BRD4 bromodomain (Ki = 30-50 nM)

MS436, through a sequence of water-mediated interactions, has low nanomolar affinity (estimated Ki of 30-50 nM) with preference for the first bromodomain over the second. MS436 efficiently suppresses BRD4 activity in NF-κB-directed generation of NO and pro -inflammatory cytokine interleukin-6 in murine macrophages. MS436 represents a novel class of bromodomain inhibitors and will permit further exploration of the biological functions of the two bromodomains of BRD4 in gene expression. MS436 demonstrates high affinity with an estimated Ki=30-50 nM for the BRD4 BrD1 and a 10-fold selectivity over the BrD2, which is achieved by a unique series of water-mediated intermolecular interactions[1].

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Notably, MS436 (compound 27) is the most potent compound of this set of diazobenzene compounds as determined in an in vitro fluorescent anisotropy assay with Ki better than 85 nM for the BRD4 BrD1 (limited by the binding affinity of the assay probe; the estimated Ki is 30–50 nM), and an approximately 10-fold selectivity for the BrD1 over the BrD2 of BRD4 (Table 2). This is the first reported low nanomolar affinity BrD inhibitor with clear selectivity between these two BrDs of BRD4 as far as we know. MS436 is ten times more potent than the model compound MS435 towards the BRD4 BrD1. Interestingly, MS267 (compound 28), a “hybrid” compound incorporating the key features of MS435 and MS436 exhibited an about 2-fold reduction in potency than MS436, although it is still much better than MS435. In addition, placing a Cl at the ortho position with respect to the hydroxyl group in the A ring in another hybrid compound MS363 (compound 29) showed a 2-fold decrease in binding affinity for both BrDs of BRD4. Nevertheless, both MS267 and MS363 maintained the selectivity for the BRD4 BrD1 over the BrD2. Notably, change of the methyl group at R3 in MS436 to methoxy (30) resulted in a 15-fold drop of binding affinity to either BRD4 BrD1 or BrD2. This explains the importance of this methyl group in mimicking the methyl group of acetyl-lysine in a biological ligand. Finally, our endeavors to employ heterocycles in the A ring were unfruitful. Introduction of a nitrogen atom into the A ring aromatic system led to a 3-fold drop in affinity (31 and 32 vs. MS435). [1]

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Activity of Diazobenzene Compounds against the Bromodomain Panel [1]
We profiled four lead diazobenzene-based BET BrD inhibitors, MS435, MS436, MS267 and MS363 against a panel of bromodomains that represent different subgroups of the human bromodomain family (Table 3). All four inhibitors preferentially bind to the BrDs of BRD4 and BRD3 over the other BrDs tested. Notably, MS436 activity towards CBP BrD is four times better than the initial compound MS120. It is worth mentioning that these diazobenzene compounds have a different bromodomain activity profile from diazepine-based BrD inhibitors such as JQ1, MS417, and I-BET. Specifically, MS267 showed binding affinity, Ki of <1.43 µM, <1.70 µM, and 2.26 µM for the BrDs of BRD7, BPTF and BAZ2b, respectively. MS435 showed Ki of <6.56 µM for SMARCA4 BrD. These unique activity profiles make them potentially useful probes for studies of these non-BET BrDs.

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Activities of Diazobenzene Compounds in Murine Macrophage RAW264.7 Cells [1]
These four lead diazobenzene bromodomain inhibitors exhibited little observable cytotoxicity on cell growth and proliferation, as determined in a MTT assay in murine macrophage RAW264.7 cells. As shown by this MTT study, the cell viability was fairly stable with these bromodomain inhibitors at concentrations up to 100 µM (Figure 3A). We next evaluated the cellular efficacy of these bromodomain inhibitors in blocking BRD4 functions in gene transcription. Nitric oxide synthase (NOS) catalyzes a stoichiometric production of nitric oxide (NO) from L-arginine in cells. NO production in macrophages is mediated by inducible nitric oxide synthase (iNOS), which is regulated by the NF-κB pathway. As shown in our recent study, NF-κB transcriptional activity for target gene activation is dependent upon its lysine-acetylation-mediated interactions with BRD4, which recruits the activated NF-κB to its target gene sites by binding to di-acetylated histone H4, particularly H4K5ac/K8ac. Indeed, we observed that treatment with the diazobenzene compounds in a dose-dependent manner blocked NF-κB-directed NO production in RAW264.7 cells upon LPS stimulation (Figure 3B). In agreement with their in vitro binding affinity to the BRD4 BrD1, MS436 showed more profound inhibitory activity on NO production than MS435. Consistent with the fact that the BET proteins are functionally vital for macrophage inflammatory responses, these diazobenzene bromodomain inhibitors effectively block LPS-induced pro-inflammatory cytokine interleukin-6 expression in the macrophage cells as illustrated in an ELISA assay (Figure 3C), of which MS436 and MS363 showed most profound inhibitory activity similar to their activity in the NO inhibition study. Taken together, these results demonstrate that these lead diazobenzene bromodomain inhibitors, particularly MS436 can effectively modulate BRD4 functions in gene transcriptional activation in cells.

