Cell-Permeable Bicyclic Peptidyl Inhibitors against NEMO-IκB Kinase Interaction Directly from a Combinatorial Library

Abstract

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.

NF-κB Signaling in Ovarian Cancer

“…NEMO is required for the stability and activity of the IKKα/β complex that activates canonical signaling [84]. A novel bicyclic peptidyl inhibitor was developed against NEMO that demonstrated reasonable stability, cell permeability and potency against IKKβ phosphorylation in vitro [85]. This inhibitor, described as ‘peptide 7′, also affected the cell viability of OC cell lines in a dose-dependent manner without affecting the viability of a normal human ovarian surface epithelial cell line OSE at the same concentration [85].…”

Modulating Protein–Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples

“…In the second approach, Pei’s group merges randomized cyclic hexapeptides, composed of natural and non-proteinogenic residues, with diverse CPP sequences to generate a large library of bicyclic peptides [ 80 ]. This library was checked for inhibition of the NF-κB essential modulator (NEMO)-IκB kinase interaction.…”

“…They identify cell-permeable bicyclic peptide 20 ( Figure 5 ) as the most potent binder of NEMO protein reported to date (IC 50 = 1 µM). This bicyclic peptide inhibits de NF-kB signaling and shows anticancer activity in A2780 and CP70 tumor cell lines [ 80 ].…”

Small Molecule NF-κB Pathway Inhibitors in Clinic

“…Unfortunately, due to the low plasma stability and druggability issues of this peptide, it was never taken to clinic. Even modifications of NBD [ 117 ] such as covalently linking it to either cell penetrating peptides or Drosophila Antennapedia domain or even macrocyclization to make it into a proteolytically stable bicyclic peptide could not take any NEMO inhibitor to clinical trials. To succeed, IKKγ inhibitors have to overcome the proteasomal degradation and address drug delivery issues.…”