Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A

Botulinum neurotoxin serotype A (BoNT/A)
causes a debilitating
and potentially fatal illness known as botulism. The toxin is also
a bioterrorism threat, yet no pharmacological antagonist to counteract
its effects has reached clinical approval. Existing strategies to
negate BoNT/A intoxication have looked to antibodies, peptides, or
organic small molecules as potential therapeutics. In this work, a
departure from the traditional drug discovery mindset was pursued,
in which the enzyme’s susceptibility to metal ions was exploited.
A screen of a series of metal salts showed marked inhibitory activity
of group 11 and 12 metals against the BoNT/A light chain (LC) protease.
Enzyme kinetics revealed that copper (I) and (II) cations displayed
noncompetitive inhibition of the LC (Ki ≈ 1 μM), while mercury (II) cations
were 10-fold more potent. Crystallographic and mutagenesis studies
elucidated a key binding interaction between Cys165 on BoNT/A LC and
the inhibitory metals. As potential copper prodrugs, ligand-copper
complexes were examined in a cell-based model and were found to prevent
BoNT/A cleavage of the endogenous protein substrate, SNAP-25, even
at low μM concentrations of complexes. Further investigation
of the complexes suggested a bioreductive mechanism causing intracellular
release of copper, which directly inhibited the BoNT/A protease. In
vivo experiments demonstrated that copper (II) dithiocarbamate and
bis­(thiosemicarbazone) complexes could delay BoNT/A-mediated lethality
in a rodent model, indicating their potential for treating the harmful
effects of BoNT/A intoxication. Our studies illustrate that metals
can be therapeutically viable enzyme inhibitors; moreover, enzymes
that share homology with BoNT LCs may be similarly targeted with metals.