Probe		Negative control
GSK6853		GSK9311

• SMILES:
• GSK-6853: C[C@@H]1CNCCN1c1cc2c(cc1NC(c1ccccc1OC)=O)N(C)C(N2C)=O
• GSK-9311: CCN(C(c1ccccc1OC)=O)c1cc2c(cc1N1CCNC[C@H]1C)N(C)C(N2C)=O
• InChI:
• >GSK-9311: InChI=1S/C24H31N5O3/c1-6-28(23(30)17-9-7-8-10-22(17)32-5)20-13-18-19(27(4)24(31)26(18)3)14-21(20)29-12-11-25-15-16(29)2/h7-10,13-14,16,25H,6,11-12,15H2,1-5H3/t16-/m1/s1
• InChIKey:
• GSK-6853: FQWDVNSBYDXPIO-CQSZACIVSA-N
• GSK-9311: WFXIHQFRQPGCCR-MRXNPFEDSA-N

Temperature Shift

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

BROMO scan^TM

Bromodomain selectivity was evaluated in the BROMO scan™ panel (DiscoveRx Corp., Fremont, CA, USA, http://www.discoverx.com). This screen measures competition against reference immobilized ligands for 35 DNA-tagged bromodomains. GSK6853 is more than 1600 selective for BRPF1B over tested bromodomains.

GSK6853 was tested in a panel of 48 unrelated targets only weak off-target activity has been observed compared to the activity on BRPF1B.

No potent off-targets identified

In a NanoBRET^TM cellular target engagement assay using isolated BRPF1B BRD with NLS and Halo-tagged histone H3.3 BRPF1 showed a dose-dependent displacement from histone H3.3, with cellular IC₅₀ of 20 nM, but no effect of the less active control GSK9311.

Chemoproteomic competition binding assay in HUT-78 cell lysate shows binding to endogenous BRPF-1 with selectivity over BRD3 (8).

Work on this probe has been published in 'GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain’.

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.
8. Bamborough P. et al., GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain. ACS Med Chem Lett. 2016, 7, 552-7. doi: 10.1021/acsmedchemlett.6b00092. eCollection 2016 Jun 9.

4 chains in asymmetric unit (not yet released on PDB)

X-ray structure of BRPF-1 (cyan) with GSK6853, overlayed with BRPF-2 apo (magenta) (not yet released on PDB)

NanoBRET assay

HEK293 cells (8 x 10⁵) were plated in each well of a 6-well plate and co-transfected with Histone H3.3-HaloTag (NM_002107) and NanoLuc-BRPF1 isoform 1 (P55201-1) bromodomain amino acids 625-735 or isoform 2 (P55201-2) bromodomain amino acids 625-741. Isoform 2 has an insertion 660 -> SEVTELD in the bromodomain. 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). Cell density was adjusted to 2 x 10⁵ cells/ml and then re-plated in a 96-well assay white plate (Corning Costar #3917). Inhibitors were then added directly to media at final concentrations between 0-33 μM and the plates were incubated for 18hrs at 37 °C in the presence of 5% CO₂. NanoBRET furimazine substrate (Promega) was added to both control and experimental samples at a final concentration of 10 μM. Readings were performed within 5 minutes using the CLARIOstar BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5sec 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 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.

Chemoproteomic assay with dose-dependent competition

Chemical probe was spiked into HUT-78 nuclear and chromatin extracts and incubated for 45 min at 4 °C. Derivatized sepharose beads (35 μL beads per sample) were equilibrated in lysis buffer and incubated with cell extract pre-incubated with compound. Beads were washed with lysis buffer containing 0.2 % NP-40 and eluted with 2x SDS sample buffer supplemented with DTT. Proteins were alkylated with iodoacetamide, separated on 4–12 % NuPAGE (Invitrogen), and stained with colloidal Coomassie.