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| Targets |
KD: 28.77 μM (Skp2)[1]
Skp2 (S-phase kinase-associated protein 2). SCFSkp2-IN-2 (compound AAA-237) is a Skp2 inhibitor that binds directly to the Skp2 protein with a dissociation constant (Kd) of 28.77 uM. It inhibits the Skp2 subunit of the SCF E3 ubiquitin ligase. |
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| ln Vitro |
Skp2 can be directly bound by SCFSkp2-IN-2 (Compound AAA-237) (0.3-3 μM; 24-72 h), which will cause the proteasome to break down Skp2. In NSCLC cells, AAA-237 has a time-dependent inhibitory effect on Skp2 protein expression but no discernible effect on Skp2 mRNA expression [1]. A549 and H1299 cell proliferation is inhibited by SCFSkp2-IN-2 (0.3-3 μM; 24-72 h) in a dose- and time-dependent manner [1]. The G0/G1 checkpoint is where the cell cycle is arrested by SCFSkp2-IN-2 (0.3-3 μM; 24-72 h) because it modulates the PI3K/Akt-FOXO1 and Skp2-Cip/Kip signaling pathways [1]. A549 and H1299 cells undergo apoptosis when exposed to SCFSkp2-IN-2 (0.3-3 μM; 24-72 h) in a dose- and time-dependent manner [1]. In NSCLC cells, SCFSkp2-IN-2 (0.3 μM; 1–7 days) causes senescence [1].
SCFSkp2-IN-2 (AAA-237) is a Skp2 inhibitor with a Kd of 28.77 uM for binding to the Skp2 protein. By binding to Skp2, it inhibits the ubiquitination of Skp2 substrates. This leads to the stabilization of key cell cycle regulators (e.g., p27Kip1) and the induction of apoptosis in non-small cell lung cancer (NSCLC) cells. Its antiproliferative activity has been demonstrated in NSCLC cell lines, and it shows potential for anticancer research. |
| ln Vivo |
In a lung cancer A549 xenograft mice model, SCFSkp2-IN-2 (Compound AAA-237) (15 or 45 mg/kg; ip; daily for 14 days) exhibits anti-tumor efficacy[1].
In vitro, SCFSkp2-IN-2 induces apoptosis in NSCLC cells and shows antitumor activities. In vivo activity data are not detailed in the supplier literature. As a Skp2 inhibitor, it has potential applications in animal models of cancer, particularly in NSCLC where Skp2 is often overexpressed and correlates with poor prognosis. Further in vivo studies would be required to characterize its efficacy, pharmacokinetics, and safety profile. |
| Enzyme Assay |
For non-cellular assays, the binding affinity of SCFSkp2-IN-2 to Skp2 is measured using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). Recombinant Skp2 protein is immobilized on a sensor chip. SCFSkp2-IN-2 is flowed over at increasing concentrations (1-1000 uM). The association and dissociation rates are measured to calculate the Kd value (28.77 uM). For functional assays, in vitro ubiquitination reactions using purified SCF complex components and a ubiquitin-charged E2 are performed to test if the inhibitor blocks p27Kip1 ubiquitination.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: A549 and H1299 cells Tested Concentrations: 0.3, 1 and 3 μM Incubation Duration: 24, 48 and 72 h Experimental Results: Increased the expression levels of p21Cip1 and p27Kip1. diminished the level of Skp2. diminished the expression levels of CDK2, p-CDK2, cyclin E1, CDK4, p-CDK4 and cyclin D. Increased the levels of cleaved PARP, cleaved caspase 3, cleaved caspase 9 and Bax. diminished the expression of PARP and Bcl-2. Cell Proliferation Assay [1] Cell Types: A549 and H1299 cells Tested Concentrations: 0.3, 1 and 3 μM Incubation Duration: 24, 48 and 72 h Experimental Results: Inhibited the proliferation of A549 and H1299 cells in a dose- and time-dependent manner. The IC50 for A549 was 3 μM at 24 h, 2.5 μM at 48 h and 0.7 μM at 72 h. The IC50 for H1299 was 3.9 μM at 24 h, 1.8 μM at 48 h and 1.1 μM at 72 h. Cell Cycle Analysis[1] Cell Types: A549 and H1299 cells Tested Concentrations: 0.3, 1 and 3 μM Incubation Duration: 24, 48 and 72 h Experimental Results: Arrested the cell cycle at G0/G1 in a dose- and time-dependent manner. Apoptos Cellular assays involve treating non-small cell lung cancer (NSCLC) cells (e.g., H1299, A549) with SCFSkp2-IN-2 (10-100 uM) for 24-72 hours. Cell viability is measured by MTT or CellTiter-Glo assays. Apoptosis is assessed by Annexin V/PI staining and flow cytometry, and by measuring caspase-3/7 activity. p27Kip1, p21, and cyclin E levels are analyzed by Western blot. Cell cycle distribution is analyzed by propidium iodide staining and flow cytometry to confirm cell cycle arrest. |
