Glioblastoma (GBM) is the most common primary brain malignancy, rarely amenable to treatment with a high recurrence rate. GBM are prone to develop resistance to the current repertoire of drugs, including the first-line chemotherapeutic agents with frequent recurrence, limiting therapeutic success. Recent clinical data has evidenced the BRD2 and BRD4 of the BET family proteins as the new druggable targets against GBM. In this relevance, we have discovered a compound (pyrano 1,3 oxazine derivative; NSC 328111; NS5) as an inhibitor of hBRD2 by the rational structure-based approach. The crystal structure of the complex, refined to 1.5 Å resolution, revealed that the NS5 ligand significantly binds to the N-terminal bromodomain (BD1) of BRD2 at the acetylated (Kac) histone binding site. The quantitative binding studies, by SPR and MST assay, indicate that NS5 binds to BD1 of BRD2 with a KD value of ∼1.3 µM. The cell-based assay, in the U87MG glioma cells, confirmed that the discovered compound NS5 significantly attenuated proliferation and migration. Furthermore, evaluation at the translational level established significant inhibition of BRD2 upon treatment with NS5. Hence, we propose that the novel lead compound NS5 has an inhibitory effect on BRD2 in glioblastoma.

Introduction

The role of epigenetic regulation of gene expression is crucial in normal cellular homeostasis. Alterations in the regulation of this orchestrated control through abnormal gene expressions or mutations of key proteins have been implicated in a broad array of malignancies [1]. The bromodomain and extra-terminal (BET) family proteins are the epigenetic readers of the acetylated histones, and function as key co-regulators of transcription [2]. The BET proteins (BRD2, BRD3, BRD4, and BRDT) regulate proliferation, the cell-cycle and cell differentiation; and thus, are established therapeutic targets for treating malignancies [3,4]. BET family protein inhibitors including RVX-208, I-BET 762, OTX015, CPI-0610, and TEN-010 are being currently evaluated in clinical trials, of which OTX015 has shown encouraging results against hematologic malignancies [5–9].

Glioblastoma (GBM) is the most common primary brain tumor with a high mortality [10]. The GBM is characterized by extensive migratory and infiltrative growth which renders total resection surgically challenging with frequent relapse. The median survival of GBM patients is abysmal 12–17 months despite multi-model management [11]. Currently, standard management includes gross total resection followed by the DNA alkylating drug, temozolomide (TMZ), accompanied by radiotherapy [12,13]. Hence, the five-year survival rate is <6% [14].

Additionally, several factors can lead to therapeutic failure of GBM, including rapid proliferation, aggressive migration, and invasion [15]. Migration and invasion of glioma cells is a complex process similar to metastasis which requires proteases-mediated degradation of extracellular matrix. Therefore, novel therapeutic agents targeting different pathways to inhibit multiple facets of GBM is compelling.

Epigenetic modifications, which are considered to be a key mechanism in GBM development [16], are gaining more relevance as it can be a clinical biomarker for stratification and classification of GBM, and can serve as potential drug targets as evidenced by recent clinical trials [17]. The mechanistic links between BET activity and brain tumors, especially GBM is beginning to emerge, and the recent study revealed that the expression level of BRD2 and BRD4 is high in GBM [18]. This offers an avenue for discovering novel small molecule inhibitors for epigenetic targets against GBM.

In this study, we discovered a novel inhibitor of hBRD2, (NSC 328111; hereafter, NS5), a pyrano 1,3 oxazine derivative, by the rational structure-based method. The structure of the complex, refined to 1.5 Å resolution, revealed that the compound binds to BD1 (N-terminal bromodomain) of BRD2 at the Kac binding site. Moreover, our in vitro biochemical studies, evaluation of proliferation and migration suggests that the discovered compound exhibits an inhibitory effect on GBM cells. Furthermore, treatment of GBM cells with NS5 inhibited hBRD2 at the translational level.

Materials and methods

Virtual screening of NCI diversity set III

The three-dimensional structure of hBRD2–BD1–MB3 inhibitor complex (PDB ID: 4A9F) [19] was used for docking studies. Protein preparation steps for docking studies were carried out by removing all water molecules, extraneous ions occurring due to crystallization reagents, and the ligand from the binding site. Next, hydrogen atoms were added to proteins and performed bond optimization and energy minimization step. The grid box size of 20 × 21 × 20 Å occupying the entire Kac binding pocket with centering on the ligand, 1-methylpyrrolidin-2-one (MB3) was created by Autodock/Vina plugin for PyMol [20].

The active site residues, notably, W97, P98, F99, L108, Y113, and N156 encompassed this grid box. The validation protocol for the docking run was performed by re-docking the co-crystallized ligands into their respective crystal structures and determining the RMSD. The optimized grid parameters were used for virtual screening of the library compounds in AutoDock Vina 1.1.2. The docking parameters such as exhaustiveness was set to 50, and all other parameters were set to default values.

The NCI diversity set III database comprising 1630 compounds was retrieved. The pdbqt format of compounds bearing 3D co-ordinates, polar hydrogens, and partial charges was prepared in OpenBabel 2.3.1. The docked poses were analyzed based on cut-off docking score values. For molecular visualization, docking poses generated by AutoDock Vina were directly loaded into PyMol through PyMol Autodock/Vina Plugin. The top-ranked compounds were inspected visually on the graphics for right chemical geometry and interactions.

An additional measure of the binding performance of the docked compounds in relation to the number of heavy atoms/non-hydrogen atoms is given by ligand efficiency values. It quantifies size and lipophilicity of small molecules that are required to gain overall interactions and thereby the binding affinity to a drug target. The ‘ligand efficiency’ (LE) is defined by LE = −ΔG/HA, where −ΔG is the free energy of binding and HA is the number of non-hydrogen atoms of the ligand. The compounds showing LE > 0.29 kcal mol−1 HA−1 (based on a <10 nM molecule having HA of 38 (∼500 Da) and cLogP of <37) were accepted as good starting hits for lead optimization [21]. The LE value for each ligand was calculated from the MGL tools based on docking energy values obtained from Autodock/Vina. The docking energy scores as well as the LE values for the top compounds obtained from hBRD2–BD1 screening have shown in Table 1. Pictures of the docked protein–ligand complexes were generated by PyMol. The selected ligands from the in silico analysis were obtained from the National Cancer Research Institute, NIH, U.S.A.. The ADME properties of these compounds were determined using the SwissADME (http://www.swissadme.ch/index.php) server [22].

Table 1
In-Silico Ranking for the seven compounds selected through Virtual Screening of NCI Diversity Set III.
S. noCompound IDChemical NameDocking Energy (kcal/mol)Ligand Efficiency2D Representation
1. Lig1 (NSC 116702) 1-(4-(4-methylphenyl)-5-phenyl-1,3-oxazol-2-yl)isoquinoline −8.3 −0.30  
2. Lig2 (NSC 127133) 2-[2-[(6-hydroxyphenanthridin-3-yl)carbamoyl]phenyl]benzoic −8.1 −0.27  
3. Lig3 (NSC 43998) N-(benzhydrylideneamino)-5-nitro-pyridin-2-amine −7.7 −0.32  
4. Lig4 (NSC 116709)  −7.3 −0.27  
5. Lig5 (NSC 328111)  −6.8 −0.32  
6. Lig6 (NSC 135168)  −6.4 −0.26  
7. Lig7 (NSC 326375)  −6.1 −0.30  
S. noCompound IDChemical NameDocking Energy (kcal/mol)Ligand Efficiency2D Representation
1. Lig1 (NSC 116702) 1-(4-(4-methylphenyl)-5-phenyl-1,3-oxazol-2-yl)isoquinoline −8.3 −0.30  
2. Lig2 (NSC 127133) 2-[2-[(6-hydroxyphenanthridin-3-yl)carbamoyl]phenyl]benzoic −8.1 −0.27  
3. Lig3 (NSC 43998) N-(benzhydrylideneamino)-5-nitro-pyridin-2-amine −7.7 −0.32  
4. Lig4 (NSC 116709)  −7.3 −0.27  
5. Lig5 (NSC 328111)  −6.8 −0.32  
6. Lig6 (NSC 135168)  −6.4 −0.26  
7. Lig7 (NSC 326375)  −6.1 −0.30  

They also showed favorable binding in the SPR screening process.