Fluorescence Anisotropy Binding Assay [1]
Binding affinity of the newly synthesized diazobenzene compounds for various bromodoamins was assessed in a fluorescence anisotropy competition assay using a fluorescein isothiocyanate (FITC)-labeled MS417 as an assay probe as described previously10. Competition experiments were performed with a BrD protein (0.25–1 µM) and the fluorescent probe (80 nM), and increasing concentration of unlabeled competing ligand in a PBS buffer (pH 7.4) in total volume of 80 µL Measurements were obtained after a 1 hour incubation of the fluorescent ligand and the protein at 25°C with Safire 2 microplate reader (Tecan). In a competition-binding assay, fluorescent ligand concentration was ≤ 2Kd, and protein concentration was set at which 50–80% of fluorescent ligand is bound. Dissociation constant of a competing ligand was calculated with the correction to Cheng-Prussoff equation introduced by Nicolovska-Coleska and colleagues. Assuming one-site competitive binding model, the equation used to calculate Ki’s from IC50 values recovered from fitting data using Prism: where [I50] is the concentration of free inhibitor at 50% inhibition, [L50], the concentration of free labeled ligand at 50% inhibition, and [P0], concentration of free protein at 0% inhibition. Note that Kd for each protein-probe pair is the limit of resolvable Ki in a competition assay.

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Protein Crystallization, X-ray Diffraction Data Collection and Structure Determination [1]
Purified BRD4-BrD1 protein (14 mg/mL) was mixed with a diazobenzene BrD inhibitor at 1:10 molar ratio of protein:ligand. The complex was crystallized using the sitting drop vapor diffusion method at 20°C by mixing 1 µL of protein solution with 1 µL of the reservoir solution that contains 15–30% PEG 4000, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5. Crystals were soaked in the corresponding mother liquor supplemented with 20% ethylene glycerol as cryoprotectant before freezing in liquid nitrogen. X-ray diffraction data were collected at 100K at beamline X6A of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. Data were processed using the HKL-2000 suite. The BRD4-BD1 structure was solved by molecular replacement using the program MOLREP, and the structure refinement was done using the program Refmac. Graphics program COOT was used for model building and visualization. Crystal diffraction data and refinement statistics for the structure are displayed in Supplemental Table S2 (see Supplemental Information).

Cell Viability Study of Murine Macrophage Cells [1]
Murine macrophage RAW264.7 cells were plated at a density of 1 × 104 cells per well in a 96-well plate and incubated at 37 °C for 18 h. The cells were then treated with the diazobenzene bromodomain inhibitors up to 100 µM for 24 hours. At the end of the 24 hr incubation, 10 µL of the MTT solution (4 mg/ml) was added to each well and incubated at 37°C for 4 h. The supernatants were then removed and the cells were solubilized in 100 µl of 100% DMSO. The diazobenzene compounds were first dissolved in DMSO then diluted with culture medium to concentrations that ranged from 0.28 to 50000 nM. The final concentration of DMSO was adjusted to 0.05% (v/v). The extent of the reduction was measured by the absorbance at 570/630 nm using EnVison 2104 Multilabel Reader.

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Assessing LPS-induced IL-6 and Nitric Oxide Levels in Murine Macrophage Cells Murine macrophage RAW264.7 cells were cultivated in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. Cells in 96-well plates (0.1 mL, 3 × 105 cells/mL) were treated with lead diazobenzene inhibitors. After 30 min, all supernatants were removed and cells were treated with LPS (1 µg/mL) and lead diazobenzene inhibitors. After 24 h, the supernatant was collected and measured using mouse IL-6 ELISA assay kit. The lead diazobenzene bromodomain inhibitors were first dissolved in DMSO then diluted with culture medium to concentrations that ranged from 0.28 to 50,000 nM. The final concentration of DMSO was adjusted to 0.05% (v/v). The assay was measured by an absorption reading at 570 nm using EnVison 2104 Multi-label Reader. For assessing LPS-induced nitric oxide release, the nitrite production was measured by spectrophotometry at 520 nm using EnVison 2104 Multilabel Reader. Each experiment was performed in triplicate and plotted using Prism 5.0. The curve-fitting equation used was “log(inhibitor) vs. response – variable slope (four parameters)”.

