A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A.

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC50 values ⩽1µM and 11 effective at ⩽2µM in an ex vivo assay.

Fungal bis-Naphthopyrones as Inhibitors of Botulinum Neurotoxin Serotype A.

An in silico screen of the NIH Molecular Library Small Molecule Repository (MLSMR) of ∼350000 compounds and confirmatory bioassays led to identification of chaetochromin A (1) as an inhibitor of botulinum neurotoxin serotype A (BoNT A). Subsequent acquisition and testing of analogues of 1 uncovered two compounds, talaroderxines A (2) and B (3), with improved activity. These are the first fungal metabolites reported to exhibit BoNT/A inhibitory activity.

Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A.

The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to have comparable activity, the absolute structures of (+)-(R)-1 and (-)-(S)-1 were determined by comparison of calculated and observed circular dichroism spectra. Molecular modeling studies were undertaken in an effort to understand the observed bioactivity and revealed different ensembles of binding modes, with roughly equal binding energies, for the two enantiomers.

Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A.

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-nitro-anilino)(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 microM. 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.