Mono-acetylated H4 peptide and protein production of hBRD2 and hBRD4 bromodomains, BD1 and BD2. The FITC-tagged histone H4 tail peptide acetylated at K12 (H4K12ac; residues 1–15; SGRGKGGKGLGK(Ac)GGA), used for competitive binding analysis, was purchased from GL Biochem (Shanghai, China). Expression and purification of the BD1 bromodomain of hBRD2 was performed as described elsewhere [23,24].

Briefly, the cDNA fragment of the sequences corresponding to the hBRD2–BD1 bromodomain (aa: 74–194). The bromodomain was sub-cloned into a pET28a vector (Invitrogen) and transformed into E. coli. The IPTG concentration (1 mM) was standardized to obtain the soluble bromodomain proteins in supernatant. The cells were harvested and subjected to sonication. After the centrifugation, the supernatant was loaded on to a pre-equilibrated Ni-NTA column (GE Healthcare, U.S.A.) and the protein was eluted in elution buffer containing 50 mM Tris–HCl pH 7.5, 300 mM NaCl, 250 mM imidazole. The His-tag was removed subsequently by using thrombin (Sigma). Finally, size-exclusion chromatography was performed using the gel filtration column Hi-Load 16/600 Superdex (GE Healthcare, U.S.A.) to obtain purified protein over 95% homogeneity. Samples were taken at appropriate intervals and loaded on to the SDS–PAGE for analysis. The recombinant bromodomain protein was concentrated and stored in −80°C until further usage.

Co-crystallization and X-ray crystallography

Apo-crystallization setups yielded rod-shaped BD1 crystals, in a condition of 1 µl protein (5 mg/ml) equilibrated against 1 µl of reservoir buffer (800 mM ammonium Sulfate, 100 mM MES pH 6.5, 5% Glycerol, 10% PEG 3350) within a week's time at 20°C. For co-crystallization studies, a saturated solution of selected 20 compounds from in silico screening was prepared in 5% DMSO and protein buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM DTT). They were individually mixed with protein to achieve a final concentration of 5 mg/ml and kept for overnight incubation at 4°C. Crystals were briefly soaked in a cryo-protectant solution containing 10% glycerol in reservoir solution before flash freezing them.

Preliminary X-ray crystallography studies were performed on the in-house X-ray diffractometer source (Rigaku MicroMax-007) integrated with HyPix 6000 detector, at NIMHANS. For high-resolution data, X-ray data for these crystals were collected on the beamline, ID30A at ESRF, Grenoble, France. The diffraction datasets were integrated and scaled using iMOSFLM [25] and Aimless [26]. The crystal structures were solved by molecular replacement using Phaser-MR [27] in the CCP4 software package (Collaborative Computational Project, Number 4, 1994). A monomer of BD1 (PDB ID: 4UYF) [28] was used as the search model for structure determination. For all the 20 crystals, the molecular replacement results yielded three BD1 molecules in the asymmetric unit. By analyzing the 2mFo–DFc and difference Fourier mFo–DFc maps of the corresponding crystal structures, it showed an apparent electron density for only one compound, NS5, in the binding site. The absence of other two NS5 molecules in the asymmetric unit may be due to moderate binding affinity with the protein. The energy minimized co-ordinates and crystal information files (CIF) for NS5 were generated in Jligand [29].

The structure of the BD1–NS5 complex was refined using the Phenix.refine followed by few refinement cycles in REFMAC [30]. The Coot [31] program was used to visually inspect and built the structures. The final refined structure of the BD1–NS5 complex in the asymmetric unit possesses 2761 protein atoms, 1 PEG molecule, 1 DTT molecule, 2 DMSO molecules, 2 sulfate molecules and 465 water molecules, with a final Rwork of 18.6% and Rfree of 22.6% at 1.5 Å resolution. The stereochemistry of the protein structure was satisfactory, as assessed with MOLPROBITY [32]. The X-ray data collection, scaling, and refinement statistics are summarized in Table 2. The structural co-ordinates of the BD1–NS5 complex were deposited in the RCSB (PDB ID: 6JKE).

Table 2
Summary of data collection and refinement statistics for hBRD2–BD1–NS5 complex
BD1–NS5 complex
Space group C2 
Cell dimensions (Å; °) a = 114.37, b = 55.75, c = 67.85; β = 94.16 
Wavelength (Å) 0.9724 
Resolution (Å)a 67.67–1.5 
Unique reflections 66,509 (3209) 
Rmeasb (%) 6.1 (51.2) 
I/σ(I)⟩ 12.4 (1.9) 
Completeness (%) 97.5 (95.8) 
Multiplicity 2.9 (2.7) 
CC1/2 0.991 (0.723) 
Wilson plot B-factor (Å219.87 
Resolution (Å) 67.67–1.5 
No. of reflections 66408 
cRwork/dRfree (%) 18.6/22.6 
Total no. of atoms 3293 
Protein atoms 2761 
Water molecules 465 
Glycerol molecules 
PEG molecules 
Ligand (NS5) 
DTT 
DMSO 
SO4 
Average B-factor (Å2
 Protein atoms 24.17 
 Ligand, NS5 40.0 
 Water molecules 52.8 
RMSD Bonds (Å) 0.006 
RMSD Angles (°) 1.04 
BD1–NS5 complex
Space group C2 
Cell dimensions (Å; °) a = 114.37, b = 55.75, c = 67.85; β = 94.16 
Wavelength (Å) 0.9724 
Resolution (Å)a 67.67–1.5 
Unique reflections 66,509 (3209) 
Rmeasb (%) 6.1 (51.2) 
I/σ(I)⟩ 12.4 (1.9) 
Completeness (%) 97.5 (95.8) 
Multiplicity 2.9 (2.7) 
CC1/2 0.991 (0.723) 
Wilson plot B-factor (Å219.87 
Resolution (Å) 67.67–1.5 
No. of reflections 66408 
cRwork/dRfree (%) 18.6/22.6 
Total no. of atoms 3293 
Protein atoms 2761 
Water molecules 465 
Glycerol molecules 
PEG molecules 
Ligand (NS5) 
DTT 
DMSO 
SO4 
Average B-factor (Å2
 Protein atoms 24.17 
 Ligand, NS5 40.0 
 Water molecules 52.8 
RMSD Bonds (Å) 0.006 
RMSD Angles (°) 1.04 
a

Numbers in parentheses are values in the highest resolution shell;

b

Rmeas = Σhkl{N(hkl)/[N(hkl) − 1]}1/2 × Σi|Ii(hkl) − <I(hkl)>|/Σhkl ΣiIi(hkl), where N(hkl) is the multiplicity of I(hkl) and<I(hkl)> is the mean intensity of that reflection;

c

Rwork = Σhkl||Fobs| − |Fcalc||/Σhkl|Fobs|, where |Fobs| and |Fcalc| are the observed and calculated structure-factor amplitudes, respectively;

d

Rfree was calculated with 5.0% of reflections in the test set.

Surface plasmon resonance (SPR) assay

In vitro evaluation of the selected 20 in silico screened compounds (Table 1) against hBRD2–BD1 was carried out by SPR assay method using Biacore 3000 (GE Healthcare, U.S.A.) at 25°C. The flow cells of a CM5 amine-coupling chip was used to prepare the surface for protein immobilizing using EDC and NHS chemistry, as per the chip activation protocol mentioned in the Handbook (GE Healthcare). The immobilization on the CM5 chip was performed using purified BD1 (15 µg/ml). The reference flow cell on the sensor chip was prepared by blocking carboxyls, and no protein was added. Two different ligand concentrations (250 µM and 500 µM) were allowed to flow over the immobilized-protein surface, and their binding responses to protein were recorded in RU (response units). 1× PBS, 5% DMSO with 0.005% Tween 20 was used as both running and analyte binding buffer. The chip surface was regenerated by running a regeneration buffer. The maximum RU with each analyte reflected relative binding affinity to hBRD2–BD1. Kinetics analysis was performed for those compounds for which the binding was found to be satisfactory.

The kinetic assay was performed for the compound NS5 by using different serially diluted concentrations (from 350 µM to 4 µM) in a running buffer. The final response of bound protein was calculated by the subtraction of the reference RU. Sensograms were analyzed with BIAevaluation software (GE Healthcare) using a 1 : 1 binding model. The analysis of sensograms and determination of binding constants, KD were performed with BIAevaluation software (GE Healthcare).

