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.

The Ras subfamily of small GTPases is mutated in ∼30% of human cancers and represents compelling yet challenging anticancer drug targets owing to their flat protein surface. We previously reported a bicyclic peptidyl inhibitor, cyclorasin B3, which binds selectively to Ras–GTP with modest affinity and blocks its interaction with downstream effector proteins in vitro but lacks cell permeability or biological activity. In this study, optimization of B3 yielded a potent pan-Ras inhibitor, cyclorasin B4-27, which binds selectively to the GTP-bound forms of wild-type and mutant Ras isoforms (K D = 21 nM for KRasG12V–GppNHp) and is highly cell-permeable and metabolically stable (serum t 1/2 > 24 h). B4-27 inhibits Ras signaling in vitro and in vivo by blocking Ras from interacting with downstream effector proteins and induces apoptosis of Ras-mutant cancer cells. When administered systemically (i.v.), B4-27 suppressed tumor growth in two different mouse xenograft models at 1–5 mg/kg of daily doses.