title: Bis-imidazoles as molecular probes for peripheral sites of the zinc endopeptidase of botulinum neurotoxin serotype A.
abstract: 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 angstroms2. 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 microM. 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.
authors.affiliation: Computer-Aided Molecular Design Laboratory, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
authors.forename: Isidro
authors.initials: I
authors.lastname: Merino
authors.forename: Jason D
authors.initials: JD
authors.lastname: Thompson
authors.forename: Charles B
authors.initials: CB
authors.lastname: Millard
authors.forename: James J
authors.initials: JJ
authors.lastname: Schmidt
authors.forename: Yuan-Ping
authors.initials: YP
authors.lastname: Pang
mesh_terms: D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000737:chemistry*; D003198:Computer Simulation; D010450:Endopeptidases / Q000737:chemistry* / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D007093:Imidazoles / Q000737:chemistry* / Q000494:pharmacology; D008954:Models, Biological; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry* / Q000494:pharmacology; D015394:Molecular Structure; D015032:Zinc / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D015335:Molecular Probes; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc