Diamide Insecticide Target Site Specificity in the Heliothis and Musca

Article pubs.acs.org/JAFC

Diamide Insecticide Target Site Specificity in the Heliothis and Musca Ryanodine Receptors Relative to Toxicity Suzhen Qi,†,#,⊥ Peter Lümmen,§ Ralf Nauen,§ 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 § Small Molecules Research, Pest Control, Bayer CropScience AG, Alfred Nobel Strasse 50, Monheim, Germany 40789 # Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, China 100193 S Supporting Information *

ABSTRACT: Anthranilic and phthalic diamides act on the ryanodine receptor (RyR), which constitutes the Ca2+-activated Ca2+ channel and can be assayed as shown here in Heliothis thoracic muscle tissue with anthranilic diamide [3H]chlorantraniliprole ([3H]Chlo), phthalic diamide [3H]flubendiamide ([3H]Flu), and [3H]ryanodine ([3H]Ry). Using Heliothis with [3H]Chlo or [3H]Flu gives very similar anthranilic and phthalic diamide binding site structure−activity correlations, indicating a common binding site. The anthranilic and phthalic diamide stimulation of [3H]Ry binding in Heliothis generally parallels their inhibition of [3H]Chlo and [3H]Flu binding. In Musca adults [3H]Ry binding site stimulation is a good predictor of in vivo activity for anthranilic but not phthalic diamides, and no high-affinity [3H]Flu specific binding site is observed. These relationships establish species differences in diamide target site specificity important in structure optimization and target site-based resistance mechanisms. KEYWORDS: ryanodine receptor, anthranilic diamides, phthalic diamides, structure−activity relationship, toxicity

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INTRODUCTION

relationships are important in insecticide design, species specificity, and resistance management.

1−6

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Diamides are the newest major class of insecticides. They combine outstanding potency on many major agricultural pests with low mammalian toxicity. The current diamides are of two chemotypes. Anthranilic diamides are exemplified by chlorantraniliprole (Chlo) and cyantraniliprole (Cyan)3−6 and phthalic diamides by flubendiamide (Flu).1,2 Their mode of action is different from that of all other insecticides so target site cross resistance is not a problem.7 Both chemotypes are Ca2+ channel modulators and ryanodine (Ry) receptor (RyR) activators in essentially the same way; that is, no differences are apparent in their electrophysiological effects on insect muscle preparations.1−6 Cross resistance between Chlo and Flu in control of Plutella xylostella8−10 further suggests a common mode of action. The molecular targets of Ry and the diamides in insects are partially characterized with appropriate radioligands, that is, [3H]Ry,11,12 [3H]Chlo,13 and [3H]Flu2,14 (Figure 1). Species differences are apparent in these binding sites. The [3H]Flu, [3H]Chlo, and [3H]Ry sites have been studied in muscle tissues of Musca, Apis, Heliothis, and Agrotis15 with major differences between species: [3H]Ry binding and [3H]Chlo binding have common characteristics in all of these insects based on Ca2+ and ATP effects and interactions with unlabeled Ry, Chlo, Cyan, and Flu, but no high affinity [3H]Flu binding site was evident in Musca and Apis under our test conditions.15 The goal of this investigation is to establish if the anthranilic and phthalic diamides have a common target site in some species but not others (e.g., Heliothis versus Musca) and if radioligand binding studies reflect the in vivo actions and toxic effects. These © 2014 American Chemical Society

MATERIALS AND METHODS

Chemicals. [ 3 H]Ry (95 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA, USA). [3H]Chlo was synthesized in house.13 Twenty-six anthranilic and phthalic diamides with generic structures shown in Figure 2 were used to compare the chemotype−activity relationships of binding assays with toxicity. Although the structures of individual compounds were not revealed for reasons of patenting status, their classification into two distinct chemotypes provided the opportunity to establish the relevance of the binding site assay results to toxicity in both the anthranilic and phthalic diamide series. [3H]Flu and 11 analogues of Chlo were provided by Bayer CropScience (Monheim, Germany), and 12 analogues of Flu were supplied by Nihon Nohyaku (Tokyo, Japan). The compounds are numbered on the general basis of potency from the most to the least in the Heliothis thoracic muscle binding assay with [3H]Chlo for the anthranilic diamide set and [3H]Flu for the phthalic diamides as described later. [3H]Chlo, [3H]Flu, and [3H]Ry Binding Assays in Vitro. Membranes with associated RyR from Heliothis and Musca thoracic flight muscle were prepared as described and stored at −80 °C.15 Each reaction mixture contained 1 nM [3H]Chlo, [3H]Flu, or [3H]Ry, 200 μg of membrane protein,13,15 and 30 nM test compound (in 2 μL of dimethyl sulfoxide) in binding buffer (0.8 mM CaCl2, 2 mM ATPMg2+ salt, and 1.5 M KCl in 10 mM Hepes, pH 7.4) with a final volume of 412 μL. Incubations were for 120 min at 37 °C. Nonspecific binding was determined with 10 μM Chlo for [3H]Chlo, 10 μM Flu for [3H]Flu, and 10 μM Ry for [3H]Ry binding. Bound radioligand Received: December 20, 2013 Accepted: April 18, 2014 Published: April 18, 2014 4077

