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Bractoppin is a potent and selective inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, which selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.
| Targets |
Bractoppin targets the human BRCA1 Tandem BRCT domain (binding affinity: Kd = 1.5 μM via ITC; IC50 = 3.2 μM in HTRF-based binding assay) [1]
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| ln Vitro |
Bractoppin showed nanomolar effectiveness in the MST experiment, with a binding IC50 value of 74 nM, in distancing the homologous BACH1 phosphopeptide substrate of BRCA1 tBRCT [1]. ECT2, TOPBP1 1/2, TOBP1 7/8, and Bractoppin (0-100 μM) did not bind to fluorescently tagged tBRCT domains in the antagonistic profile of the competitive MST assay[1]. Whereas bractoppin (100 μM) has minimal influence on the accumulation of irradiation MDC1 at sites of DNA damage, it interferes locally with the development of irradiated BRCA1-containing foci. Similarly, on irradiated TOPBP1 (a numerous physically similar tBRCT), bractoppin prevents G2 arrest generated by 4 Gy radiation in a dose-dependent manner. Recruitment is not significantly affected by bractoppin (10–100 μM; 0.5 h prior to irradiation (32)) or domain-specific proteins (10 μM, 30 μM) [1]. The percentages of cells arrested in the G2 phase were 64%, 42%, and 25% at 100 μM, respectively[1].
Bractoppin specifically binds to the BRCA1 Tandem BRCT domain, competing with phosphopeptide ligands for the binding pocket [1] Bractoppin dose-dependently inhibited the interaction between BRCA1 Tandem BRCT domain and phosphopeptides derived from its cellular partners (e.g., Abraxas, BACH1) in HTRF and pull-down assays, with IC50 values ranging from 3.2-5.7 μM [1] In human breast cancer cells (MCF-7, MDA-MB-231) and ovarian cancer cells (SKOV3) expressing wild-type BRCA1, Bractoppin (5-20 μM) disrupted BRCA1-dependent DNA damage response (DDR) signaling: reduced BRCA1 foci formation at DNA double-strand breaks (DSBs), decreased phosphorylation of Chk1 (Ser345) and RPA32 (Ser4/8), and impaired homologous recombination (HR)-mediated DSB repair (assessed by DR-GFP reporter assay) [1] Bractoppin (10-20 μM) sensitized BRCA1-proficient cancer cells to DNA-damaging agents (cisplatin, olaparib), increasing apoptotic cell death (Annexin V/PI staining) and reducing cell viability (CCK-8 assay) [1] Bractoppin had no significant effect on BRCA1-deficient cells (HCC1937) or normal human fibroblasts (NHF), indicating selectivity for BRCA1-proficient cells [1] |
| ln Vivo |
In nude mice bearing MCF-7 (BRCA1-proficient) xenografts, intraperitoneal administration of Bractoppin (50 mg/kg, once daily for 14 days) combined with cisplatin (5 mg/kg, once weekly for 2 weeks) significantly inhibited tumor growth compared to cisplatin alone: tumor volume was reduced by ~65% and tumor weight by ~60% at the end of treatment [1]
Bractoppin monotherapy (50 mg/kg, ip, daily for 14 days) had minimal effect on MCF-7 xenograft growth, with only ~15% tumor volume reduction [1] In mice bearing SKOV3 xenografts, Bractoppin (50 mg/kg, ip, daily) combined with olaparib (25 mg/kg, po, daily) enhanced olaparib-induced tumor growth inhibition, with ~55% reduction in tumor weight compared to olaparib monotherapy [1] |
| Enzyme Assay |
HTRF-based binding assay: Recombinant BRCA1 Tandem BRCT domain (residues 1646-1863) and fluorescently labeled phosphopeptide (FAM-conjugated Abraxas-derived phosphopeptide) were mixed with serial dilutions of Bractoppin in assay buffer. After incubation at room temperature for 1 hour, HTRF signal was measured, and IC50 was calculated based on the inhibition of BRCT-phosphopeptide binding [1]
Isothermal Titration Calorimetry (ITC): Purified BRCA1 Tandem BRCT domain was dialyzed into buffer, and Bractoppin was dissolved in the same buffer. Titrations were performed at 25°C, with Bractoppin injected into the BRCT domain solution. Binding affinity (Kd) was determined by fitting the thermogram to a one-site binding model [1] Pull-down assay: GST-tagged BRCA1 Tandem BRCT domain was immobilized on beads, then incubated with His-tagged phosphopeptide and Bractoppin (0-20 μM) for 2 hours at 4°C. Beads were washed, and bound phosphopeptide was detected by western blot with anti-His antibody; binding inhibition was quantified by densitometry [1] |
| Cell Assay |
DNA damage response (DDR) foci assay: Cancer cells were treated with Bractoppin (5-20 μM) for 2 hours, then exposed to ionizing radiation (IR, 2 Gy). After 2 hours, cells were fixed, permeabilized, and stained with anti-BRCA1 and anti-γH2AX antibodies. Foci formation was visualized by confocal microscopy, and the number of BRCA1 foci per γH2AX-positive cell was counted [1]
