Article pubs.acs.org/JAFC
Insect γ‑Aminobutyric Acid Receptors and Isoxazoline Insecticides: Toxicological Profiles Relative to the Binding Sites of [3H]Fluralaner, [3H]-4′-Ethynyl-4‑n‑propylbicycloorthobenzoate, and [3H]Avermectin Chunqing Zhao† and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720-3112, United States S Supporting Information *
ABSTRACT: Isoxazoline insecticides, such as fluralaner (formerly A1443), are noncompetitive γ-aminobutyric acid (GABA) receptor (GABA-R) antagonists with selective toxicity for insects versus mammals. The isoxazoline target in house fly (Musca domestica) brain has subnanomolar affinity for [3H]fluralaner and a unique pattern of sensitivity to isoxazolines and avermectin B1a (AVE) but not to fipronil and α-endosulfan. Inhibitor specificity profiles for 15 isoxazolines examined with Musca GABA-R and [3H]fluralaner, [3H]-4′-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB), and [3H]AVE binding follow the same structure−activity trends although without high correlation. The 3 most potent of the 15 isoxazolines tested in Musca [3H]fluralaner, [3H]EBOB, and [3H]AVE binding assays and in honeybee (Apis mellifera) brain [3H]fluralaner assays are generally those most toxic to Musca and four agricultural pests. Fluralaner does not inhibit [3H]EBOB binding to the human GABA-R recombinant β3 homopentamer, which is highly sensitive to all of the commercial GABAergic insecticides. The unique isoxazoline binding site may resurrect the GABA-R as a major insecticide target. KEYWORDS: avermectin, chloride channel, GABA receptor, honeybee, house fly, human β3 homopentamer, isoxazoline
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INTRODUCTION
Insecticide radioligands and receptor binding assays play a major role in defining the mode of action and resistance mechanisms. One of the best predictors of GABA-R antagonist insecticidal activity is the [3H]-4′-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) (Figure 1) binding assay with Musca head membranes. 10 With several different chemotypes (polychlorocycloalkanes, fiproles, bicycloorthobenzoates, dithianes, and picrotoxinin analogues) there is a close correlation between IC50 in the [3H]EBOB assay and toxicity to Musca pretreated with a cytochrome P450 inhibitor to minimize detoxification.10 [3H]EBOB is the most widely used radioligand for the insect and mammalian GABA receptor noncompetitive blocker site, but it is not the first or necessarily the most relevant. The first was [3H]dihydropicrotoxinin used with rat brain synaptosomes.11 Three trioxabicyclooctanes were subsequently developed for this purpose, sequentially [35S]-tertbutylbicyclophosphorothionate ([35S]TBPS),12 [3H]-tert-butylbicycloorthobenzoate ([3H]TBOB),13 and [3H]EBOB.10,14 Two chlorinated hydrocarbon insecticides were also introduced as radioligands, i.e., [3H]-α-endosulfan15 and [3H]BIDN,16 which were high in relevance but poor in measurement of coupled systems. [3H]Ivermectin and [3H]avermectin B1a ([3H]AVE) (Figure 1) were used to measure the chloride channel activator site in Musca head membranes.17,18 [3H]Fluralaner was the first radioligand shown to detect the isoxazoline binding site of the insect GABA-R.19
The γ-aminobutyric acid (GABA)-gated chloride channel and GABA receptor (GABA-R) are the targets for an expanding number and variety of insecticides, including the polychlorocycloalkanes (e.g., α-endosulfan), fiproles (e.g., fipronil), and avermectins.1−4 The isoxazolines are the latest addition with outstanding potency on pests of agriculture and pets and low mammalian toxicity.4−9 They are effective on Lepidoptera, Hemiptera, Coleoptera, thrips, and mites.5−7 Isoxazoline fluralaner (formerly A1443) (Figure 1) is under investigation as an ectoparasiticide for control of cat fleas and dog ticks.8,9 There is also keen interest in this chemotype because of apparently no cross-resistance with insecticide-tolerant strains, particularly dieldrin-resistant (rdl) house flies (Musca domestica).8,9
Received: Revised: Accepted: Published:
Figure 1. Radioligands studied. © 2014 American Chemical Society
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The goals of this study were first to directly compare the isoxazoline toxicological profiles of the [3H]fluralaner binding site with that of [3H]EBOB and [3H]AVE in the Musca receptor and second to examine the relationship in Musca and honeybee (Apis mellifera) between binding site specificity and pest toxicity. The action of fluralaner in mammalian brain was also considered.
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Membrane Receptor Preparation. Musca adults were frozen in liquid nitrogen and the heads recovered by shaking and sieving with a yield of about 1.0 g of heads per 1000 flies. The heads (1.0 g) were homogenized in 10 mL of ice-cold buffer A (250 mM sucrose, 10 mM Tris−HCl, pH 7.5) containing Haltphosphatase inhibitor cocktail (Thermo Scientific, Rockford, MA) (1%, v/v). The homogenate was filtered through four layers of nylon mesh (64 μm) and centrifuged at 700g for 30 min and the supernatant filtered again through four layers of nylon mesh. The membranes were then recovered by centrifugation at 25000g for 30 min. The pellet was resuspended in buffer A, kept on ice for 10 min, and then centrifuged again at 25000g for 30 min. The final membrane pellet was resuspended in buffer B [300 mM NaCl and 10 mM phosphate-buffered pH 7.5 saline (PBS)], filtered through four layers of nylon screen to remove particles, and employed for the binding assay after protein determination.22 The same procedure was used to prepare Apis brain membranes from bees obtained at a local apiary. Binding Assays. The receptor binding assays were based on our original studies with Musca for three radioligands, [3H]fluralaner,19 [3H]EBOB,10 and [3H]AVE.17 The binding assays consisted of sequential addition to incubation tubes (13 × 100 mm) of candidate inhibitor in DMSO (0.5 μL) and membrane preparation (200 μg of Musca or Apis protein) in PBS (1.0 mL). The radioligand [final concentration 0.1 nM [3H]fluralaner, 0.5 nM [3H]EBOB, or 0.5 nM [3H]AVE] was introduced into 10 μL of buffer B with mixing. Incubations were for 70 min at 22 °C followed by addition of ice-cold PBS (5 mL) and filtration with GF/B glass-fiber filters and then two 5 mL rinse washes of cold PBS. Radioactivity on the filters was counted with 10 mL of scintillation fluid Safety-Solve cocktail (Research Products International Corp., Elk Grove, IL) after the filters were allowed to stand in the dark overnight. Nonspecific binding was determined with 5 μM fluralaner for [3H]fluralaner, 5 μM fipronil for [3H]EBOB, and 5 μM AVM for [3H]AVE. Each experiment was repeated 3−5 times, and the results reported are the means with standard errors. Mammalian GABA-R preparations and assays were as previously reported for rat brain membranes23 and recombinant β3 homopentamer.24,25
MATERIALS AND METHODS
Chemicals and Radioligands. Three radioligands were used: [3H]fluralaner (14 Ci/mmol),19 [3H]EBOB (26 Ci/mmol),10 and [3 H]AVE (11 Ci/mmol).17 Fluralaner (1) was prepared as described,19 and isoxazolines 2−15 (Figure 2) were provided by DuPont Crop Protection (Newark, DE). Other compounds were obtained from commercial sources.
