PRC1
Polycomb Repressive Complex 1 (PRC1), specifically targeting the E3 ubiquitin ligase activity mediated by its core components RING1B and BMI1 (IC50: ~2.5 μM, determined by luciferase-coupled ubiquitination assay for RING1B/BMI1 E3 ligase activity; no activity data reported for other PRC subunits or unrelated E3 ligases)[1]

PRT4165 is a potent inhibitor of PRC1-mediated H2A ubiquitylation. According to in vitro E3 ubiquitin ligase activity tests, PRT4165 inhibits RING 1A and RNF2 but not RNF8 or RNF168. Either RNF2 or RING1 can contribute to the E3 ubiquitin ligase activity, but PRT4165 can completely inhibit H2A ubiquitylation. When cells are treated with 50 μM PRT4165 for 60 minutes, the amount of total ubiquitylated histone H2A is dramatically reduced.Additionally, it is discovered that unirradiated cells with longer exposure times to PRT4165 (30 and 60 min) have higher levels of γ-H2AX. When compared to mock-treated cells, PRT4165 inhibits double-strand break (DSB) repair at the 8-hour mark. The number of cells in G2/M increases in cells treated with increasing PRT4165 concentrations[1].

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1. Inhibition of PRC1-mediated H2A ubiquitination (H2Aub1): In recombinant PRC1 (RING1B/BMI1 complex) assays, PRT4165 dose-dependently inhibited RING1B/BMI1-catalyzed H2Aub1. At 2.5 μM, the inhibition rate of H2Aub1 was ~50% (consistent with IC50), and at 10 μM, the inhibition rate exceeded 80% (vs. vehicle control). No significant effect on the protein levels of RING1B or BMI1 was observed.
2. Effect on H2Aub1 in cells: HeLa cells treated with 10 μM PRT4165 for 24 hours showed a ~70% reduction in nuclear H2Aub1 levels (detected by Western blot), while the levels of total H2A, H3, and H4 histones remained unchanged, indicating specific inhibition of H2A ubiquitination.
3. Impairment of DNA double-strand break (DSB) repair: HeLa cells were treated with 10 μM etoposide (to induce DSBs) for 1 hour, then incubated with 10 μM PRT4165. At 48 hours post-etoposide withdrawal, the number of γH2AX foci (a DSB marker) per cell was ~3.2-fold higher in PRT4165-treated group than in vehicle control; meanwhile, 53BP1 foci (a DSB repair factor) accumulated ~2.8-fold, suggesting delayed DSB repair.
4. Inhibition of cell proliferation: MTT assay showed that PRT4165 inhibited the proliferation of HeLa and U2OS cells (72-hour treatment) with IC50 values of ~3.8 μM and ~4.2 μM, respectively. At 20 μM, the proliferation inhibition rates were >90% for both cell lines.[1]

Inhibition of PRC1 causes alteration of gene regulation and polyploidy development during decidualization in vivo.

1. Recombinant protein preparation: RING1B and BMI1 proteins were expressed in prokaryotic cells and purified by affinity chromatography. The active PRC1 core complex was formed by mixing RING1B and BMI1 at a 1:1 molar ratio and incubating at 4℃ for 1 hour.
2. Ubiquitination reaction setup: The 50 μL reaction system contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM ATP, 10 μM ubiquitin, 0.5 μM E1 enzyme, 2 μM E2 enzyme (UbcH5c), 1 μM PRC1 complex, and PRT4165 at different concentrations (0.1, 0.5, 1, 2.5, 5, 10, 20 μM). The system was pre-incubated at 37℃ for 30 minutes.
3. Substrate addition and reaction termination: 2 μM histone H2A (substrate) was added to the system, and the reaction was continued at 37℃ for 1 hour. The reaction was terminated by adding 5×SDS loading buffer and boiling at 95℃ for 5 minutes.
4. Detection and data analysis: H2Aub1 levels were detected by Western blot using an anti-H2Aub1 antibody. The band intensity was quantified with ImageJ, and the inhibition rate at each PRT4165 concentration was calculated relative to the vehicle control. The IC50 was derived from nonlinear regression of the concentration-inhibition curve.[1]

Cells are treated with different concentrations of the PRT4165 60 min prior to irradiation (2 Gy). Two hours after IR, the cells are harvested. After twice washing the cells in PBS, they are fixed for ten minutes at 37°C using 1% paraformaldehyde. The cells are permeabilized with 90% methanol and kept at -20°C for an entire night after being cooled on ice for one minute. After two PBS washes, fixed cells are blocked for ten minutes using FACS incubation buffer (0.5% BSA in PBS). After that, the cells are stained for one hour at room temperature using an anti-phosphohistone H3 (serine 10) antibody at a 1:500 dilution in FACS incubation buffer[1].

