Molibresib (GSK525762) is an inhibitor of BET proteins (IC50: about 35 nM).

BET proteins:35 nM

At day 2 of differentiation, I-BET-762 treatment altered cytokine production in CD4+ T cells, up-regulating the expression of some anti-inflammatory gene products and down-regulating the expression of some pro-inflammatory cytokines.I-BET-762 inhibited the binding of bromodomain and additional terminal domain proteins and BRD2/3/4 to the tandem bromodomain of the BET (Kd: 50.5-61.3 nM). In FRET analysis, displacement of the BET tandem bromodomain a tetraacetylated H4 peptide (IC50: 32.5-42.5 nM).

At day 2 of differentiation, I-BET-762 treatment altered cytokine production in CD4+ T cells, up-regulating the expression of some anti-inflammatory gene products and down-regulating the expression of some pro-inflammatory cytokines.I-BET-762 inhibited the binding of bromodomain and additional terminal domain proteins and BRD2/3/4 to the tandem bromodomain of the BET (Kd: 50.5-61.3 nM). In FRET analysis, displacement of the BET tandem bromodomain a tetraacetylated H4 peptide (IC50: 32.5-42.5 nM).

Fluorescence resonance energy transfer (FRET) titrations: Fluorescence resonance energy transfer (FRET) titrations. I-BET is titrated against BRD2 (200 nM), BRD3 (100 nM) and BRD4 (50 nM) in 50 mM HEPES pH7.5, 50 mM NaCl, 0.5 mM CHAPS in the presence of tetra-acetylated Histone H4 peptide (200 nM). After equilibrating for 1 hour, the bromodomain protein : peptide interaction is detected using FRET following the addition of 2 nM Europium cryptate labelled streptavidin and 10 nM XL-665-labelled anti-6His antibody in assay buffer containing 0.05% (v/v) BSA and 400 mM KF. Plates are read using an Envision Plate reader (excitation 320 nm, emission 615 nm and 665 nm).

CD4+ T cells are isolated from lymph nodes and spleens of 10- to 12-wk old mice and activated with plate bound anti-CD3 and anti-CD28 antibodies in the presence of indicated cytokines. I-BET-762 compounds is included during the 60–72 h of initial activation. Over the course of 5 d of T-cell culture and expansion, the compounds is diluted 12-fold relative to the starting concentrations.(Only for Reference)