Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions. Screening of a bicyclic peptide library against tumor necrosis factor-alpha (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions.

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” by the conventional small-molecule approach due to absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library followed by structure-activity relationship analysis, we discovered a family of cyclic peptides possessing both Ras-binding and cell-penetrating properties. These cell-permeable cyclic peptides inhibited Ras signaling by binding to Ras-GTP and blocking its interaction with downstream proteins and induced apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for inhibition of intracellular protein-protein interactions and direct Ras inhibitors as a novel class of anticancer agents.

Cyclic peptides have great potential as therapeutic agents and research tools. However, their applications against intracellular targets have been limited, because cyclic peptides are generally impermeable to the cell membrane. It was previously shown that fusion of cyclic peptides with a cyclic cell-penetrating peptide resulted in cell-permeable bicyclic peptides that are proteolytically stable and biologically active in cellular assays. In this work, we tested the generality of the bicyclic approach by synthesizing a combinatorial library of 5.7 × 106 bicyclic peptides featuring a degenerate sequence in the first ring and an invariant cell-penetrating peptide in the second ring. Screening of the library against oncoprotein K-Ras G12V followed by hit optimization produced a moderately potent and cell-permeable K-Ras inhibitor, which physically blocks the Ras-effector interactions in vitro, inhibits the signaling events downstream of Ras in cancer cells, and induces apoptosis of the cancer cells. Our approach should be generally applicable to developing cell-permeable bicyclic peptide inhibitors against other intracellular proteins.

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely "undruggable" by the conventional small-molecule approach due to absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library followed by structure-activity relationship analysis, we discovered a family of cyclic peptides possessing both Ras-binding and cell-penetrating properties. These cell-permeable cyclic peptides inhibited Ras signaling by binding to Ras-GTP and blocking its interaction with downstream proteins and induced apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic

A combinatorial library of 6 × 106 cyclic peptides was synthesized in the one bead-two compound format, with each bead displaying a unique cyclic peptide on its surface and a linear peptide encoding tag in its interior. Screening of the library against K-Ras identified compounds that bound K-Ras with submicromolar affinity and disrupted its interaction with effector proteins.