TR-FRET BRD9 Binding Assay BRD9 TR-FRET Assay
A proprietary bromodomain binding small molecule containing a pendant primary amine was tagged with Alexa Fluor 647 (GSK2833930A). All assay components were dissolved in buffer (50 mM HEPES pH7.4, 50 mM NaCl, 5% Glycerol, 1 mM DTT and 1 mM CHAPS). The final concentration of BRD9 protein was 10 nM and the Alexa Fluor647 ligand was at Kd (~100 nM for BRD9). 5 uL of this reaction mixture was added to all wells containing 50 nl of various concentrations of test compound or DMSO vehicle (0.5% DMSO final) in 384 well microtitre plates and incubated in dark for 30 min at room temperature. Eu-W1024 Anti-6xHis Antibody (AD0111 PerkinElmer) at 1.5 nM FAC was used as a detection reagent. The plates were read on the Envision reader and the donor and acceptor counts were determined and the ratio of acceptor/donor was calculated.
Chemoproteomic Profiling
Nuclear extract was produced from fresh HuT78 cells. Affinity profiling assays were performed by derivatising sepharose beads with 2.0 mM of a proprietary bromodomain binding small molecule containing a pendant primary amine (GSK2893910A). I-BRD9 was spiked into HuT78 mixed nuclear and chromatin extracts and incubated for 45 minutes at 4 °C. Derivatised sepharose beads (35 μl beads per sample) were equilibrated in lysis buffer and incubated with cell extract pre-incubated with compound. Beads were washed with lysis buffer containing 0.2 % NP-40 and eluted with 2x SDS sample buffer supplemented with DTT. Aliquots of the eluates from chemoproteomic assays were separated on 4–12 % gel (NuPAGE, Invitrogen) and this was used for Western Blot analysis with antI-BRD9 (Abcam, ab-66443) and anti-BRD3 (Santa Cruz, SC-81202) antibodies.
NanoBRET Assay
HEK293 cells were plated in each well of a 6-well plate and co-transfected with Histone H3.3-HaloTag (NM_002107) and NanoLuc-BRD9 (Q9H8M2) BD domain amino acids 120-240. 20 hrs post-transfection the cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100nM NanoBRET 618 fluorescent ligand (Promega). Cell density was adjusted, re-plated in a 96-well plate and inhibitor added directly to media at final concentrations between 0-33 μM and the plates were incubated for 18 hours at 37 °C in the presence of 5% CO2. NanoBRET furimazine substrate (Promega) was added to both control and experimental samples at a final concentration of 10 μM and readings were performed within 5 minutes using the CLARIOstar (BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5sec reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/450 nm for experimental samples (i.e. those treated with NanoBRET fluorescent ligand) subtracted by and the emission at 610 nm/450 nm for control samples (not treated with NanoBRET fluorescent ligand).
