Peptide Phage Display Library as Source for Inhibitors of Clostridial Neurotoxins

Abstract

Clostridial neurotoxins are the most powerful toxins known. There are no available antidotes to neutralize neurotoxins after they have been internalized by neuronal cells. Enzymatic domains of clostridial neurotoxins are zinc-endopeptidases specific for protein components of the neuroexocytosis apparatus. Thus, attempts were made to find such antidotes among molecules possessing chelating properties. Subsequently, it was proposed that the process of interaction between clostridial neurotoxins and their substrates might be more complex than viewed previously and may include several separate regions of interaction. Phage display technology is free from bias toward any particular model. This technology in combination with recombinantly produced light chains of botulinum neurotoxins serotypes A, B, and C was used to identify potential inhibitors of clostridial neurotoxins. Identified sequences did not show substantial similarity with substrate proteins of clostridial neurotoxins. Nevertheless, three peptides chosen for further analysis were able to inhibit enzymatic activity of all clostridial neurotoxins tested. This work demonstrates that at least one of these peptides could not be cleaved by clostridial neurotoxin. Attempts to delete amino acid residues from this peptide resulted in dramatic loss of its inhibitory activity. Finally, this work presents a novel approach to searching for inhibitors of clostridial neurotoxins.

The Medicinal Chemistry of Botulinum, Ricin and Anthrax Toxins

“…Zdanovsky and co-workers [77] [47]. The SI sub region of BoNT/B has structure similar to that predicted for VAMP-2 [68].…”

“…These results suggest that the BoNT/A active site can enzyme activity of BoNT. All of these analogs exhibited accommodate two large aromatic ring systems (containing inhibition in the low ItM range [77]. two to three fused aromaticiheteroaromatic rings).…”

The Strange Case of the Botulinum Neurotoxin: Using Chemistry and Biology to Modulate the Most Deadly Poison

“…[112,113] Three different peptides were identified and found to inhibit protease activity in vitro. [113] However, of all the LC serotypes studied, only moderate binding by the peptides to LC/A, /B, and /E was exhibited (250 mm for complete inhibition).…”

“…[112,113] Three different peptides were identified and found to inhibit protease activity in vitro. [113] However, of all the LC serotypes studied, only moderate binding by the peptides to LC/A, /B, and /E was exhibited (250 mm for complete inhibition). Nonetheless, this study provides an example of the utility of phage display for identifying lead peptides that can potentially be exploited both for further clarification of the molecular requirements necessary for BoNT inhibition and for insight into the BoNT mechanism of action.…”

Der seltsame Fall des Botulinum‐Neurotoxins: chemische und biologische Modulierung des tödlichsten aller Gifte

“…[112,113] Drei Peptide wurden identifiziert, die in vitro als Inhibitoren der Proteaseaktivität wirken, [113] allerdings konnte lediglich eine moderate Bindung der Peptide an LC/A, /B und /E nachgewiesen werden (vollständige Inhibierung bei 250 mm). Trotz der eingeschränkten Aktivitäten belegt dieses Beispiel, dass Phagen-Display zur Identifizierung von Leitpeptiden verwendet werden kann, die anschließend weiter optimiert werden und Einblicke in die Wirkungsweise des Toxins geben können.…”