Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Abstract

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.

Discovery of Species-selective and Resistance-breaking Anticholinesterase Insecticides for the Malaria Mosquito

“…A later study used yeast cells to express the catalytic domains of both WT and G119S Ag AChE [22]. This study confirmed the high V max of WT Ag AChE (2,700 U/mg, cf.…”

“…However, the measured k cat (7.4 × 10 3 min −1 ) of the WT Ag AChE enzyme was significantly lower than that obtained by other groups (cf. Table 2) [21,22]. Finally, transcript levels of ace -1 and ace -2 Ag AChE were studied in An.…”

“…In contrast, the agricultural insecticide aldicarb measurably inhibited G119S Ag AChE ( k i = 3.15 ± 0.08 mM −1 min −1 ) and had an SR of only 4.2 [22]. Aldicarb proved quite toxic to Akron strain An.…”

“…Molecular modeling studies of the tetrahedral intermediates of aldicarb with WT and G119S Ag AChE showed that aldicarb suffered no steric interactions in binding to the catalytic serine (S199) of G119S Ag AChE [22]. In contrast, 3-butylphenol-derived methylcarbamate 9 was projected to suffer unfavorable steric interactions with S119 on binding to S199 of G119S Ag AChE.…”

“…As can be seen in Table 4, pyrazol-4-yl methylcarbamates 14 and 15 very rapidly inactivated WT Ag AChE, and they also rapidly inactivated G119S Ag AChE, with k i values ranging from 137 to 290 mM −1 min −1 [22]. Consequently the SR values of these compounds (30–32) was much more favorable than that of the phenol carbamates Consistent with their rapid inhibition of G119S Ag AChE, these compounds proved quite toxic to Akron strain An gambiae , with RR = 0.8 to 1.5 [22]. The considerably lower RR value for these compounds relative to their SR values again points to the role of metabolism and transport in modulating toxicity.…”