dx.doi.org/10.1021/jf501236h | J. Agric. Food Chem. 2014, 62, 4077−4082

Journal of Agricultural and Food Chemistry

Article

Figure 1. Diamide and ryanodine radioligands showing 3H-labeling positions for [3H]Chlo and [3H]Ry (labeling site for [3H]Flu not disclosed). poisoning) for each individual concentration tested; for example, in tests at 1000, 200, 40, 8, 1.6, 0.32, 0.064, and 0.0128 ppm and corresponding efficacy values of 100, 100, 100, 100, 100, 70, 20, and 0%, the toxicity index is 590. Musca Poisoning Signs and [3H]Chlo and [3H]Ry Binding Assays in Vivo. Twenty adult Musca anesthetized with carbon dioxide for 10 min were individually injected with a discriminating dose of each compound of 0.1 μg/fly (about 5 μg/g) into either the thorax or the abdomen (two sets of flies). Two hours after treatment, poisoning signs were observed for each fly to obtain a total rating for the set of 20 flies using the following system: 0 for normal, 1 for impaired movement, and 2 for dead. The final poisoning signs were equal to the number of impaired flies plus twice the number of dead flies multiplied by 2.5, that is, 100% impaired movement = 50 and 100% mortality = 100 poisoning rating. Thoraces were recovered 2 h after injection to determine ex vivo inhibition or stimulation of binding. Ten thoraces were homogenized by a Polytron (Brinkmann Instruments) in 1 mL of membrane preparation buffer (10 μM phenylmethanesulfonyl floride, 0.8% bovine serum albumin, and 303 mM sucrose in 20 mM Tris-maleate, pH 7.0) for 40 s.15 Each homogenate was filtered through four layers of 64 μm mesh nylon screen after centrifuging at 700g for 30 min, and then 200 μL was used for radioligand binding assay as described above for binding in vitro.15 Statistical Analysis. Linear regression was performed to calculate the correlation among [3H]Chlo, [3H]Flu, and [3H]Ry binding or versus toxicity for each compound with Heliothis or [3H]Chlo and [3H]Ry binding in vitro versus in vivo and binding in vitro/in vivo versus toxicity for each compound with Musca. GraphPad Prism 5.0 software was used to plot and analyze data.

Figure 2. Diamides with various substituents (R1, R2, and R3). The phthalic diamides are also referred to as 1,2-benzenedicarboxamides (BDCAs). was harvested by filtration and quantitated according to earlier studies, and the percentage inhibition or stimulation versus control was determined.13,15 Heliothis and Spodoptera Bioassays. The efficacy of diamides against insecticide-susceptible strains of Heliothis virescens and Spodoptera frugiperda was tested using an artificial diet-based bioassay as described elsewhere.16 Briefly, cylinders of artificial diet (height, 0.6 cm; diameter, 1.5 cm) were placed in 24-well tissue culture plates. Insecticide stock solutions were prepared by dissolving active ingredient in a 3:1 (v/v) mixture of dimethylformamide and emulsifier W. Subsequently, the stock solution was diluted with water. Twelve diet cylinders were each applied with 100 μL per concentration and represented one replicate. The experiment was repeated three times with two replicates per concentration. One second-instar larva was transferred to each treated cylinder. The plates were covered with tissue paper, sealed with a ventilated lid, and stored at room temperature in the dark. Percentage efficacy was assessed after 7 days. The toxicity ratings were generated by summing the assessed percentage efficacy (larvae dead or showing irreversible symptoms of

Figure 3. Heliothis toxicological profiles for anthranilic (A) and phthalic (B) diamide inhibition of [3H]Chlo and [3H]Flu binding assayed at 30 nM test compound. 4078



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Article

RESULTS

Heliothis RyR. Diamide Toxicological Profiles for [3H]Chlo and [3H]Flu Binding Sites. The 11 anthranilic analogues plus Chlo and Cyan were tested at a discriminating concentration (30 nM) for inhibition of [3H]Chlo and [3H]Flu binding (Figure 3A), and the same experiment was made with the 12 phthalic analogues plus Flu at 30 nM (Figure 3B). The anthranilic and phthalic diamides have the same efficacy for inhibiting binding at the Chlo site as for the Flu site (Figure 4).

