The structures of the quinolinol BADGE derivatives under investigation contain additional basic moieties including 2-amino or 3-amino pyridine. The presence of these structural motifs suggests that these molecules may interact with the hydrophobic pocket DOI hydrochloride located in the active site of the BoNT/A-LC and interact with Tyr366 and Val258. The quinolinol moiety alone in the presence of zinc does not inhibit the proteolytic activity of BoNT serotypes A and B as described by Adler et al.. Data obtained from in silico docking along with the in vitro inhibition at 100 mM concentration of quinolinol derivatives used in the study is summarized in table S2. As shown in figure 1, NSC 84087 is docked in the large hydrophobic pocket of the BoNT/A-LC active site, and its hydroxy quinoline moiety coordinates with zinc. The methoxy group of aniline ring can form a hydrogen bond with His227, which coordinates with zinc, and may contribute to the specificity and potency of this inhibitor. Additionally, the phenyl group is found to fit between Glu164 and Cys165 which are reported to participate in substrate binding. This could explain the importance of hydroxy group in inhibiting BoNT/A-LC, and suggests that the quinolinols inhibit BoNT/A by blocking the active site zinc. It should be noted that the crystal structures of the complexes of known small-molecule and peptide inhibitors with BoNT/A-LC have shown that chelation to zinc is involved in the binding and inhibition of the light chain in both cases. BoNT-LCs are remarkable among proteases for the extremely long substrate required for efficient proteolysis, whereas other microbial metalloproteases have been found to display activity against as short as dipeptides. The catalytic LC domain of BoNT/A is a compact globule consisting of a mixture of a-helices, b-sheets, and a -strands with a zinc-containing metalloprotease active site bound deeply inside a large open cavity. The remarkable substrate selectivity of BoNT/A for SNAP-25 has been explained to be a consequence of extensive interactions with two exosite domains distinct from the active site. A model for substrate recognition has been proposed in which a-exosite binding occurs first via helix formation in the appropriate region of SNAP-25, followed by b-exosite recognition and subsequent conformational changes in the enzyme to facilitate efficient substrate cleavage. This model argues that, without exosite binding, BoNT/A-LC is a significantly less efficient enzyme, and thus these regions could be targeted for lead development. To examine the lead compounds in vivo, a wellestablished mouse bioassay was used. This model is the Food and Drug Administration standard for assessing BoNT levels and the universally accepted method for the study of BoNT antagonists. For evaluation of inhibitory potential of small-molecules, they were injected into test animals as described in materials and methods.