This application is in response to NOT-AI-04-044, Biodefense Countermeasure Development: Project Bioshield. Among the listed CDC Category A agents, botulinum neurotoxins (BoNTs) are the most toxic proteins known with microgram quantities of the protein causing severe morbidity and mortality. The goal of this proposal is to develop small molecule inhibitors of the light chain proteases of the BoNTs; in effect, these would inactivate the toxin thereby increasing the corresponding survival rate. To achieve this goal, a group of recognized investigators have been assembled to identify inhibitors in a high-throughput fashion. All compound libraries planned for testing are sets of privileged scaffolds developed in the laboratories of Professors Kim Janda and Dale Boger. Based on preliminary in silico docking studies using BoNT serotype A, it is anticipated that these compounds will be potent inhibitors of the light chain proteases. In our proposed studies, we will collaborate with Professors Joseph Barbieri and Eric Johnson to develop optimized light chain variants of the most common serotypes (i.e., BoNT A, B, and E). Subsequently, synthesized compound libraries will be screened using these optimized proteases for inhibition in a high throughput format previously developed by the Barbieri and Johnson laboratories. Upon the discovery of a suitable target, neutralization studies will also be conducted to validate our in vitro inhibition studies. Finally, in order to optimize any lead compounds, we will undertake structural studies of the light chain proteases and holotoxins bound to inhibitors both in silico and by crystallographic analysis in collaboration with Professor Raymond Stevens. In total, our studies could provide new compounds capable of inhibiting the BoNT light chain proteases, as well as preclinical evaluation of these molecules in relevant models. Furthermore, these studies may provide new pharmacophores which can be rapidly translated into novel drugs capable of treating BoNT exposure.
