Modulation of virus-induced NF-κB signaling by NEMO coiled coil mimics

Abstract

Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.

Fig. 1. Overview of vFLIP-mediated activation of NF-κB signaling pathway.

a Binding of vFLIP, a viral oncoprotein, to NEMO activates NF-κB signaling. NEMO adopts a helical coiled coil motif to bind to vFLIP (PDB Code 3CL3). We explored small molecule libraries, stabilized α-helices, and crosslinked helix dimers (CHDs) to inhibit NEMO–vFLIP complex formation. Potent inhibition required a CHD motif that captured critical contacts from both helices of NEMO coiled coil. b Cartoon depicts various binding partners of NEMO responsible for activating and repressing NF-κB. vFLIP shown here in green binds the second helical domain (HLX2) downstream of the IKKα/β binding region.

Fig. 2. Rational design of inhibitors of the NEMO-vFLIP interaction.

a Helical wheel diagram depicting native (top) NEMO coiled coil and optimized (bottom) sequences. A crosslinker is placed at the e and g positions to constrain the dimer. b Peptide sequences of designed compounds and their respective inhibitory constants from the TR-FRET assay. Superscript ‘a’ denotes incomplete dissociation of the NEMO-vFLIP complex observed at 100 μM concentrations (Fig. 3a). c Cartoon depicts four high-ranking pockets identified by AlphaSpace and corresponding residues from CHD3^NEMO. The changes in %pocket occupancy as a result of three mutations are highlighted.

Fig. 3. Biophysical characterization, stability and cellular uptake of NEMO mimics.

a The potential of NEMO mimics to inhibit vFLIP-NEMO interaction was evaluated in a TR-FRET assay by monitoring the fluorescence emission of the acceptor. The data represents the mean ± SD of two independent experiments (n = 2) each performed with at least three replicates. These studies illustrated the potency of the optimized derivative CHD3^NEMO. b The specific association of the FITC-derivatized CHD3^NEMO with vFLIP over full length NEMO was further probed in a fluorescence polarization assay. The data represents the mean ± SD of a single experiment performed in triplicates. This experiment was repeated twice (n = 2) with similar results. c The conformation of the coiled coil mimics was investigated by circular dichroism spectroscopy in aqueous buffer. d The conformational stability of CHD3^NEMO was further evaluated in a thermal denaturation study. The circular dichroism spectra were collected at regular intervals between 5–95 °C. e CHD3^NEMO was found to resist serum proteases. Peptide degradation was probed over 24 h by HPLC. This data represents mean ± SD (n = 2) f Cellular uptake of FITC-labeled CHD3^NEMO into live BC-1 cells. Cells were visualized by fluorescence microscopy after 1 h incubation. Effect of temperature and 10 mM sodium azide on the cellular uptake of the NEMO mimic was also explored. Hoechst stain was used to detect the nuclei. Representative images are shown from 2 independent experiments (n = 2). Scale bar = 20 μm.

Fig. 4. CHD3^NEMO suppresses vFLIP-mediated NF-κB transcriptional activity and disrupts NEMO-vFLIP complex formation.

a CHD3^NEMO, but not control peptide CHD2^NEMO or CHD4^NEMO, inhibits NF-κB transcriptional activity significantly in BC-3 NF-κB-luc PEL cells in a dose-dependent manner at (a) t = 5 h and (b) t = 24 h post-treatment. BC-3 reporter cell line was treated with increasing concentrations of the different NEMO mimetics or in the presence of the NF-κB inhibitor Bay 11-7082 or HSP90 inhibitor PU-H71. Luciferase assays were performed at the indicated time points. Statistical analysis was performed using two-tailed unpaired t-test comparing treated samples to DMSO control (****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 and non-significant p > 0.05). The data represents mean ± SEM of two independent experiments (n = 2) performed in triplicates. c CHD3^NEMO disrupts vFLIP/NEMO interaction in live cells. Co-immunoprecipiation with an anti-FLAG M2 beads was performed using a vFLIP–FLAG doxycycline-inducible Namalwa cell line. Results show a dose-dependent reduction in the levels of interacting NEMO upon treating cells with the vFLIP inhibitor CHD3^NEMO but not with CHD2^NEMO or CHD4^NEMO peptide. WT uninduced: Namalwa WT vFLIP stable cell line without induction with doxycycline; Mock: parental Namalwa cell line; Mut vFLIP: Namalwa stable cell line carrying vFLIP NF-κB dead mutant that lacks the ability to bind to NEMO. Mut vFLIP and WT vFLIP cell lines were treated with doxycycline to induce vFLIP expression 24 h prior to treating WT vFLIP cell line with DMSO or the different NEMO mimetics.