Microscale thermophoresis (MST) assay

The hBRD2 bromodomain, BD1 was labeled with the NT-647 reactive dye (NT-647 labeling kit, NanoTemper Technologies, Germany) as per their protocol. The labeled protein at a concentration of 20 µM were used. The protein-dye mixture was incubated for 30 min in the dark at room temperature. Unreacted dye was removed with the supplied dye removal columns equilibrated with the MST buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2). The label/protein ratio was determined by photometry at 650 and 280 nm to check the fluorescence intensity of the labeled protein. Protein was diluted to 1 : 10, if the fluorescence count signal of the labeled protein was beyond 400–500 fluorescence units. The ligand stock solution was serially diluted in 16-steps in PBST buffer (2% final DMSO concentration). An amount of 1 mM of NS5 was used as the highest concentration for the serial dilution. The samples were loaded into the Monolith standard capillaries, and thermophoresis was measured on a Monolith NT.115 equipment (NanoTemper Technologies, Germany). All the experiments were carried out in triplicate. The acquired data were analyzed by the MO Affinity Analysis 2.2.7 software. The binding constants (KD) were calculated by fitting the data with a 1 : 1 stoichiometry ratio condition.

Competitive binding assay

The FITC-tagged acetylated H4 peptide (H4K12ac) and NS5 were used to assess the competitive inhibition of the hBRD2–BD1 bromodomain. The assay mixture contained 300 nM protein, 60 nM peptide, and 1 µM of NS5. Assay mixture was added into 96-well plates and incubated for 45 min at room temperature in the dark. Data was collected and analyzed using the microplate reader (Tecan M200). Experiments were carried out in triplicate.

Cell culture experiments

Human glioblastoma cell line U87MG was obtained from National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in Dulbecco's Modified Eagle's Medium-High glucose (DMEM) (D5648-1L, Sigma–Aldrich, U.S.A.) supplemented with 10% foetal bovine serum (FBS) (Gibco), PenStrep (Gibco) [penicillin (100 U/ml) and streptomycin (100 mg/ml)] at 37°C in a humidified air atmosphere containing 5% CO2. Culture medium was exchanged twice a week. Once cell growth reached sub-confluence, cells were detached from flask using 0.05% trypsin-EDTA (Gibco). They were washed with DPBS, centrifuged at 2000 rpm for 5 min followed by trypsinization. The cell pellet was suspended in fresh culture media before use.

Cell proliferation assay

The effect of the compound NS5 on glioma cell proliferation was assessed in U87MG glioblastoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were washed with DPBS, trypsinized, and counted in a Neubauer chamber slide using trypan blue dye exclusion method. Viable cells were plated at a density of 1 × 104 cells per well in 96-well plates in a final volume of 0.1 ml media containing 10% FBS. After 24 h, cells were washed with DPBS and treated with various concentrations of NS5 (0.1 µM, 1 µM, 10 µM and 100 µM). This experiment was conducted in triplicates. The plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. After 24 h incubation, 100 µl of 2 mg/ml MTT was added to each well and incubated for 4 h at 37°C. Then, the medium was replaced with 200 µl of DMSO (dimethylsulfoxide) to solubilize the formazan crystals in each well and mixed gently. Absorbance was measured at 570 nm using a TECAN Infinite M200 multi-well plate reader. Data shown in the figure is representative of three independent experiments carried out in triplicates.

Trans-well migration assay

The glioma cell migration was investigated by trans-well migration method. The cells were gently washed with DPBS, before trypsinization. The cells were counted using a Neubauer chamber, and 2.5 × 104 cells were added to the upper chamber in FBS-free DMEM. Before transferring onto the upper chamber, the cells were treated with NS5 (10 µM). The DMEM with 10% FBS was added to the lower chamber of the insert as a chemo-attractant. Cell migration was allowed to proceed for 24 h at 37°C in a standard tissue culture incubator. The migrated cells were fixed and stained with 0.5% (w/v) crystal violet stained cells and the photomicrographs were taken at least in five random fields using an inverted microscope. Experiments were conducted in duplicate. Data shown in the figure is representative of three independent experiments.

Western blot assay

Total proteins were extracted using a PBS buffer containing complete protease inhibitor and phosphatase inhibitor. Protein concentration was determined by Bradford method. The protein extracts were resolved in 10% SDS–PAGE and transferred to nitrocellulose (NC) membrane using the semi-dry method (Bio-Rad). Then the membrane was blocked in 3% skimmed milk solution dissolved in PBS buffer. The samples were incubated with corresponding primary antibodies (BRD2 from Abnova, Taiwan, Cat. No. ABBIN562664 β-Tubulin from DSHB, The U.S.A., Cat. No. E7-C) overnight at 4°C. The membrane was washed with PBS and incubated with IgG-HRP secondary antibody (Jackson Lab, The U.S.A., Cat. No.140193 and Merck, Germany, Cat. No. AP132P) at room temperature for 1 h. The blots were visualized using chemiluminescence HRP substrate (GE Healthcare), and Chemi-Doc imagine system (Bio-Rad) was used for the image acquisition. Data shown are representative of three individual experiments.

Statistical analysis

All the values were expressed as mean ± SD. Data were analyzed using GraphPad Prism 6.0 software by student t-test. P value <0.05 was considered as significant.

Results

Virtual screening studies and Surface plasmon resonance assay

The docking analysis of the in silico screening of NCI diversity set III library (∼1630 compounds) against hBRD2–BD1 was performed. Twenty short-listed compounds were procured from National Cancer Institute (NCI), NIH, U.S.A. and dissolved in 100% DMSO to prepare 100 mM stock solutions. Amongst the 20 compounds, seven compounds (Supplementary Figure S1) illustrated robust interactions with BD1 as confirmed by SPR assay (Figure 1A). Intriguingly, the compound, NS5 (Figure 1B) showed a significant binding towards BD1. The SPR assay showed a concentration-dependent binding at different concentrations of NS5 (Figure 1C). The steady-state analysis yielded a KD value of 5 µM, suggesting that the discovered compound indeed binds substantially to the protein (Figure 1C). The physicochemical properties of NS5 have been evaluated (Supplementary Table S1). The predicted pharmacokinetic profile was also determined (Supplementary Table S2). The ADME properties of NS5 indicate that it is likely to be a lead compound.

Experimental in vitro evaluation of NS5 compound.

Figure 1.
Experimental in vitro evaluation of NS5 compound.

(A) SPR screening results of some NCI compounds with BRD2-BD1 that were shortlisted via in-silico analysis. (B) Chemical structure of the NS5 compound. (C) SPR kinetics sensograms of different concentrations of NS5 over immobilized BRD2-BD1.

Figure 1.
Experimental in vitro evaluation of NS5 compound.

(A) SPR screening results of some NCI compounds with BRD2-BD1 that were shortlisted via in-silico analysis. (B) Chemical structure of the NS5 compound. (C) SPR kinetics sensograms of different concentrations of NS5 over immobilized BRD2-BD1.

Microscale thermophoresis (MST) assay

To further validate the SPR results of BD1–NS5 binding, MST was also performed to confirm the binding from thermophoresis curves obtained as a function of time. The BD1 protein was labeled with NT-467 fluorescent dye and 16 different concentrations of NS5 from 1 mM to 30 nM were prepared to evaluate the binding interactions of BD1–NS5 as elaborated in the material and methods section. The interaction between BD1 and NS5 was measured using 20% LED-power and medium MST-power. MST traces showed smooth appearance and ligand concentration-dependent shift in magnitude. A concentration-dependent binding of NS5 with BD1 protein was observed (Figure 2). The binding affinity (KD) based on the directional movement of molecules along the temperature gradient and the protein–ligand interactions were measured using thermophoretic properties of the interacting molecules. The NS5 ligand binding to BD1 results in the binding affinity of 1.3 µM, suggesting that NS5 significantly binds to BD1.

MST analysis of NS5 binding towards hBRD2–BD1.

Figure 2.
MST analysis of NS5 binding towards hBRD2–BD1.

Concentration-response dependent behavior of the binding interaction between BRD2-BD1 and NS5 shows significant binding with a KD value of 1.3 µM. The KD values were obtained after fitting the binding curves between fraction bound plotted as a function of the ligand concentration (0 = unbound, 1 = bound).

Figure 2.
MST analysis of NS5 binding towards hBRD2–BD1.

