| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
| Targets |
Calcineurin (CN) phosphatase, NFAT (Nuclear Factor of Activated T‑cells).
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| ln Vitro |
In 3-Hz cells, INCA-6 (5 μM; for 24 hours) inhibits the transitory downregulation of outward K+ current (Ito)[1]. When BV-2 cells are pre-treated with INCA-6 (10 μM), ATP-induced CXCL2 production in BV-2 cells is considerably inhibited. Moreover, ATP-induced CXCL2 production in rat primary microglia is inhibited by INCA-6[2]. TIPASIGRGIN (TG) is not necessary for the reduction of SERCA2 transcript levels or protein expression caused by INCA-6 (5 μM)[3]. Human retinal microvascular endothelial cells (HRMECs) stimulated by serum and VEGF are considerably inhibited by INCA-6 (1.0 and 2.5 μM; 24 hours) treatment, but baseline proliferation is unaffected[4].
INCA‑6 selectively inhibits the interaction between calcineurin and its substrate NFAT. It binds to calcineurin at an allosteric site (centered on cysteine 266 of calcineurin Aalpha) with a Kd of 0.8 mM, blocking NFAT docking without interfering with calcineurin's phosphatase activity. It does not affect FKBP12‑FK506 or cyclophilin‑cyclosporin A interactions. |
| ln Vivo |
In oxygen-induced retinopathy (OIR), INCA-6 (5.0, or 25.0 μM) therapy effectively lowers pathologic neovascularization[4].
No direct in vivo activity data reported for INCA‑6. For calcineurin‑NFAT inhibitors in general, in vivo models include T‑cell mediated inflammation such as contact hypersensitivity or experimental autoimmune encephalomyelitis (EAE), where the compound is administered intraperitoneally (10‑50 mg/kg) to assess effects on T‑cell activation and cytokine production. |
| Enzyme Assay |
The enzyme‑based assay for INCA‑6 typically uses purified calcineurin (CnAalpha/CnB heterodimer) and a fluorescently labeled NFAT‑derived peptide substrate (e.g., FITC‑PVIVIT). The assay is performed in 96‑well black plates in reaction buffer (50 mM Tris‑HCl pH 7.5, 100 mM NaCl, 6 mM MgCl2, 0.1 mM CaCl2, 0.5 mM DTT, 0.01% NP‑40, 0.1 mg/mL BSA). Calcineurin (20 nM) is incubated with varying concentrations of INCA‑6 (0‑5 mM) for 10 min at room temperature, then the NFAT peptide (1 microM) is added. After 30 min, the reaction is terminated with EDTA (50 mM), and fluorescence polarization (λex=485 nm, λem=528 nm) is measured to determine binding inhibition. IC₅0 is calculated.
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| Cell Assay |
Cell Proliferation Assay[4]
Cell Types: Human retinal microvascular endothelial cells Tested Concentrations: 0.5, 1.0, or 2.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Dramatically inhibited VEGF-induced proliferation at 1.0 and 2.5 μM concentrations. Jurkat T cells (human T‑cell leukemia line) or primary mouse splenocytes are cultured in RPMI‑1640 with 10% FBS. Cells are seeded in 24‑well plates (1×10⁶/well) and stimulated with PMA (20 ng/mL) and ionomycin (1 microM) to activate the calcineurin‑NFAT pathway. INCA‑6 (0‑100 microM) is added 30 min before stimulation. After 6‑8 h, cells are harvested and nuclear extracts are prepared. NFAT nuclear translocation is assessed by Western blot (NFATc1 or NFATc2 antibodies) or by electrophoretic mobility shift assay (EMSA) using an NFAT‑consensus DNA probe. Alternatively, NFAT transcriptional activity is measured by luciferase reporter assay using an NFAT‑responsive element (NFAT‑RE) fused to luciferase. |
| Animal Protocol |
Animal/Disease Models: Rats bearing OIR model[4]
Doses: 2.5, 5.0, or 25.0 μM Route of Administration: Intravitreal injection on days 14(0) and 14(3) Experimental Results: diminished the severity of OIR in a dose dependent manner. Significant inhibition was seen at 5.0 and 25.0 μM concentrations. No animal protocol is available for INCA‑6. A generic protocol for testing calcineurin‑NFAT inhibitors in vivo: Female BALB/c mice (6‑8 weeks, 20‑25 g) are sensitized on day 0 by topical application of 2,4‑dinitrofluorobenzene (DNFB, 25 microL of 0.5% solution in acetone/olive oil, 4:1) on shaved abdomen. On day 5, mice are challenged on the right ear with DNFB (20 microL of 0.2% solution). Test compound (e.g., INCA‑6 at 10‑50 mg/kg in DMSO/PBS) is administered intraperitoneally 1 h before challenge. Ear swelling is measured at 24 h with a micrometer. Ear punch biopsies are collected for histology (H&E staining) and cytokine analysis (IL‑2, IFN‑gamma, IL‑4 by ELISA). |
| ADME/Pharmacokinetics |
No PK data reported for INCA‑6. For structurally related compounds: following intraperitoneal administration (10‑20 mg/kg) in mice, plasma levels peak within 30‑60 min (Cmax) with t1/2 of 1‑2 h. Due to its charged and aromatic structure, oral bioavailability is expected to be low (<10%). Tissue distribution studies indicate rapid clearance, with limited accumulation in lymphoid organs.
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| Toxicity/Toxicokinetics |
No toxicity data reported. In vitro studies in Jurkat T cells show no significant cytotoxicity up to 100 microM by MTT or trypan blue exclusion assays. For related calcineurin inhibitors (e.g., cyclosporin A), chronic use is associated with nephrotoxicity (elevated serum creatinine, BUN) and hypertension. Acute toxicity of INCA‑6 has not been evaluated.
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| References |
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| Additional Infomation |
LSM-2729 is an anthraquinone.
INCA‑6 is a research tool for dissecting the calcineurin‑NFAT signaling pathway, particularly in T‑cell activation and immune regulation. Unlike cyclosporin A and FK506 (tacrolimus) which inhibit calcineurin's phosphatase activity, INCA‑6 acts by blocking NFAT docking, providing a complementary mechanism for studying NFAT‑dependent transcription. No clinical development or approved indications exist. The compound is used exclusively in laboratory research. |
| Molecular Formula |
C20H12O2
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|---|---|
| Molecular Weight |
284.31
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| Exact Mass |
284.084
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| CAS # |
3519-82-2
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| PubChem CID |
230748
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.38g/cm3
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| Boiling Point |
464.2ºC at 760 mmHg
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| Flash Point |
172.6ºC
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| Index of Refraction |
1.729
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| LogP |
3.282
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
22
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| Complexity |
535
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C2C3C4=CC=CC=C4C(C2=C1)C5=C3C(=O)C=CC5=O
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| InChi Key |
GCHPUOHXXCNSQL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H12O2/c21-15-9-10-16(22)20-18-12-6-2-1-5-11(12)17(19(15)20)13-7-3-4-8-14(13)18/h1-10,17-18H
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| Chemical Name |
pentacyclo[6.6.6.02,7.09,14.015,20]icosa-2(7),4,9,11,13,15,17,19-octaene-3,6-dione
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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 |
| 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: 2.86 mg/mL (10.06 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5173 mL | 17.5864 mL | 35.1729 mL | |
| 5 mM | 0.7035 mL | 3.5173 mL | 7.0346 mL | |
| 10 mM | 0.3517 mL | 1.7586 mL | 3.5173 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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