Introduction and Objectives: Botulinum neurotoxin type A (BoNT/A) is extremely toxic, possessing an estimated intravenous LD50 of 1-2 ng/kg and as such has been designated a category A bioterrorism agent.1,2 BoNT/A also possesses an extremely long half-life and persists within muscle neurons for months to >1 year.3 Because of BoNT/A longevity, we have utilized covalent inhibition as a means to abrogate BoNT/A’s toxicity. To this end, we describe an approach to designing inhibitors that possess both electrophilic warheads and metal-binding groups for the bifunctional inhibition of BoNT/A.

Methods: Small molecule inhibitors that possessed electrophilic moieties were designed, using X-ray crystallography as guidance, to target both the zinc metal-binding region and Cys165 within the active site of BoNT/A. Synthesized compounds were evaluated for covalent inhibition using a continuous SNAPtide FRET assay4, and exhaustive dialysis. Compounds were also evaluated against a C165A variant. Compound reactivity, stability, MMP selectivity, and cellular efficacy/toxicity were also evaluated.

Results: Several electrophilic warhead types were confirmed to inhibit BoNT/A LC covalently with substantial differences in time-dependent inhibition between the WT and C165A variant. A trend in warhead reactivity was reflected in inhibitor stability and toxicity. Compounds exhibited moderate potency in a BoNT/A neuronal cellular assay but were not further explored due to undesirable therapeutic potential.

Conclusions: A fundamental framework for the bifunctional covalent inhibition of BoNT/A LC has been established. This approach has potential to be translated to other small molecule metal-binding inhibitors of BoNT/A LC with the vision that different pharmacophores, possessing improved physicochemical properties, will address BoNT/A’s toxicity and longevity within cells.

Funding: This work was funded by NIH grants R01 AI153298 and R21 AI137709 and F32; the Fulbright Scholar Program; the Natural Sciences and Engineering Research Council of Canada PGSD3-502274; and the Skaggs Institute for Chemical Biology.