Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library

Peptide libraries allow selecting new molecules, defined as mimotopes, whic h are able to mimic the structural and functional features of a native prot ein. This technology can be applied for the development of new reagents, wh ich can interfere with the action of specific ligands on their target recep tors. In the present study we used a combinatorial library approach to prod uce synthetic peptides mimicking the snake neurotoxin binding site of nicot inic receptors. On the basis of amino acid sequence comparison of different alpha -bungarotoxin binding receptors, we designed a 14 amino acid combina torial synthetic peptide library with five invariant, four partially varian t, and five totally variant positions. Peptides were synthesized using SPOT synthesis on cellulose membranes, and binding sequences were selected usin g biotinylated a-bungarotoxin. Each variant position was systematically ide ntified, and all possible combinations of the best reacting amino acids in each variant position were tested. The best reactive sequences were identif ied, produced in soluble form, and tested in BIACORE to compare their kinet ic constants. We identified several different peptides that can inhibit the binding of alpha -bungarotoxin to both muscle and neuronal nicotinic recep tors. Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences.