Hit identification of IKKβ natural product inhibitor
© Leung et al.; licensee BioMed Central Ltd. 2013
Received: 15 November 2011
Accepted: 21 December 2012
Published: 7 January 2013
The nuclear factor-κB (NF-κB) proteins are a small group of heterodimeric transcription factors that play an important role in regulating the inflammatory, immune, and apoptotic responses. NF-κB activity is suppressed by association with the inhibitor IκB. Aberrant NF-κB signaling activity has been associated with the development of cancer, chronic inflammatory diseases and auto-immune diseases. The IKK protein complex is comprised of IKKα, IKKβ and NEMO subunits, with IKKβ thought to play the dominant role in modulating NF-κB activity. Therefore, the discovery of new IKKβ inhibitors may offer new therapeutic options for the treatment of cancer and inflammatory diseases.
A structure-based molecular docking approach has been employed to discover novel IKKβ inhibitors from a natural product library of over 90,000 compounds. Preliminary screening of the 12 highest-scoring compounds using a luciferase reporter assay identified 4 promising candidates for further biological study. Among these, the benzoic acid derivative (1) showed the most promising activity at inhibiting IKKβ phosphorylation and TNF-α-induced NF-κB signaling in vitro.
In this study, we have successfully identified a benzoic acid derivative (1) as a novel IKKβ inhibitor via high-throughput molecular docking. Compound 1 was able to inhibit IKKβ phosphorylation activity in vitro, and block IκBα protein degradation and subsequent NF-κB activation in human cells. Further in silico optimization of the compound is currently being conducted in order to generate more potent analogues for biological tests.
The nuclear factor-κB (NF-κB) proteins are a small group of heterodimeric transcription factors that play an important role in regulating inflammatory, immune, and apoptotic responses [1–3]. NF-κB is ubiquitously present in the cytoplasm and its activity is normally suppressed by association with inhibitor IκB . The intracellular NF-κB signaling cascade is initiated by a variety of inducers including proinflammatory cytokines TNF-α, IL-1 or endotoxins [5, 6]. The aberrant activity to the NF-κB signaling pathway has been implicated in the development of a number of human diseases including cancer, auto-immune and chronic inflammatory conditions [3, 7, 8]. Therefore, inhibitors of the NF-κB signaling pathway could offer potential therapeutic value for the treatment of such diseases [9, 10].
The IκB kinase is a multi-component complex composed of two catalytic subunits, IKKα and IKKβ and a regulatory unit NF-κB essential modulator (NEMO) [11–13]. Although both catalytic units are able to phosphorylate IκB, IKKβ has been shown to play the dominant role in activating NF-κB signaling in response to inflammatory stimuli [14, 15]. Phosphorylated IκB is subsequently tagged by the E1 ubiquitin enzyme and degraded by the proteasome to liberate active NF-κB. Free NF-κB then translocates into the nucleus, where it binds to its cognate DNA site and enhances the expression of a number of genes related to the immune response, cell proliferation and survival [16, 17]. Consequently, IKKβ represents an attractive target in the NF-κB pathway for the development of anti-inflammatory or anti-cancer therapeutics.
Virtual screening (VS) has emerged as a powerful tool in drug discovery complementing the vast array of popular but relatively costly high-throughput screening technologies [18, 19]. Using virtual screening, the number of compounds to be evaluated in vitro could be dramatically decreased, which could greatly reduce the time and resource costs of drug discovery efforts. Meanwhile, natural products (NPs) have long provided a valuable source of inspiration to medicinal chemists due to the diversity of their molecular scaffolds, favourable biocompatibility and evolutionarily validated bioactive substructures [20, 21]. Combining these two ideas, our group has previously identified natural product or small molecule inhibitors antagonizing cancer or inflammation-related targets using virtual screening [22–28]. For example, we have successfully identified natural product or natural product-like compounds targeting the c-myc oncogene G-quadruplex, tumor necrosis factor-alpha (TNF-α) and NEDD8-activating enzyme (NAE) [29–34].
Results and Discussion
High-throughput virtual screening
Molecular modeling analysis
Our molecular docking analysis revealed that the top-scoring binding mode of the natural product derivative 1 to the IKKβ complex is similar to that of the reference compound. The bound inhibitor in the co-crystal structure of IKKβ interacts with the ATP binding pocket in shape-driven manner . While the structure of the reference compound contains the anilinopyrimidine motif that is found in other kinase inhibitors such as imatinib , no detectable hydrogen bonds between the hinge region of IKKβ and the anilinopyrimidine moiety of the reference compound were recorded. The aromatic rings of the reference compound span the hinge loop while its terminal chlorine atom points towards the gatekeeper residue Met96 (Figure 2b).
By comparison, the benzoic acid moiety of 1 is situated at the end of the hinge loop with predicted hydrogen bonding interactions between the carboxyl oxygen and amide oxygen atoms of 1 with the phenolic hydrogen atom of Tyr98 and the backbone amino group of Gly102, respectively (Figure 2c). The pendant side chain of 1 is predicted to be situated in a hydrophobic binding pocket also occupied by the reference compound. We envisage that 1 could act as a reversible inhibitor of IKKβ by blocking the nucleotide recognition domain that binds ATP . The binding score for 1 with the IKKβ complex was calculated to be -35.28 kcal/mol, reflecting a strong interaction between the compound and the IKKβ binding site.
The other eleven compounds were also predicted to situate in the hinge region of the binding pockets in the docking analysis. Most of the compounds could form hydrogen bonds with the hinge residues including Glu97, Cys99 and Glu100. Furthermore, several of the compounds formed additional hydrogen bonds with the residues in the solvent accessible region (Arg31 and Lys106). The lowest energy binding pose of the other compounds are summarized in Additional file 1: Table S2.
