Bis-imidazoles as molecular probes for peripheral sites of the zinc endopeptidase of botulinum neurotoxin serotype A.
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.
Merino
Isidro
I
Computer-Aided Molecular Design Laboratory, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
Thompson
Jason D
JD
Millard
Charles B
CB
Schmidt
James J
JJ
Pang
Yuan-Ping
YP
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*
D004791:Enzyme Inhibitors; D007093:Imidazoles; D015335:Molecular Probes; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc