Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.

Botulinum neurotoxins (BoNTs) are the deadliest of microbial toxins. The enzymes' Zinc(II) metalloprotease, referred to as the light chain (LC) component, inhibits acetylcholine release into neuromuscular junctions, resulting in the disease botulism. Currently, no therapies counter BoNT poisoning post-neuronal intoxication; however, it is hypothesized that small molecules may be used to inhibit BoNT LC activity in the neuronal cytosol. Herein, we describe the pharmacophore-based design and chemical synthesis of potent (non-Zinc(II) chelating) small molecule (non-peptidic) inhibitors (SMNPIs) of the BoNT serotype A LC (the most toxic of the BoNT serotype LCs). Specifically, the three-dimensional superimpositions of 2-[4-(4-amidinephenoxy)-phenyl]-indole-6-amidine-based SMNPI regioisomers (K(i) = 0.600 µM (± 0.100 µM)), with a novel lead bis-[3-amide-5-(imidazolino)-phenyl]-terephthalamide (BAIPT)-based SMNPI (K(i) = 8.52 µM (± 0.53 µM)), resulted in a refined 4-zone pharmacophore. The refined model guided the design of BAIPT-based SMNPIs possessing K(i) values = 0.572 µM (± 0.041 µM) and 0.900 µM (± 0.078 µM).