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RESULTS
Musca and Apis [3H]Fluralaner Binding Assays. Optimization of the [3H]fluralaner binding assay proved to be a challenge because of relatively high levels of nonspecific binding and possibly multiple binding sites.8,19 The method described is acceptable but still far from ideal using 0.1 nM [3H]fluralaner to give an adequate level of specific binding. Total and specific binding and percent specific binding for Musca averaged 2000 ± 100 dpm, 600 ± 100 dpm, and about 30%, respectively. The corresponding values for Apis averaged 1650 ± 100 dpm, 800 ± 50 dpm, and about 45%, respectively. [3H]EBOB and [3H]AVE were assayed with the same Musca preparation and method as for [3H]fluralaner except with 0.5 nM radioligand. Total, nonspecific, and specific binding for Musca with [3H]EBOB were 1200 ± 100 dpm, 600 ± 50 dpm, and about 50%, respectively, and with [3H]AVE they were 2700 ± 100 dpm, 2100 ± 100 dpm, and about 75%, respectively. Binding Site Profiles. Musca [3H]Fluralaner, [3H]EBOB, and [3H]AVE Binding Sites. Four Insecticide Chemotypes (Figure 3). The four representative inhibitors used were isoxazoline (fluralaner), phenylpyrazole (fipronil), organochlorine (α-endosulfan), and macrocyclic lactone (AVE) tested at 0.1, 1, 10, 100, and 5000 nM (final concentration). The Musca [3H]fluralaner site is highly sensitive to fluralaner and AVE with IC50 values of about 0.4 and 3 nM, respectively, but not to fipronil or α-endosulfan.19 The [3H]EBOB site is sensitive to fluralaner, fipronil, and α-endosulfan (IC50 values of
Figure 2. Structures of isoxazolines studied. Insecticidal activities are reported by Lahm et al.6 for all compounds except 1, 4, 10, 11, and 12. Toxicity to Insects. Adult Musca (about 25 mg each) 3−5 days after emergence from the pupae (obtained from Benzon Research Inc., Carlisle, PA) were treated by intrathoracic injection with 0.03 μg of isoxazoline in 0.2 μL of DMSO−water (1:3), and the toxicity was determined at 24 h. Poisoning signs of this 1.2 μg/g isoxazoline dose were rated for each fly in groups of 40 as 0 (no effect), 1 (unable to walk or fly), and 2 (no response to prodding) following the reported method.20,21 The assay was repeated two more times and averaged on a percentage basis. The values were calculated as follows: number of impaired flies (score 1) plus twice the number of dead flies (score 2); i.e., all impaired was scored 50, and complete mortality was scored 100. The insecticidal activity of nine isoxazolines studied here was previously reported as ppm LC50 with four pest species, i.e., fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), potato leafhopper (Empoasca fabae), and western flower thrips (Frankliniella occidentalis).6 Potency values for these pests were expressed as 1/LC50. 1020
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Figure 4. Potency relationships in Musca of 15 isoxazolines at 10 nM as inhibitors of [3H]fluralaner versus [3H]EBOB (A), [3H]fluralaner versus [3H]AVE (B), and [3H]EBOB versus [3H]AVE (C) binding.
[3H]fluralaner binding was similar (r2 = 0.55) for Apis and Musca. Toxicity to Musca and Four Pest Species Relative to Musca and Apis [3H]Fluralaner Binding Affinity. Musca (Figure 6). The set of 15 isoxazolines was assayed for toxicity to Musca adults by intrathoracic injection of a standard 1.2 μg/g dose. Only three of the isoxazolines (1−3) showed high toxicity, and they were the same ones most effective as inhibitors of [3H]fluralaner binding and among the better inhibitors of [3H]EBOB and [3H]AVE binding. Pest Species. The findings on toxicity to Musca and four pest species were compared with each other (Figure 7) and with the structure−activity relationships for inhibition in Apis of [3H]fluralaner binding (Figure 5B−E). Isoxazoline poisoning of Musca was at best a moderate predictor of toxicity to Spodoptera and Helicoverpa and even poorer with Frankliniella and Empoasca. Compounds 2, 3, 7, and 8 were highly active on all species, whereas 5 and 14 were intermediate in toxicity and 6, 9, and 13 had the lowest activity. The correlation of receptor binding with toxicity was always better for Apis (Figure 5 B−E) (r2 = 0.75−0.84) than for Musca (r2 = 0.30−0.68) (Supporting Information). The best correlation was for Apis [3H]fluralaner binding inhibition with Empoasca toxicity (r2 = 0.84). Mammalian GABAA-R (Figure 8). Fluralaner, fipronil, and heptachlor epoxide were compared for potency in the [3H]EBOB binding assay with rat brain membranes and recombinant human β3 homopentamer. Fluralaner gave 37% inhibition at 104 nM with rat brain membrane, consistent with
Figure 3. Effect in Musca of fluralaner, fipronil, α-endosulfan, and AVE on binding of [ 3 H]fluralaner, [3 H]EBOB, and [ 3H]AVE. A concentration of 5000 nM is shown as 103.7.
