Isothermal Titration Calorimetry
Experiments were carried out on a VP-ITC titration microcalorimeter from MicroCalTM, LLC (Northampton, MA), at 15 °C while stirring at 295 rpm, in ITC buffer (50 mM HEPES, 150 mM NaCl). 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/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. MicroCalTM Origin software was used to calculate enthalpies of binding (ΔH) and binding constants (KB). 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.
Differential Scanning Fluorimetry (DSF)
Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered (10 mM HEPES, 500 mM NaCl) and assayed at a final concentration of 2 µM. 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 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. Data was analysed as previously reported [6]
Biolayer interference assays
BAZ2A and BAZ2B were expressed in frame with a C-terminal Avi tag and a His-TEV tag enabling enzymatic conjugation of a single biotin using the biotin ligase (BirA) which had been co-expressed in E. coli. Cells grown in the presence of 0.1 mM biotin were lysed and purified using a similar procedure. Incorporation of biotin was confirmed by ESI-MS spectroscopy. The biotin labelled BAZ2B and BAZ2A was immobilized on SuperStreptavidin BLI sensors. BioLayer Interferometry (BLI) experiments were performed on a 16-channel ForteBio Octet RED384 instrument at 25°C (25 mM HEPES, pH 7.5, 0.05% Tween and 100 mM NaCl buffer). The reference sensors without attached bromodomain were used to subtract background binding to SuperStreptavidin sensors. Data were processed and analysed using ForteBio Analysis software.
CEREP assay
GSK2801 (10 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).
Fluorescence Recovery After Photobleaching (FRAP) Assay
FRAP studies were performed using U20S cells expressing full-length BAZ2A or BRD4 protein fused with an N-terminal eGFP as previously described [4]. Six hours after transfection 2.5uM SAHA (to increase global histone acetylation) was added and GSK2801 was added 1 hour before imaging. Imaging was carried out 24 hours after transfection. Percent inhibition was calculated between the DMSO treated (0%) and N1873F expressing mutant (100%)
Cytotoxicity Assay
U20S cells were harvested from exponential phase cultures and plated in a 96-well opaque flat bottomed plates at a cell density of 3 x 103 cells/well (100uL). Compounds were dissolved in DMSO at 10 uM and serial dilutions performed. 5 uL of compound solution was added to each well and incubated for 24 or 72 hours at 37˚C in a humidified atmosphere containing CO2 (5%). 10 uL of WST-1 (Roche) was added and the plates returned to the incubator. Plates were read on a plate reader at 450 nm after 2 h for cells treated with GSK2801 for 24 h. Results plotted as % of DMSO control.
Pharmacokinetics
These PK studies were performed by Shanghai ChemPartner Co. Ltd. The IP and PO dosing solutions were prepared in 0.5% CMC+1% Tween 80. No clinical symptoms were observed during the entire in-life study. Male CD-1 mice, 28-32g were used (with free access to food and water). A dose of 30 mg/kg (10 mL/kg) was used via intraperitoneal injection (N=9) and for oral dosing 30 mg/kg (10 mL/kg) via gavage (N=9). The animals were restrained manually for blood collection and approx. 110 μL blood/time point was collected, centrifuged at 4°C (2000 g, 5min) to obtain plasma within 15 min after sample collection. Plasma samples were stored at -70℃ until analysis. An UPLC/MS-MS-002 (API-4000) was used for sample analysis, with Dexamethasone as internal standard. An aliquot of 20 μL sample was added with 200 μL ACN containing 50 ng/mL dexamethasone. The mixture was vortexed for 2 min and centrifuged at 14000 rpm for 5 min. An aliquot of 1 μL supernatant was injected for LC-MS/MS analysis.
