AllD-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

Abstract

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D-amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.

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.…”

Inhibition of Melittin Activity Using a Small Molecule with an Indole Ring

“…The IC 50 of 5CT was determined to be 66 μM (13 μg/mL). The reported IC 50 values of hexapeptide inhibitors range from several μg/mL to more than a dozen μg/mL. , Despite the small size and simple molecular structure of 5CT, its IC 50 was comparable with those of previous studies. 5CT was used as an inhibitor in the following experiments to study the mechanism of melittin inhibition.…”

“…The labile conformation of melittin is sensitive to solution conditions (e.g., ionic strength, pH, and solution concentration) . Indeed, Knoppel et al reported that melittin has a random structure in dilute aqueous solution, while it has an α-helical structure in methanol solution and when it is embedded in a lipid membrane. − Four (or more) melittin monomers can self-assemble to form a tetramer (or oligomer), which becomes a transmembrane pore on cell membranes, leading to cytolysis and hemolysis. − Despite the various studies on melittin, there are limited studies on melittin inhibitors (peptide-based inhibitors − and polymer-based inhibitors − ) likely because of the labile conformation of melittin. Inspired by the above studies, we conjectured that a novel inhibitor with controlled chirality can be designed by considering the primary structure of melittin.…”

Novel, potent calmodulin antagonists derived from an all‐d hexapeptide combinatorial library that inhibit in vivo cell proliferation: activity and structural characterization

“…8). For example, the tetrameric self‐aggregation of the naturally occurring bee venom peptide melittin was found to be completely inhibited by individual all‐ d ‐amino acid hexapeptides derived from SCLs (9). Such studies support the ability of protein‐like complexes to specifically bind to short peptides having an all‐ d configuration.…”