Physical and chemical properties
Molecular weight	380.45
Molecular formula	C21H24N4O3
IUPAC name	N-(1,3-dimethyl-2-oxo-6-(pyrrolidin-1-yl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2-methoxybenzamide
clogP	2.23
PSA	65.12
No. of chiral centres	0
No. of rotatable bonds	5
No. of hydrogen bond acceptors	4
No. of hydrogen bond donors	1
Storage	Stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolution	Soluble in DMSO

• SMILES:
• CN1C2=CC(=C(C=C2N(C1=O)C)N3CCCC3)NC(=O)C4=CC=CC=C4OC
• InChI:
• InChI=1S/C21H24N4O3/c1-23-17-12-15(22-20(26)14-8-4-5-9-19(14)28-3)16(25-10-6-7-11-25)13-18(17)24(2)21(23)27/h4-5,8-9,12-13H,6-7,10-11H2,1-3H3,(H,22,26)
• InChIKey:
• QCIJLRJBZDBVDB-UHFFFAOYSA-N

Dissociation constants of PFI-4 and BRDF1B or CECR2 interactions measured by isothermal titration calorimetry (ITC).

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

Thermal-stability of BRPF1A/B determined by CESTA at 1 µM PFI-4.

BRET assay using BRPF1A or BRPF1B-BRD with NLS (nuclear localisation signal) and H3.3 after treatment with PFI-4.

Half-times of fluorescence recovery (t1/2) after photo-bleaching measured for BRPF1B and BRPF1A treated either with or without SAHA and PFI-4 at indicated concentrations.

Confocal pictures of U2OS cells transfected with triple BRD–GFP construct of BRPF1B or BRPF1A containing NLS treated either with or without 1 μM PFI-4.

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). PFI-4 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 GFP-bound BRPF1a or BRPF1B (triple bromodomain). 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 PFI-4 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).