Acetylcholinesterase (AChE) is a key enzyme for the transmission of the nerve impulse. This enzyme has a hydrophobic gorge of approximately 20 Å separating an acylation catalytic site, and a peripheral site located at the entry of the gorge. Organophosphorous Nerve agents (pest control or warfare agents) act as AChE irreversible inhibitors. Enzyme inhibition occurs through phosphorylation of the serine of the acylation site, forming a phosphonic diester. This diester can be hydrolized, more or less effectively according to the organophosphorus nerve agents used via '-nucleophilic like oximes. Without external intervention, the phosphonic diester is transformed by the enzyme into phosphonic acid (so called 'ageing' of the poisoned AChE). Yet, after more than 50 years of investigation there is no broad spectrum reactivator; and the few existing efficient reactivators do not effectively reactivate AChE inhibited by every type of nerve agent. The aim of this project is thus, through two complementary approaches, rational design in a one hand and a target guided combinatorial synthesis associated with a hightroughput screening in the second hand, to find new and more efficient reactivators, with the ultimate goal in mind to find a broad spectrum reactivator able to penetrate the Blood Brain Barrier . The selected approach lies in the observation that the most effective AChE reactivators are homo or hetero-dimers, bearing a reactive oxime function, and able to bind to both enzyme catalytic sites. The goal is thus to use this synergy between the various binding sites of the enzyme to lead to an optimized positioning of the nucleophile towards the phosphorylated serine. It is thus based on several aspects: 1 - The synthesis of two libraries, one of alpha nucleophiles, able to bind poisoned AChE phosphorylated catalytic site, and the other one of peripheral site (or hydrophobic gorge)ligands. These PAS ligands (either based on known structures -phenanthridiniums, isoquinolines- or original ones as coumarines, aflatoxin or hybrids derived from cytisine) will be chosen for their ability to induce a significant conformational change of catalytic site of the enzyme, which should allow the nucleophile to adopt a reactive position towards the phosphorylated serine. 2 - The evaluation of the binding mode of the different targeted ligands via docking experiments onto the poisoned enzyme active site, to determine the accessibility of the active site on the one hand and on the other hand the relative influence of each binding mode on the positioning of nucleophilic towards the phosphorylated serine. 3 - The synthesis of heterodimers displaying a synergetic binding ability. This will be done using two strategies, the first one based on a rational design based on the known structure of AChE active sites, the second one via an adaptation of the principle of in situ click chemistry. Both strategies will be prepared via the addition of linker attachment points on both libraries members. 4 - The production of recombinant human or mouse AChE, their poisoning by differtent nerve agents, and the evaluation of their reactivation efficiency via HTS assays. 5 - The crystallization of the complexed enzymes (recombinant human or mùouse AChE) with the new reactivators, in order to determine its position in the enzyme biding site, and assess the changes which could be made in order to further optimize their properties. The expected results in this study are three-fold: on the one hand provide a more effective treatment for organophosphorus nerve agents poisonning, whether chronic (pesticides), or acute (chemical warfare), either via new reactivating functions, or via allosteric acting heterodimers. On the other hand broaden the possibilities offered by in situ click chemistry. Finally, the modeling of interactions and the crystallization of reactivator-enzyme complex should help to understand the mode of action and to open up avenues for new and more efficient reactivators. The project is strongly backed by the opportunity to gather in a single collaborative effort the whole spectrum of the required tasks : molecular modelling, organic chemistry, recombinant enzymes production, ability to work with the real nerve agents, and thus to obtain reasonable quantity of the phosphorylated enzymes, X ray diffraction facility