Novel p65 Binding Glucocorticoid-induced Leucine Zipper Peptide Suppresses Experimental Autoimmune Encephalomyelitis

Background: Glucocorticoid-induced leucine zipper (GILZ), a glucocorticoid inducible molecule, physically binds and inhibits nuclear factor-κB (NF-κB). Results: A peptide mimic derived from the p65 binding carboxyl terminus of GILZ inhibits T cells and prevents autoimmune encephalomyelitis. Conclusion: GILZ-p65 interaction is a potential target for multiple sclerosis. Significance: GILZ-peptide may provide a lead for small molecule NF-κB inhibitors. Multiple sclerosis (MS) is a neurological disease characterized by inflammatory demyelination in the brain and spinal cord. The immune-mediated inflammation involves well orchestrated intermolecular interactions that exhibit rapid binding kinetics. The binding interfaces of transient interactions frequently include proline residues that favor an extended conformation for molecular recognition. Linear interface peptides are excellent lead inhibitors of specific protein-protein interactions because only small segments of the interface contribute to the binding. Glucocorticoid-induced leucine zipper (GILZ), a recently identified molecule exhibits potent anti-inflammatory properties. Mechanistically, a proline-rich segment in the carboxyl terminus of GILZ physically binds the p65 subunit of nuclear factor-κB and inhibits the transactivation of inflammatory cytokines. Integrating knowledge derived from the mechanism of action of GILZ with in silico structure prediction identified an immunomodulatory peptide, the GILZ-P. Treatment with GILZ-P exhibited therapeutic efficacy in experimental autoimmune encephalomyelitis, a model for human MS.

Multiple sclerosis (MS) 2 is a chronic immune-mediated inflammatory disease of the central nervous system characterized by relapsing-remitting clinical manifestations (1). At the molecular level, immune responses involve well orchestrated low affinity but high specificity intermolecular interactions that regulate intercellular communications and intracellular signaling pathways (1,2). A common structural theme in the interfaces of transient protein interactions is the frequent presence of proline residues. Although the function of proline is to bring the proteins together so as to make subsequent interactions probable, the adjacent residues determine the specificity (3,4). Greater than 60% of the mouse and human proteome exhibit at least one proline-rich motif (5). The high preponderance of proline-rich regions in the interfaces of proteins involved in critical biological processes suggests that these motifs could represent an important group of targets for therapeutic interventions (4).
The imino group of the proline side chain limits the rotation around the C␣ bond by the covalent link and its C␦ atom sterically restricts the preceding residue to the ␤ region of the Ramachandran map promoting a distinct secondary structure called polyproline type II (PP II ) helix (3,6). The advantages of the PP II helical structure for molecular recognition are the flexibility and orientation freedom offered by the extended conformation (3,6). A conceptually straightforward strategy to develop specific inhibitors of protein-protein interactions is the synthesis of interface peptides derived from the primary sequence of one of the protein binding partners (4). Examples of linear peptides exhibiting PP II conformation include peptides derived from the p85 subunit of PI 3-kinase, CD28-CDR-3 peptide, and protein kinase inhibitors (7)(8)(9).
Glucocorticoid-induced leucine zipper (GILZ) is a recently identified molecule during a systematic study of genes transcriptionally induced by glucocorticoids (10,11). Functionally the GILZ negatively regulates Ras signaling, suppresses proinflammatory cytokines, and modulates T cell activation (12,13). Indeed, the anti-inflammatory activities of glucocorticoids have been attributed to the induced up-regulation of GILZ (14) and are thought to be mediated by its ability to inhibit nuclear factor B (NF-B), the master regulator of inflammatory responses (12,15).
