Selective and Irreversible Inhibitors of Mosquito Acetylcholinesterases for Controlling Malaria and Other Mosquito-Borne Diseases

Abstract

New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 µM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast (∼30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.

Novel Selective and Irreversible Mosquito Acetylcholinesterase Inhibitors for Controlling Malaria and Other Mosquito-Borne Diseases

“…Our previously reported methanethiosulfonates form a disulfide bond with the insect-specific cysteine1215, and the methanethiosulfonate adducts are unstable in the presence of a disulfide-bond–cleavage agent during the digestion process. For this reason, we set out to develop maleimide-containing inhibitors that form a carbon-sulfur bond to Cys286, thereby their adducts are stable during the digestion process.…”

“…The PYn series was designed purposely to have reduced affinity for the active site to investigate the effect of the inhibitor affinity on the inhibitor reactivity toward Cys286. The use of long alkylene chains in the prototypes was based on the chain-length–activity relationship of our reported irreversible AP-AChE inhibitors1215 and supported by 100 10-ns-long molecular dynamics simulations (each with unique initial velocities and a 1.0-fs time step) of agAP-AChE in reversible complex with PM20 using an explicit water model282930. These simulations predicted that PM20 was capable of spanning the active site of agAP-AChE with its pyridinium group forming cation-pi interactions with Trp84, Tyr121, Tyr130, and Tyr328 and with its maleimide alkene carbon atom located 3.6 Å away from the sulfur atom of Cys286 (Fig.…”

“…Following this reasoning, we developed methanethiosulfonate derivatives that selectively and irreversibly inhibit insect AP-AChEs presumably through conjugation to the insect-specific cysteine1215. However, no direct proof of the conjugation of a sulfhydryl agent to the insect-specific cysteine in AP-AChE has been presented to date, and doubts have remained about whether the insect-specific cysteine is accessible for conjugation and whether cysteine-targeting AP-AChE inhibitors can be developed with kinetic properties comparable to those of insecticides.…”

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

“…Incubation with 6 μM 3d – n for 1 h caused >95% inhibition of Ag AChE, 75–90% inhibition of C. pipiens AChE, and 60–80% inhibition of Ae, aegypti AChE (Fig. 3) [33]. Treatment of 3l ( n = 18)-treated Ag AChE with 2-mercaptoethanol again led to substantial recovery of enzymatic activity.…”

“…To assess the level of reactivity needed to ligate Cys286, the less reactive 1° bromide 4 was then assessed as an inhibitor. But following a 2.5 h incubation at 1 μM, only a 10% reduction in Ag AChE activity was observed [33]. …”