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Published June 8, 2015 | public
Journal Article Open

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.

Additional Information

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Received: March 16, 2015; Revised: April 9, 2015; Article first published online: 29 Apr. 2015. This work was supported by the Institute for Collaborative Biotechnologies (W911NF-09-0001) from the U.S. Army Research Office and the Defense Advanced Research Projects Agency (DARPA) through the Cooperative Agreement HR0011-11-2-0006, and the Jean Perkins Foundation. B.F. is supported by an HHMI International Student Research Fellowship. The following reagents were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Polyclonal anti- BoNT/A1 produced in sheep, NR-9584.

Attached Files

Supplemental Material - anie_201502451_sm_miscellaneous_information.pdf

Accepted Version - nihms703153.pdf


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August 22, 2023
August 22, 2023