Potency against Target

Selectivity Within Target family

DSF screen

GSK2801 showed significant thermal shifts of 4.1 and 2.7 °C for BAZ2A and BAZ2B respectively. GSK2801 did not show significant thermal shifts against all other bromodomains, except for BRD9 (ΔTm: 2.9 °C) and TAF1L (ΔTm: 3.4 °C) was observed. The K_D of GSK2801 binding to BRD9 and TAF1L were determined to be 1.2 and 3.2 μM respectively by ITC.

BLI

Because of the limited sensitivity of BAZ2A/B in the thermal stability assay, the selectivity of GSK2801 was measured by BLI. Forty-two bromodomains modified with a Bir A biotin ligase targeting sequence were co-expressed in bacteria with the enzyme. The biotinylated proteins were used in BLI experiments probing the interaction of GSK2801 at two concentrations (0.2 and 1.0 μM). In agreement with the ΔTm data, BRD9 and TAF1(L) were detected as the major off-targets, while no other significant interactions were detected within the bromodomain family.

Click to enlarge

The structurally related GSK8573 proved to be inactive against BAZ2A/B and all other bromodomains except BRD9 in the BLI assay

Click to enlarge

FRAP

To investigate whether GSK2801 can displace BAZ2 bromodomains from chromatin in living cells, we performed a fluorescence recovery after photobleaching (FRAP) assay utilizing GFP-tagged BAZ2A full length protein transfected into human osteosarcoma cells (U2OS). Mutant BAZ2A (N1873F) was used as a control that does not bind acetylated lysine containing peptides and therefore mirrors the behaviour of inhibitor bound BAZ2A. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) was used to increase overall levels of histone acetylation, resulting in a sufficient assay window to enable the measurement of differences in recovery time and demonstrating the acetylation dependence of the FRAP experiments. GSK2801 at 1 μM GSK2801 accelerated FRAP half-recovery time to the same extent as observed for the mutant construct indicating that the compound was able to displace BAZ2A from chromatin. Conversely, the inactive GSK-8573 did not have any effect on the half-recovery time of GFP-BAZ2A

Cytotoxicity

GSK2801 showed no significant toxicity against U02S or HeLa cells 

1. A Novel Family of Bromodomain Genes,
    

   Jones, M. H.; Hamana, N.; Nezu, Ji.; Shimane, M., Genomics, 63, 40– 45 (2000)

2. Identification of a sudden cardiac death susceptibility locus at 2q24.2 through genome-wide association in European ancestry individuals,
    

   Arking, D. E.; Junttila, M. J.; Goyette, P.; Huertas-Vazquez, A.; Eijgelsheim, M.; Blom, M. T.; Newton-Cheh, C.; Reinier, K.; Teodorescu, C.; Uy-Evanado, A.; Carter-Monroe, N.; Kaikkonen, K. S.; Kortelainen, M. L.; Boucher, G.; Lagace, C.; Moes, A.; Zhao, X.; Kolodgie, F.; Rivadeneira, F.; Hofman, A.; Witteman, J. C.; Uitterlinden, A. G.; Marsman, R. F.; Pazoki, R.; Bardai, A.; Koster, R. W.; Dehghan, A.; Hwang, S. J.; Bhatnagar, P.; Post, W.; Hilton, G.; Prineas, R. J.; Li, M.; Kottgen, A.; Ehret, G.; Boerwinkle, E.; Coresh, J.; Kao, W. H.; Psaty, B. M.; Tomaselli, G. F.; Sotoodehnia, N.; Siscovick, D. S.; Burke, G. L.; Marban, E.; Spooner, P. M.; Cupples, L. A.; Jui, J.; Gunson, K.; Kesaniemi, Y. A.; Wilde, A. A.; Tardif, J. C.; O’Donnell, C. J.; Bezzina, C. R.; Virmani, R.; Stricker, B. H.; Tan, H. L.; Albert, C. M.; Chakravarti, A.; Rioux, J. D.; Huikuri, H. V.; Chugh, S. S., PLoS Genet., 7, e1002158 (2011)

3. The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats

   Guetg, C.; Lienemann, P.; Sirri, V.; Grummt, I.; Hernandez‐Verdun, D.; Hottiger, M.O.; Fussenegger, M.; Santoro, R., The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats, Embo J., 29, 2135-2146, (2010)
    

4. Structure Enabled Design of BAZ2-ICR, A Chemical Probe Targeting the Bromodomains of BAZ2A and BAZ2B, Drouin, L.; McGrath, S.; Vidler, L.R.; Chaikuad, A.; Monteiro, O.; Tallant, C.; Philpott, M.; Rogers, C.; Fedorov, O.; Liu, M.; Akhtar, W.; Hayes, M.; Raynaud, F.; Müller, S.; Knapp, S.; Hoelder. S., J. Med. Chem., 58, 2553–2559, (2015)
    
5. Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2B, Chen, P.; Chaikuad, A.; Bamborough, P.; Bantscheff, M.; Bountra, C.; Chung, C.; Fedorov, O.; Grandi, P.; Jung, D.; Lesniak, R.; Lindon, M.; Müller, S.; Philpott, M.; Prinjha, R.; Rogers, C.; Selenski, C.; Tallant, C.; Werner, T.; Willson, T.M.; Knapp, S.;  Drewry, D.H., J. Med. Chem., 2015, DOI: 10.1021/acs.jmedchem.5b00209
    
6. Selective inhibition of BET bromodomains, Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W.B.; Fedorov, O.; Morse, E.M.; Keates, T.; Hickman, T.T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M.R.; Wang, Y.; Christie, A.L.; West, N.; Cameron, M.J.; Schwartz, B.; Heightman, T.D.; La Thangue, N.; French, C.A.; Wiest, O.; Kung, A.L.; Knapp, S.; Bradner, J. E.,  Nature 468, (7327) 1067-1073 ( 2010)

The pharmacokinetic parameters GSK2801 for in vivo experiments were measured after intraperitoneal and oral dosing in male CD1 mice. This PK study showed that GSK2801 has reasonable in vivo exposure after oral dosing, modest clearance, and reasonable plasma stability. These properties should allow GSK2801 to be used as a BAZ2A/B bromodomain inhibitor in vivo.

The co-crystal structure of GSK2801 and BAZ2B has been solved (pdb id: 4RVR).

• The structure showed that the 2-substitution at the phenyl ring produced a 90° rotation of the phenyl ring incompatible with potent BET binding. In BAZ2B, the rotated phenyl ring makes an end-on π-stacking interaction with W1887 and orients the 2-sulphonylmethyl group to produce a hydrogen-bond with the ZA-loop backbone (N1894 amine). In addition, the sulfonyl oxygen groups also make a pair of hydrogen bonds to the backbone NH of N1894.

Click to enlarge
 

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. MicroCal^TM 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.