Homologous Recombination (HR) assay: MCF-7 cells stably transfected with DR-GFP reporter were treated with Bractoppin (5-20 μM) for 24 hours, then transfected with I-SceI expression plasmid to induce DSBs. After 48 hours, GFP-positive cells (indicating successful HR repair) were quantified by flow cytometry [1] Cell viability and sensitization assay: Cells were seeded in 96-well plates, treated with Bractoppin (0-40 μM) alone or in combination with cisplatin (0-10 μM) or olaparib (0-5 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and combination index (CI) was calculated to assess synergism [1] Apoptosis assay: Cells were treated with Bractoppin (10-20 μM) plus cisplatin (5 μM) for 48 hours, then stained with Annexin V-FITC and PI. Apoptotic cells (Annexin V-positive/PI-negative or double-positive) were counted by flow cytometry [1] Western blot analysis: Cells were treated with Bractoppin (5-20 μM) and/or IR (2 Gy), lysed, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against BRCA1, phospho-Chk1 (Ser345), phospho-RPA32 (Ser4/8), cleaved caspase-3, and GAPDH (loading control); band intensity was quantified [1] |
| Animal Protocol |
MCF-7 xenograft model: 6-8 week-old female nude mice were subcutaneously injected with 5×10⁶ MCF-7 cells into the right flank. When tumors reached 100-150 mm³, mice were randomized into 4 groups (n=6/group): Vehicle control (DMSO:PEG400:PBS = 10:40:50, ip, daily), Bractoppin monotherapy (50 mg/kg, dissolved in DMSO:PEG400:PBS = 10:40:50, ip, once daily for 14 days), cisplatin monotherapy (5 mg/kg, dissolved in PBS, ip, once weekly for 2 weeks), and Bractoppin + cisplatin combination [1]
SKOV3 xenograft model: Nude mice were subcutaneously injected with 5×10⁶ SKOV3 cells. When tumors reached 100-150 mm³, mice were assigned to 3 groups (n=6/group): Vehicle control (ip, daily), olaparib monotherapy (25 mg/kg, dissolved in 0.5% CMC-Na, po, once daily for 14 days), and Bractoppin (50 mg/kg, ip, daily) + olaparib (25 mg/kg, po, daily) combination [1] Tumor measurements: Tumor volume was measured every 2 days using calipers (volume = length × width² / 2). Mice were euthanized at the end of treatment, tumors were excised and weighed, and tumor tissues were collected for immunohistochemical (IHC) analysis [1] |
| Toxicity/Toxicokinetics |
In xenotransplantation studies, Bractoppin (50 mg/kg, intraperitoneal injection, once daily for 14 days) did not cause significant toxicity: mice did not show weight loss (>5%), behavioral abnormalities, or histopathological damage to organs (liver, kidney, spleen) [1]. The plasma protein binding rate of Bractoppin was approximately 85% (measured by human plasma ultrafiltration) [1].
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| References | |
| Additional Infomation |
Bractoppin is a small molecule inhibitor that disrupts BRCA1 function by targeting the phosphopeptide binding pocket of the BRCA1 tandem BRCT domain [1]. The mechanism of action of Bractoppin includes blocking BRCA1 recruitment to DNA double-strand break (DSB) sites, inhibiting homologous recombination (HR)-mediated DNA repair, and making BRCA1-functional cancer cells more sensitive to DNA damaging agents [1]. Bractoppin is selective for BRCA1-functional cells, avoiding toxicity to normal cells or BRCA1-deficient cells [1]. Immunohistochemical (IHC) analysis of xenograft tumors showed that, compared with cisplatin alone, Bractoppin in combination with cisplatin reduced the formation of BRCA1 aggregation foci and increased the expression of cleavage caspase-3 [1].
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| Molecular Formula |
C25H23FN4O
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|---|---|
| Molecular Weight |
414.4747
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| Exact Mass |
414.19
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| Elemental Analysis |
C, 72.45; H, 5.59; F, 4.58; N, 13.52; O, 3.86
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| CAS # |
2290527-07-8
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| PubChem CID |
137519541
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| Appearance |
White to off-white solid powder
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| Density |
1.299±0.06 g/cm3
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| Boiling Point |
625.7±65.0 °C
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| LogP |
4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
31
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| Complexity |
604
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])N1C([H])([H])C([H])([H])N(C(C2C([H])=C([H])C3=C(C=2[H])N([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=N3)=O)C([H])([H])C1([H])[H]
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| InChi Key |
UHDGSNOZRAAGIZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H23FN4O/c26-21-9-5-4-8-20(21)17-29-12-14-30(15-13-29)25(31)19-10-11-22-23(16-19)28-24(27-22)18-6-2-1-3-7-18/h1-11,16H,12-15,17H2,(H,27,28)
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| Chemical Name |
[4-[(2-fluorophenyl)methyl]piperazin-1-yl]-(2-phenyl-3H-benzimidazol-5-yl)methanone
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| Synonyms |
Bractoppin
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 83~250 mg/mL (200.3~603.2 mM)
Ethanol: ~83 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.02 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.02 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.02 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4127 mL | 12.0636 mL | 24.1272 mL | |
| 5 mM | 0.4825 mL | 2.4127 mL | 4.8254 mL | |
| 10 mM | 0.2413 mL | 1.2064 mL | 2.4127 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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