Fig. 5. CHD3^NEMO induces cell death in vFLIP-expressing PEL cell lines.

a CHD3^NEMO but not CHD2^NEMO or CHD4^NEMO controls induces cell death in a panel of PEL cell lines. vFLIP (−) Namalwa cell line was used as a control. BC-3, BCBL-1, BC-1, and Namalwa cells were treated with increasing concentrations of CHD2^NEMO, CHD3^NEMO, CHD4^NEMO, and cytotoxicity was assayed using the CellTiter-Glo assay at t = 24 h and t = 72 h, respectively. Data represents mean ± SEM of (n = 3) independent experiments performed in duplicates. The IC₅₀s (µM) of the CHDs in the tested cell lines are displayed in the table below the graphs. b–c Flow cytometry analysis showing that CHD3^NEMO induces apoptosis in vFLIP (+) BC-1 PEL cell line, but not vFLIP (−) Namalwa. CHD2^NEMO and CHD4^NEMO had no effect. BC-1 cells were treated with DMSO, 5 µM, 25 µM or 50 µM of CHD3^NEMO for 48 h. After staining for DAPI and Annexin V, cells were examined using flow cytometry. Results were quantified into percentages of live (Annexin V−, DAPI−), early apoptotic cells (Annexin V+) or late apoptotic cells (Annexin V+/ DAPI + and DAPI+). Results shown are the mean ± SEM of (n = 3) independent experiments. Statistical analysis was performed using two-tailed unpaired t-test comparing treated samples to DMSO control. DMSO vs 25μM CHD3^NEMO early apoptotic population p = 0.0124, DMSO versus 50 μM CHD3^NEMO late apoptotic p = 0.0010 (*p ≤ 0.05, ***p ≤ 0.001 respectively). One-way ANOVA analysis was performed comparing 25 μM dose of each of CHD2^NEMO, CHD3^NEMO, CHD4^NEMO peptides and was found to be significant with p = 0.0087, **p ≤ 0.01).

Fig. 6. Effect of CHD3^NEMO on tumor growth in a traceable reporter BC-3-Luc PEL xenograft mouse model.

a Box- and Whisker plots of relative tumor burden at day 20 post-engraftment quantified using bioluminescence imaging. Statistical analysis was performed using two-sided Mann Whitney test (p = 0.012, *p ≤ 0.05). One representative mice for each group is presented (right). Box and whisker plots represent all individual data points within the /min-max range (vehicle n = 10 and treated n = 5). b Bioluminescence quantitation representing tumor burden (mean ± SEM) after 20 days of tumor engraftment in the vehicle-treated (blue line, n = 10) versus CHD3^NEMO treated group (red line, n = 5). Statistical analysis was performed using two-sided Mann Whitney test (p = 0.012, *p ≤ 0.05). c Kaplan-Meier survival analysis showing that mice treated with the CHD3^NEMO peptide (in red) has a survival advantage compared to the control group (in blue). The difference in survival curves was analyzed by log-rank (Mantel-Cox) test (P = 0.002). d Mice treated with CHD3^NEMO for 24 h have attenuated NF-κB signaling in tumor cells.

Fig. 7. Model for CHD3^NEMO mechanism of action.

a vFLIP activates NF-κB by engaging NEMO. b CHD3^NEMO (NEMO mimic) can directly bind vFLIP and compete with NEMO-vFLIP complex formation leading to destabilization of the IKK signalosome and inhibition of vFLIP-induced NF-κB activation in PEL cells.