39−41 nM) but much less so to AVE. The [3H]AVE site is poorly sensitive to fluralaner, apparently stimulated to a small degree (20−50%) by fipronil, insensitive to α-endosulfan, and highly sensitive to AVE (IC50 ≈ 20 nM). Fifteen Isoxazolines (Figure 4). Fifteen isoxazolines were examined for their inhibitory activity to [3H]fluralaner, [3H]EBOB, and [3H]AVE binding. The most active compounds were isoxazolines 1 (fluralaner), 2, and 3. The others were much weaker inhibitors. The binding site profiles for [3H]fluralaner relative to [3H]EBOB give a correlation coefficient of 0.53, about the same as that for [3H]fluralaner versus [3H]AVE (r2 = 0.50) and much better than that for [3H]EBOB versus [3H]AVE (r2 = 0.12). There are clearly common features of the target or coupled binding sites but not identity for isoxazolines interacting in the three radioligand assays. Apis [3H]Fluralaner Binding Site with 15 Isoxazolines (Figure 5A). The specificity of 15 isoxazolines for inhibition of 1021
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Figure 6. Potency in Musca of 15 isoxazolines at 10 nM as inhibitors of [3H]fluralaner (A), [3H]EBOB (B), and [3H]AVE (C) binding relative to their toxicity.
Figure 5. Potency relationships in Apis and Musca of 15 isoxazolines at 10 nM as inhibitors of [3H]fluralaner binding (A) and for 9 isoxazolines relative to toxicity in four pest insects (B−E). The corresponding r2 values for Musca were 0.56 for Spodoptera, 0.30 for Helicoverpa, 0.52 for Empoasca, and 0.68 for Frankliniella (see the Supporting Information).
an earlier report, 8 but it had almost no effect on the recombinant β3 homopentamer. Heptachlor epoxide and fipronil showed high affinity for rat brain membranes with IC50 values of 200 and 800 nM, respectively, and even higher affinity for the recombinant human β3 subunit with IC50 values of 5.4 and 3.4 nM, respectively.
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DISCUSSION The GABA receptor is the target for the isoxazoline insecticides based on several types of evidence. The most complete studies were for fluralaner acting on insect ligand-gated ion channels using MdGBCl and MdGluCl cDNAs, which encode the subunits of Musca GABA- and glutamate-gated chloride channels, respectively.8 Two-electrode voltage clamp electrophysiology was used to confirm that fluralaner blocks GABAand glutamate-induced chloride currents in Xenopus oocytes expressing MdGBCl or MdGluCl channels, with IC50 values of 5.3 and 80 nM, respectively. Blockade by fluralaner was observed in A2′S-MdGBCl and S2′A-MdGluCl mutant channels at levels similar to those of the respective wild types, and Musca expressing A2′S-MdGBCl channels were as susceptible to fluralaner as standard house flies. These findings indicated that fluralaner is a novel and specific blocker of insect ligand-gated chloride channels.8
Figure 7. Toxicity of nine isoxazolines to Musca relative to four pest species.
