Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1–3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.


Supplementary
: Temperature shift data for human bromodomains in the presence of OF-1, NI-57 and PFI-4 at 10 µM compound concentration. Shifts larger than 1 o C and smaller than 2 o C are highlighted in yellow, shifts larger than 2 o C and smaller than 5 o C in orange and ∆Tm shifts larger than 5 o C are highlighted in dark red. Thermal melting experiments were carried out using a Stratagene Mx3005p Real Time PCR machine (Agilent Technologies). Proteins were purified as described (Filippakopoulos et al., 2012). 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 as described (Filippakopoulos et al., 2010).  Assays were performed as described previously with minor modifications (Philpott et al., 2011). 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 doseresponse 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. IC 50 values were calculated in Prism 5 (GraphPad Software, USA) after normalization against corresponding DMSO controls.  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.

BRD
Example of an ITC experiment measuring off target activity of NI-57 with BRD4 (1).

Crystallographic Methods
Crystallization BRPF1 (residues 626-746) was used for the apo and co-crystal structures and BRPF2 (residues 563-688) was used for co-crystallization with OF-1 probe complex. Aliquots of the purified proteins were set up for crystallization using a mosquito® crystallization robot (TTP Labtech). All crystallizations were carried out using the sitting drop vapour diffusion method at 4°C. Apo-crystals BRPF1: Crystals of BRPF1 in the free-form were obtained by mixing 200 nL of the protein (28 mg/ml) and 100 nL of crystallization buffer (0.1M bis tris propane pH 7.8, 0.15M sodium nitrate, 10% ethylene glycol, 20% PEG3350). Co-crystallization BRPF1 with histone peptides: protein at 15.4 mg/mL was mixed 1:10 molar ratio with H3 and H4 peptides. Co-crystals in complex with H3K14ac (K-S-T-G-G-Kac-A-P-R-K-Q) were obtained by mixing 150 nL of protein and 150 nL of 25% PEG 3350, 0.2M MgCl 2 , 0.1 M bistris pH 5.5. Co-crystals in complex with H4K5acK8ac (S-G-R-G-Kac-G-G-Kac-G-L) were obtained by mixing 100 nl of protein with 200 nL of 0.2 M NaCl, 25% PEG 3350, 0.1 M Hepes pH 7.5. Co-crystallization BRPF1 with compounds: protein at 17.5 mg/ml was mixed with a molar ratio of 1:4 with the compounds. Crystals were obtained by mixing 100 nL protein with 200 nL crystallization buffer. Co-crystals with the probe OF-1 have grown in 0.1M bis tris propane pH 7.4, 0.15M sodium nitrate, 5% ethylene glycol, 25% PEG3350. Cocrystals with the probe PFI-4 have grown in 15% PEG 3350, 0.25 M sodium nitrate condition. Co-crystallization BRPF2 with OF-1: protein at 18.5 mg/mL was mixed with a molar ratio of 1:2 with OF-1. Crystals appeared by mixing 200 nL of protein and 100 nL of 0.1 M tri-sodium citrate dihydrate pH 5.6, 1 M ammonium dihydrogen phosphate condition.

Data Collection and Structure Determination
Crystals were cryo-protected using the well solution supplemented with additional 20% ethylene glycol and were flash frozen in liquid nitrogen. Data were collected at the Diamond Light Source beamline I03 and I04 at a wavelength of 0.9763Å. Indexing and integration was carried out using XDS (Kabsch, 2010). Initial phases were calculated by molecular replacement with PHASER (Bunkoczi et al., 2013) using an ensemble of known bromodomain models (PDB IDs 2OSS, 2OUO, 2GRC, 2OO1, 3DAI, 3D7C, 3DWY, 3G0L). Unique and initial solutions were improved in a total of 50 cycles of automated protein chain tracing starting from existing model and computed using ARP/wARP (Cohen et al., 2008). Further manual building with COOT (Emsley and Cowtan, 2004) and refinement against maximum likelihood target using REFMAC5 (Murshudov et al., 2011). Thermal motions were analysed using TLSMD (Painter and Merritt, 2006) and hydrogen atoms were included in late refinement cycles. PRODRG was used to generate compound coordinates and cif. Files (Schuttelkopf and van Aalten, 2004). All model validations were carried out using MolProbity (Hintze et al., 2016). Data collection and refinement statistics are compiled in Supplementary Table 5. The models and structure factors have been deposited with PDB accession codes: 4LC2.pdb, 5FFV.pdb, 5FFW.pdb, 5FFY.pdb, 5FG4.pdb, 5FG5.pdb and 5FG6.pdb. Relative IC 50 values were estimated by non-linear regression analysis of (log) concentration of each inhibitor versus milliBRET ratios (GraphPad Prism).

