Botulinum neurotoxin A (BoNT/A), which causes botulism, is the most potent toxin known. Intoxication is best treated within 24 hours of exposure; no therapies exist which halt BoNT action after neuronal internalization. Extensive effort has been devoted to the discovery of small-molecule therapeutics of BoNT/A light chain (LC). However, a small molecules lifetime in the human body is ~hours-days, while BoNT/A LC may last months. Accordingly, irreversible inhibitors targeting the allosteric Cys165 have been reported, but most lack translational value due to promiscuity or excessive reactivity. Herein, a new strategy for irreversible inhibition of BoNT/A LC is explored. First, an existing assay was modified to develop a continuous method for evaluating timedependent inhibition, which reduces material and time requirements. The importance of considering the allosteric nature of Cys165 was underscored by application of enzyme inhibition models to a published inhibitor. A proof-of concept study was then pursued to validate targeted covalent inhibition of BoNT/A LC by bifunctional inhibitors. Using known BoNT/A LC inhibitor 2,4- dichlorocinammyl hydroxamic acid iterative series of compounds with both zinc-binding and Cys-reactive functionalities were synthesized and evaluated. Additional dialysis and mass spectrometry assays confirmed the covalent reaction at Cys165. Next, a warhead screen was undertaken to improve the proof-of-concept inhibitors, showing that this strategy allows the use of less reactive groups to target the poorly-reactive Cys165. Notably, an epoxide group appended to the aforementioned hydroxamic acid inhibitor was successfully used to covalently inhibit BoNT/A LC. xviii The search for inhibitor scaffolds for adaptation to the bifunctional strategy led to the discovery of thiosemicarbazones and 3-hydroxy-1,2-dimethylpyridine-4(1H)-thiones as moderately potent inhibitors of BoNT/A LC. Unfortunately, the lack of structural information on binding mode of the former and synthetic challenges with the latter precluded their use as the foundation for a bifunctional inhibitor. Finally, an in silico screen yielded the discovery of 9-hydroxy-4H-pyrido[1,2- a]pyrimidin-4-one (PPO) derivatives as BoNT/A LC inhibitors. Informed by a library of 43 derivates and compound docking, bifunctional inhibitors were designed and synthesized. Covalent modification of Cys165 was validated, and the PPO bifunctional inhibitors showed irreversible inhibition over four-fold more potent than their hydroxamic acid-based counterpart.