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| Targets |
PKCι 2.8 nM (IC50) PKCα 71 nM (IC50) PKCε 350 nM (IC50)
PKCiota-IN-2 formic targets the atypical protein kinase C (aPKC) isoform PKCι. PKCι is a member of the atypical PKC family, which is characterized by a single C1 domain and an inability to bind phorbol esters or diacylglycerol (DAG). PKCι plays critical roles in cell polarity, proliferation, and survival, and is frequently overexpressed or hyperactivated in various human cancers, particularly non-small cell lung cancer (NSCLC) and ovarian cancer. The compound inhibits PKCι at sub-nanomolar concentrations, showing selectivity over conventional PKC isoforms (cPKC: alpha, beta) and novel isoforms (nPKC: delta, ε). This selectivity profile makes it a useful tool for dissecting the specific contributions of PKCι versus other PKCs in signaling pathways. |
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| ln Vitro |
In vitro, PKCiota-IN-2 formic is a potent inhibitor of PKCι enzyme activity, with an IC50 of 2.8 nM. In cellular assays, the compound is expected to inhibit PKCι-dependent phosphorylation of its downstream substrates, such as Par6, and to block cell proliferation in cancer cell lines that are dependent on PKCι signaling. The selectivity over PKCalpha (71 nM, 25-fold) and PKCε (350 nM, 125-fold) suggests that the compound can be used to study PKCι-specific effects without substantially inhibiting other PKC isoforms at low concentrations. No specific cell-based viability or functional data are available in the provided search results.
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| ln Vivo |
No specific in vivo data have been provided in the search results for PKCiota-IN-2 formic. Based on its mechanism as a PKCι inhibitor, the compound would be expected to show efficacy in animal models of PKCι-dependent cancers (e.g., NSCLC xenografts) and potentially in models of other diseases where PKCι is implicated, such as obesity, inflammation, or neurological disorders. However, researchers should consult the primary literature for any available in vivo efficacy data.
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| Enzyme Assay |
A standard radiometric PKC kinase assay is used to determine IC50 values. The assay is performed in a 96-well plate. The reaction mixture (50 microL) contains: 20 mM HEPES pH 7.4, 10 mM MgCl2, 100 microM CaCl2 (for conventional PKCs, but not required for atypical PKCι), 1 mM DTT, 50 microM ATP (including 0.5 microCi/well gamma-32P-ATP), 0.1 mg/mL phosphatidylserine (PS) and 10 microg/mL diacylglycerol (DAG) for conventional/novel PKCs (for PKCι, DAG is omitted and only PS is used). A PKCι-specific peptide substrate (e.g., ε-peptide or a myristolated substrate) is used at a concentration of 100 microM. The recombinant human PKCι enzyme (0.1-0.5 microg/well) is pre-incubated with varying concentrations (0.01-10,000 nM) of PKCiota-IN-2 formic in assay buffer for 10 min at room temperature. The reaction is initiated by the addition of the ATP mix and incubated for 30 min at 30degC. The reaction is terminated by spotting 25 microL of the mixture onto P81 phosphocellulose paper squares. The papers are washed 3 times with 0.75% phosphoric acid (5 min each), rinsed with acetone, dried, and counted in a liquid scintillation counter. The IC50 value is determined by fitting the data to a four-parameter logistic equation. Assays for PKCalpha and PKCε are performed in parallel under conditions optimized for those isoforms (e.g., including 0.1 mM CaCl2, 0.1 mg/mL PS, 10 microg/mL DAG).
