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

Abstract: Methods for screening protein–protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein–protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH₂‐X₁X₂GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide ‘hits’ were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide?mAb interactions; 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 NH₂‐VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor–ligand or other protein–protein interactions, or with enzymatic activity.     

Structure-based design of a potent and selective small peptide inhibitor of Mycobacterium tuberculosis 6-hydroxymethyl-7, 8-dihydropteroate synthase: a computer modelling approach

In an attempt to design novel anti-TB drugs, the target chosen is the enzyme 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS), which is an attractive target since it is present in microorganisms but not in humans. The existing drugs for this target are the sulfa drugs, which have been used for about seven decades. However, single mutations in the DHPS gene can cause resistance to sulfa drugs. Therefore, there is a need for the design of novel drugs. Based on the recently determined crystal structure of Mycobacterium tuberculosis (M.tb) DHPS complexed with a known substrate analogue, and on the crystal structures of E. coli DHPS and Staphylococcus aureus DHPS, we have identified a dipeptide inhibitor with the sequence WK. Docking calculations indicate that this peptide has a significantly higher potency than the sulfa drugs. In addition, the potency is 70-90 times higher for M.tb DHPS as compared to that for the pterin and folate-binding sites of key human proteins. Thus, the designed inhibitor is a promising lead compound for the development of novel antimycobcaterial agents.