Mimotopes of the Nicotinic Receptor Binding Site Selected by a Combinatorial Peptide Library

Abstract

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

Peptide–protein interactions studied by surface plasmon and nuclear magnetic resonances

“…In the present report the favorable opportunity to discuss a good wealth of dynamic and structural data for such similar molecular systems will be exploited in order to correlate complex a¤nity with structural features. A good agreement of SPR derived K A values with IC 50 and K A conventional methods has been recently discussed for the investigated system [6]. The SPR characteristics of these mimotopes and of some of their analogs will be also analyzed.…”

“…Among the various peptide^protein interactions, the complex formation between acetylcholine receptor (AChR) mimotopes and K-bungarotoxin (K-btx) has been analyzed in detail both by SPR [6] and NMR [7,8,9]. Thus, the structure of three slightly di¡erent complexes are available in the Protein Data Bank (PDB) with the 1JBD, 1HAA and 2BTX codes [10], where K-btx is bound respectively to the peptides WRYYESSLKPYPD, HRYYESSLEPWYPD and MRYYESSLKSYPD, henceforth called HaPep, p6.7 and LLPep.…”

A serum-stable branched dimeric anti-VEGF peptide blocks tumor growth via anti-angiogenic activity

“…The use of peptide and its derivatives for the application to basic and medicinal research has been extensively studied (Bruckdorfer et al, 2004;Park et al, 2009). Synthetic combinatorial peptide libraries have been utilized successfully to discover bioactive peptides such as antimicrobial peptides (Blondelle et al, 1996), ligands for cell surface receptors (Bae et al, 2003), protein kinase inhibitors and substrates (Wu et al, 1994;Songyang et al, 1995), and peptide mimotopes of receptor binding sites (Bracci et al, 2001). Moreover, a variety of modification methods have been developed and successfully applied to increase the activity and serum stability of the original peptides such as pegylation and acetylation (Sahu et al, 2000;Lee et al, 2005).…”

Protein microarrays: new tools for pharmaceutical development

“…Peptide arrays have been used for identification of α-bungarotoxin inhibitors [56], mapping metal-binding sites [57] and for discovery of peptides that act as antagonists to proteins that are likely drug targets [58]. Small molecule binders were screened by immobilizing organic molecule libraries followed by incubation with fluorescently tagged proteins and detection of the fluorescence to measure binding [52].…”