 1029 1030 1031 1032 1033 1034

1035 1036 1037 1038 1039 1040 1041

March 9, 2018

specific binding. C. Quantitation of affinity (Kd), and amount (Bmax) and association and dissociation rates (not shown). D. Competitive binding with natural and synthetic compounds. E. Relevance established by physiological effect in vivo consistent with in vitro observations (not shown) and by structure-activity relationships (SAR) to establish that the binding assay faithfully reproduces the organismal effect. F. Specificity compares the binding site in resistant strains or species of pests and insects versus mammals.

Figure 3. GABA-R binding sites for insecticide chloride channel blockers (NCAs), modulators (AVE) and therapeutics GABA (positive allosteric modulators). Only three of the five transmembrane subunits of the chloride channel are shown. Based in part on Casida40. Other ligand-gated ion channels (nAChR, Glu-R) also have coupled sites for agonists, antagonists and allosteric modulators.



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1042 neonic IMI (23) Insect nAChR

Nicotinoid DN-IMI (18) Mammal nAChR

Aplysia AChBP

Lymnaea AchBP

W = tryptophan, Y = tyrosine, S = serine, C-C = cysteine disulfide 1043 1044 1045 1046 1047 1048 1049

1050 1051 1052

Figure 4. Proposed selective interactions of the neonic IMI (23) negatively-charged nitro tip (N NO2) with insect nAChR and Aplysia AChBP compared with nicotinoid DN-IMI (18) cationic region (=NH2+) with mammalian nAChR and Lymnaea AChBP. Supporting data for these relationships are given in references 112 and 113. Table 1. Radioligand isotope half lives and specific activities

a

635 keV positron energy used for positron emission tomography (PET).


Page 35 of 45



1054 1055 1056 1057 1058 1059 1060

March 9, 2018

Table 2. GABA-R [3H, 14C, 18F and 35S]radioligands

a

Mammalian (M) brain or insect (I) head. SAR in vitro consistent with toxicity or physiological effect. c TMS = trimethylsilyl. d TFMD = trifluoromethyldiazirinyl. b



Page 36 of 45

J. Agric. Food Chem. 1061 1062 1063

March 9, 2018

Table 2 Structures (continued) Cl Cl CN

* *

Cl

CF3 CF3

CN

Cl

S O

O O S

*

Cl

*

6

7

O

*

R

S

P

O

R2

O

O

2

R3

9A

R

8

N

R'

O

N N

S

O

CF3

N

O

N

Cl

R1

O

O

N Cl

R1 = CN

R2 =

*

Cl O

R3 = NH2

S

R' CF3

3

H

(CH3)3C-

4A

*CH3CH * 2CH2

4B

*CH3CH * 2CH2

*

N

9B C C

R1 = H

R3 = H *

R2 = N

CH

CF3

F F O N F

C Si(CH3)3

H

* O

N

Cl

O S S

Cl

F

CH3

5A

O

5B R =

F

F F

N3

R2

F H N

CH2OCH2CR

*

R1 N

O

SCH2CH3SCH2

N H

10 O

S S

O

*

CF3 F CF3

O R3

11A R1 = H R2 = CH3 11B R1 = CH3 R2 = CF3

R3 = CH3 R3 = Br


O

Page 37 of 45



March 9, 2018

Table 3. Glu-R [3H]radioligands

1065 1066 1067 1068 1069 1070

a

Mammalian (M) brain or insect (I) head. p-Glycoprotein mammalian brain. c SAR in vitro consistent with toxicity or physiological effect. Table 3 structures (continued) Table 4. nAChR [3H]radioligands b

R H3C

OCH3

O

OCH3

O

22/23

CH3

H3C

O

O

CH3

O

CH3

O

H H3C

CH2CH3 O

O

OH

O

*

CH3

H OH

12A R =

OH

12B R =

OH, 22,23-dihydro**

12C R =

NHCH 3

OCH3 HO CH3 H3C

CH3

O

O

O O

H3C O

O

OH

O

14

CH3 H

NOH




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1071 1072 1073 1074

a b

Mammalian (M) brain or insect (I) head. SAR in vitro consistent with toxicity or physiological effect.


Page 39 of 45


J. Agric. Food Chem. 1075 1076

March 9, 2018

Table 4 Structures (continued).




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Page 41 of 45



1080 1081 1082 1083 1084 1085

March 9, 2018

Table 5. Na+ channel modulator insecticides and related compounds

a b





Page 42 of 45


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H3C Br O

CH3 N H

42 Cl

N

43

H HO

CH3

CH3

CH3

O H3CO

OH

H H

O

O OH

H3CO

CO2CH3 O

H OH CH3 OH OH

OCF3

CN

N N N O

44A 44B

R

N

R = CO2CH3 R=H

H N

NH

45

CF3

O

OCF3


Page 43 of 45



1090 1091 1092 1093

March 9, 2018

Table 6. RyR, NADH: ubiquinone oxidoreductase and various insecticide target radioligands

a b




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J. Agric. Food Chem. 1094 1095 1096

March 9, 2018

Table 6 Structures (continued)..

*

*

O

*

O

CH3

R

O

* *

O

54A R =

OH O OH

*

NHC(S)NHC(CH3)3

CH3

NHC(SO)NHC(CH3)3 54B R =

O

CH3

O

55A

*

CH3 O

55B

N C NC(CH3)3


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March 9, 2018

Table 7. Some interacting compounds at coupled binding sites

1098 1099

a

Insecticides are considered in other tables and the text.


Radioligand Recognition of Insecticide Targets - American Chemical

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