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.

Bicyclic peptides have greater conformational rigidity and metabolic stability than linear and monocyclic peptides and are capable of binding to challenging drug targets with antibody-like affinity and specificity. Powerful combinatorial library technologies have recently been developed to rapidly synthesize and screen large bicyclic peptide libraries for ligands against enzymes, receptors, and protein-protein interaction targets. Bicyclic peptides have been developed as potential therapeutics against a wide range of diseases, drug targeting agents, imaging/diagnostic probes, and research tools. In this minireview, we provide a summary of the recent progresses on the synthesis and applications of bicyclic peptides.

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. Here, we report a general, reversible bicyclization strategy to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell-penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell-permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction.

Macrocyclic peptides are capable of binding to flat protein surfaces such as the interfaces of protein–protein interactions with antibody-like affinity and specificity, but generally lack cell permeability in order to access intracellular targets. In this work, we designed and synthesized a large combinatorial library of cell-permeable bicyclic peptides, in which the first ring consisted of randomized peptide sequences for potential binding to a target of interest, while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the IκB kinase α/β (IKKα/β)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKKβ interaction with low μM IC50 values. Further optimization of one of the hits led to a relatively potent and cell-permeable NEMO inhibitor (IC50 = 1.0 μM), which selectively inhibited canonical NF-κB signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells. The inhibitor provides a useful tool for investigating the biological functions of NEMO/NF-κB and a potential lead for further development of a novel class of anti-inflammatory and anticancer drugs.

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. Here, we report a general, reversible bicyclization strategy to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell-penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell-permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was Supporting information for this article is given via a link at the end of the document HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction. Drug deliveryPeptide bicyclization via a pair of disulphide bonds increases its proteolytic stability and cell permeability and yet allows for regeneration of the functional linear peptide once inside the cytosol of the cell. KeywordsCell-penetrating peptide; cyclic peptide; NEMO inhibitor; bicyclization; protein-protein interactionCompared to small-molecule drugs, peptides are highly selective and efficacious and, at the same time, relatively safe and well tolerated. A particularly exciting application of peptides is the inhibition of protein-protein interactions (PPIs), which remain challenging targets for small molecules. [1] Consequently, there is an increased interest in peptides in pharmaceutical research and development, and ~140 peptide therapeutics are currently being evaluated in clinical trials. [2] However, peptides are inherently susceptible to proteolytic degradation. Additionally, peptides are generally impermeable to the cell membrane, largely limiting their applications to extracellular targets. Although N-methylation of the peptide backbone and formation of intramolecular hydrogen bonds have been shown to improve the proteolytic stability and membrane permeability of certain cyclic peptides, [3,4] alternative strategies to increase both the metabolic stability and cell permeability of peptide drugs are clearly needed.NF-κB is a transcription factor that controls the expression of numerous gene products involved in immune, stress, inflammatory responses, cell proliferation, and apoptosis. [5] Aberrant activation of NF-κB signaling has been implicated in a number of autoimmune diseases (e.g., rheumatoid arthritis) and cancer (e.g., diffuse large B-cell lymphoma), among others. [6] Canonical NF-κB signaling is mediated by the interaction between the inhibitor of κB (IκB)-kinase (IKK) complex and regulatory protein NF-κB essential modifier (NEMO). [7] Binding to NEMO activates IKK, which in turn phosphorylates IκB, promoting the proteasomal degradation of IκB and release of active NF-κB. Modulators targeting various steps of the NF-κB signaling pa...