We also investigated the selectivity of compound 1 for IKKβ over four other kinases (PKCα, PAK4, CaMK2α and JAK2) using molecular modeling. While compound 1 was predicted to bind at the ATP binding sites of the four other kinases, the ICM docking energies of the 1-kinase complexes were significantly less negative than that for IKKβ (Additional file 1: Table S3). Molecules exhibiting such weak binding energies would be expected to be inactive in vitro.
1 inhibits IκBα phosphorylation in vitro
1 inhibits TNF-α induced NF-κB signaling in a HepG2 cell line
Based on the results of the IKKβ assay and the molecular modeling analysis, we envisage that the inhibition of TNF-α-induced NF-κB signaling by 1 could be attributed, at least in part, to the inhibition of IKKβ activity in vitro, thus preventing the degradation of the NF-κB repressor IκBα. The slightly higher potency of 1 in the cell-based luciferase assay compared to the enzyme assay is possibly due to a multi-target effect of 1, suggesting that this compound could potentially influence other steps involved in NF-κB activation.
In conclusion, we have discovered a new small molecule IKKβ inhibitor from a large natural product library of 90,000 compounds using high-throughput structure-based molecular docking. The benzoic acid derivative 1 is able to inhibit IKKβ activity in both cell-free and system with micromolar potency. Furthermore, compound 1 could inhibit IKKβ-mediated NF-κB signaling pathway in human cancer cells. We envisage that compound 1 attenuates the in cellulo transcriptional activity of NF-κB, at least in part, by abrogating the activity of IKKβ. The discovery of this natural product-like derivative provides medicinal chemists with a structurally interesting scaffold, facilitating further chemical modifications in order to sample greater regions of the chemical space of potential IKKβ inhibitors. We are currently investigating the effects of 1 on the proteins involved in NF-κB signaling and conducting in silico lead optimization to generate more potent analogues of 1 for in vitro biological testing.
Materials and cell lines
The NP/NP-like compound collection, which includes compound 1 and the other tested compounds, was obtained from InterBioScreen (Moscow, RUS). The K-LISA™ IKKβ Inhibitor Screening Kit was obtained from Calbiochem (Darmstadt, Germany). Passive lysis buffer and luciferase assay reagent were obtained from Promega Corporation (Madison, WI, USA). HepG2 and HepG2-NF-κB-Luc cells were provided by Prof. Y.C. Cheng (Department of Pharmacology, Yale University School of Medicine, USA). Cells cultured in Minimum Essential Media containing 10% fetal bovine serum were incubated at 37°C/5% CO2 and passaged three times a week.
IKKβ enzymatic activity
IKKβ activity was determined using the ELISA-based (K-LISA™) IKKβ Inhibitor Screening Kit according to the manufacturer’s instructions. The GST-IκBα 50-amino acid peptide that includes the Ser32 and Ser36 IKKβ phosphorylation sites was used as a substrate and was incubated for 30 min at 30°C with human recombinant IKKβ in the presence of DMSO vehicle or different concentrations of 1 in a glutathione-coated 96-well plate. The phosphorylated GST-IκBα substrate was subsequently detected using anti-phospho-IκBα (Ser32/Ser36) antibody and a horseradish peroxidase-conjugated secondary antibody. The samples were finally incubated with TMB solution, and the color development was monitored at 450 nm on a plate reader (Bio-Rad).
NF-κB transactivation activity
Exponentially growing HepG2-NF-κB-Luc cells were seeded overnight at 1 × 104 cells/well in a 48-well plate. On the next day, the cells were pre-incubated with the indicated concentrations of 1 for 1 h before stimulation by 5 ng/mL of TNF-α for an additional 3 h. Passive lysis buffer (50 μL) was added to each well and the plate was incubated for 15 min with shaking. A 20 μL aliquot from each well was mixed with 70 μL luciferase assay reagent in a 96-well white plate. The transcriptional activity was determined by measuring the activity of firefly luciferase in a multi-well plate luminometer (Fusion α-FP, Perkin-Elmer).
where Evw, Eel, Ehb, Ehp, and Esf are Van der Waals, electrostatic, hydrogen bonding, and nonpolar and polar atom solvation energy differences between bound and unbound states, respectively. Eint is the ligand internal strain, ΔSTor is its conformational entropy loss upon binding, and T = 300 K, and αi are ligand- and receptor independent constants. The initial model of IKKβ was built from the X-ray crystal structure of the Inhibitor of kappaB kinase beta (PDB: 3RZF) according to a previously reported procedure. Hydrogen and missing heavy atoms were added to the receptor structure followed by local minimization by using the conjugate gradient algorithm and analytical derivatives in the internal coordinates. In the docking analysis, the binding site was assigned across the entire structure of the protein complex. Each compound was assigned the MMFF force field atom types and charges and was then subjected to Cartesian minimization. The ICM docking was performed to find the most favorable orientation. The resulting trajectories of the complex between the small molecules and protein complex were energy minimized, and the interaction energies were computed. Each compound was docked three times and the minimum of the three scores was used. The 12 highest scoring compounds were utilized for biological testing without further selection. The crystal structures of PAK4 (4APP), PKCα (3IW4), CAMK2α (2VZ6) and JAK2 (3IOK) were also prepared and compound 1 was docked to these molecular models individually using the aforementioned procedures.
This work is supported by Hong Kong Baptist University (FRG2/11-12/009), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU), the Health and Medical Research Fund (HMRF/11101212), the Research Grants Council (HKBU/201811 and HKBU/204612), the Science and Technology Development Fund, Macao SAR (001/2012/A) and the University of Macau MYRG091(Y1-L2)-ICMS12-LCH and MYRG121(Y1-L2)-ICMS12-LCH).
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