High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.
Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 μM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified.
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Kristin M
KM
Bompiani
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Dejan
D
Caglič
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Michelle C
MC
Krutein
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Galit
G
Benoni
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Morgan
M
Hrones
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Luke L
LL
Lairson
Fox Chase Chemical Diversity Center , 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States.
Haiyan
H
Bian
Fox Chase Chemical Diversity Center , 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States.
Garry R
GR
Smith
Department of Chemistry, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Tobin J
TJ
Dickerson
D000818:Animals; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D051379:Mice; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity; D011720:Pyrazoles / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013830:Thiadiazoles / Q000737:chemistry
D011480:Protease Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D011804:Quinolines; D054852:Small Molecule Libraries; D013830:Thiadiazoles; C118531:pyrazolopyridine; C037219:quinoline; D001905:Botulinum Toxins
activator; botulinum neurotoxin; high-throughput screening; inhibitor; small molecule