The present study confirms the potency of fluralaner as a high-affinity inhibitor of the Musca [3H]EBOB site8 and shows directly the subnanomolar affinity of [3H]fluralaner for Musca19 and Apis GABA-R. The ecological impact of the isoxazolinesensitive GABA-R site not only in target pests but also in Apis remains to be determined. The Apis GABA-R assay is better 1022
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Park, NC) for helpful suggestions on preparing Musca membranes for [3H]EBOB binding assays.
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ABBREVIATIONS USED AVE, avermectin B1a; [3H]EBOB, the radioligand 4′-ethynyl-4n-propylbicycloorthobenzoate; fluralaner or A1443, 4-[5-(3,5dichlorophenyl)-4,5-dihydro-5-(trifluoromethyl)-3-isoxazolyl]2-methyl-N-[2-oxo-2-(phenylamino)ethyl]benzamide; GABAR, γ-aminobutyric acid receptor; PBS, phosphate-buffered saline; [3H]TBOB, the radioligand tert-butylbicycloorthobenzoate; [35S]TBPS, the radioligand tert-butylbicyclophosphorothionate
Figure 8. Potency in rat brain membranes and the recombinant human GABA-R β3 homopentamer of fluralaner, fipronil, and heptachlor epoxide as inhibitors of [3H]EBOB binding.
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(1) Casida, J. E. Insecticide action at the GABA-gated chloride channel: Recognition, progress, and prospects. Arch. Insect Biochem. Physiol. 1993, 22, 13−23. (2) Buckingham, S. D.; Sattelle, D. B. GABA receptor in insects. In Comprehensive Molecular Insect Science; Gilbert, L. K., Gill, S. S., Eds.; Elsevier: Amsterdam, 2005; Vol. 5, pp 107−142. (3) Ozoe, Y.; Takeda, M.; Matsuda, K. γ-Aminobutyric acid receptors: A rationale for developing selective insect pest control chemicals. In Biorational Control of Arthropod Pests; Ishaaya, S., Horowitz, A. R., Eds.; Springer: New York, 2009; pp 131−162. (4) Ozoe, Y. γ-Aminobutyrate- and glutamate-gated chloride channels as targets of insecticides. Adv. Insect Physiol. 2013, 44, 211−286. (5) Lahm, G. P.; Shoop, W. L; Xu, M. Isoxazolines for controlling invertebrate pests. U.S. Patent 8231888, 2012. (6) Lahm, G. P.; Cordova, D.; Barry, J. D.; Pahutski, T. F.; Smith, B. K.; Long, J. K.; Benner, E. A.; Holyoke, C. W.; Joraski, K.; Xu, M.; Schroeder, M. E.; Wagerle, T.; Mahaffey, M. J.; Smith, R. M.; Tong, M.-H. 4-Azolylphenyl isoxazoline insecticides acting at the GABA gated chloride channel. Bioorg. Med. Chem. Lett. 2013, 23, 3001−3006. (7) Mita, T.; Kikuchi, T.; Mizukoshi, T.; Yaosaka, M.; Komoda, M. Isoxazoline-substituted benzamide compound and noxious organism control agent. WO 2005-085126; JP 2007-308471. (8) Ozoe, Y.; Asahi, M.; Ozoe, F.; Nakahira, K.; Mita, T. The antiparasitic isoxazoline A1443 is a potent blocker of insect ligandgated chloride channels. Biochem. Biophys. Res. Commun. 2010, 391, 744−749. (9) 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, in press. (10) Deng, Y.; Palmer, C. J.; Casida, J. E. House fly brain γaminobutyric acid-gated chloride channel: Target for multiple classes of insecticides. Pestic. Biochem. Physiol. 1991, 41, 60−65. (11) Matsumura, F.; Ghiasuddin, S. M. Evidence for similarities between cyclodiene type insecticides and picrotoxinin in their action mechanisms. J. Environ. Sci. Health 1983, B18, 1−14. (12) Cohen, E.; Casida, J. E. Effects of insecticides and GABAergic agents on a house fly [35S]t-butylbicyclophosphorothionate binding site. Pestic. Biochem. Physiol. 1986, 25, 63−72. (13) 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. (14) 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. (15) Cole, L. M.; Saleh, M. A.; Casida, J. E. House fly head GABAgated chloride channel: [3H]α-Endosulfan binding in relation to polychlorocycloalkane insecticide action. Pestic. Sci. 1994, 42, 59−63. (16) Rauh, J. J.; Benner, E.; Schnee, 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
