Rational design of cell-permeable cyclic peptides containing a D-Pro-L-Pro motif

Cyclic peptides are capable of binding to challenging targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to intracellular targets because of poor membrane permeability. In this work, we discovered a conformationally constrained cyclic cell-penetrating peptide (CPP) containing a D-Pro-L-Pro motif, cyclo(AF Phi rpPRRFQ) (where Phi is L-naphthylalanine, r is D-arginine, and p is D-proline). The structural constraints provided by cyclization and the D-Pro-L-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo ((YVNF)-V-p Phi rpPRR) (where Y-p is L-phosphotyrosine), against the Grb2 SH2 domain. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant beta-hairpin character surrounding the D-Pro-L-Pro motif. These results demonstrate cyclo(AF Phi rpPRRFQ) as an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain).