Differential metabolism of sulfoximine and neonicotinoid insecticides by Drosophila melanogaster monooxygenase CYP6G1

https://doi.org/10.1016/j.pestbp.2012.05.006Get rights and content

Abstract

Sulfoxaflor [N-[methyloxido[1-[6-(trifluoromethyl)-3-pyridinyl]ethyl]-λ4-sulfanylidene] cyanamide] is in development as the first product from the new sulfoximine class of insect control agents. Highly effective against a variety of sap-feeding pest insects, available data indicate no cross-resistance to sulfoxaflor in pest insect strains that exhibit high levels of resistance to neonicotinoids and other insecticides. In vitro studies of the cytochrome P450 monooxygenase CYP6G1 from Drosophila melanogaster, expressed in a Drosophila cell line, show very high levels of metabolism for a variety of neonicotinoids, but not for sulfoxaflor and its chloropyridine-analog. A sulfoxaflor analog with nitrogen in place of the carbon in the bridge between the pyridine and sulfoximine moiety shows a modest degree of metabolism. In silico homology modeling of the CYP6G1 with the sulfoximines and neonicotinoids suggests that steric effects may limit interactions of the sulfoximines with the reactive heme-oxo complex. A distinct relationship was identified for the summed Hückel charges and the degree of metabolism observed. These observations help explain the lack of sulfoxaflor metabolism by CYP6G1, and in turn provide a basis for the lack of cross-resistance to sulfoxaflor in insecticide resistant strains of pest insects.

Highlights

► The monooxygenase CYP6G1 was cloned and expressed in D.mel-2 cells. ► Sulfoxaflor (SFX) and its chloropyridine-analog were not metabolized by CYP6G1. ► All neonicotinoids tested were metabolized by CYP6G1. ► Homology models of CYP6G1 suggest differences in binding between SFX and neonicotinoids. ► Differences in metabolism may explain the lack of cross-resistance to SFX.

Introduction

Integrated pest management (IPM) programs require an array of options for insect control. Since the early 1990s the control of many sap feeding insects, such as aphids and whiteflies, has increasingly relied on the neonicotinoid insecticides [1]. However, following two decades of extensive use, there are now a number of cases of resistance to the neonicotinoids, especially imidacloprid [2], [3], [4]. Thus, there is a need for alternative insecticides to control sap-feeding insect pests.

The new insect control agent sulfoxaflor [N-[methyloxido[1-[6-(trifluoromethyl)-3-pyridinyl]ethyl]-λ4-sulfanylidene] cyanamide; Fig. 1] is in development as the first product from a new class of insecticides built around a novel chemical moiety, sulfoximine [5]. Sulfoxaflor is active on a broad spectrum of sap-feeding insects including aphid, whitefly, hopper, mealy bug, and Lygus pests [5], [6], [7]. Like the neonicotinoids, the spinosyns, and the nereistoxin analogs, the sulfoximines interact with the nicotinic acetylcholine receptor (nAChR), but in a manner that is distinct from other chemistries [5], [8]. Additionally, the sulfoximines exhibit excellent efficacy against insects resistant to neonicotinoids [5], [6].

Studies with target site mutants of Drosophila melanogaster indicate that sulfoxaflor is little affected by mutations in the nAChR conferring resistance to a variety of neonicotinoids and spinosyns [9]. However, with one exception [4], resistance to date for the neonicotinoids in the field is associated with enhanced metabolism, usually by cytochrome P450 monooxygenases [3], [10], [11]. The novel chemical structure of sulfoxaflor and the lack of cross-resistance in strains of aphids, whiteflies and planthoppers resistant to the neonicotinoids [5], [6] suggest that sulfoxaflor may not be good substrate for the cytochrome P450 monooxygenases involved in neonicotinoid resistance [5]. Therefore, we examined the in vitro cell-based metabolism of a selected set of sulfoximines and neonicotinoids (Fig. 1) by a cytochrome P450 monooxygenase (CYP6G1) linked to neonicotinoid resistance in D. melanogaster [12], [13], [14]. For these studies we cloned CYP6G1 and expressed the gene in a D.mel-2 cell line. In silico homology modeling along with other physicochemical parameters, were then used to provide a conceptual basis for the in vitro metabolism results.

Section snippets

Chemicals

The sulfoximines were prepared at Dow AgroSciences and the neonicotinoids were purchased from Chem Service (West Chester, PA). All other chemicals were from conventional commercial sources.

CYP6G1 PCR amplification

The CYP6G1 gene was amplified from adult D. melanogaster 1st strand cDNA synthesized from larval mRNA (Clontech Laboratories, Mountain View, CA), using Superscript™ III reverse transcription kit (Invitrogen, Carlsbad, CA) [5]. Briefly, the primers added Bam HI sites to both ends of the CYP6G1 gene and a

CYP6G1-mediated metabolism

Incubation of the sulfoximines or neonicotinoids with D.mel-2 cells lacking the CYP6G1 transgene resulted in near complete recovery of all of the compounds (Table 1). When imidacloprid or acetamiprid were incubated with D.mel-2 cells expressing the CYP6G1 gene, there was little recovery of the parent compounds. LCMS analyses of D.mel-2 extracts showed metabolism of acetamiprid to N-demethyl acetamiprid (Fig. 3), and the metabolism of imidacloprid to a hydroxylated metabolite and the related

CYP450 metabolism and resistance

Altered cytochrome P450 monooxygenases are important mechanisms of resistance for a variety of insecticides in a number of insect species [13], [19], [20]. The lack of cross-resistance observed for sulfoxaflor in a range of neonicotinoid resistant insect species (5,6) suggested that the resistance mechanisms for the neonicotinoids in these insects may not be effective against the sulfoximine insecticides. Because neonicotinoid resistance has typically been associated with the presence of

Conclusion

Sulfoxaflor is effective against a variety of insect populations resistant to the neonicotinoids and other insecticides (5–7). The present investigation establishes that, compared to the neonicotinoids examined, sulfoxaflor is relatively unaffected by a D. melanogaster cytochrome P450 monooxygenase, CYP6G1, known to be involved in resistance to the neonicotinoids and other insecticides. The present result suggests that the unique chemical structure of sulfoxaflor may be one factor that limits

Acknowledgments

We thank Drs. Dave Demeter, Chaoxian Geng and Frank Wessels for useful discussions and comments on the manuscript.

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