than the Musca GABA-R assay as a predictor of insecticidal activity, a finding rationalized as a higher ratio of the most relevant isoxazoline high-affinity binding site in the Apis assay. A marked difference is observed in the inhibitor specificity of the Musca [3H]fluralaner and [3H]EBOB sites, establishing that they are distinct but coupled. The AVE agonist site is coupled to both the fluralaner and EBOB sites but each in a unique way. The three distinct and coupled sites are all targets for insecticide action, but the isoxazoline site provides two unique features. It is much more important in insects than mammals, providing a high level of selective toxicity. The sites have no features in common that confer cross-resistance. This is an exciting observation stimulating interest in isoxazolines as possibly the next generation of GABAergic insecticides.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1 showing the relation for nine isoxazolines between [3H]fluralaner binding data in Musca and toxicity to four pest species. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Phone: 510-642-5424. Fax: 510-642-6497. E-mail: ectl@ berkeley.edu. Present Address †
C.Z.: College of Science, China Agricultural University, Beijing, China 100193.
Funding
C.Z. was supported in part by State Scholarship Fund No. 2011635139 provided by the China Scholarship Council. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are greatly indebted to George P. Lahm, James D. Barry, and Thomas F. Pahutski of DuPont Crop Protection Stine Haskell Research Center (Newark, DE) for providing a set of 14 isoxazolines critical for the validating experiments on in vitro structure−activity relationships relative to insecticidal activity. C.Z. thanks Professor Lihong Qiu of the China Agricultural University for academic counsel and Berkeley laboratory colleagues Amanda Ly, Breanna Ford-Morris, Madhur Garg, and Stephen Lantz for assistance and advice. We thank Vincent Salgado and Brecht London of BASF Corp. (Research Triangle 1023
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bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates. Br. J. Pharmacol. 1997, 121, 1496−1505. (17) Deng, Y.; Casida, J. E. House fly head GABA-gated chloride channel: Toxicologically relevant binding site for avermectins coupled to site for ethynylbicycloorthobenzoate. Pestic. Biochem. Physiol. 1992, 43, 116−122. (18) Orr, N.; Shaffner, A. J.; Richey, K.; Crouse, G. D. Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites. Pestic. Biochem. Physiol. 2009, 95, 1−5. (19) García-Reynaga, P.; Zhao, C.; Sarpong, R.; Casida, J. E. New GABA/glutamate receptor target for [3H]isoxazoline insecticide. Chem. Res. Toxicol. 2013, 26, 514−516. (20) Shao, X.; Swenson, T. L.; Casida, J. E. Cycloxaprid insecticide: Nicotinic acetylcholine receptor binding site and metabolism. J. Agric. Food Chem. 2013, 61, 7883−7888. (21) Shao, X.; Xia, S.; Durkin, K. A.; Casida, J. E. Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 17273−17277. (22) Bradford, M. M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (23) Cole, L. M.; Casida, J. E. GABA-gated chloride channel: Binding site for 4′-ethynyl-4-n-[2,3- 3 H 2 ]propylbicycloorthobenzoate ([3H]EBOB) in vertebrate brain and insect head. Pestic. Biochem. Physiol. 1992, 44, 1−8. (24) Ratra, G. S.; Kamita, S. G.; Casida, J. E. Role of human GABAA receptor β3 subunit in insecticide toxicity. Toxicol. Appl. Pharmacol. 2001, 172, 233−240. (25) Chen, L.; Durkin, K. A.; Casida, J. E. Structural model for γaminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5185−5190.
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