Improved Aminopyridine Analogs for Symptomatic Treatment of Botulism

About This Grant

PROJECT SUMMARY/ABSTRACT Botulinum neurotoxins (BoNTs) are the most potent biological threats known, with estimated human lethal doses as low as 0.1–10 ng/mL depending on serotype and exposure route. BoNTs act by entering motor neurons and cleaving SNARE proteins essential for neurotransmitter release, resulting in flaccid paralysis and, in severe cases, lethal respiratory failure. Although naturally occurring cases of botulism are rare, the potential for deliberate or accidental exposure presents a serious public health risk. BoNTs are classified as Tier 1 Select Agents due to their extreme potency, ease of dissemination, and capacity to overwhelm health care infrastructure. Currently, the only FDA-approved treatment is an equine-derived immunoglobulin antitoxin administered post-exposure to neutralize circulating toxin. However, antitoxin has a narrow therapeutic window and cannot reverse paralysis once toxin is internalized into neurons—consequently, most patients still require prolonged mechanical ventilation to survive. We recently demonstrated that the FDA-approved voltage-gated potassium channel (VGKC) blocker, 3,4-diaminopyridine (3,4-DAP), reverses symptoms of botulism by increasing acetylcholine release at intoxicated neuromuscular junctions. In rodent and non-human primate models of lethal BoNT exposure, 3,4-DAP improves respiratory function, restores neuromuscular transmission, and prolongs survival. Based on these findings, we are currently funded by the Department of Defense to pursue regulatory approval for 3,4-DAP under the FDA Animal Rule. However, two key limitations restrict its broader clinical use: (1) seizure liability due to CNS penetration, and (2) highly variable blood levels due to polymorphisms in N-acetyltransferase-2 (NAT2), its primary metabolic enzyme. These limitations are primary bottlenecks to its use as a medical countermeasure for botulism exposure under emergency conditions or in austere settings. To address this critical gap, we propose to develop next-generation aminopyridine (AP) analogs that retain the therapeutic efficacy of 3,4-DAP while overcoming its pharmacological and pharmacogenetic liabilities. Our preliminary studies in BoNT-intoxicated rodents demonstrate that structurally diverse AP analogs lacking NAT2-susceptible arylamines can reverse respiratory paralysis in vivo. These studies establish preliminary safety and efficacy for new AP drugs and support the underlying hypothesis that the core AP pharmacophore is amenable to structural modifications that optimize therapeutic benefits. Many of these analogs show efficacy at doses that are safe and well-tolerated in mice. Here, we propose a systematic structure-guided screening and optimization strategy to identify, characterize, and advance AP analogs for eventual regulatory approval.