Epitope targeting of tertiary protein structure enables target-guided synthesis of a potent in-cell inhibitor of botulinum neurotoxin.
Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target-guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate-mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope-targeting in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active-site-adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an in vitro inhibition constant of 165 pM against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model.
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 (USA).|Department of Applied Physics and Materials Science, California Institute of Technology (USA).
Blake
B
Farrow
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 (USA).
Michelle
M
Wong
Indi Molecular, 6162 Bristol Parkway, Culver City, CA 90230 (USA).
Jacquie
J
Malette
Indi Molecular, 6162 Bristol Parkway, Culver City, CA 90230 (USA).
Bert
B
Lai
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 (USA).
Kaycie M
KM
Deyle
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 (USA).
Samir
S
Das
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 (USA).
Arundhati
A
Nag
Indi Molecular, 6162 Bristol Parkway, Culver City, CA 90230 (USA).
Heather D
HD
Agnew
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125 (USA). heath@caltech.edu.
James R
JR
Heath
D000595:Amino Acid Sequence; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D020134:Catalytic Domain; D002454:Cell Differentiation; D002478:Cells, Cultured; D057930:Click Chemistry; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D004791:Enzyme Inhibitors; D000939:Epitopes; D008024:Ligands; D010455:Peptides; D019274:Botulinum Toxins, Type A
botulinum neurotoxin; combinatorial screening; epitope targeting; peptides; target-guided synthesis