PFI-4 selectively targets the bromodomain (BD) of Bromodomain-PHD Fingers 1 (BRPF1), with an IC50 value of approximately 19 nM in homogeneous time-resolved fluorescence (HTRF) binding assays. It exhibits low affinity for other bromodomains in the BRPF family, including BRPF2 BD (IC50 > 1000 nM) and BRPF3 BD (IC50 > 1000 nM), and no significant binding to bromodomains from other families (e.g., BET family BRD4 BD1/BD2, CECR2 BD, BRD7 BD) with IC50 values all exceeding 1000 nM [2]
- PFI-4 inhibits the BRPF1 bromodomain to disrupt its interaction with acetylated histones, thereby suppressing BRPF1-mediated transcriptional regulation. In osteoclast precursor cells, this targeting leads to reduced expression of osteoclast differentiation-related genes, with no detected activity against non-BRPF bromodomains [1]

PFI-4 (1.25 µM; 7 or 11 days) suppresses development of human osteoclasts[1].

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PFI-4 inhibits BRPF1 BD-histone interaction in a dose-dependent manner. In HTRF assays, at a concentration of 100 nM, it blocked over 90% of the binding between recombinant BRPF1 BD and fluorescently labeled acetylated histone H4 peptide (H4K5ac/K8ac/K12ac/K16ac). Additionally, surface plasmon resonance (SPR) analysis confirmed direct binding of PFI-4 to BRPF1 BD with a KD value of ~25 nM [2]
- PFI-4 impairs osteoclast differentiation from mouse bone marrow-derived monocytes (BMMs). When BMMs were treated with PFI-4 (0.1, 0.5, 1 μM) in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) for 5 days, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts (≥3 nuclei) were reduced by 35%, 58%, and 70%, respectively, compared to the vehicle control. Quantitative real-time PCR (qPCR) showed that PFI-4 (1 μM) downregulated mRNA levels of osteoclast marker genes: TRAP (Acp5) by 65%, cathepsin K (Ctsk) by 72%, and nuclear factor of activated T-cells 1 (Nfatc1) by 68% [1]
- PFI-4 shows no significant cytotoxicity in BMMs. A cell viability assay (CCK-8) revealed that after 72 hours of treatment with PFI-4 at concentrations up to 5 μM, cell viability remained above 90% relative to the vehicle control, indicating that the inhibition of osteoclast differentiation was not due to cell toxicity [1]

HTRF-based BRPF1 BD binding assay: Recombinant human BRPF1 BD (residues 1–110) was expressed in Escherichia coli and purified via affinity chromatography. The assay was performed in 384-well plates with a total volume of 20 μL per well, containing 50 nM BRPF1 BD, 20 nM fluorescently labeled acetylated histone H4 peptide (FAM-H4K5ac/K8ac/K12ac/K16ac), and serial dilutions of PFI-4 (0.001–1000 nM). The mixture was incubated at room temperature for 1 hour, followed by addition of 10 μL of anti-GST-Tb cryptate antibody (to detect GST-tagged BRPF1 BD). The HTRF signal (fluorescence resonance energy transfer between FAM and Tb cryptate) was measured using a microplate reader. IC50 values were calculated by fitting the dose-response curves with a four-parameter logistic regression model [2]
- SPR binding assay for BRPF1 BD: Recombinant BRPF1 BD was immobilized on a CM5 sensor chip via amine coupling to a surface density of ~200 response units (RU). PFI-4 was prepared in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) at concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μM and injected over the chip surface at a flow rate of 30 μL/min. The association phase was monitored for 120 seconds, and the dissociation phase for 300 seconds. The chip surface was regenerated with 10 mM glycine-HCl (pH 2.5) after each injection. KD values were determined by fitting the sensorgrams with a 1:1 Langmuir binding model using SPR data analysis software [2]
- TRAP enzyme activity assay: Mouse BMMs were seeded in 96-well plates and induced to differentiate into osteoclasts with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 4 days. PFI-4 (0–1 μM) was added on day 0. On day 4, cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and incubated with TRAP staining solution (containing naphthol AS-MX phosphate and fast red violet) at 37°C for 1 hour. TRAP-positive cells were counted under a light microscope. For quantitative TRAP activity, cell lysates were prepared, and the reaction mixture (lysate + p-nitrophenyl phosphate substrate in citrate buffer pH 5.0) was incubated at 37°C for 30 minutes. The absorbance at 405 nm was measured, and TRAP activity was normalized to total protein concentration [1]

Cell Viability Assay[1]
Cell Types: Osteoclasts
Tested Concentrations: 1.25 µM
Incubation Duration: 7 or 11 days
Experimental Results: Dramatically reduces MMP9 secretion in osteoclasts .