Figure 4. Heliothis toxicological profiles for anthranilic and phthalic diamide inhibition of [3H]Chlo and [3H]Flu binding assayed at 30 nM test compound. Individual compounds are identified in Figure 3.

Diamide Toxicological Profiles Relative to Insecticidal Activity. The binding site profiles were compared with the insecticidal activities for the anthranilic diamides with [3H]Chlo (Figure 5A) and [3H]Flu (Figure 5B). The potency of the anthranilic diamides to inhibit [3H]Chlo and [3H]Flu binding is consistent with their insecticidal activity. Because [3H]Chlo and [3H]Flu binding data are very similar, this also means that with Heliothis the use of either radioligand predicts toxicity in both diamide chemotypes. Diamides Increase [3H]Ry Binding. The two sets of diamides inhibitory to [3H]Chlo and [3H]Flu binding (Figure 5A, B) were assayed for effect on [3H]Ry binding (Figure 5C) with a much lower correlation to toxicity (r2 = 0.08). Inhibition at the diamide binding site assayed with [3H]Chlo and [3H]Flu is generally associated with increased binding at the [3H]Ry site, that is, they are negatively correlated (Figure 6). Musca RyR. Diamide Toxicological Profiles for [3H]Chlo and [3H]Ry Binding Sites. The experiments for in vitro inhibition of [3H]Chlo and [3H]Ry binding by 13 anthranilic diamides and 13 phthalic diamides described above with Heliothis RyR were also performed with Musca RyR under the same conditions. The toxicological profile was defined for the [3H]Chlo binding site but not for the [3H]Flu site because the latter radioligand does not undergo sufficient specific binding for meaningful determinations. Some anthranilic diamides inhibit [3H]Chlo binding and can greatly stimulate [3H]Ry binding (Figure 7A), whereas phthalic diamides can double [3H]Chlo binding with less effect on [3H]Ry binding (Figure 7B,A+B). The diamides were assayed both in vitro and in vivo, giving a good correlation between the findings for [3H]Chlo binding with anthranilic diamides but a poor correlation with phthalic diamides (Supporting Information Figure 1) and the

Figure 5. Heliothis and Spodoptera insecticidal activity of anthranilic diamides relative to potency for inhibition of Heliothis [3H]Chlo (A) and [3H]Flu (B) binding and stimulation of [3H]Ry (C) binding assayed at 30 nM test compound. Toxicity data for Heliothis are used for eight compounds (○) and for Spodoptera with the other three compounds (5, 6, and 8) (△) where Heliothis data were not available. The Heliothis value averaged 90 ± 30% of the Spodoptera value for the eight compounds assayed with both species (see Supporting Information Table 1).

same trend for [3H]Ry binding assays (Supporting Information Figure 2). Diamide Toxicological Profiles for [3H]Chlo and [3H]Ry Binding Relative to Insecticidal Activity. [3H]Chlo (Figure 8A,B) and [3H]Ry (Figure 8C,D) binding site profiles were compared with the insecticidal activities for the anthranilic and phthalic diamide sets. The insecticidal activity of the anthranilic and phthalic diamides is poorly related to their inhibition of [3H]Chlo binding but better correlated with their stimulation of [3H]Ry binding. Generally similar results were observed with in vivo binding studies (Supporting Information Figures 1 and 2). Species Differences in Binding Sites for [3H]Chlo and 3 [ H]Ry. In the binding studies with Heliothis and Musca, 4079



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Figure 7. Musca toxicological profiles for anthranilic (A) and phthalic (B) diamides or both (A+B) relative to effects on [3H]Chlo and [3H]Ry binding assayed at 30 nM test compound.

Figure 6. Heliothis toxicological profiles for anthranilic diamide and phthalic diamide inhibition of [3H]Chlo (A) and [3H]Flu (B) binding versus stimulation of [3H]Ry binding assayed at 30 nM test compound.

diamides.19,20 To date, neither Chlo21 nor Flu22 has been found to exhibit cross resistance with other commercial insecticides. All field-evolved resistant lepidoptera populations that have been tested against the two diamides have shown cross-resistance between Chlo and Flu, which appears to be due to a single amino acid mutation in the RyR C-terminal transmembrane domain, which encodes the proposed binding site of diamides.10 There is high homology between lepidoptera RyR genes; thus, the conserved transmembrane region might contribute to the same binding sites for Chlo and Flu. A set of anthranilic diamides and another of phthalic diamides were evaluated with [3H]Chlo and [3H]Flu binding assays as predictors of toxicity using data from ingestion of the anthranilic diamides by lepidoptera larvae in the Bayer laboratory and injection of both diamide chemotypes into Musca in the Berkeley laboratory. In Heliothis, the [3H]Chlo and [3H]Flu binding assays were good predictors of toxicity for both the anthranilic and phthalic diamides. In Musca, the binding site−toxicity correlations were less favorable or absent, possibly due in part to diamide detoxification. Although the data are not shown, the in vivo RyR inhibition in Musca was nearly the same for injections into either the thorax or the abdomen, indicating that thoracic muscle RyR binding occurred mostly in vivo rather than during the binding assay. The potency of diamides as inhibitors of [3H]Chlo and 3 [ H]Flu binding is generally correlated with their effectiveness in stimulating [3H]Ry binding; that is, binding at the Chlo/Flu site opens or increases the open time of the Ca2+ channel. A

