Epitope Targeting of Tertiary Protein Structure Enables Target‐Guided Synthesis of a Potent In‐Cell Inhibitor of Botulinum Neurotoxin

Abstract

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. We combine in situ click target-guided synthesis 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 targeted 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 inhibition constant of 165 pM in vitro 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. Keywordstarget-guided synthesis; botulinum neurotoxin; combinatorial screening; epitope targeting; peptides Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and is produced by some species of the bacterial genus Clostridium. BoNT/A is a chemodenervating zincdependent protease that prevent the Ca 2+ -triggered release of acetylcholine in neuromuscular junctions, by cleaving one of the three SNARE proteins required for synaptic vesicle

Kinetic Target-Guided Synthesis in Drug Discovery and Chemical Biology: A Comprehensive Facts and Figures Survey

“…In the context of the search of nontraditional drugging approaches, this technique was also successfully performed on Botulinum neurotoxin serotype A (BoNT/A), the most lethal known toxin. [77] The direct inhibition of its active site is prevented because of its occlusion by a belt subdomain. To circumvent this, an epitope-targeting strategy combined with in situ click chemistry was used to find a macrocycle that binds to an active site-adjacent epitope of BoNT/A.…”

Facile de Novo Sequencing of Tetrazine‐Cyclized Peptides through UV‐Induced Ring‐Opening and Cleavage from the Solid Phase

“…[18] For the click-cyclized library, five amino acids spaced between an alkynecontaining unnatural amino acid and an azide-containing unnatural amino acid were found to produce the ideal ring size to produce homogeneous monomeric cycles on resin. [19] Screening this library yielded ligands that detected cancer biomarkers, malarial biomarkers, [18] ligands that inhibited kinase activity, inhibited heme sequestration, [20] inhibited botulinum neurotoxin uptake in cells; [21] or modulated protein folding. [22] However, sequencing of both the click-cyclized and the RCMcyclized libraries required use of the time-consuming and arcane Edman peptide sequencing technology.…”

“…GA [a] C(Mmt)AK [a] GC(Mmt)G 31.0 GA(CAKGC) Tetr G 21 27.9 90.0 [a] Indicated that the amino acid had a side chain protecting group (PG) present during the cyclization. For W, K, the PG was Boc; for S, T, Y, E, D, the PG was tBu; for H, N, the PG was Trt; for R, the PG was Pbf.…”