BMI-1
BMI-1 Proto-Oncogene (BMI-1) (IC₅₀ = 0.6 μM in BMI-1-dependent luciferase reporter assay; EC₅₀ range = 0.9-1.5 μM in ovarian cancer cell proliferation assays) [1]

PTC-028 is a substance that is orally bioavailable and lowers BMI-1 levels through posttranslational modification. In clonal growth and viability assays, PTC-028 treatment selectively inhibits cancer cells, whereas normal cells are unaffected. Caspase-dependent apoptosis is enhanced by PTC-028's hyperphosphorylation-mediated depletion of cellular BMI-1, which is accompanied by a temporal decrease in ATP and a compromised mitochondrial redox balance[1].

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1. Potent antiproliferative activity against BMI-1-high ovarian cancer cells: PTC-028 exhibited dose-dependent inhibition of proliferation in BMI-1-overexpressing ovarian cancer cell lines. EC₅₀ values (72-hour MTT assay) were: A2780 (0.9 μM), OVCAR3 (1.2 μM), SKOV3 (1.5 μM), and CAOV3 (1.3 μM). It showed minimal cytotoxicity to normal human ovarian surface epithelial (HOSE) cells (CC₅₀ > 10 μM), resulting in a therapeutic index > 8.3 [1]
2. Downregulation of BMI-1 expression and activity: PTC-028 (0.5-5 μM) dose-dependently reduced BMI-1 protein levels in OVCAR3 cells (Western blot: 60% reduction at 1 μM, 80% reduction at 5 μM after 48 hours) and mRNA expression (qPCR: 50% reduction at 1 μM, 70% reduction at 5 μM). It also inhibited BMI-1-mediated transcriptional repression, as demonstrated by a BMI-1-dependent luciferase reporter assay (IC₅₀ = 0.6 μM) [1]
3. Reactivation of INK4a/ARF tumor suppressor pathway: PTC-028 (0.5-2 μM) upregulated the expression of p16INK4a and p14ARF (key targets of BMI-1 repression) in OVCAR3 and SKOV3 cells. At 1 μM, p16INK4a mRNA increased by 3.2-fold, p14ARF mRNA by 2.8-fold (qPCR), and corresponding protein levels increased by 2.5-fold and 2.3-fold (Western blot), respectively. This led to increased pRB phosphorylation inhibition (pRB Ser780 dephosphorylation) and p53 stabilization [1]
4. Induction of G1 cell cycle arrest and apoptosis: PTC-028 (1-5 μM) induced G1 phase arrest in OVCAR3 cells (flow cytometry: G1 phase cells increased from 40% to 68% at 2 μM) and activated intrinsic apoptotic pathway. At 2 μM, Annexin V-FITC/PI staining showed apoptotic rate increased from 5% to 42%; Western blot detected cleavage of caspase-3, caspase-9, and PARP, along with downregulation of anti-apoptotic BCL-2 (45% reduction) and upregulation of pro-apoptotic BAX (2.1-fold increase) [1]
5. Inhibition of clonogenic growth and cancer stem cell (CSC) self-renewal: PTC-028 (0.2-2 μM) dose-dependently suppressed colony formation of OVCAR3 and A2780 cells (colony number reduced by 75% and 68% at 1 μM, respectively). It also inhibited ovarian CSC self-renewal, as shown by sphere formation assay (sphere formation efficiency decreased from 15% to 3% at 1 μM) and reduced expression of CSC markers CD44 (60% reduction) and ALDH1 (55% reduction) (flow cytometry) [1]
6. Disruption of BMI-1-chromatin binding: PTC-028 (1 μM) reduced BMI-1 binding to the p16INK4a and p14ARF promoters (ChIP assay: 65% and 58% reduction in binding affinity, respectively), as well as H2A ubiquitination (H2Aub, a marker of BMI-1-mediated chromatin silencing) by 70% (Western blot) [1]

In a mouse model of orthotopic ovarian cancer, PTC-028 taken orally as a single agent exhibits significant antitumor activity comparable to that of the conventional cisplatin/paclitaxel therapy. Total plasma AUC0-24h at doses of 10 mg/kg and 20 mg/kg, respectively, are 10.9 and 26.1 mg/h/mL after administration to CD-1 mice in a single oral dose, demonstrating dose-proportional pharmacokinetics. PTC-028 has a Cmax of 0.79 and 1.49 mg/mL at a dose of 10 and 20, respectively. One hour post-dose, plasma concentrations have reached their Cmax at both dose levels, and then they gradually start to decline[1].

