Identification of Inhibitors of Melittin Using Nonsupport-bound Combinatorial Libraries

Abstract

A strategy has been developed for the identification of inhibitors of toxins or regulatory proteins. This approach is based on blocking the access of such proteins to their biological targets during their solution transport. This approach uses the strength of nonsupport-bound synthetic combinatorial libraries (SCLs) for the study of acceptor-ligand interactions. A non-receptor assisted toxin, melittin, was selected for the present study to illustrate this application of the SCL approach. Hexapeptide SCLs were assayed for their ability to inhibit the cytolytic activity of melittin toward bacterial and erythrocyte cells. Over 20 inhibitory hexapeptides were identified following the screening and deconvolution processes from millions of sequences. The identified inhibitory peptides appeared to interact directly with melittin. These interactions appear to decrease melittin's ability to undergo lipid- and/or polysaccharide-induced conformational changes, and are demonstrated by fluorescence and circular dichroism spectroscopy.

The Current Status of Heterocyclic Combinatorial Libraries

“…Peptide combinatorial libraries have led to the identification of a wide range of bioactive peptides, including novel antibacterials, 41 potent agonists and antagonists to opioid receptors, [44][45][46] trypsin inhibitors, 47 compounds that inhibit melittin's hemolytic activity, 48 and antigenic peptides recognized by monoclonal antibodies. [49][50][51][52] Along with linear peptide sequences, our laboratory and other groups have also developed combinatorial libraries of cyclic peptides.…”

Combinatorial Library Screening with Liposomes for Discovery of Membrane Active Peptides

“…The one-bead-one-compound (OBOC) combinatorial library method is one such approach that affords the following advantages: (i) the high number of possible hits allows for classification of motifs in peptide sequence associated with membrane activity, aiding in the elucidation of mechanisms that are currently poorly defined, (ii) vesicle composition (lipids, cholesterol content, membrane proteins) and buffer conditions can be built into the screening conditions to better mimic various stages of the endosomal pathway, and (iii) inclusion of unnatural, d - and β-amino acids can overcome limitations of quick degradation, immunogenicity, and low membrane permeability associated with peptides made of natural amino acids (e.g., recognition by degradative enzymes or the sterically dependent MHC-antigen T-cell receptor) . There has been some success in applying combinatorial techniques to peptide discovery, − but none have approached the potential throughput of the OBOC screening presented here. In the OBOC methodology, each polymer microbead displays a unique peptide, leading to a library of millions potential MAPs via a combinatorial chemistry approach.…”

Homogeneous Oligomeric Ligands Prepared via Radical Polymerization that Recognize and Neutralize a Target Peptide

“…Melittin, a 26‐amino acid peptide, contains consecutive positively charged and hydrophobic sequences (Scheme a) . Since melittin has a strong cytolytic activity, it has been used as a model toxic target in the development of ligands that act as antidotes . To prepare multifunctional ligands that bind to and neutralize the toxicity of melittin, we selected negatively charged acrylic acid (AAc, later referred to as A) and hydrophobic tert ‐butyl acrylamide (TBAm, later referred to as T) as the functional monomers, expecting electrostatic and hydrophobic interactions with the positively charged and hydrophobic surface of the peptide.…”