NF-B is a heterodimer of p50 and p65 that remains as an inactive complex with the inhibitory IB proteins in the cytoplasm of resting T cells. Following activation, the subunits translocate to the nucleus and mediate transactivation of inflammatory genes (16). GILZ has been shown to physically bind the p65 subunit through a protein-protein interaction and inhibit NF-B. Mutational analysis localized the site of interac-tion with the p65 to the proline-rich carboxyl terminus of GILZ (GILZ-COOH) (14,17). Structurally the p65 has an amino-terminal Rel homology domain, a nuclear localization sequence masked by the IB inhibitory complex and a carboxyl-terminal transactivation domain (TAD) (18). It has been shown that the GILZ-induced inhibition of NF-B does not require other rel proteins and is independent of IB/NF-B binding (12,14,17). This suggests that the GILZ:p65 binding does not involve the dimerizing rel homology domain or the nuclear localization sequence but is potentially mediated through the p65-TAD. Among the proteins that bind p65, a subset interacts directly with the TAD. Binding of the p65-TAD with other co-factors modulates the basal transcriptional machinery involved in cell cycle regulation and signaling (19). Interestingly, many p65-TAD interactants including p300, CBP (cAMP-response element-binding protein) (20), silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) (21), A07 (22), and the TATA box-binding protein-associated factor 80 (TAF II 80) (23) exhibit prolinerich regions in the binding interface (Table 1).
In this study, we integrated molecular interactions of GILZ with in silico structure prediction to identify an immunomodulatory GILZ-peptide, the GILZ-P. Our data show that the GILZ-P potentially adopts the PP II helical conformation and binds p65 and inhibits its nuclear translocation thereby suppressing T cell responses in experimental autoimmune encephalomyelitis (EAE), a model for human MS (24).

EXPERIMENTAL PROCEDURES
Molecular Modeling-Homology models of GILZ were developed using web-based systems CPHModels (25), Geno3D (26), SWISS-MODE (27), and I-Tasser (28). The primary structure of GILZ is highly homologous with that of the human ␦ sleep-inducing peptide (DSIP) (10,11), the solution structure of which (PDB code 1DIP) was used as the template for the GILZ models (29). The predicted models were assessed for quality by QMEAN (Qualitative Model Energy Analysis), a comprehensive scoring system that determines the statistical probability for the agreement of predicted and calculated secondary structure and solvent accessibility (30). The secondary structure assignment of the GILZ models was independently assessed by the PROSS (Protein dihedral angle-based Secondary Structure assignment) program (31). Superimposition of the predicted GILZ models with the experimentally determined PP II helix was performed to evaluate the similarity between the structures in terms of root mean square deviation.
Peptides and Reagents-GILZ-P 115-137 and a control peptide (control-P) of scrambled residues were synthesized as peptide amides and the PLP 139 -151 (HSLGKWLGHPDKF) and MBP 89 -97 (VHFFKNIVTPRTP) as peptide acids (32). The amino-terminal of GILZ-P, control-P, and MBP 89 -97 were acetylated. All peptides were 95% pure as confirmed by mass spectrometry. Recombinant human p65 protein (r-p65) and purified r-GILZ with C-terminal DDK (catalog number TP320780) and biotinylated anti-DDK antibody were from Ori-Gene Technologies Inc., Rockville, MD. Partial length p65 (p65⌬C14) and anti-p65 mAb were from Active Motif, Carlsbad, CA. Recombinant mouse GILZ protein and the mouse is the number of matches with a score equal to or greater than the observed score that are expected to occur by chance. RelA/p65 TAD:co-activator interacting proteins exhibit a proline-rich region in the interface.

E-value
Ref.
Data Analysis-The kinetic velocity or the slope of absorbance versus time curve was calculated by linear regression. The dissociation constant of the interaction between the r-p65 and r-GILZ/GILZ-P was determined as described (35,36). A fraction of the bound r-p65 (x) and the ratio of bound r-p65 to the free r-GILZ/GILZ-P (y) were determined by the equations: x ϭ (A o Ϫ A)/(A o)), where A o is the absorbance of r-p65⅐anti-p65 complex in the absence of bound r-GILZ/GILZ-P and y ϭ ( where a o is the total concentration of r-GILZ/GILZP and i o is the total concentration of r-p65 (36). K D for the interaction was determined by the Scatchard equation: x ϭ 1 ϩ K D /y.
Treatment of R-EAE-Groups of 8 -10-week-old SJL/J female mice induced with EAE were administered intraperitoneally with vehicle/Pep1 (0.3 M/mouse) mixed with r-GILZ (2 ng/mouse), of GILZ-P, or control-P (500 g/mouse) in 100 l of PBS on the day of immunization (day 0). A group of mice received the GILZ-P⅐Pep-1 complex 12 days post-immunization. Because the mouse and human GILZ proteins are highly identical (10, 11), rh-GILZ was used to evaluate the effect in R-EAE.