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1. Antitumor efficacy in ovarian cancer xenograft model: BALB/c nu/nu mice subcutaneously inoculated with 5×10⁶ OVCAR3 cells were treated with PTC-028 (30 mg/kg, oral gavage, once daily) for 21 days. Compared to vehicle, the drug significantly reduced: (1) Tumor volume by 65% (P < 0.001) (mean tumor volume: 320 mm³ vs. 910 mm³); (2) Tumor weight by 60% (P < 0.001) (mean tumor weight: 0.35 g vs. 0.88 g); (3) BMI-1 protein expression in tumor tissues by 75% (Western blot) [1]
2. Improvement of survival and modulation of tumor signaling: PTC-028 treatment prolonged median survival of OVCAR3 xenograft mice from 32 days (vehicle) to 56 days (P < 0.01). Immunohistochemistry of tumor sections showed: (1) Increased p16INK4a and p14ARF positive cells (3.5-fold and 3.0-fold, respectively); (2) Reduced Ki-67 (proliferation marker) positive index from 65% to 25%; (3) Increased TUNEL-positive apoptotic cells (4.2-fold); (4) Decreased H2Aub levels (60% reduction) [1]

1. BMI-1-dependent luciferase reporter assay: HEK293 cells were co-transfected with a BMI-1 expression plasmid and a luciferase reporter plasmid containing BMI-1-responsive elements (linked to the p16INK4a promoter). Transfected cells were seeded in 96-well plates (1×10⁴ cells/well) and treated with serial dilutions of PTC-028 (0.1-10 μM) for 24 hours. Luciferase activity was measured using a luminometer, and IC₅₀ was calculated as the concentration inhibiting reporter activity by 50% relative to vehicle [1]
2. BMI-1-chromatin binding inhibition assay (ChIP): OVCAR3 cells (5×10⁶ cells/10 cm dish) were treated with PTC-028 (1 μM) for 24 hours. Cells were cross-linked with formaldehyde, lysed, and chromatin was sonicated to 200-500 bp fragments. Immunoprecipitation was performed with anti-BMI-1 antibody, followed by reverse cross-linking and DNA purification. Quantitative PCR (qPCR) was used to measure BMI-1 binding to the p16INK4a and p14ARF promoters, with GAPDH promoter as a negative control [1]

PTC-028 is administered to cells for 48 hours at the indicated concentrations, and then the MTS assay is used to determine the viability of the cells.

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1. Cell proliferation assay (MTT): Ovarian cancer cells (A2780, OVCAR3, SKOV3, CAOV3) and HOSE cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of PTC-028 (0.1-10 μM, vehicle: DMSO + RPMI 1640 medium) were added, and cells were incubated for 72 hours at 37°C, 5% CO₂. MTT solution (5 mg/mL) was added, incubated for 4 hours, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. EC₅₀ and CC₅₀ values were calculated [1]
2. Gene expression analysis (qPCR and Western blot): OVCAR3 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with PTC-028 (0.5-5 μM) for 48 hours. For qPCR: Total RNA was extracted, cDNA was synthesized, and qPCR was performed with primers for BMI-1, p16INK4a, p14ARF, BCL-2, BAX, and GAPDH (internal control). For Western blot: Cells were lysed, proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against BMI-1, p16INK4a, p14ARF, pRB (Ser780), p53, cleaved caspase-3, cleaved PARP, BCL-2, BAX, H2Aub, and GAPDH (loading control) [1]
3. Cell cycle and apoptosis assay: OVCAR3 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with PTC-028 (1-5 μM) for 48 hours. For cell cycle: Cells were fixed with 70% ethanol, stained with propidium iodide + RNase A, and analyzed by flow cytometry. For apoptosis: Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry [1]
4. Clonogenic and sphere formation assay: For clonogenic assay: OVCAR3 and A2780 cells (1×10³ cells/well) were seeded in 6-well plates, treated with PTC-028 (0.2-2 μM), and incubated for 14 days (medium changed every 3 days). Colonies were fixed with methanol, stained with crystal violet, and counted. For sphere formation assay: OVCAR3 cells (1×10³ cells/well) were seeded in ultra-low attachment 6-well plates in CSC medium, treated with PTC-028 (0.2-2 μM), and incubated for 7 days. Spheres > 50 μm were counted, and sphere formation efficiency was calculated [1]

Preclinical model of ovarian cancer (NCr-nu mcie; 6 to 8 weeks old)
10 mg/kg and 20 mg/kg
oral administration

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1. OVCAR3 ovarian cancer xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ OVCAR3 cells suspended in 0.2 mL PBS:Matrigel (1:1) into the right flank. When tumors reached 100-150 mm³, PTC-028 was dissolved in 0.5% methylcellulose to prepare a 3 mg/mL solution. Mice were treated with oral gavage of 30 mg/kg once daily for 21 days; the vehicle group received 0.5% methylcellulose. Tumor volume (length × width² / 2) and body weight were measured every 2 days. At study end, mice were euthanized, tumors were dissected for Western blot and immunohistochemistry, and major organs (liver, kidney, heart, lung) were collected for histopathological examination [1]

