A DSF screen against Human bromodomains reveals only one significant off-target; BRD9:

ITC showed good potency and sufficient selectivity against BRD9 to meet SGC probe criteria:

Materials and Methods

Thermal stability assay

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 μM in 20 μL volume. Compounds were added at a final concentration of 10 μM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval.

AlphaScreen

All bromodomain proteins were prepared according to the published procedures (Filippakopoulos at al, 2012). Assay was performed as described previously (Philpott et al, 2011). All reagents were pre-diluted in 25 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 and 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates.  Plates filled with 5 uL of the assay buffer followed by 7 uL of biotinylated peptide [H-YSGRGKacGGKacGLGKacGGAKacRHRK(Biotin)-OH and His-tagged protein to achieve final assay concentrations of 25 nM. Plates were sealed and incubated for a further 60 minutes, before the addition of 8 μl of the mixture of streptavidin-coated donor beads (12.5 μg/ml) and nickel chelate acceptor beads (12.5 μg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set.

Isothermal Titration Calorimetry (ITC)

Experiments were carried out on a VP-ITC microcalorimeter (MicroCal™). All experiments were performed at 15 °C in 25 mM HEPES pH 7.4, 150 mM NaCl, 500 μM TCEP. 50 mM stocks of compound was thawed and diluted in 2 mL of buffer to a final concentration of 10 µM in the ITC cell. The protein titrations were conducted using an initial injection of 2 µl followed by 30 identical injections of 6 µl. The dilution heats were measured on separate experiments and were subtracted from the titration data. Thermodynamic parameters were calculated using ∆G = ∆H - T∆S = -RTlnKB, where ∆G, ∆H and ∆S are the changes in free energy, enthalpy and entropy of binding respectively. In all cases a single binding site model was employed.

TP-238 and its negative control were tested for target engagement in HEK cells using NanoBRET^TM. Significant inhibition by TP-238 against BPTF (n = 3, EC₅₀ = 228 nM) and CECR2 (n = 3, EC₅₀ = 289 nM) whereas the negative control TP-422 exhibited no significant activity at the tested concentrations:

Further, no significant inhibition against BRD9 was found in a NanoBRET^TM assay in HEK293 cells:

FRAP assays were also performed which showed significant inhibition by TP-238 against BPTF and CECR2:

FL-BPTF FRAP:

FL-CECR2 FRAP:

Materials and Methods

HEK cells were reverse transfected with NL-FL CECR2 or NL-BPTF BD. Cells were treated with a concentration range of TP-238, TP-422 (negative control), with BD070434a tracer (1 µM) for BPTF or BRD-02 tracer (0.5 µM) for CECR2 for 3 hrs before BRET measurements were taken.

For BRD9, the full length protein was used. HEK2983 cells were transfected with NL-BRD9 and treated with tracer (2 µM) and the respective compounds, followed by addition of Nano-Glo substrate and extracellular inhibitor. BRET was determined at 450 and 610nM.

FRAP assays were performed using U2OS cells reverse transfected with GFP-FL FALZ WT or #PHE or GFP-FL CECR2 WT or #ALA for 6-8 hrs. Transfection reagents were replaced with media or 2.5 µM SAHA + incubated O/N. Cells were treated with 1 µM test cpds for 1 hr before imaging. 6 repeat assays were performed for each protein.

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2. Lee SK, Park EJ et al. Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein. Mol Cells, 2012, 34(1):85-91
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A close homologue to TP-248 was co-crystallised to investigate the possible binding mode of the probe: