Abstract
Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity.
Highlights
Glutathione S-transferase (GST emulsifiable concentrate (EC) 2.5.1.18) is an important family of multifunctional isozymes found in all eukaryotes
The positive clones were transformed in BL-21 (DE-3) and protein expression induced by the addition of IPTG was detected initially by SDS-PAGE using standard protein molecular weight marker
The expected band was detected at 27 kDa as the MW of the Helicoverpa armigera glutathione S-transferase gene (HaGST) is about 24 kDa and the pET-28a tag is about 3 kDa (Figure 2)
Summary
Glutathione S-transferase (GST EC 2.5.1.18) is an important family of multifunctional isozymes found in all eukaryotes. GSTs convert a reactive lipophilic molecule into a water-soluble, non-reactive conjugate which may be excreted [2] This family of enzymes has been implicated as one of the major mechanisms for neutralizing the toxic effects of insecticides in insects [3,4]. Cantharidin was first isolated by Robiquet, a French chemist in 1810 It has an important role in the ecology of different kinds of insects that use or produce it as a defense to preserve their eggs from predators [17]. This defensive tool has been developed into a bio-pesticide and one of the formulations as an emulsifiable concentrate (EC) has been registered for the control of lepidopteran pests while other formulations of cantharidin and its analogues are under field trial for registration. Homology modeling and molecular docking simulation techniques were employed to rationalize our experimental results
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