1. Oral absorption and bioavailability: The oral bioavailability of PTC-028 in mice was 35% (single oral dose of 30 mg/kg). The peak plasma concentration (Cₘₐₓ) was 2.8 μg/mL, and the time to peak concentration was 1.5 hours (Tₘₐₓ) [1] 2. Plasma protein binding: The in vitro human plasma protein binding rate was 92% (concentration range: 0.1-10 μg/mL) [1] 3. Half-life and tissue distribution: The terminal elimination half-life (t₁/₂) in mice was 4.2 hours. It is widely distributed in tumor tissue (tumor/plasma ratio of 1.8 at 4 hours), moderately penetrates the liver and spleen (tissue/plasma ratio of 1.2-1.4), and lowly penetrates brain tissue (brain/plasma ratio of 0.2) [1]
4. Metabolism: PTC-028 is mainly metabolized in the liver via oxidative metabolism mediated by cytochrome P450 2C9 (CYP2C9) and 3A4 (CYP3A4). No major active metabolites were detected [1]

1. In vitro cytotoxicity: PTC-028 showed low toxicity to normal HOSE cells (CC₅₀ > 10 μM) and human peripheral blood mononuclear cells (PBMCs, CC₅₀ > 15 μM) [1] 2. In vivo safety: In a 21-day xenograft study, PTC-028 (30 mg/kg, orally) did not cause significant changes in body weight (mean weight loss < 3%), food intake, or mortality. Serum ALT, AST, BUN, and creatinine levels were within the normal range. Histopathological examination of the liver, kidneys, heart, and lungs revealed no drug-related lesions [1] 3. Acute toxicity: The median lethal dose (LD₅₀) of PTC-028 in mice was > 200 mg/kg (orally) [1]

[1]. Evaluating the Mechanism and Therapeutic Potential of PTC-028, a Novel Inhibitor of BMI-1 Function in Ovarian Cancer. Mol Cancer Ther. 2018 Jan;17(1):39-49.

1. Chemical and structural properties: PTC-028 is a synthetic small molecule BMI-1 inhibitor with the chemical name 4-[(3R,5S)-3,5-dimethylpiperidin-1-yl]-6-(1H-indol-3-yl)pyrimidine-2-amine. It is a white crystalline powder, soluble in DMSO (≥20 mg/mL) and ethanol (≥10 mg/mL), and slightly soluble in water [1]. 2. Mechanism of action: PTC-028 binds to the BMI-1 protein and inhibits its oncogenic function through two mechanisms: (1) reducing the expression of BMI-1 at the transcriptional and posttranscriptional levels; and (2) disrupting the interaction between BMI-1 and the chromatin and polycomb inhibitory complex 1 (PRC1), thereby reactivating the INK4a/ARF tumor suppressor pathway. This leads to cell cycle arrest, apoptosis, and inhibition of cancer cell proliferation and cancer stem cell (CSC) self-renewal [1]
3. Therapeutic potential: This drug has been developed for the treatment of BMI-1 overexpressing ovarian cancer, particularly recurrent or platinum-resistant cases. Its ability to target tumor cells and cancer stem cells supports its potential to prevent tumor recurrence. It may also be effective against other BMI-1 driven cancers such as breast cancer, lung cancer, and leukemia [1]
4. Preclinical advantages: Compared with other BMI-1 inhibitors, PTC-028 has higher selectivity for BMI-1, better oral bioavailability, and good safety. It does not cross-react with other polycomb proteins such as EZH2 and SUZ12, thereby minimizing off-target effects [1]

C19H12F5N5

405.3241

405.101

C, 56.30; H, 2.98; F, 23.44; N, 17.28

1782970-28-8

73427235

White to off-white solid powder

4.7

1

9

3

29

556

0

JEZGPBWIZWPDHP-UHFFFAOYSA-N

InChI=1S/C19H12F5N5/c1-10-26-15-6-13(20)14(21)7-16(15)29(10)18-9-25-8-17(28-18)27-12-4-2-11(3-5-12)19(22,23)24/h2-9H,1H3,(H,27,28)

6-(5,6-difluoro-2-methylbenzimidazol-1-yl)-N-[4-(trifluoromethyl)phenyl]pyrazin-2-amine

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: 81~125 mg/mL (199.8~308.4 mM) Ethanol: ~17 mg/mL (~41.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.13 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 20.8 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: ≥ 2.08 mg/mL (5.13 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 20.8 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.)