Project Summary Botulinum neurotoxin serotype A (BoNT/A) is the most lethal known human poison, exhibiting a potency1011 times that of cyanide (LD50 ~ 1-5 ng/kg). In addition to this remarkable toxicity, the ease of BoNT/Aproduction and the absence of effective post-exposure interventions have prompted the Centers for DiseaseControl (CDC) to classify BoNT/A as a biodefense threat of the highest risk to national security. Althoughimmunotherapies exist to protect against BoNT/A toxicity, their effectiveness is lost once the toxin isinternalized into neuronal cells (<12 h post-exposure). Compounding this fact, BoNT/A persists intraneuronallyon the order of months to >1 year in its toxic form. Small molecules offer the only possibility of inhibitingintraneuronal BoNT/A as these compounds can cross neuronal membranes; however, the development ofclinically-relevant therapies has been limited by the short half-lives of these inhibitors, which are only a fractionof the timescale of BoNT/A persistence. In this proposal, we present two pharmacotherapeutic strategies for overcoming BoNT/A persistence inneurons. In the first approach, ubiquitination and proteasomal degradation of BoNT/A can be induced using abifunctional small molecule known as a proteolysis targeting chimera, or PROTAC. These molecules rely onconjugating a vetted BoNT/A antagonist to an E3 ubiquitin ligase-recruiting ligand. In the cell, these PROTACstether BoNT/A to the cell?s protein degradation machinery. In the second approach, we will convert known zincchelating inhibitors of BoNT/A into bifunctional compounds that also covalently engage cysteine 165 of theBoNT/A active site. Covalent modification of this residue will permanently modify the conformation of the activesite, inhibiting BoNT/A catalysis and thus, toxicity. To validate these strategies, we will design and synthesizePROTACs (Aim 1) and covalent inhibitors (Aim 2) based on previously reported hydroxamate BoNT/Ainhibitors developed by our laboratory. After synthesis, we will extensively evaluate these compounds in in vitroand cellular assays for BoNT/A inhibition. The proposed work will help to achieve our long-term goal ofdelivering a clinically-relevant pharmacotherapeutic strategy for treating post-exposure BoNT/A intoxication.The objective here is to deliver the first small molecules that effectively overcome BoNT/A persistence in theneuronal compartment. This work is important because we will deliver tools to probe mechanisms of BoNT/Apersistence; in addition to, offering novel strategies in BoNT/A therapeutic design. Upon completion of thisproposal, the most efficacious and ?drug-like? molecules will be prime for evaluation in relevant in vivo models.This goal is aligned with the mission of the NIAID to support research aimed at developing state-of-the-arttreatments for biowarfare agents.
Strategies for the Irreversible Inhibition of Botulinum Neurotoxin
Objective
Investigators
Janda, Kim
Institution
Scripps Research Institute
Start date
2018
End date
2020
Funding Source
Project number
1R21AI137709-01A1
Accession number
137709
Categories