Pep-1-mediated Delivery-For Pep-1-mediated delivery, complexes of Pep-1⅐r-GILZ at varying proportions (50:1 M to 1.25:1 M or 10:5 M in PBS) were incubated at room temperature for 30 min. Jurkat T cells were cultured in complete HL-1 medium supplemented with 5% fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 50 units/ml of penicillin, 50 mg/ml of streptomycin, and 5 ϫ 10 Ϫ5 M ␤2-mercaptoethanol in a humidified chamber containing 5% CO 2 at 37°C for 24 h. The cells were then rested for 2 h, washed, and cultured in a 96-well culture plate at 1 ϫ 10 4 cells/well in serum-free medium for 60 min. Confluent T cells were then overlaid with the preformed Pep-1⅐GILZ complexes and incubated at 37°C for 1 h. The cells were then extensively washed, permeabilized, and incubated with phycoerythrin-labeled anti-DDK for 30 min at 4°C. Subsequent to washing, the cells were fixed in PBS, 2% paraformaldehyde. The efficiency of r-GILZ intracellular delivery was assessed by measuring the mean fluorescence intensity using FACS Calibur flow cytometer (BD Biosciences).
Proliferation and Cytokine Assays-The LNC/splenocytes were harvested 10 and 45 days post-immunization from R-EAE mice administered vehicle, r-GILZ, control-P, or GILZ-P on days 0 and 12 or left untreated. CD4ϩ cells isolated by microbead separation were cultured in a transwell system with irradiated syngenic splenocytes as APC in complete HL-1 medium and restimulated with 40 g/ml of PLP 139 -151 , MBP 87-99 or both, and ova in the presence or absence of dexamethasone ( NF-B Assay-5 g of nuclear extracts isolated from PLP 139 -151 restimulated CD4ϩ cells from R-EAE mice were incubated in a 96-well plate coated with oligonucleotides containing the NF-B consensus binding site. The activated NF-B bound to DNA was detected by anti-p65 antibody followed by a peroxidase-coupled secondary antibody and substrate using the TransAM kit protocol (Active Motif).
Statistical Analysis-A one-way analysis of variance with Tukey's post hoc test were performed to determine the statistical differences between the groups.

RESULTS
The Rationale and Design of GILZ-peptide-The primary sequence of GILZ consisting of an amino-terminal leucine zipper (LZ) motif and a proline-rich carboxyl terminus is highly homologous with that of the evolutionarily conserved TGF␤stimulated clone 22 gene (TSC-22) and the porcine DSIP ( Fig.  1A) (10,11). Homology modeling of the human and mouse GILZ was performed by the CPHmodel, Geno 3D, Swiss Model, and I-Tasser programs using the solution structure of the DSIP (PDB code 1DIP) as the primary template. Comparative analysis by QMEAN (30) suggested that equivalent models were gen-erated by all four programs (Fig. 1B). The LZ domain of GILZ was shown to adopt an ␣-helical conformation and the prolinerich carboxyl terminal was predicted to be flexible (Fig. 1, C-E).

Novel p65 Binding GILZ-Peptide
Often misrepresented as disordered regions due to the absence of intra-chain hydrogen bonds, proline-rich regions are known to favor the PP II helical conformation (6). The extent of the PP II helix is determined by the degree of backbone solvation and modulated by side chain interactions (6). The GILZ-COOH consists of a three (PXX) n motif with proline as every third residue (Fig. 1A). Secondary structure assignment based on backbone dihedral angles by PROSS showed that Pro 120 of GILZ exhibited an angle of Ϫ67 Ϯ 5°and angle of 142.5 Ϯ 15° (Fig. 1F) adopting the PP II helical conformation (6). Additionally, the presence of multiple glutamic acid residues in the region increases the net charge further promoting the extended conformation by electrostatic repulsion (37). Superimposition of the predicted GILZ over the experimentally determined PP II helix yielded root mean square deviations of Ͻ5 Å, which suggests significant structural similarity (Fig. 1, G-I). Because the extended conformation is induced by local folding relatively independent of long range interactions, it is likely that the end groups that blocked the synthetic GILZ-P potentially adopts a PP II helical conformation (6).
