Modulating the Affinities of Phosphopeptides for the Human Pin1 WW Domain Using 4-Substituted Proline Derivatives

Human Pin1 is involved in cancer developments and has been a pharmaceutical target. Thus, finding a high-affinity inhibitor of Pin1 has become an attractive topic. The WW domain of human Pin1 can recognize the phosphoserine/phosphothreonine-proline (pS/pT-P) motifs, while its PPIase domain catalyzes the cis/trans isomerization of prolyl bonds to regulate the cell cycle. Here we incorporated a series of 4-substituted proline derivatives into the phosphopeptides and investigated their affinities for the WW domain of Pin1 to develop better inhibitors of Pin1. On the basis of the ligand Myt1-T412 [PPA(pT)PP], we synthesized several phosphopeptides in which the proline residue in the pT-P motif was replaced with various 4-substituted proline derivatives. Isothermal titration calorimetry and fluorescence anisotropy analyses show that the replacement of proline with (2S,4R)-4-fluoroproline increases the binding affinity of the peptide. Circular dichroism measurements suggest that a more PPII-like structure of phosphopeptides makes them bind to the WW domain more tightly. Chemical shift perturbation experiments also indicate that (2S,4R)-4-fluoroproline interacts with Trp34 of the WW domain in the binding site. Results of molecular modeling further suggested that a strong C-H···π interaction induced by (2S,4R)-4-fluoroproline is important in enhancing the affinity of the peptide for the WW domain. The results of this study provide new valuable information for designing and developing effective inhibitors of human Pin1.