• competitive assays;
  • enzyme-linked immunosorbent assay;
  • simplified combinatorial peptide libraries;
  • surface plasmon resonance

Most biological processes involve permanent and temporary interactions between different proteins: protein complexes often play key roles in human diseases and, as a consequence, molecules that prevent protein–protein interactions can be potential new therapeutic agents to treat diseases. Here, we describe a simplified approach by which small synthetic peptide libraries were screened to identify the inhibitors of the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes15 (PED/PEA15) and D4α, a functional domain of the phospholipase D1, that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. By using an enzyme-linked immunosorbent assay (ELISA)-based screening, performed on a fully automated platform, we analyzed two simplified peptide libraries in a positional scanning format. This screening led to the identification of small peptides able to inhibit PED/PEA15–D4α interaction. The selection of inhibitors was carried out employing combined competitive and direct experiments, through ELISA and surface plasmon resonance techniques, providing peptides with IC50 values in the micromolar range. Our results showed that the protein complex PED/PEA15-D4α is susceptible to peptides having H-donor groups and aromatic rings on specific positions. These small sequences can be considered as promising scaffolds that could be converted into higher-affinity inhibitor compounds.