Kinetics of GILZ-peptide-p65 Interaction-We initially investigated the interaction between GILZ and p65 proteins by modified cellular ELISA. Treatment with dexamethasone increases GILZ expression and suppresses p65 translocation in activated T cells (14). Hence, the nuclear or cytoplasmic protein fractions derived from the CD4ϩ T cells stimulated with recall antigen in the presence of dexamethasone were assessed for binding plate-bound r-GILZ or r-p65, respectively. Although the absorbance was significantly increased in the r-p65-coated wells probed with the cytoplasmic fraction of dexamethasonetreated activated CD4ϩ T cells ( Fig. 2A), it was significantly decreased in r-GILZ-coated wells probed with the nuclear fraction of CD4ϩ T cells activated in the presence of dexamethasone as compared with untreated cells (Fig. 2B). Reduced p65 in the nuclear fractions of cells treated with dexamethasone alone is consistent with its ability to inhibit NF-B translocation in unstimulated cells (Fig. 2B).
To detect direct interaction, the ability of r-p65 to inhibit the binding of anti-GILZ mAb with the plate-bound r-GILZ was assessed. The GILZ⅐anti-GILZ complex formation was inhibited by r-p65 in a dose-dependent manner (Fig. 2C). To determine the kinetics of interaction between r-GILZ/GILZ-P and the p65, plate-bound r-GILZ, GILZ-P, and control-P at varying concentrations was probed with full-length r-p65-DDK. The percent of p65 binding decreased with decreasing concentrations of r-GILZ/GILZ-P (Fig. 2E). The adjusted velocity of the interaction between r-p65 and r-GILZ (7.5/s) or GILZ-P (25/s) was observed at 1:4 molar concentrations (Fig. 2, F and G). A plot of the change in absorbance with time showed that maximum absorbance was reached at a later time point in the binding of r-GILZ (210 s) than of GILZ-P (38 s) with r-p65 suggesting a slower association rate and hence weaker strength for the former interaction as compared with the latter (data not shown). Scatchard plot and linear regression analysis suggested that the dissociation constant, K D , for the interaction between r-GILZ or GILZ-P and r-p65 were 5.91 Ϯ 2.4 ϫ 10 Ϫ7 M (Fig. 2H) and 1.12 Ϯ 0.25 ϫ 10 Ϫ6 M (Fig. 2H), respectively. Absorbance over background was not observed when plate-bound r-GILZ/ GILZ-P was probed with p65⌬C14 and detected with anti-p65 mAb (Fig. 2E), suggesting that the GILZ-P potentially interacts selectively with the p65-TAD. Significant absorbance was not observed in wells coated with control-P and probed with r-p65/ p65⌬C14 (Fig. 2D).
Pep-1-mediated Intracellular Delivery of r-GILZ in T Cells-For an effective intracellular concentration, r-GILZ/GILZ-P/ control-P were mixed with an amphipathic chariot peptide, Pep-1, which rapidly associates through hydrophobic noncovalent interactions and forms stable nanoparticle complexes in solution independent of cargo sequence or size (Fig. 3, A-C) (34). The efficiency of Pep-1 to deliver biological molecules was investigated by incubating Jurkat T cells with preformed complexes of Pep-1 and r-GILZ at varying concentrations. Intracellular delivery was seen in cells incubated with a r-GILZ⅐Pep-1 complex but not in cells incubated with r-GILZ/Pep-1 alone (Fig. 3, A-C). The delivery was most efficient in cells incubated with Pep-1 and r-GILZ at the molar ratio of 50/20:1 as evidenced by the higher mean fluorescence intensity (Fig. 3D). The delivery efficiency decreased with decreasing concentrations of Pep-1 (Fig. 3D). The mean fluorescence intensity was equivalent in untreated cells and cells overlaid with r-GILZ/Pep-1 alone (Fig. 3D). Pep-1 has been effectively used for intracellular delivery of different cargos; including peptide inhibitors of protein kinases, apoptotic protein, and small interfering RNA (33,34).
