Discovery of Dipeptides as Potent Botulinum Neurotoxin A Light-Chain Inhibitors

Abstract

The botulinum neurotoxin, the caustic agent that causes botulism, is the most lethal toxin known to man. The neurotoxin composed of a heavy chain (HC) and a light chain (LC) enters neurons and cleaves SNARE proteins, leading to flaccid paralysis, which, in severe occurrences, can result in death. A therapeutic target for botulinum neurotoxin (BoNT) intoxication is the LC, a zinc metalloprotease that directly cleaves SNARE proteins. Herein we report dipeptides containing an aromatic connected to the N-terminus via a sulfonamide and a hydroxamic acid at the C-terminus as BoNT/A LC inhibitors. On the basis of a structure-activity relationship study, 33 was discovered to inhibit the BoNT/A LC with an IC₅₀ of 21 nM. X-ray crystallography analysis of 30 and 33 revealed that the dipeptides inhibit through a competitive mechanism and identified several key intermolecular interactions.

Scheme 1. Synthesis of Dipeptides

Reagents and conditions: (a) Fmoc-protected amino acid, HOBt, DIC, DMF; (b) 25% PIP in DMF; (c) sulfonyl chloride, 2,6-lutidine, DCM; (d) 50% TFA in DCM.

Figure 1

X-ray crystal structure of 30 bound to the BoNT/A LC active site (PDB 7KYF). Green dashed lines represent intermolecular contacts generated with ChimeraX.

Figure 2

X-ray crystal structure of 33 bound to the BoNT/A LC active site (PDB 7KYH). Green dashed lines represent intermolecular contacts generated with ChimeraX.