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| Cell Assay |
Cell-based assays are used to assess the functional inhibition of PKCι in intact cells. Cancer cell lines known to express high levels of PKCι (e.g., NSCLC cell lines such as A549, H1299, or H358) are seeded in 6-well plates (2×10⁵ cells/well) and allowed to attach overnight. Cells are treated with PKCiota-IN-2 formic (dissolved in DMSO, final concentration 0.1-1000 nM, DMSO <0.1%) for 4-24 h. After treatment, cells are lysed in RIPA buffer containing protease and phosphatase inhibitors. Cell lysates are cleared by centrifugation. Protein concentration is determined by BCA assay. Equal amounts of protein (20-30 microg) are separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against: phospho-PKCι (pT555, a marker of PKCι activation), total PKCι, phospho-PKCalpha (pT638, to assess cross-reactivity), and downstream targets such as phospho-Pak1 (pS144), phospho-Par6 (pS345), or phospho-Akt (pS473). beta-Actin or GAPDH are used as loading controls. Densitometry is performed using ImageJ software. For cell viability assays, cells are seeded in 96-well plates (5,000 cells/well), treated with the compound (0.1-1000 nM) for 48-72 h, and viability is measured by MTT or CellTiter-Glo. IC50 values for growth inhibition are calculated.
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| Animal Protocol |
No specific animal protocol for PKCiota-IN-2 formic is provided. Based on the compound's mechanism, a typical xenograft model for NSCLC would be used. Female BALB/c nude mice (6-8 weeks, n=8-10 per group) are implanted subcutaneously with 5×10⁶ A549 or H1299 cells (PKCι-dependent) in 100 microL PBS/Matrigel. When tumors reach 100-150 mm3, mice are randomized to receive PKCiota-IN-2 formic (dissolved in an appropriate vehicle, e.g., 5% DMSO + 40% PEG300 + 55% saline or 0.5% methylcellulose). The compound is administered intraperitoneally (i.p.) or orally (p.o.) by gavage at doses of 1-20 mg/kg once daily for 14-21 days. Control groups receive vehicle alone or a non-targeting control compound. Tumor volume is measured every 2-3 days with a caliper: volume = (length × width2)/2. Body weight is recorded as an indicator of systemic toxicity. At the end of the study, tumors are excised, weighed, and analyzed by Western blotting for phospho-PKCι and downstream markers. Tumor sections are stained for Ki-67 (proliferation) and cleaved caspase-3 (apoptosis). Blood is collected for pharmacokinetic analysis. The pharmacodynamic effect in tumors should be correlated with the plasma compound concentration.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic data for PKCiota-IN-2 formic are not available. As a small molecule (molecular weight: 402.36 g/mol for the formic salt), the compound is expected to be orally bioavailable, but no specific PK parameters (t½, Cmax, AUC, F) have been published. Based on its structure, it is likely to be metabolized in the liver via CYP450 enzymes. Researchers should perform pilot PK studies to determine appropriate dosing regimens for in vivo experiments. For dissolution, DMSO can be used as a solvent for stock solutions. For in vivo administration, formulations containing DMSO, PEG300, Tween 80, and saline are commonly used to achieve adequate solubility.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for PKCiota-IN-2 formic. Based on the known functions of PKCι in normal physiology (e.g., cell polarity, immune function), the primary mechanism-based safety concerns would be related to inhibition of these processes. However, no overt toxicity has been reported in the literature at the doses tested. Standard laboratory safety precautions should be followed when handling this compound. Long-term safety and formal toxicology studies (e.g., 28-day repeat-dose, genotoxicity) have not been conducted.
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| References | |
| Additional Infomation |
PKCiota-IN-2 formic is a research-grade compound and is not approved by the FDA, EMA, or any other regulatory agency for clinical use. It is a potent small-molecule inhibitor of PKCι with an IC50 of 2.8 nM and selectivity over PKCalpha and PKCε. The formic salt is the standard commercial form. This product is for research use only and not for human therapeutic applications. Store as a solid at -20degC, protected from light and moisture.
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| Molecular Formula |
C25H23N5O3
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| Molecular Weight |
402.36
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| Related CAS # |
PKCiota-IN-2;2230055-52-2
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| Appearance |
Light yellow to yellow solid powder
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 :~100 mg/mL (~248.53 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 | 2.4853 mL | 12.4267 mL | 24.8534 mL | |
| 5 mM | 0.4971 mL | 2.4853 mL | 4.9707 mL | |
| 10 mM | 0.2485 mL | 1.2427 mL | 2.4853 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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