DiSSiMiL: Diverse Small Size Mini-Libraries applied to simple and rapid epitope mapping of a monoclonal antibody

Methods for screening protein-protein interactions are useful in protein sc ience and for the generation of drug leads. We set out to develop a simplif ied assay to rapidly test protein-protein interactions, with a library of 4 00 pentapeptides comprising the 20 natural amino acids at two variable posi tions followed by three glycines (NH2-X(1)X(2)GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against t he HOXD4 protein. Three pentapeptide 'hits' were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide mAb interacti ons; and from assays using pentapeptides in solution to competitively block HOXD4 mAb interactions. Alignment of the three 'hit' pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH2-VYPWMK. Synthesis of N H2-VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOX D4 ablated binding by mAb 10D11 when amino acids in the putative epitope we re mutated. We propose that these simplified hut diverse libraries can be u sed for rapid epitope mapping of some mAbs, and for generating lead small p eptide analogs that interfere with receptor-ligand or other protein-protein interactions, or with enzymatic activity.