Synthesis of Combinatorial Chemical Libraries Containing Potential Inhibitors of Botulinum Neurotoxin Protease Activity

SOLUS biodefense proposes to develop novel combinatorial synthesis methods for generating librariesof botulinum metalloprotease inhibitors suitable for potential pre-clinical testing. Recent advances in 3Dcrystallography of botulinum toxin structure and inthe understanding of toxin-peptide substrate interactions and structure-function relationships have provided key leads into likely optimal pharmacophore structure required for effective BoNT inhibition. This, in turn, offers insight into innovativestrategies for design of combinatorial synthesis schemes directed at the high-throughput solid-state synthesis of 1D, 2D and 3D combinatorial libraries of pharmacophore type compounds suitable for screening. A starting molecular backbone based on the knownsafety of FDA-approved metalloprotease inhibitor will be used to synthesize 1D libraries with key functional moieties: sulfhydryl, hydroxylamine, or N-carboxyalkyl, each with or without of a guanidino functional group. Compound efficacy will be screened ina high- throughput in vitro fluorescence microplate assay developed at USAMRIID and targeted against specifically developed BoNT/A and BoNT/B peptide substrates. Promising compounds will be refined in a second round leading to a library of 2D candidatesto be tested in cellular models of cytotoxicity and ACh release in order to identify potential drugs with low in vitro toxicity profiles.Development of the proposed combinatorial method and metalloprotease inhibitor library offers significantcommercialpotential beyond the contained market range of the botulinum applications field. Metalloprotease-mediated pathophysiology is an expanding field of biomedical research and clinicalapplication. This is particularly true in the tumor angiogenesis field.Moreover, in the era of antibioticresistance, bacterial metalloprotease inhibition is an attractive pharmaceutical development opportunity fortreatment of a spectrum of infectious processes via suppression of related toxin pathophysiology. Thus,both theadvances in combinatorial synthetic design and strategy, as well as the knowledge of structurefunction relationships inherent in the identification of new metalloprotease inhibitor drug candidates wouldcontribute significantly to research and developmentfor commercial applications in an expanding market.