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.


B. Farrow, M. Wong, J. Malette, B. Lai, K. M. Deyle, S. Das, A. Nag, H. D. Agnew, and J. R. Heath
Angewandte Chemie International Edition
Volume: 54
Number: 24
Pages: 7114-7119
Date: June, 2015
ICB Affiliated Authors: James R Heath