An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists.
Among the agents classified as "Category A" by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.
Eubanks
Lisa M
LM
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Hixon
Mark S
MS
Jin
Wei
W
Hong
Sukwon
S
Clancy
Colin M
CM
Tepp
William H
WH
Baldwin
Michael R
MR
Malizio
Carl J
CJ
Goodnough
Michael C
MC
Barbieri
Joseph T
JT
Johnson
Eric A
EA
Boger
Dale L
DL
Dickerson
Tobin J
TJ
Janda
Kim D
KD
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors*; D005260:Female; D020128:Inhibitory Concentration 50; D051379:Mice; D009465:Neuromuscular Agents / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
D009465:Neuromuscular Agents; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A