Novel selective and irreversible mosquito acetylcholinesterase inhibitors for controlling malaria and other mosquito-borne diseases

Abstract

We reported previously that insect acetylcholinesterases (AChEs) could be selectively and irreversibly inhibited by methanethiosulfonates presumably through conjugation to an insect-specific cysteine in these enzymes. However, no direct proof for the conjugation has been published to date, and doubts remain about whether such cysteine-targeting inhibitors have desirable kinetic properties for insecticide use. Here we report mass spectrometric proof of the conjugation and new chemicals that irreversibly inhibited African malaria mosquito AChE with bimolecular inhibition rate constants (k(inact)/K(I)) of 3,604-458,597 M(-1)sec(-1) but spared human AChE. In comparison, the insecticide paraoxon irreversibly inhibited mosquito and human AChEs with k(inact)/K(I) values of 1,915 and 1,507 M(-1)sec(-1), respectively, under the same assay conditions. These results further support our hypothesis that the insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides with reduced insecticide resistance and low toxicity to mammals, fish, and birds for the control of mosquito-borne diseases.

Figure 1. New chemicals that target an insect-specific cysteine of AP-AChEs.

Upper left panel: cross-section of the AP-AChE and AChE active sites showing the locations of the insect-specific cysteine and the corresponding residue in non-insect species; upper right panel: chemical structures of PMn, PYn, PYS18, and PMS20; middle panel: two-step quiescent affinity labeling mechanism for PMn and PYn and definition of kinetic parameters; lower panel: syntheses of PMn, PYn, PYS18, and PMS20. DCM: CH₂Cl₂; DMF: N,N′-dimethylformamide; NBS: N-bromosuccinimide; TFA: CF₃CO₂; THF: tetrahydrofuran; : reflux.

Figure 2. Close-up view of agAP-AChE in reversible complex with PM20 predicted by microsecond molecular dynamics simulations.

The nitrogen, oxygen, and sulfur atoms are in blue, red, and green, respectively. The carbon atoms in agAP-AChE and PM20 are in tangerine and yellow, respectively. The mesh depicts the portion of PM20 that is inserted in the active site of agAP-AChE. The simulation protocol is provided in the Supplementary Information.

Figure 3. Time-course and dilution experiments for the inhibition of agAP-AChE and hAChE by PY18, PM20, PMS20, paraoxon, and NEM.

% enzyme activity: the enzyme activity compared to that without inhibitor treatment. Before the 10-fold dilution, the concentrations of PY18, PM20, PMS20, paraoxon, and NEM were 0.1, 0.001, 1.67, 0.2, and 100 μM for the agAP-AChE inhibition assays and 6.67, 0.5, 0.833, 0.2, and 100 μM for the hAChE inhibition assays, respectively.

Figure 4. Mass spectrometric proof of the conjugation of PM20 to Cys286 in agAP-AChE.

Upper panel: high-resolution Fourier transform mass spectrometry survey scan of the [M+H]⁺² ion (m/z of 1082.6122) corresponding to the PM20-labeled fragment of agAP-AChE with calculated (cal.) m/z of 1082.6120. Middle panel: supporting tandem mass spectrum showing the observed b- and y-type ions resulting from fragmentations of the [M+H]²⁺ ion. Lower panel: mechanisms for the oxazolone formation and b-ion cyclization.

Figure 5. Progressive AChE inhibition as a function of time and inhibitor concentration indicating the two-step quiescent affinity labeling mechanism for the test inhibitors.

% enzyme activity: the enzyme activity compared to that without inhibitor treatment. Left: linear plots of natural log of % AChE activity versus time in second at different inhibitor concentrations; right: nonlinear plots of the observed inhibition rate in 1/second versus inhibitor concentration in nanomolar.

Figure 6. Determination of equilibrium dissociation constants (K_i).

Left: reciprocal hydrolysis rate (1/v in second per optical density) was plotted against reciprocal substrate concentration (1/[ATCh] in 1/mM) in the absence and presence of an inhibitor at varying concentrations; right: the slope of the double reciprocal plot was plotted against inhibitor concentration ([I] in μM). K_i was obtained from the negative x intercept of the slope replot.

Figure 7. Effects of 50 μM NEM and 6.7 nM PM20 on agAP-AChE and hAChE activities.

% enzyme activity: the enzyme activity compared to that without inhibitor treatment.