Concentration-response dependent behavior of the binding interaction between BRD2-BD1 and NS5 shows significant binding with a KD value of 1.3 µM. The KD values were obtained after fitting the binding curves between fraction bound plotted as a function of the ligand concentration (0 = unbound, 1 = bound).

Competitive binding assay

To investigate the binding specificity of NS5 with the hBRD2–BD1 in the presence of the N-terminal acetylated H4 peptide, competitive binding experiment was performed using FITC-tagged H4 peptide acetylated at Lys12 (1–15aa, H4K12ac). As expected, NS5 substantially inhibits the H4K12ac peptide binding competitively with the BRD2-BD1 bromodomain (Figure 3). An amount of 1 µM concentration of NS5 binding with BRD2-BD1 bromodomain results in 30–35% H4K12ac peptide displacement competitively compared with the control.

Competitive binding assay for BD1 of BRD2.

Figure 3.
Competitive binding assay for BD1 of BRD2.

The FITC-tagged H4 acetylated peptide (H4K12ac) binding with the BRD2 bromodomain was displaced by NS5 (1 µM). The percent inhibition calculated with Graphpad Prism and the P-value was calculated using unpaired two-tailed t-test in comparison with control.

Figure 3.
Competitive binding assay for BD1 of BRD2.

The FITC-tagged H4 acetylated peptide (H4K12ac) binding with the BRD2 bromodomain was displaced by NS5 (1 µM). The percent inhibition calculated with Graphpad Prism and the P-value was calculated using unpaired two-tailed t-test in comparison with control.

Crystal structure of the BD1–NS5 complex

The seven compounds, short-listed from the SPR assay, were used for X-ray studies (Table 1). High-resolution X-ray diffraction data, from BD1 co-crystals with these seven compounds, were collected and their structures were determined by molecular replacement. Amongst them, the NS5 complex yielded an extra electron density for the ligand. The BD1 in complex with NS5, diffracted to 1.5 Å resolution, was crystallized in the C2 space group with three BD1 molecules and one ligand in the asymmetric unit. The two chains, A and B, form an intact dimer while the third chain, C form a dimer with the symmetry-related molecule (not shown). The BD1 tertiary structure formed a closely packed left-handed α-helical bundle of four helices (αZ, αA, αB, and αC) (Figure 4A). The deep hydrophobic binding pocket could be delineated within the loop regions of the helical bundle by the flexible and longer ZA loop (W97-D104), and shorter BC loop (N156-D160) regions. The extensive hydrogen-bonding network of six water molecules buried deep into the Kac site was also observed in our structure.

The crystal structure of BRD2-BD1 in complex with NS5 (PDB ID: 6JKE).

Figure 4.
The crystal structure of BRD2-BD1 in complex with NS5 (PDB ID: 6JKE).

(A) NS5 occupies the chain A of the BRD2-BD1 binding pocket. (B) A closer view of the electron density of NS5-|2Fo|–|Fc| map observed at 1σ in the binding pocket of BD1. (C) The major interactions of the NS5 are via N3, O6 and dione oxygen at C5 with conserved asparagine, namely N156 of the BRD2-BD1. A water molecule is engaged in a two-way hydrogen bond to P102 and NS5-N9. The conserved water molecule bridges dione oxygen atoms of NS5 and Y113. The -Cl at C7 position is involved in π-cation interaction with the sidechain of K107. Cyan ball and sticks represent ligand NS5, and blue cartoon represents the BD1 protein. The direct hydrogen bonds are indicated by blue dashes, and red spheres indicate interacting water molecules. I162 is observed in double conformation in our structure. Pictures of protein–ligand complex created using PyMol.

Figure 4.
The crystal structure of BRD2-BD1 in complex with NS5 (PDB ID: 6JKE).

(A) NS5 occupies the chain A of the BRD2-BD1 binding pocket. (B) A closer view of the electron density of NS5-|2Fo|–|Fc| map observed at 1σ in the binding pocket of BD1. (C) The major interactions of the NS5 are via N3, O6 and dione oxygen at C5 with conserved asparagine, namely N156 of the BRD2-BD1. A water molecule is engaged in a two-way hydrogen bond to P102 and NS5-N9. The conserved water molecule bridges dione oxygen atoms of NS5 and Y113. The -Cl at C7 position is involved in π-cation interaction with the sidechain of K107. Cyan ball and sticks represent ligand NS5, and blue cartoon represents the BD1 protein. The direct hydrogen bonds are indicated by blue dashes, and red spheres indicate interacting water molecules. I162 is observed in double conformation in our structure. Pictures of protein–ligand complex created using PyMol.

The difference Fourier map showed unambiguous electron density at the Kac binding site for the compound NS5 (NSC 328111; 7-chloro-2-(3-chloroanilino) pyrano[3,4-e][1,3]oxazine-4,5-dione) (Figure 4B). In the Kac binding pocket, NS5 bound via pyrano 1,3 oxazine moiety to the carboxamide side chain of N156 of chain A. A tightly-bound conserved water molecule bridges Y113 and the O5 atom of pyrano 1,3 oxazine of NS5 (Figure 4C). This oxygen occupied the same position as the -OH of the acetyl group of K12ac. The second dione at the C4 position of NS5, was observed to attract other water molecules to the binding site. Thus, the pyrano 1,3 oxazine behaved as the acetyl lysine mimicking moiety. The gatekeeper residue I162 is observed in double conformation in our structure and elicited necessary hydrophobic interactions with the pyrano 1,3 oxazine core of NS5.

NS5 extended towards the WPF shelf region via C2 of the pyrano 1,3 oxazine core. The lone benzene ring, forming the extension of NS5, was sandwiched between P102, K107, L108 termed the ‘ZA channel'. The most important tethering interaction for this portion of NS5 was the hydrogen bonding interactions to the backbone oxygen of P102 through a bridged water molecule interaction (Figure 4C). The conserved water molecule bridging the backbone carbonyl of P102 of the ZA loop preferred slightly displaced position in our structure (see below). This aided the lone pair of linker N9 of NS5 to form stronger hydrogen bond interactions (2.70 Å) to a conserved water molecule. Thus, the strategically positioned linker nitrogen N9 interacted via relatively weaker hydrogen bonding interaction to the carbonyl backbone of P98. P98 engaged in direct hydrogen bonding interaction (2.9 Å) to the N3 of NS5. The electron donor group, O1, uniquely attracted (2.9 Å) another water molecule to the binding site, strengthening the overall binding potential of the pyrano 1,3 oxazine core. Such interactions of NS5 to the backbone atoms of P98 and the conserved water molecule near P102 are unique to our inhibitor bound structure. We also observed several other water molecules at the edge of the binding cavity. Two of which, are drawn in the vicinity of −Cl at C7 of the pyrano 1,3 oxazine, culminating in the tight binding of the molecule to BD1 binding site.

Effect of NS5 on proliferation of glioma cells

To evaluate the effect of NS5 compound on proliferation, U87MG glioma cells were treated with NS5 for 24 h at various concentrations (0.1, 1, 10, and 100 µM) and the proliferation was evaluated by MTT assay. The NS5 compound significantly decreased glioma cell proliferation compared with vehicle-treated (DMSO) control at all the concentration studied in a dose-dependent manner (Figure 5). We used this assay to pre-optimize the effective dose for further study by determining the IC50. The IC50 value was found close to 10 µM after NS5 treatment in U87MG glioma cells. This concentration was considered for further downstream experiments.

Effect of NS5 on the proliferation in U87MG cells.

Figure 5.
Effect of NS5 on the proliferation in U87MG cells.

Effect of NS5 on proliferation of U87MG cells. The U87MG cells were seeded for 12 h and thereafter, treated with NS5 compound in various concentrations (0.1, 1, 10, and 100 µM) for 24 h. DMSO was used as vehicle control. Shown are the averages of the minimum three independent experiments performed in triplicate. Error bars indicate the standard deviation. Error bars represent the SD calculated over three independent experiments. *P ≤ 0.05; The P-value has been calculated using unpaired two-tailed t-test in comparison with control.

Figure 5.
Effect of NS5 on the proliferation in U87MG cells.

