A DARPin promotes faster onset of botulinum neurotoxin A1 action

Abstract

In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.

Emerging Trends of Immunosensors Development for Detection of Food Toxins

“…Using particular antibodies conjugated to HRP, the eluate containing the released peptide fragment is transferred to microwell plates coated with streptavidin and detected. To determine the serotype A of botulinum neurotoxin, a straightforward dot blot immunoassay was created that may be used as a portable device and an automated screening tool ( (Leka et al, 2023;Mechaly et al, 2022;Nguyen et al, 2024).…”