Inhibition of NF-κB Activation by Peptides Targeting NF-κB Essential Modulator (NEMO) Oligomerization

Abstract NF-κB essential modulator/IKK-γ (NEMO/IKK-γ) plays a key role in the activation of the NF-κB pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861–27869). To search for drugs inhibiting NF-κB activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-κB activation was quantified using an NF-κB-dependent β-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-κB activation with an IC50 in the μm range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-κB activity. Our results provide the “proof of concept” for a new and promising strategy for the inhibition of NF-κB pathway activation through targeting the oligomerization state of the NEMO protein.