Physiochemical Properties of the Top Diazobenzene Bromodomain Inhibitors [1]
We evaluated the physicochemical properties including CLogP, ligand efficiency (LE) and lipophilic ligand efficiency (LLE) of our top four diazobenzene-based bromodomain inhibitors, MS435, MS436, MS267 and MS363 to further understand their potential in cellular study. Of these, MS436 shows the best physiochemical properties (Table 4). As suggested in recent toxicology studies, compounds with CLogP <3 have much lower potential adverse effects in vivo. MS436 has CLogP of 2.65, thus belonging to the low-risk category. MS436 also displays higher ligand efficiency than recently reported 3,5-dimethoxylisoxazole-based bromodomain inhibitors, striking an excellent balance between lipophilicity and potency. The lipophilic ligand efficiency of MS436 (LLE = 3.42) is also well above the other diazobenzene analogs and in line with the most probable LLE distribution (2–4) as reported in a recent statistical analysis of successful drug molecules.

[1]. Structure-Guided Design of Potent Diazobenzene Inhibitors for the BET Bromodomains. J Med Chem. 2013 Nov 27;56(22):9251-64.

BRD4, characterized by two acetyl-lysine binding bromodomains and an extra-terminal (ET) domain, is a key chromatin organizer that directs gene activation in chromatin through transcription factor recruitment, enhancer assembly, and pause release of the RNA polymerase II complex for transcription elongation. BRD4 has been recently validated as a new epigenetic drug target for cancer and inflammation. Our current knowledge of the functional differences of the two bromodomains of BRD4, however, is limited and is hindered by the lack of selective inhibitors. Here, we report our structure-guided development of diazobenzene-based small-molecule inhibitors for the BRD4 bromodomains that have over 90% sequence identity at the acetyl-lysine binding site. Our lead compound, MS436, through a set of water-mediated interactions, exhibits low nanomolar affinity (estimated Ki of 30-50 nM), with preference for the first bromodomain over the second. We demonstrated that MS436 effectively inhibits BRD4 activity in NF-κB-directed production of nitric oxide and proinflammatory cytokine interleukin-6 in murine macrophages. MS436 represents a new class of bromodomain inhibitors and will facilitate further investigation of the biological functions of the two bromodomains of BRD4 in gene expression. [1]

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In this study, we report our structure-guided development of a new class of diazobenzene-based small molecule inhibitors for the BET bromodomains. MS436 is the best inhibitor yielded from our extensive lead optimization using a combined medicinal chemistry and structure-activity relationship study. We improved the affinity of the diazobenzene compounds toward the BRD4 BrD1 by over 100 fold. MS436 has an estimated Ki of 30–50 nM for the BRD4 BrD1 with a 10-fold selectivity over the BrD2. These lead diazobenzene compounds possess preferable drug-like properties. We further demonstrated the cellular efficacy of four lead diazobenzene BrD inhibitors in inhibiting BRD4 transcriptional activity in LPS-activated, NK-κB-directed production of nitric oxide and pro-inflammatory cytokine IL-6 in murine macrophage RAW264.7 cells. To our knowledge, MS436 is the first low nanomolar small-molecule bromodomain inhibitor that is selective between the two structurally highly similar BrDs of BRD4. It compares favorably to the other recently reported BRD4 BrD inhibitors such as 3,4-dihydro-3methyl-2(1H)-quinazolinones and 3,5-dimethoxyl-isoxazole,14 which do not show selectivity between the two BrDs of BRD4. [1]

C18H17N5O3S

383.424281835556

383.105

C, 56.39; H, 4.47; N, 18.27; O, 12.52; S, 8.36

1395084-25-9

135566899

Pink to red solid powder

1.4±0.1 g/cm3

673.7±65.0 °C at 760 mmHg

361.3±34.3 °C

0.0±2.1 mmHg at 25°C

1.686

1.78

3

8

5

27

601

0

S(C1C=CC(=CC=1)/N=N/C1C=C(C)C(=CC=1N)O)(NC1C=CC=CN=1)(=O)=O

DZTGIRNXWSZBIM-QURGRASLSA-N

InChI=1S/C18H17N5O3S/c1-12-10-16(15(19)11-17(12)24)22-21-13-5-7-14(8-6-13)27(25,26)23-18-4-2-3-9-20-18/h2-11,24H,19H2,1H3,(H,20,23)/b22-21+

(E)-4-[2-(2-Amino-4-hydroxy-5-methylphenyl)diazenyl]-N-2-pyridinylbenzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1 mg/mL (2.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)