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1. Assay for nuclear H2Aub1 levels in HeLa cells: - HeLa cells were seeded in 6-well plates at a density of 5×10⁵ cells/well and cultured in complete medium for 24 hours. PRT4165 was added to final concentrations of 0.1, 1, 2.5, 5, 10, 20 μM, and the cells were cultured for another 24 hours.
- Cells were harvested, and nuclear proteins were extracted using a nuclear protein extraction kit. Equal amounts of nuclear proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against H2Aub1, total H2A, and H3 (loading control).
- The ratio of H2Aub1 to total H2A was quantified with ImageJ to evaluate the inhibitory effect of PRT4165 on H2A ubiquitination.
2. Assay for DSB repair-related foci (γH2AX and 53BP1): - HeLa cells were seeded on coverslips in 24-well plates at 1×10⁵ cells/well and cultured for 24 hours. Cells were treated with 10 μM etoposide for 1 hour to induce DSBs, then etoposide was removed, and 10 μM PRT4165 was added. Cells were cultured for 0, 24, or 48 hours.
- Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour. They were then incubated with primary antibodies against γH2AX and 53BP1 at 4℃ overnight, followed by fluorescent secondary antibodies for 1 hour at room temperature. Nuclei were stained with DAPI.
- Foci were observed under a laser confocal microscope, and the number of γH2AX and 53BP1 foci per cell was counted (50 cells per group) to assess DSB repair efficiency.
3. Cell proliferation inhibition assay (MTT): - HeLa and U2OS cells were seeded in 96-well plates at 5×10³ cells/well and cultured for 24 hours. PRT4165 was added to final concentrations of 0.1, 0.5, 1, 2.5, 5, 10, 20, 50 μM (3 replicate wells per concentration), and cells were cultured for 72 hours.
- 20 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at 37℃ for 4 hours. The supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured with a microplate reader.
- Cell viability was calculated as (absorbance of drug group / absorbance of vehicle control) × 100%, and IC50 values were obtained by fitting the concentration-viability curve with nonlinear regression.[1]

Dissolved in sesame oil containing 10% ethanol; 4 mg/kg and 8 mg/kg; intraluminally injection CD-1 mice

[1].A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks. J Biol Chem. 2013 Sep 13;288(37):26944-54.

PRT4165 is the first reported small molecule inhibitor that specifically targets the E3 ubiquitin ligase activity of the PRC1 complex (RING1B/BMI1 subcomplex) rather than disrupting the assembly of PRC1 subunits.
2. The inhibitory effect of PRT4165 on DSB repair indicates that PRC1-mediated H2A ubiquitination is a key regulatory step in the DSB repair process, providing a new tool for studying the functional role of PRC1 in DNA damage response.
3. PRT4165 has a selective inhibitory effect on PRC1: In vitro experiments showed that at a concentration of 20 μM PRT4165, the activities of other E3 ubiquitin ligases (such as MDM2 and TRAF6) were not significantly inhibited, indicating that its cross-reactivity with other unrelated E3 ligases is low. [1]

C15H9NO2

235.240

235.063

C, 76.59; H, 3.86; N, 5.95; O, 13.60

31083-55-3

207893

Yellow solid powder

1.4±0.1 g/cm3

452.3±45.0 °C at 760 mmHg

227.0±35.1 °C

0.0±1.1 mmHg at 25°C

1.710

2.7

0

3

1

18

373

0

O=C1/C(C(C2=C1C=CC=C2)=O)=C/C3=CC=CN=C3

OMHZFEWYVFWVLI-UHFFFAOYSA-N

InChI=1S/C15H9NO2/c17-14-11-5-1-2-6-12(11)15(18)13(14)8-10-4-3-7-16-9-10/h1-9H

2-(3-Pyridinylmethylene)-1H-Indene-1,3(2H)-dione

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)

DMSO : 25~11 mg/mL ( 106.27~46.76 mM)
Ethanol : ~4 mg/mL
Water : Insoluble

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.63 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: 2.5 mg/mL (10.63 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: 5%DMSO+Corn oil: 1.2mg/ml

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