PROJECT SUMMARY/ABSTRACT Botulinum neurotoxin serotype A (BoNT/A), which causes the disease botulism, is the most potent toxin known to man. BoNTs are most commonly encountered as BotoxTM, the increasing use of which has made iatrogenic botulism a major concern. BoNTs are one of only six pathogens designated by the CDC as a category A bioterrorism agent due to its toxicity and ease of production. Furthermore, the spread of botulism among heroin users is a growing concern. Despite the potential threat and the severity of the disease, there is no therapeutic available for rescuing the neuronal intoxication that causes botulism. At best, the progression of the disease is mitigated by treatment with a heptavalent antitoxin, which still requires months of hospitalization. Our long-term goal is to develop a clinically viable therapeutic capable of reversing the effects of botulinum neurotoxin, in addition to arresting progress. As BoNT intoxication is a solitary event, we posit that an irreversible covalent inhibitor capable of entering muscle neurons could permanently compromise its catalytic machinery, providing a solution to the discrepancy between the lifetime of a small molecule in neurons and the persistence of the neurotoxin. In contrast to the numerous reports of irreversible inhibitors of serine/cysteine proteases, irreversible inhibition of metalloproteinases is rare, a result of differences in catalytic mechanisms. As such we have devised a strategy wherein a covalent warhead that targets an allosteric reactive residue is tethered to a potent active site inhibitor, thus creating a “bifunctional” inhibitor. This in essence skirts enzyme mechanistic issues and now allows covalent targeting of the BoNT/LC. Based on promising preliminary data, we propose four specific aims that will lead to the identification of potent, reactive, and selective molecules. 1) Using docking and structure activity relationship (SAR) data we will adapt previously identified reversible inhibitors of BoNT/A light chain to the bifunctional covalent strategy. 2) We will screen covalent fragments in the presence of reversible inhibitors to select for warheads that tolerate the presence of the reversible inhibitor scaffold, and to account for active site conformational changes induced by the reversible inhibitor. 3) We will obtain and analyze crystallographic, cell and pharmacokinetic data to iteratively improve our inhibitors, prioritizing potency, selectivity, and safety in order to maximize the chance for success during in vivo studies. 4) Finally, by testing our compounds in the FDA gold standard mouse lethality model, we will assess the efficacy of our developed compounds and their suitability for pre-clinical and clinical studies.