Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening.

Methods Development


Botulinum neurotoxin serotype A (BoNT/A) is the most lethal toxin among the Tier 1 Select Agents. Development of potent and selective small molecule inhibitors against BoNT/A zinc metalloprotease remains a challenging problem due to its exceptionally large substrate binding surface and conformational plasticity. The exosites of the catalytic domain of BoNT/A are intriguing alternative sites for small molecule intervention, but their suitability for inhibitor design remains largely unexplored. In this study, we employed two recently identified exosite inhibitors, D-chicoric acid and lomofungin, to probe the structural features of the exosites and molecular mechanisms of synergistic inhibition. The results showed that D-chicoric acid favors binding at the α-exosite, whereas lomofungin preferentially binds at the β-exosite by mimicking the substrate β-sheet binding interaction. Molecular dynamics simulations and binding interaction analysis of the exosite inhibitors with BoNT/A revealed key elements and hotspots that likely contribute to the inhibitor binding and synergistic inhibition. Finally, we performed database virtual screening for novel inhibitors of BoNT/A targeting the exosites. Hits C1 and C2 showed non-competitive inhibition and likely target the α- and β-exosites, respectively. The identified exosite inhibitors may provide novel candidates for structure-based development of therapeutics against BoNT/A intoxication.


Hu, Xin; Legler, Patricia M; Southall, Noel; Maloney, David J; Simeonov, Anton; Jadhav, Ajit;


  • Binding Sites
  • Botulinum Toxins, Type A/ antagonists & inhibitors
  • Botulinum Toxins, Type A/ chemistry
  • Botulinum Toxins, Type A/ toxicity
  • Caffeic Acids/ chemistry
  • Computer Simulation
  • Humans
  • Molecular Conformation
  • Molecular Dynamics Simulation
  • Phenazines/ chemistry
  • Protein Binding
  • Structure-Activity Relationship
  • Succinates/ chemistry

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