Effect of NS5 on proliferation of U87MG cells. The U87MG cells were seeded for 12 h and thereafter, treated with NS5 compound in various concentrations (0.1, 1, 10, and 100 µM) for 24 h. DMSO was used as vehicle control. Shown are the averages of the minimum three independent experiments performed in triplicate. Error bars indicate the standard deviation. Error bars represent the SD calculated over three independent experiments. *P ≤ 0.05; The P-value has been calculated using unpaired two-tailed t-test in comparison with control.

Effect of NS5 on migration of glioma cells

Next, to determine the role of NS5 on the migration of glioma cells, U87MG glioma cells were treated with NS5 at 10 µM concentrations and the migration was evaluated by trans-well migration assay as described in materials and methods. As shown in Figure 6, there was a 25–28% decrease in the migration of cells in comparison with the vehicle-treated control (P < 0.05) (Figure 6A,B).

Effect of NS5 on the migration of U87MG cells.

Figure 6.
Effect of NS5 on the migration of U87MG cells.

(A) Representative pictures are showing invaded cells (concentration of 10 µM of NS5). (B) Effect of NS5 on the migration of U87MG cells. The glioma cells were incubated with 10 µM of NS5 with or without NS5 and allowed to migrate through polycarbonate coated filters for 24 h. Each treatment group was repeated in duplicate, and each experiment in triplicate. Data shown are the averages of three independent experiments. *P ≤ 0.05; The P-value has been calculated using unpaired two-tailed t-test in comparison with control.

Figure 6.
Effect of NS5 on the migration of U87MG cells.

(A) Representative pictures are showing invaded cells (concentration of 10 µM of NS5). (B) Effect of NS5 on the migration of U87MG cells. The glioma cells were incubated with 10 µM of NS5 with or without NS5 and allowed to migrate through polycarbonate coated filters for 24 h. Each treatment group was repeated in duplicate, and each experiment in triplicate. Data shown are the averages of three independent experiments. *P ≤ 0.05; The P-value has been calculated using unpaired two-tailed t-test in comparison with control.

Western blot assay

To validate the effect of NS5 on protein expression level of BRD2, western blot assay was performed. The U87MG cells were treated with 10 µM NS5 and incubated for 24 h in CO2 incubator. The cell lysis, protein extraction, and blotting experiments were carried out as mentioned in the materials and method section. Findings from immunoblot analysis clearly showed that NS5 compound significantly down-regulated the expression level of hBRD2 protein (Figure 7).

Effect of NS5 on the protein expression level of BRD2.

Figure 7.
Effect of NS5 on the protein expression level of BRD2.

(A) Representative BRD2 immunoblot. (B) Immunoblot analysis representing decrease in the BRD2 protein expression level after treatment with NS5 (10 µM). *P ≤ 0.05; The P-value has been calculated using unpaired two-tailed t-test in comparison with control. Blot is representative of at least three independent experiments.

Figure 7.
Effect of NS5 on the protein expression level of BRD2.

(A) Representative BRD2 immunoblot. (B) Immunoblot analysis representing decrease in the BRD2 protein expression level after treatment with NS5 (10 µM). *P ≤ 0.05; The P-value has been calculated using unpaired two-tailed t-test in comparison with control. Blot is representative of at least three independent experiments.

Discussion

Glioblastoma (GBM) is the most common primary brain malignancy and poses grave therapeutic challenge attributed to its invasive behavior. This heterogeneous tumor is characterized by genetic and epigenetic alterations that modify multiple signaling pathways resulting in growth, progression, and therapeutic resistance [33]. Evidence suggests epigenetic processes modulate the structural and functional complexity of the nervous system, with perturbations in these mechanisms being implicated in cerebral pathologies [34,35]. The bromodomains are the epigenetic readers, integral to many chromatin-associated proteins, recognize and bind to the acetylated histones, and regulate transcription. The BET family proteins BRD2 and BRD4, with conserved bromodomains, are exciting therapeutic targets for developing drug molecules against GBM. Currently, temozolomide (TMZ) and lomustine are the only FDA approved first-line chemotherapeutics but is marred by frequent drug resistance [36]. There is a dire need to address the current chemotherapeutic scenario, including identifying new molecules against GBM.

Our work on discovering potential compounds by rational structure-based approach yielded a new scaffold structure of hBRD2–BD1 inhibitor from the NCI diversity set III library (Table 1). The discovered compound, NS5, binds substantially at the Kac binding site of hBRD2–BD1. The pyrano 1,3 oxazine core contributes significant interactions with the conserved residues; I162, N156, and water molecules. Moreover, the uniqueness of pyrano 1,3 oxazine core of NS5 lies in its capability to draw additional water molecules to the binding site. The chlorobenzene ring extends into the ZA channel and WPF shelf, making brief interactions with P98 and P102 and the surrounding water molecules. Secondly, −Cl at position 12 of ring C allows subtle engagement of the ZA loop by mediating long range electrostatic interactions via long side chain of K107.

The key linker atom N9 of NS5, presents exciting chemical features and plays a vital role as reinforcing the binding of the pyrano 1,3 oxazine core in the Kac binding pocket. We speculate that the two single bonds on either side of N9 make allowances for high degree of rotations for the attached aromatic rings. The rotation about the bond connecting N9 and the chlorobenzene ring may be more apparent due to the presence of moderate intermolecular interactions in this region. It is reflected in the partial electron density in the corresponding region of the crystal structure (Figure 4B). Such internal changes of the molecule are countered by the hydrophobic interactions of the chloroanilino group with V103, L108, and the WPF shelf, particularly, W97 (T-shape π–π stacking interactions) that makes the binding of the choloroanilino group possible. Overall, the significant number of hydrophilic and hydrophobic interactions contribute towards the strong binding of the compound to BD1. It correlates well with the substantial binding affinity in low micromolar range as observed by SPR and MST studies.

Of the various BET family inhibitors reported till date, only three- JQ1, OTX015, and I-BET151 have shown to inhibit the growth of GBM cells [18,37–39]. The other inhibitor, I-BET726, was shown to be active in neuroblastoma [28]. Recently, the fragment F1, obtained by automated ligand screening pipeline, was shown to have a potent inhibition against hBRD4–BD1 [40]. Therefore, a comparison of NS5 with these established inhibitors of BRD2 and BRD4 is worthy of mention (Figure 8).

Comparison of the BRD2–BD1–NS5 complex.

Figure 8.
Comparison of the BRD2–BD1–NS5 complex.

(A) Superposition of the BRD2–BD1–NS5 complex (cyan ball and stick) on to the mutated BRD4–BD1–OTX015 complex (yellow ball and stick; PDB ID: 5WMD) and BRD4-BD1-JQ1 complex (orange ball and stick; PDB ID: 3MXF). The pyrano 1,3 oxazine scaffold of NS5 and the triazolo moiety of the triazolodiazepine (TzD) core in OTX015 and JQ1 show necessary interactions with the conserved asparagine residue in the respective complex. The tert-butyl ester group substituted to this core in JQ1 does not particularly show hydrophilic interactions in the BD1 binding site. The same position in OTX015 is substituted by the phenyl acetamide group in OTX015 and likewise, does not exhibit notable interactions within the BD1 binding site residues. (B) Superposition of the NS5 complex onto the I-BET151 complex. Pyrano 1,3 oxazine scaffold of NS5 (cyan ball and stick model) and isoxazole core of (I-BET151 in PDB ID: 4ALG) superpose to mediate hydrophilic interactions with N156 of the binding site. They both extend towards the WPF shelf. The water molecule in the ZA channel that enhances the overall binding of NS5 in the BRD2-BD1 binding site is shown as a light pink sphere. (C) Superposition of the NS5 complex onto the I-BET726 complex (PDB ID: 4UYF) highlights their presence in the ZA channel, by means of the chlorophenyl ring in NS5 and benzoic acid group of I-BET726. (D) Superposition of the NS5 complex with F1 fragment complex (PDB ID: 6FNX) displays similarity in the binding mode of their respective scaffolds.

Figure 8.
Comparison of the BRD2–BD1–NS5 complex.

