Botulinum neurotoxin A (
BoNT/A) is categorized as a Tier 1
bioterrorism agent and persists within muscle neurons for months, causing
paralysis. A readily available treatment that abrogates
BoNT/A's toxicity and longevity is a necessity in the event of a widespread
BoNT/A attack and for clinical treatment of
botulism, yet remains an unmet need. Herein, we describe a comprehensive warhead screening campaign of bifunctional hydroxamate-based inhibitors for the irreversible inhibition of the
BoNT/A light chain (LC). Using the 2,4-dichlorocinnamic
hydroxamic acid (DCHA)
metal-binding pharmacophore modified with a pendent warhead, a total of 37 compounds, possessing 13 distinct warhead types, were synthesized and evaluated for time-dependent inhibition against the
BoNT/A LC. Iodoacetamides,
maleimides, and an
epoxide were found to exhibit time-dependent inhibition and their k GSH measured as a description of reactivity. The
epoxide exhibited superior time-dependent inhibition over the iodoacetamides, despite reacting with
glutathione (GSH) 51-fold slower. The proximity-driven covalent bond achieved with the
epoxide inhibitor was contingent upon the vital hydroxamate-Zn2+ anchor in placing the warhead in an optimal position for reaction with Cys165. Monofunctional control compounds exemplified the necessity of the bifunctional approach, and Cys165 modification was confirmed through high-resolution mass spectrometry (HRMS) and ablation of time-dependent inhibitory activity against a C165A variant. Compounds were also evaluated against
BoNT/A-intoxicated motor neuron cells, and their cell toxicity, serum stability, and selectivity against
matrix metalloproteinases (
MMPs) were characterized. The bifunctional approach allows the use of less intrinsically reactive electrophiles to intercept Cys165, thus expanding the toolbox of potential warheads for selective irreversible
BoNT/A LC inhibition. We envision that this dual-targeted strategy is amenable to other
metalloproteases that also possess non-catalytic cysteines proximal to the active-site
metal center.