Template-Based Synthesis of Peptidomimetic Macrocycles - Discovery of Pin1 Inhibitors by Direct Stabilization of a Consensus Binding Sequence

The constitution and ring size of peptide-derived macrocycles directly influence target binding affinity and physicochemical properties. Here, we describe means to recapitulate key features of peptide-protein interaction linear motifs (LM) in diverse, macrocyclic small molecules. Large ring-forming reactions of exceptional substrate scope are achieved using latently reactive templates that engage peptide side chain functional groups. The template is activated to transiently generate either a cinnamyl carbocation or a palladium(π-cinnamyl) complex, which rapidly form carbon-carbon or carbon-heteroatom bonds. Cyclizations occur at room temperature and are typically insensitive to peptide composition or ring size. Multiply reactive templates couple macrocyclization with additional annulation reactions to further restrict conformation, to mask polar groups, and to access complex polycyclic structures of reduced peptidic character in two or three synthetic steps. Divergent Friedel-Crafts macrocyclization reactions are of special utility for exploring multiple ring connectivities within a given peptide sequence. This method has been coupled to standard solid-phase peptide synthesis to prepare a pilot library of 1000-1700 template-bridged macrocycles, which derive from 384 sequences that mimic the aryl-rich consensus substrate LM of the mitotic regulator Pin1. A new fluorescence polarization assay has been developed to support mixture-based screening of this library in 96-well format. Hit validation, mixture deconvolution, and structure elucidation has led to the identification of two series of non-phosphorylated, macrocyclic ligands which bind the Pin1 prolyl isomerase domain (Kd = 24 or 35 nM) with similar avidity to existing phosphorylated inhibitors. We show that binding affinity is directly influenced by core ring size and connectivity, in one example leading to a greater than seven-fold difference in affinity between isomeric macrocycles of identical ring size. These findings suggest that template-based methods should be useful for surveying the pharmacological properties of composite peptide macrocycles and for identifying new bioactive chemotypes by targeting protein surfaces via cognate consensus binding motifs