Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Abstract

An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitroanilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 M. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

“…Inhibitors capable of delaying the onset of botulism by blocking BoNT-Lc activity have also been described (8,69). Although these compounds generally work better in vitro (69,70), some have led to 60% of treated mice surviving 3-fold longer than controls (8). One pitfall of these molecules is that they are only effective against a specific serotype.…”

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

“…BoNT antagonist development has been largely limited to studies of small sets of compounds or peptides chosen through rational design, computer-assisted, or other methods [54][55][56][57][58][59]. Although these studies have provided a considerable amount of information about BoNT catalytic mechanism and substrate requirements, few of the small molecule inhibitors that have emerged from these studies have sufficient potency to be effective BoNT therapeutics.…”

Structure Based Discovery of Pan Active Botulinum Neurotoxin Inhibitors

“…Pharmacological treatments that could mitigate the toxicity of the toxin once it has been internalized would be important adjuncts to existing therapeutic procedures and will be invaluable in the event of an attack by serving as antidotes to those already exposed to the toxin to prevent progression and hasten recovery. Small molecule non-peptidic inhibitors (SMNPI) are being developed [12][13][14][15][16][17][18]. Synthetic molecule based high-throughput drug discovery is also in progress [19].…”