anthranilic diamides generally inhibit [3H]Chlo binding (Supporting Information Figure 3A) and stimulate [3H]Ry binding (Supporting Information Figure 3B). In contrast, phthalic diamides generally inhibit [3H]Chlo binding in Heliothis but stimulate binding in Musca (Supporting Information Figure 3A). The stimulation of [3H]Ry binding by anthranilic diamides is much greater in Musca than in Heliothis (Supporting Information Figure 3B).

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DISCUSSION The RyR is recognized as a major target for insecticide action, and now more and more genomic analysis information is available on target and nontarget insect species.17,18 The present study is an early step in evaluating species variations in insect RyR toxicology. There appears to be more than one type of diamide site in insects. In Musca the [3H]Chlo, [3H]Ry, and [3H]Flu binding sites are conformationally different, but in Heliothis RyR, anthranilic and phthalic diamide insecticides displace each other when determined as either [3H]Chlo or [3H]Flu binding and therefore probably bind at the same or closely coupled sites. Mammalian genomes express three different isoforms of the RyR, whereas insect genomes encode only a single RyR gene, and the low amino acid identity between mammal and insect RyRs probably contributes to the high selectivity of 4080



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Article

ASSOCIATED CONTENT

S Supporting Information *

Table of the diamides studied and effects on [3H]Chlo and [3H]Flu binding. Three supplemental figures, two for Musca toxicological profiles and the third comparing [3H]Chlo and [3H]Flu toxicological profiles between Heliothis and Musca. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.E.C.) Phone: (510) 642-4524. Fax: (510) 642-6497. E-mail: [email protected]. Present Address ⊥

(S.Q.) College of Science, China Agricultural University, Beijing, China 100193.

Funding

S.Q. was supported in part by State Scholarship Fund No. 2011635138 provided by the Chinese Scholarship Council. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Masanori Tohnishi and colleagues at Nihon Nohyaku as well as Rüdiger Fischer at Bayer CropScience for kindly supplying phthalic diamides and anthranilic diamides, respectively. S.Q. thanks Professor Chengju Wang of China Agricultural University for academic counsel. Helpful advice and assistance were provided by Chunqing Zhao, Xusheng Shao, and Breanna Ford of the Berkeley Laboratory.

Figure 8. Musca insecticidal activity of anthranilic (A) and phthalic (B) diamides relative to potency for inhibition of [3H]Chlo binding (A, B) and stimulation of [3H]Ry (C, D) binding assayed at 30 nM test compound.

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ABBREVIATIONS USED Chlo, chlorantraniliprole; Cyan, cyantraniliprole; Flu, flubendiamide; Ry, ryanodine; RyR, ryanodine receptor

series of Chlo analogues was tested with Musca RyR both in vitro and in vivo for effects on [3H]Ry binding with stimulation in most cases, a relationship also observed with a number of Flu analogues, further generalizing the diamide activity as a stimulator of the [3H]Ry site. Thus, the observation of enhanced [3H]Ry binding seems to be a reliable indicator of diamide action at the Ca2+-modulated Ca2+ channel. The stimulation of [3H]Chlo binding to Musca membranes by phthalic diamides may be due to the fact that their affinity for the Musca RyR is much lower (our estimate of the apparent Kd of Flu is about 185 nM) than for the Heliothis RyR. Therefore, the phthalic diamide concentrations used in this study may not be sufficient to provide effective competition with the high-affinity radioligand [3H]Chlo, as is the case with the Heliothis receptor having a higher affinity for phthalic diamides. An increase in the open probability of the Musca RyR induced by phthalic diamides could plausibly explain the stimulatory effect on [3H]Chlo binding by increasing the number of radioligand binding sites. The underlying assumption is that diamides are conformation-sensitive ligands which preferentially bind to the open channel as shown for [3H]Flu.23 Continuing progress in defining insect RyR regions critical to both anthranilic and phthalic diamide sensitivity is helping to define the basis for diamide selectivity among insect species and between insects and mammals.24

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Diamide Insecticide Target Site Specificity in the Heliothis and Musca

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