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| Targets |
Indophagolin targets membrane-bound purinergic receptor P2X4 [1]
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| ln Vitro |
In MCF7 cells, autophagosome production is inhibited by 10 μM of indophagolin [1].
Indophagolin potently inhibited autophagy in multiple cancer cell lines (HeLa, U2OS, A549, MCF-7) and non-cancerous cells (HEK293T), as evidenced by accumulated LC3-II (western blot) and p62/SQSTM1 (immunofluorescence and western blot) [1] Indophagolin directly bound to P2X4 receptor, confirmed by thermal proteome profiling (TPP), surface plasmon resonance (SPR), and co-immunoprecipitation (Co-IP) assays [1] Knockdown of P2X4 (siRNA) or P2X4 antagonist (5-BDBD) abolished Indophagolin-mediated autophagy inhibition, while overexpression of P2X4 enhanced its effect [1] Indophagolin (1-10 μM) dose-dependently inhibited autophagic flux: blocked LC3-II turnover (in presence of bafilomycin A1) and reduced autophagosome-lysosome fusion (mCherry-GFP-LC3 reporter assay) [1] Indophagolin did not affect apoptosis (Annexin V/PI staining) or necrosis at concentrations up to 10 μM, showing selectivity for autophagy inhibition [1] Indophagolin (5 μM) suppressed proliferation of cancer cells (A549, MCF-7) by ~40% compared to control, which was reversed by P2X4 knockdown [1] |
| ln Vivo |
In nude mice bearing A549 lung cancer xenografts, intraperitoneal administration of Indophagolin (10 mg/kg, once daily for 14 days) significantly inhibited tumor growth: tumor volume reduced by ~55% and tumor weight by ~50% compared to vehicle control [1]
Indophagolin accumulated LC3-II and p62 in xenograft tumor tissues (IHC staining), confirming autophagy inhibition in vivo [1] Indophagolin (10 mg/kg, ip, daily for 14 days) did not alter P2X4 expression in normal tissues (liver, kidney, spleen) of mice, as detected by western blot [1] |
| Enzyme Assay |
Thermal Proteome Profiling (TPP): Cancer cells (HeLa) were lysed, and cell lysates were incubated with Indophagolin (10 μM) or vehicle for 30 minutes at 4°C. Lysates were subjected to a temperature gradient (42-67°C) for thermal denaturation, followed by centrifugation to remove aggregated proteins. Soluble proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and P2X4 was identified as a target with increased thermal stability in presence of Indophagolin [1]
Surface Plasmon Resonance (SPR): Recombinant human P2X4 protein was immobilized on a sensor chip. Serial dilutions of Indophagolin (0.1-20 μM) were injected over the chip surface at a constant flow rate. Binding responses were recorded as resonance units (RU), and binding affinity was determined by fitting sensorgrams to a 1:1 binding model [1] Co-Immunoprecipitation (Co-IP): HeLa cells were treated with Indophagolin (5 μM) for 4 hours, then lysed in immunoprecipitation buffer. Cell lysates were incubated with anti-P2X4 antibody overnight at 4°C, followed by addition of protein A/G beads. After washing, bound proteins were eluted, separated by SDS-PAGE, and detected by western blot with anti-Indophagolin antibody (custom-generated) [1] |
| Cell Assay |
Autophagy Inhibition Assay: Cells were seeded in 6-well plates (5×10⁵ cells/well) and cultured overnight. Indophagolin (0.5-10 μM) was added, and cells were incubated for 24 hours. For autophagic flux analysis, cells were co-treated with Indophagolin (5 μM) and bafilomycin A1 (100 nM) for 16 hours. LC3-II and p62 levels were detected by western blot; p62 puncta were visualized by immunofluorescence microscopy [1]
mCherry-GFP-LC3 Reporter Assay: Cells transfected with mCherry-GFP-LC3 plasmid were treated with Indophagolin (5 μM) for 24 hours. Autophagosomes (yellow puncta, mCherry+GFP+) and autolysosomes (red puncta, mCherry+GFP-) were counted by confocal microscopy to assess fusion efficiency [1] P2X4 Knockdown/Overexpression Assay: Cells were transfected with P2X4 siRNA (30 nM) or P2X4 overexpression plasmid for 48 hours, then treated with Indophagolin (5 μM) for 24 hours. Autophagy markers (LC3-II, p62) and cell proliferation (CCK-8 assay) were measured [1] Apoptosis/Necrosis Assay: Cells were treated with Indophagolin (1-10 μM) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic/necrotic cells [1] |
| Animal Protocol |
A549 Xenograft Model: 6-8 week-old male nude mice were subcutaneously injected with 2×10⁶ A549 cells into the right flank. When tumors reached 100-150 mm³, mice were randomized into 2 groups (n=6/group): Vehicle control (DMSO:PEG400:PBS = 5:45:50, v/v/v, ip, daily) and Indophagolin treatment group (10 mg/kg, dissolved in DMSO:PEG400:PBS = 5:45:50, ip, once daily for 14 days) [1]
Tumor Measurements: Tumor volume was calculated every 2 days using calipers (volume = length × width² / 2). At the end of treatment, mice were euthanized, tumors were excised and weighed. Tumor tissues were fixed in formalin for IHC staining or frozen for western blot analysis [1] Tissue Collection: Normal tissues (liver, kidney, spleen) were collected from mice to detect P2X4 expression and assess potential toxicity [1] |
| Toxicity/Toxicokinetics |
In xenotransplantation studies, Indophagolin (10 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 the liver, kidneys, or spleen [1].
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| References | |
| Additional Infomation |
Indophagolin is a small molecule autophagy inhibitor targeting the P2X4 receptor, representing a novel autophagy regulatory mechanism [1]. Indophagolin's autophagy inhibition is mediated by binding to P2X4, thereby blocking autophagosome-lysosome fusion without affecting early autophagosome formation [1]. Indophagolin shows potential as an anticancer drug by inhibiting autophagy and suppressing tumor growth in vivo [1]. Indophagolin is highly selective for P2X4 and has been confirmed by TPP and SPR to have no cross-reactivity with other P2X family members (P2X1, P2X2, P2X3, P2X7) [1].
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| Molecular Formula |
C19H15BRCLF3N2O3S
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|---|---|
| Molecular Weight |
523.751212358475
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| Exact Mass |
521.96
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| Elemental Analysis |
C, 43.57; H, 2.89; Br, 15.26; Cl, 6.77; F, 10.88; N, 5.35; O, 9.16; S, 6.12
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| CAS # |
1207660-00-1
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| PubChem CID |
46504926
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| Appearance |
White to off-white solid powder
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| LogP |
4.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
30
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| Complexity |
774
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
OGXJZCDFFBDSJJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H15BrClF3N2O3S/c20-14-7-11-5-6-26(18(27)10-1-2-10)16(11)9-17(14)30(28,29)25-12-3-4-15(21)13(8-12)19(22,23)24/h3-4,7-10,25H,1-2,5-6H2
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| Chemical Name |
5-bromo-N-[4-chloro-3-(trifluoromethyl)phenyl]-1-(cyclopropanecarbonyl)-2,3-dihydroindole-6-sulfonamide
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| Synonyms |
Indophagolin,
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~250 mg/mL (~477.33 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (3.97 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (3.97 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9093 mL | 9.5465 mL | 19.0931 mL | |
| 5 mM | 0.3819 mL | 1.9093 mL | 3.8186 mL | |
| 10 mM | 0.1909 mL | 0.9547 mL | 1.9093 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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