Use of Crystallography and Molecular Modeling for the Inhibition of the Botulinum Neurotoxin A Protease.

Botulinum neurotoxins (BoNTs) are extremely toxic and have been deemed a Tier 1 potential bioterrorism agent. The most potent and persistent of the BoNTs is the "A" serotype, with strategies to counter its etiology focused on designing small-molecule inhibitors of its light chain (LC), a zinc-dependent metalloprotease. The successful structure-based drug design of inhibitors has been confounded as the LC is highly flexible with significant morphological changes occurring upon inhibitor binding. To achieve greater success, previous and new cocrystal structures were evaluated from the standpoint of inhibitor enantioselectivity and their effect on active-site morphology. Based upon these structural insights, we designed inhibitors that were predicted to take advantage of π-π stacking interactions present in a cryptic hydrophobic subpocket. Structure-activity relationships were defined, and X-ray crystal structures and docking models were examined to rationalize the observed potency differences between inhibitors.