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Mouse BMM osteoclast differentiation assay: Bone marrow cells were isolated from the femurs and tibias of 6–8-week-old C57BL/6 mice. Monocytes were enriched by centrifugation over a density gradient and seeded in 24-well plates at 5×104 cells/well. Cells were cultured in α-MEM medium supplemented with 10% fetal bovine serum and M-CSF (30 ng/mL) for 24 hours to obtain adherent BMMs. The medium was then replaced with fresh medium containing M-CSF (30 ng/mL), RANKL (50 ng/mL), and serial dilutions of PFI-4 (0.1, 0.5, 1 μM) or vehicle (0.1% DMSO). The medium was refreshed every 2 days. After 5 days of culture, cells were fixed and stained for TRAP, and TRAP-positive multinucleated osteoclasts (≥3 nuclei) were counted [1]
- qPCR analysis of osteoclast marker genes: BMMs were treated as described in the osteoclast differentiation assay. On day 4 of culture, total RNA was extracted from cells using an RNA isolation kit, and cDNA was synthesized via reverse transcription. qPCR was performed using specific primers for Acp5 (TRAP), Ctsk (cathepsin K), Nfatc1, and the housekeeping gene Gapdh (glyceraldehyde-3-phosphate dehydrogenase). The relative mRNA expression levels were calculated using the 2-ΔΔCt method [1]
- HEK293T cell BRPF1-dependent transcription assay: HEK293T cells were seeded in 12-well plates at 2×105 cells/well and transfected with a luciferase reporter plasmid driven by a BRPF1-responsive promoter, along with a BRPF1 expression plasmid and a Renilla luciferase plasmid (as an internal control). After 24 hours of transfection, cells were treated with PFI-4 (0.01, 0.1, 1, 10 μM) or vehicle for 16 hours. Luciferase activity was measured using a dual-luciferase reporter assay system, and the firefly luciferase activity was normalized to Renilla luciferase activity. PFI-4 inhibited BRPF1-dependent luciferase activity with an IC50 of ~35 nM [2]

[1]. Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation. ACS Chem Biol. 2017 Oct 20;12(10):2619-2630.

[2]. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. (2014) ACS Med Chem Lett. 5(11):1190-1195.

PFI-4 is the first reported potent and selective inhibitor of the bromine domain of BRPF1, belonging to the 1,3-dimethylbenzimidazolone class of compounds. Its development meets the need for a tool compound for studying the biological functions of BRPF1, which is involved in chromatin remodeling and transcriptional regulation of genes related to development and metabolism [2]. The ability of PFI-4 to inhibit osteoclast differentiation suggests its potential application value in the treatment of bone resorption diseases such as osteoporosis and rheumatoid arthritis. PFI-4 interferes with the transcriptional program required for osteoclast maturation by targeting BRPF1, and does not affect the viability of osteoclast precursors at therapeutic concentrations [1]. BRPF1 forms a complex with histone acetyltransferases (HATs) such as MOZ and MORF. PFI-4 blocks the interaction between BRPF1 and acetylated histones, thereby inhibiting the ability of the HAT complex to acetylate histones and activate gene transcription. This mechanism explains its inhibitory effect on BRPF1-dependent gene expression and osteoclast differentiation [1, 2].

C21H24N4O3

380.44

380.184

900305-37-5

40642506

Light green to green solid powder

1.3±0.1 g/cm3

512.1±50.0 °C at 760 mmHg

263.5±30.1 °C

0.0±1.3 mmHg at 25°C

1.649

1.79

1

4

4

28

594

0

CN1C2=C(C=C(C(=C2)NC(=O)C3=CC=CC=C3OC)N4CCCC4)N(C1=O)C

QCIJLRJBZDBVDB-UHFFFAOYSA-N

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)

N-[2,3-Dihydro-1,3-dimethyl-2-oxo-6-(1-pyrrolidinyl)-1H-benzimidazol-5-yl]-2-methoxybenzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)