Development of pyrethroid-like fluorescent substrates for glutathione S-transferase

Abstract

The availability of highly sensitive substrates is critical for the development of precise and rapid assays for detecting changes in glutathione S-transferase (GST) activity that are associated with GST-mediated metabolism of insecticides. In this study, six pyrethroid-like compounds were synthesized and characterized as substrates for insect and mammalian GSTs. All of the substrates were esters composed of the same alcohol moiety, 7-hydroxy-4-methylcoumarin, and acid moieties that structurally mimic some commonly used pyrethroid insecticides, including cypermethrin and cyhalothrin. CpGSTD1, a recombinant Delta class GST from the mosquito Culex pipiens pipiens, metabolized our pyrethroid-like substrates with both chemical and geometric preference (i.e., the cis-isomers were metabolized at 2- to 5-fold higher rates than the corresponding trans-isomers). A GST preparation from mouse liver also metabolized most of our pyrethroid-like substrates with both chemical and geometric preference but at 10- to 170-fold lower rates. CpGSTD1 and mouse GSTs metabolized 1-chloro-2,4-dinitrobenezene (CDNB), a general GST substrate, at more than 200-fold higher rates than our novel pyrethroid-like substrates. There was a 10-fold difference in the specificity constant (k(cat)/K(M) ratio) of CpGSTD1 for CDNB and those of CpGSTD1 for cis-DCVC and cis-TFMCVC, suggesting that cis-DCVC and cis-TFMCVC may be useful for the detection of GST-based metabolism of pyrethroids in mosquitoes.

Fig. 1

Chemical structures of the pyrethroid-like ester compounds generated and studied in this study. Abbreviations: TMC, 4-methyl-2-oxo-2H-chromen-6-yl, 2, 2, 3, 3-tetramethylcyclopropanecarboxylate; DMVC, 4-methyl-2-oxo-2H-chromen-6-yl, cis/trans- 2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropanecarboxylate; cis-DCVC, 4-methyl-2-oxo-2H-chromen-6-yl, cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclo propanecarboxylate; trans-DCVC, 4-methyl-2-oxo-2H-chromen-6-yl, trans-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylate; cis-TFMCVC, 4-methyl-2-oxo-2H-chromen-6-yl, cis-3-((Z)-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate; 1R trans-TFMCVC, 4-methyl-2-oxo-2H-chromen-6-yl, 1R trans-3-((Z)-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate.

Fig. 2

The effect of glutathione concentration on CpGSTD1 metabolic activity towards cis-DCVC. The reactions were performed in 90 mM sodium phosphate buffer, pH 7.4, containing 79 ng of CpGSTD1, 50 μM cis-DCVC, various concentrations of GSH (0.78 to 1,500 μM), and 2% (v/v) DEGEE at 34°C.

Fig. 3

Correlation between GST activity and protein concentration. The reactions were performed in 90 mM sodium phosphate buffer, pH 7.4, containing various amounts of CpGSTD1 (23.5 to 188 ng), 50 μM cis-DCVC, 750 μM GSH, and 2% (v/v) DEGEE at 34°C.

Fig. 4

LC/MS ESI⁺ profiles of the metabolites of the pyrethroid-like ester compounds following incubation with CpGSTD1. The reactions were performed in 90 mM sodium phosphate buffer, pH 7.4, containing 720 ng of CpGSTD1, fluorescent substrate (50 μM), 750 μM GSH, and 2% (v/v) DEGEE at 34°C. The profiles are from top to bottom: TMC, DMVC, cis-DCVC, trans-DCVC, cis-TFMCVC, and 1R trans-TFMCVC. Peaks corresponding to the predicted mass (m/z) of the GS-conjugates of DMVC, cis-/trans-DCVC, and cis-/trans-TFMCVC are indicated by the letters A (m/z 458 Da), B (m/z 498 Da), and C (m/z 532 Da), respectively. A peak corresponding to the predicted mass (m/z 432 Da) of the GSH-conjugated acid metabolite of TMC was not detected.

Fig. 5

Three potential mechanisms of the metabolism of our pyrethroid-like ester compound DCVC by GST involving halogen substitution, Michael addition, and thiolysis.