Bis-imidazoles as molecular probes for peripheral sites of the zinc endopeptidase of botulinum neurotoxin serotype A Original Research Article

Botulinum neurotoxin serotype A (BoNTA) is highly toxic, and its antidote is currently unavailable. The essential light-chain subunit of BoNTA is a zinc endopeptidase that can be used as a target for developing antidotes. However, the development of high-affinity, small-molecule inhibitors of the endopeptidase is as challenging as the development of small-molecule inhibitors of protein–protein complexation. This is because the polypeptide substrate wraps around the circumference of the endopeptidase upon binding, thereby constituting an unusually large substrate–enzyme interface of 4840 Å2. To overcome the large-interface problem, we propose using the zinc-coordination and bivalence approaches to design inhibitors of BoNTA. Here we report the development of alkylene-linked bis-imidazoles that inhibit the endopeptidase in a two-site binding mode. The bis-imidazole tethered with 13 methylene groups, the most potent of the alkylene-linked dimers, showed 61% inhibition of the zinc endopeptidase of BoNTA at a concentration of 100 μM. The results demonstrate the presence of a peripheral binding site for an imidazolium group at the rim of the BoNTA active-site cleft. This peripheral site enables the use of the bivalence approach to improve our previously reported small-molecule inhibitors that were developed according to the zinc-coordination approach.