A search query consisting of two aromatic centers and two cationic centers was defined based on previously identified small molecule inhibitors of the botulinum neurotoxin serotype A light chain (BoNT/A LC) and used to mine the National Cancer Institute Open Repository. Ten small molecule hits were identified, and upon testing, three demonstrated inhibitory activity. Of these, one was structurally unique, possessing a rigid diazachrysene scaffold. The steric limitations of the diazachrysene imposed a separation between the overlaps of previously identified inhibitors, revealing an extended binding mode. As a result, the pharmacophore for BoNT/A LC inhibition has been modified to encompass three zones. To demonstrate the utility of this model, a novel three-zone inhibitor was mined and its activity was confirmed.