Systematic discovery of molecular probes targeting multiple non-orthosteric and spatially distinct sites in the botulinum neurotoxin subtype A (BoNT/A).
The development of molecular probes targeting proteins has traditionally relied on labeling compounds already known to bind to the protein of interest. These known ligands bind to orthosteric or allosteric sites in their target protein as a way to control their activity. Binding pockets other than known orthosteric or allosteric sites may exist that are large enough to accommodate a ligand without significantly disrupting protein activity. Such sites may provide opportunities to discriminate between subtypes or other closely related proteins, since they are under less evolutionary pressure to be conserved. The Protein Scanning with Virtual Ligand Screening (PSVLS) approach was previously used to identify a novel inhibitor and a fluorescent probe against the catalytic site of the botulinum neurotoxin subtype A (BoNT/A). PSVLS screens compound databases against multiple sites within a target protein, and the results for all the sites probed against BoNT/A, not only the catalytic site, are available online. Here, we analyze the PSVLS data for multiple sites in order to identify molecular probes with affinity for binding pockets other than the catalytic site of BoNT/A. BoNT/A is a large protein with a light (LC) and a heavy (HC) chain that can be assayed separately. We used scintillation proximity assay (SPA) to test experimentally 5 probe candidates predicted computationally to have affinity for different non-orthosteric binding regions within the HC and LC, and one compound predicted not to have affinity for either domain. The binding profiles obtained experimentally confirmed the targeting of multiple and spatially distinct pockets within BoNT/A. Moreover, inhibition assay results indicate that some of these probes do not significantly interfere with the catalytic activity of BoNT/A.
Department of Chemistry at Lakehead University and Thunder Bay Regional Research Institute, Thunder Bay, ON P7B 5E1, Canada.
Saedeh
S
Dadgar
Department of Chemistry at Lakehead University and Thunder Bay Regional Research Institute, Thunder Bay, ON P7B 5E1, Canada. Electronic address: wely.floriano@lakeheadu.ca.
Wely B
WB
Floriano
D000495:Allosteric Site; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D020134:Catalytic Domain; D019295:Computational Biology; D062126:Databases, Chemical; D055808:Drug Discovery; D008024:Ligands; D015335:Molecular Probes / Q000737:chemistry*
D008024:Ligands; D015335:Molecular Probes; D019274:Botulinum Toxins, Type A
Allosteric regulation; Biomarkers; Botulinum toxin; Holistic binding; Molecular docking; Molecular probes; Non-orthosteric binding