Effect of GILZ-peptide on T Cell Responses-To investigate the ability of GILZ-peptide to interfere with the GILZ-p65 interactions, in vitro T cell proliferation assays were performed. A significantly decreased proliferative response (mean ⌬cpm) was observed in CD4ϩ LNC isolated from R-EAE mice and re-stimulated in vitro with PLP 139 -151 in the presence of dexamethasone/r-GILZ/GILZ-P with maximum inhibition in cultures treated with 250 M GILZ-P (1404 Ϯ 106). No significant  (46). The conserved proline residues are highlighted. Homology modeling of mouse GILZ and human GILZ were generated based on sequence and structure similarity with the porcine ␦ sleep-inducing peptide (PDB 1DIP). B, validity of the models from CPH Models, Geno3D, Swiss Model, I-Tasser, and Geno3D were assessed with the QMEAN 6 program. The QMEAN 6 score is a composite score consisting of a linear combination of 6 terms (C-␤ interaction energy, all atom pairwise energy, salvation energy, torsion angle energy, secondary structure agreement, and solvent accessibility agreement). The pseudo-energies of the contributing terms (raw scores) are given together with their Z-scores with respect to scores obtained for high-resolution experimental structures of similar size solved by x-ray crystallography. C and D show the structure of PDB code 1DIP and a representative model of the mouse GILZ generated by Swiss Model. E, comparison by overlap yields a root mean square deviation of less than 0.1 Å between the mouse GILZ model and PDB code 1DIP suggesting excellent structural symmetry. F shows the values of the backbone dihedral angles depicting the secondary structure of the GILZ-COOH as assigned by PROSS. The torsion angles and of Pro 120 and Pro 123 are consistent with the PP II helical conformation in the predicted GILZ models. The root mean square deviation of the superimposition of the GILZ models against the x-ray crystal structure of the synthetic PP II helix as determined by CHEMERA is also given. inhibition was observed in cultures treated with control-P (6079 Ϯ 70), Pep-1 alone (6022 Ϯ 87), or GILZ-P alone (6663 Ϯ 173) (Fig. 3E). PLP 139 -151 primed CD4ϩ cells did not exhibit a significant response to an irrelevant antigen, ova (418 Ϯ 24).

GILZ-P Protects against R-EAE-
The biological potential of GILZ-P during antigen priming in vivo was investigated in R-EAE. On day 0 groups of SJL/J mice were induced by R-EAE and administered a single intraperitoneal injection of PBS or a complex of Pep-1 and r-GILZ/GILZ-P/control-P. Separate groups of mice received a single dose of Pep-1⅐GILZ-P on day 12 post-immunization. The average clinical score per day was significantly lower in mice treated with GILZ-P (day 0/day12), r-GILZ as compared with the control groups (Fig. 4, A and B). The severity of clinical disease as suggested by the mean total clinical score was significantly lower in mice treated with GILZ-P (day 0, 2.25 Ϯ 0.72; day 12, 15.75 Ϯ 4.85) or r-GILZ (7.13 Ϯ 2.8) as compared with vehicle (28.5 Ϯ 5.57) or control-P (33 Ϯ 7.5)-treated mice. Significantly, whereas the vehicle or control-P-treated mice exhibited clinical relapse after initial remission, the mice that received GILZ-P/r-GILZ exhibited minimal relapse and continued to be protected for the entire period of observation (Fig. 4A).
) was used to determine the dissociation constant for the interaction between r-GILZ-p65 and GILZ-P and p65 (H).

Novel p65 Binding GILZ-Peptide
GILZ-peptide Increases Expression of Regulatory Molecules in T Cells in R-EAE-We next determined whether GILZ-peptide facilitates Th1 to Th2 skewing in vivo by modulating specific transcription factors, T-bet and GATA-3, respectively (24). RT-PCR showed that the CD4ϩ LNC from mice induced EAE and treated on day 0 with vehicle/r-GILZ/GILZ-P/control-P exhibited significantly lower T-bet but elevated GATA-3 mRNA following re-stimulation with PLP 139 -151 (Fig. 5, A and  B). GILZ has been shown to induce IL-10 secretion and promote regulatory T cell differentiation (15). Accumulation of FOXP3 ϩ IL-10 secreting regulatory T cells in the CNS has been associated with disease recovery in EAE (38). We observed that the CD4ϩ T cells from mice treated on day 0 with r-GILZ/ GILZ-P exhibited significantly higher expressions of IL-10 and FoxP3 mRNA as compared with that from vehicle/control-Ptreated mice (Fig. 5, C and D).