| Animal Protocol |
Animal/Disease Models: A549 xenograft model[1]
Doses: 15 or 45 mg/kg Route of Administration: intraperitoneal (ip) administration, daily for 14 days Experimental Results: Dramatically decreased tumor volume and weight. The tumor growth inhibition of the low dose and high dose was 55% and 64%, respectively. No obvious weight loss or abnormal behavior was observed. decreased the expression of Ki67 in tumor tissue. decreased the expression of Skp2 and Bcl-2 and increased the expression of p27, the cleaving of caspase 3, caspase 9, PARP and Bax. SCFSkp2-IN-2 can be formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. It would be administered intraperitoneally or orally to mice bearing subcutaneous NSCLC xenografts (e.g., H1299). Doses would need to be optimized. Tumor volume would be measured with calipers, and at the end of the study, tumors would be harvested for Western blot analysis of p27Kip1, cyclin E, and apoptosis markers. Body weight would be monitored to assess tolerability. |
| ADME/Pharmacokinetics |
SCFSkp2-IN-2 has a molecular weight of 428.57 g/mol and a molecular formula of C27H32N4O. It is soluble in DMSO (12 mg/mL). The compound is stored at 4degC, sealed, away from moisture and light. Detailed pharmacokinetic parameters (half-life, oral bioavailability) are not reported in the supplier literature. However, it is a small molecule with properties that suggest the potential for oral activity.
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| Toxicity/Toxicokinetics |
Detailed toxicological data for SCFSkp2-IN-2 are not available. It is intended for research use only and not for human consumption. Standard safety precautions for handling research chemicals should be followed, including the use of gloves and eye protection. The compound may be irritating to the skin, eyes, and respiratory tract. Off-target effects on cell cycle regulators in normal proliferative tissues could be a concern.
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| References | |
| Additional Infomation |
SCFSkp2-IN-2 (compound AAA-237) is a research-grade chemical, not an FDA-approved drug. It is used as a chemical probe to study the role of the Skp2 E3 ligase in cell cycle regulation and cancer biology. Skp2 is a well-validated oncogene and an attractive target for cancer therapy. Its inhibition leads to the accumulation of the tumor suppressor p27Kip1, which blocks cell cycle progression. This compound was identified through a high-throughput screen for inhibitors of the Skp2-Cks1 interaction.
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| Molecular Formula |
C17H20N4O2
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|---|---|
| Molecular Weight |
312.366303443909
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| Exact Mass |
312.158
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| CAS # |
1375060-02-8
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| PubChem CID |
135457298
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
2.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
23
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| Complexity |
398
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=NC=CC=C1C(N/N=C/C1=CC=C(N(CC)CC)C=C1O)=O
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| InChi Key |
HEUHCTYMFLTWAX-XDHOZWIPSA-N
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| InChi Code |
InChI=1S/C17H20N4O2/c1-3-21(4-2)15-8-7-13(16(22)10-15)12-19-20-17(23)14-6-5-9-18-11-14/h5-12,22H,3-4H2,1-2H3,(H,20,23)/b19-12+
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| Chemical Name |
N-[(E)-[4-(diethylamino)-2-hydroxyphenyl]methylideneamino]pyridine-3-carboxamide
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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: 125 mg/mL (400.17 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 | 3.2013 mL | 16.0067 mL | 32.0133 mL | |
| 5 mM | 0.6403 mL | 3.2013 mL | 6.4027 mL | |
| 10 mM | 0.3201 mL | 1.6007 mL | 3.2013 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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