De Novo Design of α-Amylase Inhibitor: A Small Linear Mimetic of Macromolecular Proteinaceous Ligands

SummaryWe report a low molecular weight inhibitor of α-amylases based on a linear peptidic scaffold designed de novo through the use of combinatorial chemistry. The inhibitory motif denoted PAMI (peptide amylase inhibitor) was selected by using L-peptide libraries and was fine-tuned by the introduction of unnatural modifications. PAMI specifically inhibits glycoside hydrolases of family 13. Its interaction with porcine pancreatic α-amylase was characterized by inhibition kinetics, fluorescence competition assays with natural α-amylase inhibitors, and isothermal titration calorimetry. We demonstrate that the critical amino acid residues in PAMI are shared with those in the macromolecular proteinaceous inhibitors that, however, bind to α-amylases through a spatially scattered set of intermolecular contacts. Thus, natural molecular evolution as well as combinatorial evolution selected the same α-amylase binding determinants for completely different spatial frameworks