# Strategies for the Irreversible Inhibition of Botulinum Neurotoxin

> **NIH NIH R21** · SCRIPPS RESEARCH INSTITUTE, THE · 2020 · $290,250

## Abstract

Project Summary
 Botulinum neurotoxin serotype A (BoNT/A) is the most lethal known human poison, exhibiting a potency
1011 times that of cyanide (LD50 ~ 1-5 ng/kg). In addition to this remarkable toxicity, the ease of BoNT/A
production and the absence of effective post-exposure interventions have prompted the Centers for Disease
Control (CDC) to classify BoNT/A as a biodefense threat of the highest risk to national security. Although
immunotherapies exist to protect against BoNT/A toxicity, their effectiveness is lost once the toxin is
internalized into neuronal cells (<12 h post-exposure). Compounding this fact, BoNT/A persists intraneuronally
on the order of months to >1 year in its toxic form. Small molecules offer the only possibility of inhibiting
intraneuronal BoNT/A as these compounds can cross neuronal membranes; however, the development of
clinically-relevant therapies has been limited by the short half-lives of these inhibitors, which are only a fraction
of the timescale of BoNT/A persistence.
 In this proposal, we present two pharmacotherapeutic strategies for overcoming BoNT/A persistence in
neurons. In the first approach, ubiquitination and proteasomal degradation of BoNT/A can be induced using a
bifunctional small molecule known as a proteolysis targeting chimera, or PROTAC. These molecules rely on
conjugating a vetted BoNT/A antagonist to an E3 ubiquitin ligase-recruiting ligand. In the cell, these PROTACs
tether BoNT/A to the cell’s protein degradation machinery. In the second approach, we will convert known zinc
chelating inhibitors of BoNT/A into bifunctional compounds that also covalently engage cysteine 165 of the
BoNT/A active site. Covalent modification of this residue will permanently modify the conformation of the active
site, inhibiting BoNT/A catalysis and thus, toxicity. To validate these strategies, we will design and synthesize
PROTACs (Aim 1) and covalent inhibitors (Aim 2) based on previously reported hydroxamate BoNT/A
inhibitors developed by our laboratory. After synthesis, we will extensively evaluate these compounds in in vitro
and cellular assays for BoNT/A inhibition. The proposed work will help to achieve our long-term goal of
delivering 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 the
neuronal compartment. This work is important because we will deliver tools to probe mechanisms of BoNT/A
persistence; in addition to, offering novel strategies in BoNT/A therapeutic design. Upon completion of this
proposal, 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-art
treatments for biowarfare agents.

## Key facts

- **NIH application ID:** 9841372
- **Project number:** 5R21AI137709-02
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Kim Janda
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $290,250
- **Award type:** 5
- **Project period:** 2018-12-21 → 2020-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9841372

## Citation

> US National Institutes of Health, RePORTER application 9841372, Strategies for the Irreversible Inhibition of Botulinum Neurotoxin (5R21AI137709-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9841372. Licensed CC0.

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