Selectivity Within Target Family
| DSF Assay | (-)JQ1 | (+)JQ1/SGCBD01 |
|---|
| Protein | ΔTm | STD | ΔTm | STD |
|---|
| ASH1L | 0.00 | 0.00 | 0.00 | 0.00 |
| ATAD2 | 0.31 | 0.23 | 0.18 | 0.68 |
| BAZ2B | 0.00 | 0.00 | 0.00 | 0.00 |
| BRD1 | 0.59 | 0.73 | 0.00 | 0.00 |
| BRD2/1 | 0.89 | 0.60 | 6.47 | 0.09 |
| BRD2/2 | 0.91 | 0.06 | 7.97 | 0.01 |
| BRD3/1 | 1.95 | 0.23 | 8.27 | 0.11 |
| BRD3/2 | 2.14 | 0.47 | 8.39 | 0.01 |
| BRD4/1 | 1.12 | 0.09 | 9.35 | 0.07 |
| BRD4/2 | 0.21 | 0.11 | 7.44 | 0.14 |
| BRD9 | 0.00 | 0.00 | 0.00 | 0.00 |
| BRDT/1 | 0.38 | 0.02 | 3.93 | 0.13 |
| BRPF1 | 0.76 | 0.18 | 0.00 | 0.00 |
| CECR2 | 0.00 | 0.00 | 0.00 | 0.00 |
| CREBBP | 1.18 | 0.21 | 1.04 | 0.11 |
| EP300 | 0.46 | 0.22 | 0.07 | 0.21 |
| FALZ | 0.00 | 0.00 | 0.00 | 0.00 |
| GCN5L2 | 0.00 | 0.00 | 0.00 | 0.00 |
| KIAA1240 | 0.14 | 0.22 | 0.05 | 0.28 |
| LOC93349 | 0.00 | 0.00 | 0.00 | 0.00 |
| PB1/1 | 0.00 | 0.00 | 0.00 | 0.00 |
| PB1/2 | 0.48 | 0.11 | 0.00 | 0.00 |
| PB1/3 | 0.98 | 0.01 | 0.79 | 0.05 |
| PB1/5 | 0.37 | 0.25 | 0.00 | 0.00 |
| PB1/6 | 0.00 | 0.00 | 0.00 | 0.00 |
| PCAF | 0.00 | 0.00 | 0.00 | 0.00 |
| PHIP/2 | 0.00 | 0.00 | 0.00 | 0.00 |
| SMARCA2 | 0.95 | 0.04 | 0.93 | 0.02 |
| SMARCA4 | 0.60 | 0.18 | 0.15 | 0.10 |
| SP140 | 0.69 | 0.80 | 0.00 | 0.00 |
| TAF1/2 | 0.56 | 0.04 | 0.28 | 0.05 |
| TAF1/3 | 0.33 | 0.03 | 0.50 | 0.04 |
| TAF1L/2 | 0.00 | 0.00 | 0.04 | 0.04 |
| TAF1L/3 | 0.14 | 0.18 | 0.00 | 0.00 |
| TIF1 | 0.00 | 0.00 | 0.00 | 0.00 |
| TRIM28/4,5 | 0.00 | 0.00 | 0.00 | 0.00 |
| WDR9 | 0.00 | 0.00 | 0.00 | 0.00 |
Isothermal titration calorimetry (ITC)
| Protein | [P]
(µM) | [L]
(µM) | Kd
(nM) | ΔHobs
(kcal/mol) | N | TΔS
(kcal/mol) | ΔG
(kcal/mol) |
|---|
| BRD2(1) | 225 | 25 | 128.4 ± 6.5 | -7.74 ± 0.003 | 1.02 ± 0.002 | 1.35 | -9.08 |
| BRD3(1) | 306 | 25 | 59.5 ± 3.1 | -6.57 ± 0.017 | 1.07 ± 0.002 | 2.97 | -9.54 |
| BRD3(2) | 280 | 30 | 82.0 ± 5.3 | -4.93 ± 0.017 | 1.03 ± 0.002 | 4.41 | -9.35 |
| BRD4(1) | 240 | 25 | 49.0 ± 2.4 | -8.42 ± 0.019 | 1.00 ± 0.001 | 1.22 | -9.64 |
| BRD4(2) | 250 | 27 | 90.1 ± 4.6 | -3.22 ± 0.009 | 1.06 ± 0.002 | 5.76 | -9.29 |
| BRDT(1) | 305 | 25 | 190.1 ± 7.6 | -8.30 ± 0.024 | 1.06 ± 0.002 | 0.56 | -8.86 |
| CREBBP | 950 | 30 | nd | nd | nd | nd | nd |
| WDR9(2) | 300 | 25 | nd | nd | nd | nd | nd |
Nd: not detected
Selectivity Beyond Target Family
CEREP Assay
Displacement of a tetra-acetylated histone H4 peptide by JQ1/SGCBD01 isomers using BRD4(1), BRD4(2) or of an acetylated H3 peptide using CREBBP.
Materials and Methods
Differential Scanning Fluorimetry (DSF)
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. The temperature dependence of the fluorescence during the protein denaturation process was approximated by the equation
where ΔuG(T) is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein respectively. The baselines of the denatured and native states were approximated by a linear fit. The observed temperature shifts, ΔTmobs, were recorded as the difference between the transition midpoints of sample and reference wells containing protein without ligand in the same plate and determined by non-linear least squares fit.
Isothermal Titration Calorimetry
Experiments were carried out on a VP-ITC titration microcalorimeter from MicroCalTM, LLC (Northampton, MA). All experiments were carried out at 15 °C while stirring at 295 rpm, in ITC buffer (50 mM HEPES pH 7.4 at 25 °C, 150 mM NaCl). The injection syringe (250 µl) was loaded with a solution of the protein sample (300 µM protein for the BETs, 950 µM protein for CREBBP and 600 µM for WDR9(2), in ITC buffer). All titrations were conducted using an initial injection of 2 µl followed by 34 identical injections of 8 µl with a duration of 16 sec (per injection) and a spacing of 250 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. The collected data were implicated in the MicroCalTM Origin software supplied with the instrument to yield enthalpies of binding (ΔH) and binding constants (KB) as previously described by Wiseman and coworkers50. Thermodynamic parameters were calculated (ΔG = ΔH - TΔS = -RTlnKB, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.
CEREP assay
JQ1/SGCBD01 (1 µm) was screened against a panel of 55 ligand receptors, ion channels and transports using an established and widely utilized commercial assay platform (ExpresSProfile; CEREP, Paris, FRANCE).
Alpha screen Assay
All reagents were diluted in 50 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150–0 µM and 4 µl transferred to low-volume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA), followed by 4 µl of HIS-tagged protein (BRD4(1), 250 nM, BRD4(2) and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 µl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH; peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 µl of streptavidin-coated donor beads (25 µg/ml) and 4 µl nickel chelate acceptor beads (25 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. IC50 values were calculated in Prism 5 after normalization against corresponding DMSO controls and are given as the final concentration of compound in the 20 µl reaction volume.