Proximity biotinylation coupled to mass spectrometry (BioID-MS)
The BioID data were generated according to Lambert et al.(Lambert et al.). Cell pellets were thawed in 1.5 ml ice cold RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA, 0.1 % SDS and 0.5 % sodium deoxcycholate. PMSF (1 mM), DTT (1 mM) and Sigma protease inhibitor cocktail (P8340, 1:500) were added immediately before use. Samples were sonicated at 4˚C using three 10 sec bursts with 2 sec pauses at 35 % amplitude. 100 units of benzonase were then added and the lysates were incubated at 4˚C for an hour with rotation and then centrifuged 20,817 x g for 20 min at 4˚C. For each sample, 60 µl of streptavidin-sepharose bead slurry (GE Healthcare, Cat 17-5113-01) were pre-washed three times with 1 ml of lysis buffer by pelleting the beads with gentle centrifugation and aspirating off the supernatant before adding the next wash. Biotinylated proteins were captured on pre-washed streptavidin beads for 3 hours at 4˚C with rotation. The beads were then gently pelleted and the unbound supernatant was saved for further analysis. The beads were then washed 2 x 1 ml with RIPA buffer and 3 x 1 ml with 50 mM ammonium bicarbonate (pH 8.0). Following the final wash, the beads were pelleted and any excess liquid was aspirated off. Beads were then resuspended in 100 µl of 50 mM ammonium bicarbonate, and 1 µg of trypsin solution was added. The samples were incubated overnight at 37˚C with rotation and then an additional 1 µg of trypsin was added, followed by a further incubation for 2-4 hours. The beads were pelleted and the supernatant was transferred to a fresh tube. The beads were rinsed with 2 x 100 µL HPLC grade water and the wash fraction was combined with the supernatant. The peptide solution was acidified with 50 % formic acid to a final concentration of 2 % and the samples were placed in a speedvac to dry. Tryptic peptides were resuspended in 25 µL of 5 % formic acid and stored at -80˚C until analyzed by mass spectrometry. Parental Flp-In T-REx HEK293 cells, and stable cells expressing BirA*-FLAG fused either to a green fluorescent protein (GFP) or to a nuclear localization sequence (NLS) were used as negative controls for the BioID experiments and processed in parallel to the BRPF bait proteins.

Mass Spectrometry and Data Analysis
BioID samples and controls were analyzed by mass spectrometry in at least two biological replicates. 5 µL of each sample were directly loaded at 400 nl/min onto a 75 µm x 12 cm emitter packed with 3 µm ReproSil-Pur C18-AQ (Dr.Maisch HPLC GmbH, Germany). Data was analyzed by mass spectrometry either on a TripleTOF 5600TM (AB SCIEX, Concord, Ontario, Canada) or, in the case of BRPF3, on a LTQ Orbitrap Velos (Thermo Fisher). For analysis on the TripleTOF 5600TM, peptides were eluted from the column over a 90 min gradient generated by a NanoLC-Ultra 1D plus (Eksigent, Dublin CA) nano-pump and analyzed on a TripleTOFTM 5600. The gradient was delivered at 200 nL/min starting from 2 % acetonitrile with 0.1 % formic acid to 35 % acetonitrile with 0.1 % formic acid over 90 minutes followed by a 15 min clean-up at 80 % acetonitrile with 0.1 % formic acid, and a 15 min equilibration period back to 2 % acetonitrile with 0.1 % formic acid for a total of 120 min. To minimize carryover between each sample, the analytical column was washed for 3 h by running an alternating "sawtooth" gradient from 35 % acetonitrile with 0.1 % formic acid to 80 % acetonitrile with 0.1 % formic acid, holding each gradient concentration for 5 min. Analytical column and instrument performance were verified after each sample by loading 30 fmol BSA tryptic peptide standard (Michrom Bioresources Inc. Fremont, CA) with 60 fmol α-Casein tryptic digest and running a short 30 min gradient. TOF MS calibration was performed on BSA reference ions before running the next sample in order to adjust for mass drift and verify peak intensity. The instrument method was set to a discovery or IDA mode which consisted of one 250 ms MS1 TOF survey scan from 400-1300 Da followed by twenty 100 ms MS2 candidate ion scans from 100-2000 Da in high sensitivity mode. Only ions with a charge of 2+ to 4+ in which exceeded a threshold of 200 cps were selected for MS2, and former precursors were excluded for 10 sec after 1 occurrence. Peptides from the BRPF3 samples were analyzed on an LTQ Orbitrap Velos (Thermo Electron) equipped with a nanoelectrospray ion source (Proxeon Biosystems) coupled to a NanoLC-Ultra 1D plus as previously described. The LTQ Orbitrap Velos was operated with Xcalibur 2.0 in datadependent acquisition mode with the following parameters: one centroid MS (mass range

Experimental:
For histochemical staining of human osteoclast-like cells for TRAP, cells were fixed in 4% formalin/PBS, and stained for at least 3 h at 37°C with equal amounts of Napthol (0.65 mg/mL, Sigma) and fast violet (1.3 mg/mL, Sigma), each in acetate-tartrate buffer. After a deep purple colour became apparent the reaction was stopped by the addition of 4 g/L NaF for 30 min. Following PBS washes, multinucleated cells (nuclei > 3) were counted under the light microscope for each well (96 wp).

FRAP Constructs and Vector map
Gateway entry clone containing triple bromodomain module was constructed by multiple LIC protocol (Aslanidis and de Jong, 1990;Gileadi et al., 2008). Briefly, pDONR221 vector was linearized by PCR using primers encoding LIC sites and NLS. BRPF1B bromodomain sequence (Supplementary Table 7) was amplified by either of three sets of primers. PCR fragments were purified using Qiagen PCR purification kit. Fragment encoding first and third repeats of BRPF1B bromodomain were treated with T4 DNA polymerase in the presence of dCTP and linearized pDONR221 vector and second bromodomain repeat were treated with T4 DNA polymerase in the presence of dGTP. Treated fragment were combined and directly transformed into E.coli MACH1 strain. Clones containing correct inserts were identified by colony PCR and confirmed by sequencing. GFP tagged triple BRPF1B bromodomain construct was constructed using Gateway LR reaction between pcDNA™6.2/N-EmGFP-DEST (Invitrogen, cat # V356-20) and entry clone encoding triple bromodomain construct.
Similar to BRPF1B Triple constructs, the full-length BRD1-GFP constructs were generated using pcDNA5_pDEST vectors (Supplementary Figure 7).