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.

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

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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.

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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.

Probe

OF-1

• SMILES:
• CN1C2=C(N(C1=O)C)C=C(C(NS(C3=C(C=C(C=C3)Br)C)(=O)=O)=C2)OC
• InChI:
• InChI=1S/C17H18BrN3O4S/c1-10-7-11(18)5-6-16(10)26(23,24)19-12-8-13-14(9-15(12)25-4)21(3)17(22)20(13)2/h5-9,19H,1-4H3
• InChIKey:
• YUNQZQREIHWDQT-UHFFFAOYSA-N

Temperature Shift Assay

Selectivity screening of chemical probe OF-1 determined by temperature shift assay.

The temperature shifts mapped onto the phylogenetic tree using red circles corresponding to ΔTm as indicated in the figure.

CESTA

Thermal-stability of BRPF1A/B determined by CESTA at 1 µM OF-1.

NanoBRET

Dose-dependent displacement of BRPF1A/B from histone H3.3 following treatment with OF-1.

FRAP Assay

Half-times of fluorescence recovery (t1/2) after photo bleaching measured for BRPF2 (BRD1) after treatment with OF-1 at different concentrations with or without SAHA.

TRAP staining for osteoclasts and measurement of pits depth.

Work on this probe has been published in 'Selective targeting of Bromodomain-PHD fingers family (BRPF) bromodomains impairs osteoclast differentiation'.

1. Laue K., et al., The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity. Development 2008, 135, 1935–194610
2. Vezzoli A. et al., Molecular basis of histone H3K36me3 recognition by the PWWP domain of Brpf1. Nat. Struct. Mol. Biol. 2010, 17, 617–61910
3. Hibiya K. et al., Brpf1, a subunit of the MOZ histone acetyl transferase complex, maintains expression of anterior and posterior Hox genes for proper patterning of craniofacial and caudal skeletons. Dev Biol. 2009, 329, 176–190
4. Johnstone R. W., et al., Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discovery 2014, 13, 673–691
5. Meier C. M., et al., Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation. ACS Chem Biol. 2017, 12, 2619–2630
6. Filippakopoulos P. et al., Histone recognition and largescale structural analysis of the human bromodomain family. Cell. 2012, 149, 214-231.
7. Philpott M., et al., Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery. Mol Biosyst. 2011, 10, 2899-908.

Isothermal Titration Calorimetry (ITC)

All calorimetric experiments were performed on a VP-ITC micro-calorimeter (MicroCalTM, LLC Northampton, MA). Protein solutions were buffer exchanged by gel filtration or dialysis into buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP). All measurements were carried out at 288.15 K. All injections were performed using an initial injection of 2 µL followed by injections of 8 µL. The data were analysed with the MicroCal ORIGIN software package employing a single binding site model. The first data point was excluded from the analysis. 

Temperature shift assay

Thermal melting experiments were carried out using a Stratagene Mx3005p Real Time PCR machine (Agilent Technologies). OF-1 was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000 as described (6).

AlphaScreen Assay

Assays were performed as described previously with minor modifications (7). All reagents were diluted in 25 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. An 11-point 1:2.0 serial dilutions of the ligands was prepared on lowvolume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA), using LabCyte Eco liquid handler. Plates filled with 5 µL of the assay buffer followed by 7 µL of biotinylated peptide [H-YSGRGKacGGKacGLGKacGGAKacRHRK(Biotin)-OH for BRD1, BRD4, BRPF1B and BRPF3 or YQTARKSTGGK(ac)APRKQLATKAK(biotin)-OH for TIF1α] and Histagged protein to achieve final assay concentrations of 25-100 nM depending on the dose-response curve for each individual protein. Plates were sealed and incubated for a further 30 minutes, before the addition of 8 µM 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. IC₅₀ values were calculated in Prism 5 (GraphPad Software, USA) after normalization against corresponding DMSO controls.

Fluorescence Recovery After Photobleaching (FRAP) Assay

FRAP studies were performed using U20S cells expressing full-length BRPF2 (BRD1). Six hours after transfection 2.5 µM SAHA (to increase global histone acetylation) was added and cells were treated with 1 µM or 5 µM of OF-1 1 hour before imaging and half recovery times from the fluorescence signal of the bleached U2OS nuclei were plotted.

NanoBRET

U2OS cells were co-transfected with Histone H3.3-HaloTag and NanoLuc-BRPF1. Twenty hours post-transfection 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 100 nM NanoBRET 618 fluorescent ligand (Promega). Cells were then treated with an increasing dose of OF-1. Five minutes prior to reading, NanoBRET furimazine substrate (Promega) was added to both control and experimental samples and plates were read on a CLARIOstar (BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5 sec 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). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000. Relative IC₅₀ values were estimated by non-linear regression analysis of (log) concentration of each inhibitor versus milliBRET ratios (GraphPad Prism).