(A) Superposition of the BRD2–BD1–NS5 complex (cyan ball and stick) on to the mutated BRD4–BD1–OTX015 complex (yellow ball and stick; PDB ID: 5WMD) and BRD4-BD1-JQ1 complex (orange ball and stick; PDB ID: 3MXF). The pyrano 1,3 oxazine scaffold of NS5 and the triazolo moiety of the triazolodiazepine (TzD) core in OTX015 and JQ1 show necessary interactions with the conserved asparagine residue in the respective complex. The tert-butyl ester group substituted to this core in JQ1 does not particularly show hydrophilic interactions in the BD1 binding site. The same position in OTX015 is substituted by the phenyl acetamide group in OTX015 and likewise, does not exhibit notable interactions within the BD1 binding site residues. (B) Superposition of the NS5 complex onto the I-BET151 complex. Pyrano 1,3 oxazine scaffold of NS5 (cyan ball and stick model) and isoxazole core of (I-BET151 in PDB ID: 4ALG) superpose to mediate hydrophilic interactions with N156 of the binding site. They both extend towards the WPF shelf. The water molecule in the ZA channel that enhances the overall binding of NS5 in the BRD2-BD1 binding site is shown as a light pink sphere. (C) Superposition of the NS5 complex onto the I-BET726 complex (PDB ID: 4UYF) highlights their presence in the ZA channel, by means of the chlorophenyl ring in NS5 and benzoic acid group of I-BET726. (D) Superposition of the NS5 complex with F1 fragment complex (PDB ID: 6FNX) displays similarity in the binding mode of their respective scaffolds.

The hBRD2–BD1–NS5 complex was superposed to the hBRD4–BD1–JQ1 complex (PDB ID: 3MXF) [41] and hBRD4–BD1–OTX015 complex (PDB ID: 5WMD) [42] to compare the binding mode of JQ1, OTX015 and NS5 in the hBRD2–BD1 binding pocket (Cα-RMSD 0.33 Å) (Figure 8A). Like JQ1, OTX015 possessed a benzodiazepine core where the tert-butyl substituent of JQ1 was replaced by the phenyl acetamide moiety in OTX015. The prominent interaction was observed with the respective conserved asparagine residue (N140 in hBRD4–BD1 and N156 in hBRD2–BD1) via hydrogen bonding that was more appreciable in our structure (2.8 Å vs. 3.14 Å). The pyrano 1,3 oxazine nucleus of NS5 mediated hydrogen bond interactions to the conserved asparagine, N156. This is analogous to the interaction of triazolo ring in the thienotriazolodiazepine nucleus of the JQ1 or OTX015. The chlorophenyl group in JQ1 and OTX015 exhibited limited hydrophobic interactions with WPF shelf (via P98 in hBRD2–BD1, P82 in hBRD4–BD1 or W81 in hBRD4–BD1) and a vital gatekeeper residue I162, while NS5 extended deeper into the WPF shelf, mediating additional interactions with W97.

The superposition of NS5 scaffold and the established inhibitor, I-BET151 (Cα-RMSD 0.33Å), bound to the hBRD2–BD1 structure (PDB ID: 4ALG) [43] showed that the NS5 binds to the hBRD2–BD1 pocket analogous to I-BET151 (Figure 8B). The I-BET151 possesses weak hydrophilic interactions compared with NS5. In I-BET151, only the oxygen moiety of the isoxazole ring forms a hydrogen bond with the conserved asparagine, whereas four hydrogen bonds are found in the corresponding region of NS5 (Figure 4C). Secondly, the quinoline nitrogen in I-BET151 contributes a weak interaction (3.4 Å) in the WPF shelf. However, in NS5, the linker nitrogen mediates substantial interactions with the help of P98 (3.0 Å). The respective groups in both the molecules further provide stabilization from behind, by means of water bridged backbone atoms of P102 in the ZA channel. The choloroanilino group in NS5 extends in the ZA channel that enhances the overall binding of NS5 in the BD1 binding site.

The NS5 scaffold and the established inhibitor of neuroblastoma, I-BET726, bound to hBRD2–BD1 (PDB ID: 4UYF) [28] present an interesting corollary (Figure 8C). The pose of the terminal benzoic acid ring, in I-BET726 (PDB ID: 4UYF) was found to be the same as the choloroanilino group of NS5. Additional interaction with K107 was found in both structures. I-BET726 via −COOH makes electrostatic interactions, while NS5 makes interactions via −Cl with K157 (Figure 4C). This is analogous to the interactions shown by the NS5 via its chlorophenyl ring. Perhaps, it may be such interactions that the tetrahydroquinoline based compound exhibits potent activity towards hBRD2 (KD = 4.4 nM), and hBRD4 (KD = 22 nM) [28].

The superposition of the hBRD2–BD1–NS5 complex with the high-resolution structure of the F1 fragment bound to hBRD4–BD1 (PDB ID: 6FNX) [40] was performed (Figure 8D). The 1,3 pyrano oxazine core of NS5 and the F1 fragment by means of 2,6 dione of the xanthine nucleus interact with the respective conserved asparagine via the same hydrogen bonding distance (2.8 Å). Moreover, the N7 and N9 of the five-membered purine ring of F1 superpose remarkably with the O1 and N3 of the oxazinium nucleus of NS5. In both NS5 and F1, the position of polar substituents (F1: dione of the sixth position and NS5: Cl at seventh position) do not mainly show any interactions with the binding site residues.

The comparison study of newly discovered pyrano 1,3 oxazine based derivative, NS5 with these known inhibitors unambiguously suggested that the NS5 inhibitor is indeed a potential and novel lead molecule in the drug discovery pipeline of BET family inhibitors targeting GBM.

The JQ1, OTX015, and I-BET151 are the known inhibitors of the BRD4 in glioblastoma. The JQ1 is highly penetrant in the brain, while I-BET151 is unlikely permeable in the brain due to its polar surface; thus it is unclear whether it can be used for the in vivo validation [18]. All these inhibitors are specific to the expression of BRD4 protein in glioblastoma. The known inhibitors of BET family proteins like JQ1, I-BET151 were able to down-regulate the expression of BRD4 in GBM and were shown to inhibit the glioma cell proliferation [18]. The BET family proteins, BRD4 and BRD2, have established the role in glioblastoma cell proliferation. The BRD2 and BRD4 may have a coinciding function or independent function in glioma cell proliferation. JQ1, OTX015, and I-BET151 were reported to predominantly target hBRD4 and showed weaker affinity towards hBRD2; whereas, NS5 was observed to specifically inhibit hBRD2 in GBM cells. Successful BET family druggable compounds resulted from intensive lead optimization efforts. The NS5 ligand, obtained from in-silico screening, which possesses scaffold 1,3 pyrano oxazine nucleus holds a great promise in further development of hBRD2 specific molecules. Hit optimization strategies are underway in the laboratory to enhance the efficacy and potency of NS5.

Our study established IC50 of NS5 in U87MG cells is 10 µM (Figure 5). Furthermore, we evaluated the biological effects of NS5 on U87MG cells. The anti-proliferative effect of NS5 at 100 µM was 75%, demonstrating its effect in a dose-dependent manner.

Invasion is the hallmark of glioblastoma [43] and migration is a prerequisite for invasion. Peritumoral migration and invasion contribute significantly towards the growth and progression of GBM [44]. During central nervous system development, overexpression of hBRD2 is reported to be essential for neural crest migration. In this relevance, we examined the effect of NS5 on the migration of GBM cells. To best of our knowledge, this is the first study to evaluate the effect of BET protein bromodomain inhibitor on the migration of glioma cells. Trans-well migration assay demonstrated that NS5 significantly attenuated the migration of GBM cells (Figure 6A,B). Our results imply that NS5 not only mitigates proliferation, but also has anti-migratory effect.

To delineate the biological effect of NS5, we evaluated the protein expression of BRD2 in U87MG cells on treatment with NS5 (Figure 7). Western blot analysis confirmed that NS5 significantly inhibited the expression of hBRD2. Therefore, as evidenced from effective inhibition of BRD2 at translational level, the implication of NS5 mediated inhibitory effect of U87MG cells through inhibition of hBRD2 can be surmised.

A recent study highlighted that BET inhibitor, dBET1 significantly reduced the expression of proinflammatory genes including IL-1β, TNF-α, MMP-9, and thereby NF-κB in microglia [45]. Also, we had demonstrated that proinflammatory milieu in glioma is crucial for migration and invasion [46,47]. Furthermore, in this context, it is worth exploring the effect of NS5 on inflammatory response and proinflammatory mediated pathway including, ERK signaling and transcription factor, NF-κB.

