A DARPin Increases the Catalytic Activity of Botulinum Neurotoxin A1

Abstract In this study, we characterized Designed Ankyrin Repat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin (BoNT) function and as potential antidotes for botulism. We selected DARPins against the catalytic domain of BoNT/A1 and characterized them by biochemical, biophysical and structural studies in combination with functional assays in cultured neurons and muscle tissue. We identified DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography revealed that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin blocked substrate cleavage of BoNT/A1 but not of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. We found that 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, resulted in a loss of inhibition of substrate cleavage by reducing DARPin affinity from pM to µM. In contrast to the in vitro results, DARPin-F5 increased BoNT/A1 activity in primary neurons. This result was confirmed by the mouse phrenic nerve hemidiaphragm assay, demonstrating faster paralysis in the presence of the DARPin. We show by functional studies in neuronal cells that DARPin-F5 increases translocation of the toxin. Our findings could have important implications for the identification of BoNTs in clinical samples as well as the development of excipients that allow BoNT treatment at a lower dosage and thereby prevent the generation of antibodies against the toxin.