COMPUTATIONAL DESIGN OF INHIBITORS AGAINST BOTULINUM NEUROTOXIN

Project SummaryBotulinum neurotoxins (BoNTs) are among the most deadly bacterial toxins, which cause the neuroparalyticdisease botulism. They are categorized as Tier 1 select agents by the CDC, and could be misused asbiological weapons. An antitoxin composed of equine-derived polyclonal IgG antibodies is currently the onlyavailable treatment for botulism. Since the equine antitoxin can cause various side effects, substantial effortshave been invested to develop monoclonal human antibody antitoxins. These antibodies are effective atneutralizing circulating BoNTs thereby preventing further disease progression. But antibodies are ineffective forBoNTs that have become internalized into neurons by the time botulism symptom is observed in patients(generally 24?48 hours following exposure). Despite extensive research, no small-molecule or peptidomimeticinhibitors for BoNTs has been advanced to clinical trials. Therefore, there is an urgent need for novelapproaches to the prevention and treatment of BoNT intoxication. In this proposal, we will focus on the type Atoxin (BoNT/A) that poses the most serious threat to humans because of its high potency and long duration ofaction. The goal is to develop the first high-throughput, large-scale computational design and screening ofsmall cyclic peptides, which inhibit the protease activity of the light chain of BoNT/A (LC/A) with high affinityand specificity. These cyclic peptides combine the specificity of antibodies with the high stability andmanufacturability of small molecules, and could be optimized to improve membrane permeability to enter thenerve termini. Furthermore, they are highly resistant to proteases and high environmental temperature, thuscould bypass the requirement for cold chain management. The specific aims are (1) to perform large-scale denovo design of hyperstable peptide inhibitors targeting LC/A; and (2) to characterize the peptide inhibitorsidentified in Aim 1 and elucidate the co-crystal structures of LC/A with the best binders. This project will becarried out using an integrated approach that combines Rosseta design, micro-chip gene synthesis, multiplexyeast cell surface display screening platform, next-generation deep sequencing, X-ray crystallography, and invitro binding and protease assays.