Using Allosteric Inhibition as a Means to Ablate Botulinum Neurotoxin Protease

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 DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs), from which there are seven serotypes are Gram-positive bacteria. The clinical signature of BoNTs is peripheral neuromuscular blockade and flaccid paralysis, which depending on the serotype can last for months. BoNTs are the most toxic proteins known to man and have been classified by the Centers for Disease Control and Prevention (CDC) as one of the six highest-risk biothreat agents. Despite their toxicity and high potential as a bioterrorist weapon, BoNTs are widely used in medical and cosmetic procedures (i.e., Botox). However, even under a controlled environment unwanted side effects have been reported causing complications for patients and in some cases severe life- threatening disorders. Currently, a botulinum heptavalent antitoxin (BAT) is the only medical intervention for BoNT poisoning and this has limited value since it can only neutralize circulating toxin, being useless once cellular poisoning takes place. At a mechanistic level botulinum intoxication proceeds through a series of three steps to produce its neuroparalytic effects: neuronal membrane binding, internalization, and intracellular poisoning. Our objective is to target the intracellular poisoning stage, which result when the toxins' zinc metalloprotease cleaves neuronal cell SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Proteolytic damage to the SNARE proteins prevents fusion of synaptic vesicles with the presynaptic membrane blocking the exocytosis of essential neurotransmitters. Botulinum neurotoxin proteases are unique among endopeptidases in that they require long substrates (SNAREs) for catalytic efficiency due to their recognition strategy. Further complicating their inhibition are allosteric patches, termed exosites that both determine specificity and also remodel the catalytic cleft upon substrate binding. The challenges in designing small molecule inhibitors to the BoNT proteases are recognized through the historic efforts against matrix metalloproteinases (MMPs) where the potential pitfalls of targeting the active site of Zn+2 proteases are well documented. We plan to develop selective molecules against BoNT serotype A (BoNT/A), the most toxic of the BoNT proteases, by targeting the enzyme's exosite. By using this approach "metalloactive" site redundancy as seen amongst this enzyme class will be avoided as will the use of compounds that chelate zinc. Our proposed research will be centered upon our discovery of the first non-peptide/protein exosite inhibitor, chicoric acid. Our chemistry efforts will be directed toward improving potency while building in drug-like properties. This effort will be assisted by NMR structure guided studies to delineate the BoNT/A exosite structural interactions essential for chicoric acid analogue binding. We anticipate that such studies will identify the key interactions required for exosite BoNT/A light chain protease (BoNT/A LC) inhibition, which in turn will allow additional structures to be prepared and tested in a more rational approach.