Human osteoclast differentiation

Primary human peripheral blood mononuclear cells (PBMCs) were collected from a Histopaque generated buffy coat after gradient centrifugation at 20 min and 500g, brakes off. The CD14+ monocyte fraction was obtained by on-column CD14+-MACS bead isolation, washed twice with MACS buffer, and seeded at a density of 50 000 c/mL in αMEM/10%FCS supplemented with 25 ng/mL MCSF. After 6 days at 37 °C, 5% CO2 treatment with OF-1 with and without 50 ng/mL RANKL was started. Media were changed with fresh compound every 3−4 days. After 14−21 days, cells were fixed and stained for TRAP.

Bone Resorption Assays.

PBMCs were isolated and seeded onto self-cut dentine slices from ivory. After 14 days of differentiation, cells were removed from dentine slices. Dentine pits were imaged with confocal microscopy.

Probe		Negative control
SGC707 (IC50=31 nM)		XY1 (IC50>100,000 nM)

Click here to download SDF file

Physical and chemical properties
Molecular weight	298.1430
Molecular formula	C16H18N4O2
IUPAC name	2-((3-aza-bicyclo[4.4.0]deca-1,3,5,7,9-pentaen-8-ylamino)-formylamino)-1-(pyrrolidin-1-yl)-ethanone
logP	1.8
PSA	59.9 A

• SMILES:
• SGC707: O=C(N1CCCC1)CNC(NC2=CC3=CC=NC=C3C=C2)=O
• XY1: O=C(N1CCCC1)CNC(NC2=CC3=CC=CC=C3C=C2)=O
• InChI:
• SGC707: InChI=1S/C16H18N4O2/c21-15(20-7-1-2-8-20)11-18-16(22)19-14-4-3-13-10-17-6-5-12(13)9-14/h3-6,9-10H,1-2,7-8,11H2,(H2,18,19,22)
• InChIKey:
• SGC707: DMIDPTCQPIJYFE-UHFFFAOYSA-N
• XY1: GSQHGSRPQHBTTP-UHFFFAOYSA-N

(C) Effect of SGC707 on the activity of 27 protein methyltransferases as well as DNMT1, DNMT3A-3L, DNMT3B-3L and BCDIN3D (an RNA-methyltransferase) were assessed.

(D) IC50 values were determined at saturating concentration of peptide substrate (1.5 µM) and SAM concentrations equal to 0.25, 0.5, 1, 2, 4, 8, 10 and 12x of the Km for SAM. No change in IC50 values at varying SAM concentrations was consistent with a noncompetitive pattern of inhibition with respect to SAM. (E) To determine the competition with peptide, the SAM concentration was kept at saturation (70 µM) and IC50 values were determined at different peptide concentrations (0.5, 1, 2, 4, 8, 12, 16, and 20 ×Km). A similar noncompetitive pattern was observed for SGC707 with respect to peptide confirming the allosteric mode of inhibition.

Western blot analysis of H4R3me2a levels.  HEK293 cells were co-transfected with FLAG tagged PRMT3 (WT) or its catalytically dead mutant E335Q (Mut) and treated with different concentrations of SGC707, as indicated. Total cell lysates were collected 24 h post inhibitor treatment and analysed for H4R3me2a levels. The total levels of exogenous histone H4 and overexpressed PRMT3 were determined with anti-GFP, anti-H4 and anti-FLAG antibodies, respectively

Kaniskan, H. Ü., Szewczyk, M. M., Yu, Z., Eram, M. S., Yang, X., Schmidt, K., Luo, X., Dai, M., He, F., Zang, I., Lin, Y., Kennedy, S., Li, F., Dobrovetsky, E., Dong, A., Smil, D., Min, S.-J., Landon, M., Lin-Jones, J., Huang, X.-P., Roth, B. L., Schapira, M., Atadja, P., Barsyte-Lovejoy, D., Arrowsmith, C. H., Brown, P. J., Zhao, K., Jin, J. and Vedadi, M. (2015), A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3).

Angew. Chem.. doi: 10.1002/ange.201412154

Main features

• Pocket
• Interactions

Pharmacological targeting of the Wdr5-MLL interaction in C/EBPa N-terminal leukemia ,Florian Grebien, Masoud Vedadi, Matthäus Getlik, Roberto Giambruno, Amit Grover, Roberto Avellino, Anna Skucha, Sarah Vittori, Ekaterina Kuznetsova, David Smil, Dalia Barsyte-Lovejoy, Fengling Li, Gennadiy Poda, Matthieu Schapira, Hong Wu, Aiping Dong, Guillermo Senisterra, Alexey Stukalov, Kilian V M Huber,Andreas Schönegger, Richard Marcellus, Martin Bilban, Christoph Bock, Peter J Brown, Johannes Zuber, Keiryn L Bennett, Rima Al-awar, Ruud Delwel, Claus Nerlov, Cheryl H Arrowsmith & Giulio Superti-Furga. Nature Chemical Biology. 10.1038/nchembio.1859

Main features

• iStructure of WDR5 with MLL peptid
• Structure of WDR5 with MLL peptide showing binding of arginine residue in WDR5 binding pocket
• Structure of WDR5 with OICR9429 showing how methylpiperazine moiety binds in pocket and antagonizes binding of MLL peptide
• Top view of OICR-9429 bound to WDR5 showing that the pyridone group fits into pocket and the morpholino group is exposed to solvent
• Close-up of OICR-9429 indicating the key H-bonds responsible for potent binding.