Peptide phage display library as source for inhibitors of clostridial neurotoxins

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.