In summary, we have discovered a novel scaffold molecule, NS5 by a rational structure-based approach. Based on our structural and biochemical binding studies, NS5 significantly binds to hBRD2–BD1 with a KD value of 1.3 µM. The crystal structure showed that the pyrano 1,3 oxazine nucleus of NS5 is capable of enunciating multiple interactions with the BD1 binding site residues. Additionally, the linker nitrogen substituent of NS5 participates in strong hydrogen bonding interactions to P98 imparting a cumulative binding effect on the ligand binding. Modeling efforts suggest that this portion of NS5 can be retained during the optimization procedure. In order to optimize the potency and activity of the compound, larger aromatic groups can be accommodated in the ZA channel, so that it may be more tightly packed against the WPF shelf. NS5 demonstrated inhibitory effect on GBM cells with an IC50 value of 10 µM and demonstrated the anti-proliferative effect in a dose-dependent manner. Additionally, the trans-migration assay demonstrated that NS5 significantly attenuated GBM cell migration. Furthermore, evaluation at the translational level established significant inhibition of BRD2 upon treatment with NS5. Thus, the discovered compound, NS5, can serve as an excellent scaffold, to identify novel pyrano 1,3 oxazine based BRD2 inhibitors targeting GBM.

Data Availability

Structural data are available in RCSB-PDB database(s) under the accession number 6JKE.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

BP is grateful to the DST (DST-FIST: SR/FST/LS-I/2017(C)), SERB (SR/SO/BB-0108/2012), and DBT, Government of India (BT/PR7079/BID/7/426/2012) for the financial support. NDN acknowledges the research grant from SERB (EMR/2014/000937).

Author Contributions

B.P. and D.N.N. designed research; B.P., D.P., M.S., G.G. and K.G. performed research; B.P., D.N.N., D.P., M.S., G.G. and K.G. analyzed data; and B.P., D.N.N., D.P., M.S., G.G. and K.G. wrote the paper.

Acknowledgements

We thank Dr. B. N. Gangadhar, The Director, NIMHANS for providing the XRD facility. GG acknowledges the junior research fellowship from the UGC, New Delhi, India. PD is thankful to ICMR for the ICMR-SRF fellowship (45/51/2018/PHA/BMS), and SM for DST WOS-A fellowship (SR/WOS-A/LS-100/2013). We thank the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Divison of Cancer Treatment and Diagnosis, NCI, U.S.A. for providing the compounds used in the study.

