MS436 1395084-25-9

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Biological ActivityMS436 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.





Fluorescence Anisotropy Binding Assay

Binding affinity of the newly synthesized diazobenzene compounds for
various bromodoamins is assessed in a fluorescence anisotropy
competition assay using a fluorescein isothiocyanate (FITC)-labeled
MS417 as an assay probe. 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.


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.

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