Project Summary/Abstract The botulinum neurotoxin (BoNT) is the most lethal toxin known to man, with a lethal dose of only 1 ng/kg of body weight. The neurotoxin acts on neurons causing irreversible termination of neurotransmission resulting in flaccid muscle paralysis diagnosed as botulism. Botulism is a serious illness that can lead to respiratory failure and death. The intentional use of the extremely deadly neurotoxin as a biological weapon is a major concern. Coupled with limited treatment options, there is an urgent need for therapeutics to treat BoNT intoxication. The goal of this project is to create new molecules that are therapeutically sound leads for the treatment of botulism. Previously, we have discovered the dipeptide scaffold as a potent source of BoNT inhibitors via enzymatic inhibition assay and X-ray crystallography. This is the first reported example of dipeptides as a BoNT inhibitor. We propose that the dipeptide is an excellent scaffold for BoNT inhibitor design and will create dipeptides with potent inhibitor activity and optimize the pharmacokinetic properties. The aims of this proposed research are 1) Synthesize dipeptide libraries to study BoNT inhibition; 2) Evaluate the dipeptides as BoNT inhibitors through enzyme inhibition assays; 3) Examine pharmacokinetic properties of the dipeptides by performing cell permeability and metabolic stability studies. Based on the results from aims 2 and 3, structural improvements will be made to the peptides to allow for the synthesis of 2nd and 3rd-generation dipeptides that are enriched with potent inhibitors, which have excellent cell permeability and metabolic stability. Overall, the proposed research will deliver true therapeutic lead compounds with potent inhibitor activity with promising pharmacokinetic profiles. This work will provide a novel scaffold that will be a sound therapeutic lead for the treatment of botulism. Undergraduate student researchers will perform all aspects of the proposed research. Research laboratory experiences will be transformative for undergraduate students and help improve their career trajectory. The research described will provide undergraduate students with laboratory training that will be valuable for their future careers in biomedical research.