Abstract
Resistance to insecticides has become a critical issue in pest management and it is particularly chronic in the control of human disease vectors. The gravity of this situation is being exacerbated since there has not been a new insecticide class produced for over twenty years. Reasoned strategies have been developed to limit resistance spread but have proven difficult to implement in the field. Here we propose a new conceptual strategy based on inhibitors that preferentially target mosquitoes already resistant to a currently used insecticide. Application of such inhibitors in rotation with the insecticide against which resistance has been selected initially is expected to restore vector control efficacy and reduce the odds of neo-resistance. We validated this strategy by screening for inhibitors of the G119S mutated acetylcholinesterase-1 (AChE1), which mediates insensitivity to the widely used organophosphates (OP) and carbamates (CX) insecticides. PyrimidineTrione Furan-substituted (PTF) compounds came out as best hits, acting biochemically as reversible and competitive inhibitors of mosquito AChE1 and preferentially inhibiting the mutated form, insensitive to OP and CX. PTF application in bioassays preferentially killed OP-resistant Culex pipiens and Anopheles gambiae larvae as a consequence of AChE1 inhibition. Modeling the evolution of frequencies of wild type and OP-insensitive AChE1 alleles in PTF-treated populations using the selectivity parameters estimated from bioassays predicts a rapid rise in the wild type allele frequency. This study identifies the first compound class that preferentially targets OP-resistant mosquitoes, thus restoring OP-susceptibility, which validates a new prospect of sustainable insecticide resistance management.ou L, Leonetti J-P, et al. (2012) Novel AChE Inhibitors for Sustainable Insecticide Resistance Management. PLoS Citation: Alout H, Labbe P, Berthomieu A, Djogbe Editor: Basil Brooke, National Institute for Communicable Diseases/NHLS, South Africa Received May 14, 2012; Accepted September 10, 2012; Published October 8, 2012 Copyright: ?2012 Alout et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ?Environnement (Ministe De ue a la `re e ?` Funding: This work received financial support by the ANR (Agence Nationale pour la Recherche), Morevol Sante ?Recherche) and Contribution 2012-XXX of the Institut des Sciences de L’Evolution de Montpellier (UMR (Unite Mixte de Recherche) CNRS (Centre National de la Recherche Scientifique) – UM2 5554). HA was supported by ANR (3- year grant) and CNRS (one-year SPV grant). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Pending is an international patent entitled “Use of compounds derived pyrimidinetrione as acetylcholinesterase inhibitors, composition containing said derivatives, and the uses thereof”, registered 10 February 2006 under the number 11/816,032. This does not alter the authors’ adherence to all the policies on sharing data and materials.
Introduction
Organophosphates (OP), carbamates (CX) and pyrethroids represent, by number, 80% of insecticides used in the field (reviewed in [1]). These molecules act on the nervous system through inhibition of acetylcholinesterase (OP and CX) or voltage-gated sodium channels (pyrethroids and DDT). The major setback of insecticide use is the selection for resistance, observed not only in the targeted pests but also in many other sympatric species [2,3]. At the physiological level, resistance is a consequence of either increased detoxication or modification of the insecticide target, the latter often resulting in very high insensitivity [4,5]. However both mechanisms may be responsible for vector control failure and have to be addressed by insecticide resistance management strategies. Resistance has spread to such an extent, particularly in mosquito vector populations, that it now represents a critical issue for the control of the diseases they transmit, e.g. malaria, dengue, filariasis, West Nile fever or Japanese encephalitis [6]. Sustainable strategies to counter resistance spread aim at maintaining resistant alleles at frequencies low enough so that current insecticides remain efficient even at moderate doses. As an example, the reasoned use of insecticides through rotations or mosaic applications takes advantage of the pleiotropic cost (i.e. the reduced fitness of resistant vs. wild type individuals in an insecticide-free environment) to maintain resistant alleles at low frequencies (reviewed in [7]). Essentially used for malaria control, fungi also represent promising tools because they kill mosquitoes at slower rate than insecticides thus reducing the risk of resistance selection [8,9,10]. Here we propose an alternative approach based on the development of “resistant killer” compounds, capable of preferentially inhibiting targets already insensitive to a given insecticide class. Combined with the fitness cost already associated with resistance, populations treated with such “resistant killers” are thus expected to regain a high frequency of susceptible wild type
alleles, a “hit where it already hurts” strategy. Ideally, the targeted protein should be highly constrained structurally to minimize its capacity to evolve through the selection of new mutations that would confer resistance to both the insecticide and the “resistant killer” compound. A good candidate is acetylcholinesterase (AChE, EC 3.1.1.7), which in Coelomates acts as a synaptic terminator of nerve impulses through hydrolysis of the neurotransmitter acetylcholine. Mosquitoes contain two AChE genes (ace-1 and ace-2), ace-1 encoding the synaptic enzyme [11,12]. So far, only three substitutions on residues lining the catalytic site confer OP and CX insensitivity to AChE1: the F331W substitution (amino-acid numbering according to the Torpedo californica AChE nomenclature [14]), found only in Culex tritaeniorhynchus [13,14], the F290V substitution, found only in C. pipiens species [13], and the universally found G119S substitution, which confers the highest level of insensitivity to a broad range of insecticides and was selected independently in several Culex and Anopheles species (C. pipiens pipiens, C. pipiens quinquefasciatus, C. vishnui, A. gambiae and A. albimanus, [15,16,17,18]).