Abbreviations

     
  • BD1

    bromodomain

  •  
  • BET

    bromodomain and extra-terminal

  •  
  • GBM

    Glioblastoma

  •  
  • LE

    ligand efficiency

  •  
  • MB3

    1-methylpyrrolidin-2-one

  •  
  • NCI

    National Cancer Institute

  •  
  • TMZ

    temozolomide

References

References
1
Arrowsmith
,
C.H.
,
Bountra
,
C.
,
Fish
,
P.V.
,
Lee
,
K.
and
Schapira
,
M.
(
2012
)
Epigenetic protein families: a new frontier for drug discovery
.
Nat. Rev. Drug Discov.
11
,
384
400
2
Belkina
,
A.C.
and
Denis
,
G.V.
(
2012
)
BET domain co-regulators in obesity, inflammation and cancer
.
Nat. Rev. Cancer
12
,
465
477
3
Andrieu
,
G.
,
Belkina
,
A.C.
and
Denis
,
G.V.
(
2016
)
Clinical trials for BET inhibitors run ahead of the science
.
Drug Discov. Today Technol.
19
,
45
50
4
Padmanabhan
,
B.
,
Mathur
,
S.
,
Manjula
,
R.
and
Tripathi
,
S.
(
2016
)
Bromodomain and extra-terminal (BET) family proteins: new therapeutic targets in major diseases
.
J. Biosci.
41
,
295
311
5
Ito
,
T.
,
Umehara
,
T.
,
Sasaki
,
K.
,
Nakamura
,
Y.
,
Nishino
,
N.
,
Terada
,
T.
, et al (
2011
)
Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors
.
Chem. Biol.
18
,
495
507
6
Dawson
,
M.A.
,
Prinjha
,
R.K.
,
Dittmann
,
A.
,
Giotopoulos
,
G.
,
Bantscheff
,
M.
,
Chan
,
W.-I.
, et al (
2011
)
Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia
.
Nature
478
,
529
533
7
Zeng
,
L.
,
Li
,
J.
,
Muller
,
M.
,
Yan
,
S.
,
Mujtaba
,
S.
,
Pan
,
C.
et al (
2005
)
Selective small molecules blocking HIV-1 Tat and coactivator PCAF association
.
J. Am. Chem. Soc.
127
,
2376
2377
8
Odore
,
E.
,
Lokiec
,
F.
,
Cvitkovic
,
E.
,
Bekradda
,
M.
,
Herait
,
P.
,
Bourdel
,
F.
, et al (
2016
)
Phase I population pharmacokinetic assessment of the oral bromodomain inhibitor OTX015 in patients with haematologic malignancies
.
Clin. Pharmacokinet.
55
,
397
405
9
Chung
,
C.-W.
,
Coste
,
H.
,
White
,
J.H.
,
Mirguet
,
O.
,
Wilde
,
J.
,
Gosmini
,
R.L.
, et al (
2011
)
Discovery and characterization of small molecule inhibitors of the BET family bromodomains
.
J. Med. Chem.
54
,
3827
3838
10
Hanif
,
F.
,
Muzaffar
,
K.
,
Perveen
,
K.
,
Malhi
,
S.M.
and
Simjee
,
S.U.
(
2017
)
Glioblastoma multiforme: a review of its epidemiology and pathogenesis through clinical presentation and treatment
.
Asian Pac. J. Cancer Prev. APJCP
18
,
3
9
11
Stupp
,
R.
,
Taillibert
,
S.
,
Kanner
,
A.A.
,
Kesari
,
S.
,
Steinberg
,
D.M.
,
Toms
,
S.A.
, et al (
2015
)
Maintenance therapy With tumor-Treating fields plus temozolomide vs temozolomide alone for glioblastoma: a randomized clinical trial
.
JAMA
314
,
2535
2543
12
Stupp
,
R.
,
Mason
,
W.P.
,
van den Bent
,
M.J.
,
Weller
,
M.
,
Fisher
,
B.
,
Taphoorn
,
M.J.B.
, et al (
2005
)
Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma
.
N. Engl. J. Med.
352
,
987
996
13
Stupp
,
R.
,
Hegi
,
M.E.
,
Mason
,
W.P.
,
van den Bent
,
M.J.
,
Taphoorn
,
M.J.B.
,
Janzer
,
R.C.
, et al (
2009
)
Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial
.
Lancet Oncol.
10
,
459
466
14
Shergalis
,
A.
,
Bankhead
,
A.
,
Luesakul
,
U.
,
Muangsin
,
N.
and
Neamati
,
N.
(
2018
)
Current challenges and opportunities in treating glioblastoma
.
Pharmacol. Rev.
70
,
412
445
15
Hochberg
,
F.H.
,
Atai
,
N.A.
,
Gonda
,
D.
,
Hughes
,
M.S.
,
Mawejje
,
B.
,
Balaj
,
L.
et al (
2014
)
Glioma diagnostics and biomarkers: an ongoing challenge in the field of medicine and science
.
Expert Rev. Mol. Diagn.
14
,
439
452
16
Gusyatiner
,
O.
and
Hegi
,
M.E.
(
2018
)
Glioma epigenetics: from subclassification to novel treatment options
.
Semin. Cancer Biol.
51
,
50
58
17
Romani
,
M.
,
Pistillo
,
M.P.
and
Banelli
,
B.
(
2018
)
Epigenetic targeting of glioblastoma
.
Front. Oncol.
8
,
448
18
Pastori
,
C.
,
Daniel
,
M.
,
Penas
,
C.
,
Volmar
,
C.-H.
,
Johnstone
,
A.L.
,
Brothers
,
S.P.
, et al (
2014
)
BET bromodomain proteins are required for glioblastoma cell proliferation
.
Epigenetics
9
,
611
620
19
Chung
,
C.-W.
,
Dean
,
A.W.
,
Woolven
,
J.M.
and
Bamborough
,
P.
(
2012
)
Fragment-based discovery of bromodomain inhibitors part 1: inhibitor binding modes and implications for lead discovery
.
J. Med. Chem.
55
,
576
586
20
DeLano
,
W.L.
(
2002
)
Pymol: an open-source molecular graphics tool
.
CCP4 Newsl. Protein Crystallogr.
40
,
82
92
https://pymol.org/2/
21
Abad-Zapatero
,
C.
(
2007
)
Ligand efficiency indices for effective drug discovery
.
Expert Opin. Drug Discov.
2
,
469
488
22
Daina
,
A.
,
Michielin
,
O.
and
Zoete
,
V.
(
2017
)
SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules
.
Sci. Rep.
7
,
42717
23
Tripathi
,
S.
,
Mathur
,
S.
,
Deshmukh
,
P.
,
Manjula
,
R.
and
Padmanabhan
,
B.
(
2016
)
A novel phenanthridionone based scaffold As a potential inhibitor of the BRD2 bromodomain: crystal structure of the complex
.
PLoS ONE
11
,
e0156344
24
Umehara
,
T.
,
Wakamori
,
M.
,
Tanaka
,
A.
,
Padmanabhan
,
B.
and
Yokoyama
,
S.
(
2007
)
Purification, crystallization and preliminary X-ray diffraction of the C-terminal bromodomain from human BRD2
.
Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun.
63
,
613
615
25
Battye
,
T.G.G.
,
Kontogiannis
,
L.
,
Johnson
,
O.
,
Powell
,
H.R.
and
Leslie
,
A.G.W.
(
2011
)
iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM
.
Acta Crystallogr. D Biol. Crystallogr.
67
,
271
281
26
Evans
,
P.R.
and
Murshudov
,
G.N.
(
2013
)
How good are my data and what is the resolution?
Acta Crystallogr. D Biol. Crystallogr.
69
,
1204
1214
27
McCoy
,
A.J.
,
Grosse-Kunstleve
,
R.W.
,
Adams
,
P.D.
,
Winn
,
M.D.
,
Storoni
,
L.C.
and
Read
,
R.J.
(
2007
)
Phaser crystallographic software
.
J. Appl. Crystallogr.
40
,
658
674
28
Gosmini
,
R.
,
Nguyen
,
V.L.
,
Toum
,
J.
,
Simon
,
C.
,
Brusq
,
J.-M.G.
,
Krysa
,
G.
, et al (
2014
)
The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor
.
J. Med. Chem.
57
,
8111
8131
29
Lebedev
,
A.A.
,
Young
,
P.
,
Isupov
,
M.N.
,
Moroz
,
O.V.
,
Vagin
,
A.A.
and
Murshudov
,
G.N.
(
2012
)
JLigand: a graphical tool for the CCP4 template-restraint library
.
Acta Crystallogr. D Biol. Crystallogr.
68
,
431
440
30
Murshudov
,
G.N.
,
Vagin
,
A.A.
and
Dodson
,
E.J.
(
1997
)
Refinement of macromolecular structures by the maximum-likelihood method
.
Acta Crystallogr. D Biol. Crystallogr.
53
,
240
255
31
Emsley
,
P.
and
Cowtan
,
K.
(
2004
)
Coot: model-building tools for molecular graphics
.
Acta Crystallogr. D Biol. Crystallogr.
60
,
2126
2132
32
Chen
,
V.B.
,
Arendall
,
W.B.
,
Headd
,
J.J.
,
Keedy
,
D.A.
,
Immormino
,
R.M.
,
Kapral
,
G.J.
et al (
2010
)
Molprobity: all-atom structure validation for macromolecular crystallography
.
Acta Crystallogr. D Biol. Crystallogr.
D66
,
12
21
33
Bulstrode
,
H.
,
Johnstone
,
E.
,
Marques-Torrejon
,
M.A.
,
Ferguson
,
K.M.
,
Bressan
,
R.B.
,
Blin
,
C.
, et al (
2017
)
Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators
.
Genes Dev.
31
,
757
773
34
Hannon
,
E.
,
Lunnon
,
K.
,
Schalkwyk
,
L.
and
Mill
,
J.
(
2015
)
Interindividual methylomic variation across blood, cortex, and cerebellum: implications for epigenetic studies of neurological and neuropsychiatric phenotypes
.
Epigenetics
10
,
1024
1032
35
Qureshi
,
I.A.
and
Mehler
,
M.F.
(
2013
)
Understanding neurological disease mechanisms in the era of epigenetics
.
JAMA Neurol.
70
,
703
710
36
Herrlinger
,
U.
,
Tzaridis
,
T.
,
Mack
,
F.
,
Steinbach
,
J.P.
,
Schlegel
,
U.
,
Sabel
,
M.
, et al (
2019
)
Lomustine-temozolomide combination therapy versus standard temozolomide therapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter (CeTeG/NOA-09): a randomised, open-label, phase 3 trial
.
Lancet
393
,
678
688
37
Pastori
,
C.
,
Kapranov
,
P.
,
Penas
,
C.
,
Peschansky
,
V.
,
Volmar
,
C.-H.
,
Sarkaria
,
J.N.
, et al (
2015
)
The bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation
.
Proc. Natl Acad. Sci. U.S.A.
112
,
8326
8331
38
Cheng
,
Z.
,
Gong
,
Y.
,
Ma
,
Y.
,
Lu
,
K.
,
Lu
,
X.
,
Pierce
,
L.A.
et al (
2013
)
Inhibition of BET bromodomain targets genetically diverse glioblastoma
.
Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.
19
,
1748
1759
39
Liu
,
C.-A.
,
Chang
,
C.-Y.
,
Hsueh
,
K.-W.
,
Su
,
H.-L.
,
Chiou
,
T.-W.
,
Lin
,
S.-Z.
et al (
2018
)
Migration/Invasion of malignant gliomas and implications for therapeutic treatment
.
Int. J. Mol. Sci.
19
,
1115
40
Hoffer
,
L.
,
Voitovich
,
Y.V.
,
Raux
,
B.
,
Carrasco
,
K.
,
Muller
,
C.
,
Fedorov
,
A.Y.
, et al (
2018
)
Integrated strategy for lead optimization based on fragment growing: the diversity-Oriented-Target-Focused-Synthesis approach
.
J. Med. Chem.
61
,
5719
5732
41
Filippakopoulos
,
P.
,
Qi
,
J.
,
Picaud
,
S.
,
Shen
,
Y.
,
Smith
,
W.B.
,
Fedorov
,
O.
, et al (
2010
)
Selective inhibition of BET bromodomains
.
Nature
468
,
1067
1073
42
Ozer
,
H.G.
,
El-Gamal
,
D.
,
Powell
,
B.
,
Hing
,
Z.A.
,
Blachly
,
J.S.
,
Harrington
,
B.
, et al (
2018
)
BRD4 profiling identifies critical chronic lymphocytic leukemia oncogenic circuits and reveals sensitivity to PLX51107, a novel structurally distinct BET inhibitor
.
Cancer Discov.
8
,
458
477
43
Seal
,
J.
,
Lamotte
,
Y.
,
Donche
,
F.
,
Bouillot
,
A.
,
Mirguet
,
O.
,
Gellibert
,
F.
, et al (
2012
)
Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A)
.
Bioorg. Med. Chem. Lett.
22
,
2968
2972
44
Nørøxe
,
D.S.
,
Poulsen
,
H.S.
and
Lassen
,
U.
(
2016
)
Hallmarks of glioblastoma: a systematic review
.
ESMO Open
1
,
e000144
45
DeMars
,
K.M.
,
Yang
,
C.
,
Castro-Rivera
,
C.I.
and
Candelario-Jalil
,
E.
(
2018
)
Selective degradation of BET proteins with dBET1, a proteolysis-targeting chimera, potently reduces pro-inflammatory responses in lipopolysaccharide-activated microglia
.
Biochem. Biophys. Res. Commun.
497
,
410
415
46
Fathima Hurmath
,
K.
,
Ramaswamy
,
P.
and
Nandakumar
,
D.N.
(
2014
)
IL-1β microenvironment promotes proliferation, migration and invasion of human glioma cells
.
Cell Biol. Int.
38
,
1415
1422
47
Ramaswamy
,
P.
,
Goswami
,
K.
,
Nanjaiah
,
N.D.
,
Srinivas
,
D.
and
Prasad
,
C.
(
2019
)
TNF-α mediated MEK-ERK signaling in invasion with putative network involving NF-κB and STAT-6: a new perspective in glioma
.
Cell Biol. Int.
43
,
1257
1266

Author notes

*

These authors contributed equally to this work.