Metal Ions Effectively Ablate the Action of Botulinum Neurotoxin A.
Paul T BremerSabine PellettJames P CarolanWilliam H TeppLisa M EubanksKaren N AllenEric A JohnsonKim D JandaPublished in: Journal of the American Chemical Society (2017)
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.
Keyphrases
- human health
- ionic liquid
- drug discovery
- health risk
- oxide nanoparticles
- risk assessment
- simultaneous determination
- escherichia coli
- health risk assessment
- single cell
- small molecule
- quantum dots
- mass spectrometry
- amino acid
- radiation therapy
- aqueous solution
- stem cells
- climate change
- mesenchymal stem cells
- high resolution
- anti inflammatory
- binding protein
- case control
- dengue virus
- solid phase extraction
- crystal structure
- locally advanced