MS436

MS436 exhibits low nanomolar affinity with preference for the first bromodomain over the second. MS436 effectively inhibits BRD4 activity in NF-κB-directed production of nitric oxide and proinflammatory cytokine interleukin-6 in murine macrophages without siginificanty inhibition on cell viability. ^[1]

• Fluorescence Anisotropy Binding Assay

  Competition experiments are 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 are obtained after a 1 hour incubation of the fluorescent ligand and the protein at 25°C with Safire 2 microplate reader. In a competition-binding assay, fluorescent ligand concentration is ≤ 2Kd, and protein concentration was set at which 50-80% of fluorescent ligand is bound. Dissociation constant of a competing ligand is 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.

• Cell lines

  Murine macrophage RAW264.7 cells

• Concentrations

  ~100 μM

• Incubation Time

  24 hours

• Method

  Murine macrophage RAW264.7 cells are plated at a density of 10000 cells per well in a 96-well plate and incubated at 37 °C for 18 h. The cells are then treated with the diazobenzene bromodomain inhibitors up to 100 μL for 24 hours. At the end of the 24 hr incubation, 10 μL of the MTT solution (4 mg/ml) is added to each well and incubated at 37°C for 4 h. The supernatants are then removed and the cells were solubilized in 100 μL of 100% DMSO. The diazobenzene compounds are first dissolved in DMSO then diluted with culture medium to concentrations that ranged from 0.28 to 50000 nM. The final concentration of DMSO is adjusted to 0.05% (v/v). The extent of the reduction is measured by the absorbance at 570/630 nm using EnVison 2104 Multilabel Reader.