The G119S-substituted AChE1 appeared as a suitable candidate for the development of reverser compounds because AChE are highly structurally constrained [19] and the G119S mutation is widely distributed worldwide in mosquitoes and associated with a substantial fitness cost in insecticide-free areas [20,21,22,23,24]. This AChE1 constraint constitutes a weakness in its adaptive capacity that might be used to develop innovative resistance management strategies: an insecticide targeting specifically the G119S AChE1 should efficiently reduce the frequency of the resistance allele, while the probability of OP-resistant mosquitoes developing a secondary resistance to the new insecticide is predicted to be quite low. To address the feasibility of this approach, compounds were screened for their capacity to inhibit more efficiently the G119S-substituted (OP-insensitive) AChE1 than the wild type (WT) AChE1. Further biochemical analysis, bioassays on mosquito larvae from susceptible and resistant A. gambiae and C. pipiens strains sharing the same genetic background, as well as gene population modeling show that application of compounds with such properties is predicted to rapidly restore OP susceptibility in field populations.
preferential inhibition on other types of OP-insensitive AChE1 (F290V and F331W substitutions) (Table S2). Last, PTFs behaved as reversible and competitive inhibitors; Loss of inhibition after dilution of the enzyme-inhibitor complex indicates that PTFs do not bind covalently to AChE1 (Figure S1); Plotting 1/v (Dixon plots) and [S]/v (Cornish-Bowden plots) against inhibitor concentration (Figure S2) indicates that inhibition is competitive since for various substrate concentrations, the enzyme maximal velocity Vmax did not vary (panels A and B) and [S]/v lines were parallel (panels C and D). Comparison of dissociation constants of the enzyme inhibitor complexes (Kic) confirmed the preferential binding to the OP-insensitive AChE1 (Table S3).PTF insecticide activity on OP-susceptible and resistant strains. The 71 analogs were screened for their toxicity on OP-Results Identification of Inhibitors of the OP-insensitive G119S AChE1
To identify inhibitors of G119S AChE1, we screened a 3,000compound chemical library using an assay adapted to a microplate format [25,26]. Nine compounds reduced the apparent hydrolysis of the acetylthiocholine substrate, of which one gave an 80?7% inhibition. The core structure of this compound is made of a heterocyclic 2,4,6-pyrimidinetrione (barbituric acid) substituted by a furan cycle (Figure 1A). To improve the efficacy, 71 commercial analogs sharing the core structure with various substitutions on R1, R2, R3, Q1 and Q2 positions were assayed (Figure 1B). For simplification, these compounds will be termed from now as PTF (PyrimidineTrione Furan-substituted) and were classified depending on the substituted positions (Table S1). PTFs exhibited a broad IC50 range on WT and G119S AChE1 (Table S1), from 0.23 mM to 4.5 mM. When sorted by their relative efficacy toward the G119S AChE1 (Table 1), 31 analogs had a RIC50 (ratio of the mean IC50 on WT over the mean IC50 on G119S AChE1) above that of the hit compound (PTF-24, RIC50 = 0.7).
Seven analogs had a RIC50 above 10, indicating a much higher efficacy toward G119S AChE1 than OP-sensitive AChE1. Six among the most efficient and specific PTFs (in bold, Table 1) also showed susceptible (Slab strain) or OP-resistant (SR stain) C. pipiens larvae. As a first approach, each PTF was applied at 300 mM for 24 hours (Table S1). Sixteen PTFs were at least 50% more efficient on OPresistant larvae (ratio Rm300 of mortality of SR larvae over mortality of Slab larvae above 1.5 in Table 1), among which thirteen were also selective as biochemical inhibitors (RIC50 ratios above 1.5, Table 1). We ensured that the mortality induced by PTFs was associated with AChE1 inhibition by measuring the residual AChE1 activity in killed C. pipiens SR larvae (Figure 2). Exposure to PTFs elicited a 55 to 70% reduction in AChE1 activity, within the range of that elicited by exposure to chlorpyrifos (75%), at a dose where it kills OP-resistant larvae through AChE1 inhibition. This demonstrates that PTF larvicidal activity is a consequence of in vivo AChE1 inhibition. To investigate further the larvicidal activities of PTFs, bioassays were performed on OP-resistant and OP-susceptible C. pipiens and A. gambiae larvae (Table 2). PTF-3, -10, -20, -25, -29 and -39 had LD50 ranging from 70.1 to 398.8 mM on OP-resistant C. pipiens larvae (SR strain) and from 160.7 to 964 mM on susceptible ones (Slab strain), with RLD50 (ratio of the mean LD50 on susceptible strain over the mean LD50 on resistant strain) ranging from 1.5 to 3.9. Anopheles gambiae had a similar pattern of susceptibility: PTFs preferentially killed OP-resistant larvae (Acerkis strain), with RLD50 values ranging from 1.3 to 7.7. Each PTF showed species-specific toxicity, but to our knowledge, PTFs are the only molecules that display a higher toxicity on OP-resistant G119S strains than on susceptible strains, both in C. pipiens and in A. gambiae. To determine the dominance of the larvicidal activity of PTFs against ace-1 alleles, six PTFs were tested on heterozygous (ace-1R/ ace-1S) larvae produced by a cross between Slab and SR strains, and also on the C. pipiens Ducos strain, harboring the duplicated ace-1 allele in which one ace-1S and one ace-1R copy are arranged in tandem at the homozygous state [27]). Although the Ducos strain showed slightly less susceptibility, all PTFs tested showed a similar efficacy on [R/S] heterozygotes phenotypes compared to [R/R] homozygotes larvae, indicating dominance of the OP-insensitive ace-1R allele (Table 2). PTFs as predicted to restore OP-susceptibility. To examine what would be the impact of PTFs or compounds of similar properties on the evolution of the frequency of resistant alleles in natural populations, we ran a simulation in which the initial infinite and panmictic population contained 10% of OPsensitive allele (ace-1S), a situation already observed in several OPtreated areas [28,29,30], and in which there was no migration.