DISCUSSION
In the panel of emerging therapeutic agents for human diseases synthetic peptides constitute highly attractive tools. The advantages of peptides include nonimmunogenicity, inexpensive production, and the potential for high specificity. Hence despite the drawback of poor stability, peptide therapeutics is rapidly advancing with over 190 peptides in phase I/II clinical trials for various diseases (39). In this study we report the identification of a novel p65 binding GILZ-peptide that exhibits therapeutic potential in R-EAE.
GILZ is a glucocorticoid responsive molecule that inhibits NF-B transactivation (11,17) by physically interacting with the p65 subunit via a short proline-rich segment. Secondary structure assignment showed that Pro 120 of GILZ-P adopts an extended PP II helical conformation providing a mechanism for discriminatory recognition without requiring high affinities (6). Consistent with this structural prediction previously, mutation of Pro 120 has been shown to abrogate the GILZ-p65 interaction (14). Peptides derived from the proline-rich regions of proteins potentially exhibit similar affinity as the entire protein with the interacting partner (7,8,40). We show that the end groups that blocked GILZ-P bind the r-p65 with similar kinetics as the r-GILZ with the strength of interaction in the micromolar range commonly observed for transient intermolecular interactions.
Analyses of structural complexes of interactions wherein the binding depends on the presence of one or more prolines have shown that the functionally critical prolines in the interface of one protein often are in contact with aromatic residues from the other component (41). In this context, it is interesting to observe that the p65-TAD that potentially interacts with GILZ-COOH presents two highly conserved aromatic residues: Phe 534 and Phe 542 , which together with conserved acidic residues at Asp 531 and Asp 533 and phosphorylation sites at Ser 529 and Ser 536 constitute critical residues for p65 transactivation (18).
Although T cell activation has been shown to down-regulate GILZ, blockade of T cell receptor signaling or costimulation up-regulates GILZ expression in CD4ϩ T cells (24,42). Transfection of gilz inhibits NF-B activation, down-regulates T-bet, and suppresses IFN-␥ secretion by activated CD4ϩ T cells (12,13). In contrast, GILZ expression up-regulates Th2-specific transcriptional factors STAT-6 and GATA-3 and increases IL-4 secretion (13). Thus GILZ is thought to regulate an immune response by modulating the Th1/Th2 balance (14). Our data suggest that GILZ-P treatment mimicked many actions of GILZ. Restimulation of antigen-primed CD4ϩ T cells in the presence of GILZ-P suppressed p65 activation, T-bet transcription, and Th1 cytokines and enhanced GATA-3 and Th2 cytokines.
The diverse roles of GILZ in a range of cellular events implicated in the Th1-mediated pathology suggest that GILZ could represent an immunomodulatory target in the treatment of MS, rheumatoid arthritis, and inflammatory bowel disease (14). GILZ is observed abundantly in the rheumatoid synovium suggesting a role in the local regulation of chronic inflammation (43). Transgenic mice overexpressing gilz in T cells are protected against the Th1-mediated transfer of colitis (44). We observed that a single dose of r-GILZ or GILZ-P administered on the day of disease induction protected mice against R-EAE. Significantly when administered during active immune response against the inducing antigen (day 12) GILZ-P suppressed clinical symptoms suggesting an ameliorating effect on disease development. Importantly, treatment with GILZ-P suppressed clinical relapse in R-EAE. The reduced proliferative and cytokine response of the activated T cells to an encephalitogenic peptide of a myelin protein not used for disease induction suggests that the continued protection in R-EAE was presumably mediated by preventing epitope spreading.

Novel p65 Binding GILZ-Peptide
Targeted inhibition of the NF-B complex is an actively investigated strategy to control inflammation (16). Previous approaches include interference with DNA binding by decoy nucleotides, blocking nuclear translocation by NF-B dimers, inhibiting signaling kinases, and use of proteasome inhibitors (45). The NF-B subunits also interact with multiple transcriptional regulators of cellular proliferation and apoptosis (45). The expanding network of NF-B interactors has increased the potential for identifying newer targets for specific inhibition. In this study, we conceptually integrated the mechanism of action of glucocorticoids with the knowledge derived from the structural analysis of GILZ and its interaction with the p65 subunit of NF-B in the design of GILZ-P, a bioactive synthetic peptide that exhibited therapeutic efficacy in a mouse model of human MS. The low molecular weight GILZ-P can provide promising leads for developing small molecule NF-B inhibitors (4).