SU9516

Catalog No.S7636 Batch:S763601

Technical Data

Formula	C₁₃H₁₁N₃O₂
Molecular Weight	241.25	CAS No.	377090-84-1
Solubility (25°C)*	In vitro	DMSO	48 mg/mL (198.96 mM)
		Ethanol	12 mg/mL (49.74 mM)
		Water	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

SU 9516 is a 3-substituted indolinone CDK inhibitor with IC50 of 22 nM, 40 nM, and 200 nM for CDK2, CDK1, and CDK4, respectively.

Targets

CDK2 ^[1]	CDK1 ^[1]	CDK4 ^[1]
22 nM	40 nM	200 nM

In vitro

SU9516 decreases cdk2-specific phosphorylation of the retinoblastoma protein pRB, increases caspase-3 activation, and alters cell cycle in RKO and SW480 cells. SU9516 also inhibits the cell proliferation, and induces cell apoptosis in both cell lines. ^[1] SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation, oxidative damage and transcriptional down-regulation of Mcl-1. ^[2] In human T-cell leukemia Jurkat cells, SU9516 significantly enhances sensitivity to methotrexate. ^[3] In addition, SU9516 also suppresses Aurora-A centrosomal localization and consequent centrosome amplification. ^[4]

Protocol (from reference)

Kinase Assay:^{^[1]}	CDK kinase assay Kinase assays are performed in 96-well polypropylene plates. Each reaction contained 2 μg of histone H1 at a final concentration of 10 μM [γ-³³P]ATP (0.2 μCi/well; approximately twice the experimentally determined Km), 10 mM MgCl₂, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol in a 40 μL volume. The reaction is initiated with the addition of 20 μL enzyme (6 ng cdk2/well resulting in a final concentration of 1.6 nM), which is previously diluted 1:50–1:200 in the same buffer, and allowed to proceed for 1 h at room temperature. Reaction is stopped by the addition of 0.01 mL 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is washed three times with 1.0% phosphoric acid, air dried, and then counted for radioactivity in a liquid scintillation counter. The cdk4 kinase assay for cyclin D1-cdk4 is carried out in a polypropylene 96-well microtiter plate format measuring the incorporation of radioactive phosphate into GST-Rb. Purified cyclin D1-cdk4 is incubated with 1 μg GST-Rb in 20 mM HEPES (pH 7.5) in the presence of 10 mM MgCl₂, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol. The final cdk4 concentration is 10 ng/well, or 1.6 nM. Kinase reaction is initiated by the addition of ATP at a final concentration of 10 μM ATP (twice the experimentally determined Km) and [γ-³³P]ATP (1.0 μCi per well) in a 60-μL volume and allowed to proceed at room temperature for 1 h. Reaction is stopped by the addition of 0.01 ml 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is treated as for Cdk1/Cdk2 assays.
Cell Assay:^{^[1]}	Cell lines RKO cells and SW480 cells Concentrations 24 hours Incubation Time ~50 μM Method RKO cells and SW480 cells are seeded in replicates in 96-well plates at 1 × 10⁴ cells/well and allowed to attach overnight. SU9516 is added in concentrations from 0.05 μM to 50.00 μM for 24 h, the cells are then washed twice with PBS, and cells are replenished with complete media. The cells are fixed at 0, 4, and 7 days post-drug removal and assayed for protein levels using a modified SRB cytotoxicity assay. The cells are fixed in 10% trichloroacetic acid for 1 h, washed in distilled H2O, and stained in 0.4% SRB/acetic acid for 30 min. The cells are then washed in 0.1% acetic acid, solubilized in 10 mM Tris (pH 9), and analyzed on a Bio-Rad 360 microplate reader at 595 nm. All experiments are repeated at least three times.

Kinase Assay:^{^[1]}

CDK kinase assay
Kinase assays are performed in 96-well polypropylene plates. Each reaction contained 2 μg of histone H1 at a final concentration of 10 μM [γ-³³P]ATP (0.2 μCi/well; approximately twice the experimentally determined Km), 10 mM MgCl₂, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol in a 40 μL volume. The reaction is initiated with the addition of 20 μL enzyme (6 ng cdk2/well resulting in a final concentration of 1.6 nM), which is previously diluted 1:50–1:200 in the same buffer, and allowed to proceed for 1 h at room temperature. Reaction is stopped by the addition of 0.01 mL 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is washed three times with 1.0% phosphoric acid, air dried, and then counted for radioactivity in a liquid scintillation counter. The cdk4 kinase assay for cyclin D1-cdk4 is carried out in a polypropylene 96-well microtiter plate format measuring the incorporation of radioactive phosphate into GST-Rb. Purified cyclin D1-cdk4 is incubated with 1 μg GST-Rb in 20 mM HEPES (pH 7.5) in the presence of 10 mM MgCl₂, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol. The final cdk4 concentration is 10 ng/well, or 1.6 nM. Kinase reaction is initiated by the addition of ATP at a final concentration of 10 μM ATP (twice the experimentally determined Km) and [γ-³³P]ATP (1.0 μCi per well) in a 60-μL volume and allowed to proceed at room temperature for 1 h. Reaction is stopped by the addition of 0.01 ml 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is treated as for Cdk1/Cdk2 assays.

Cell Assay:^{^[1]}

Cell lines
RKO cells and SW480 cells
Concentrations
24 hours
Incubation Time
~50 μM
Method
RKO cells and SW480 cells are seeded in replicates in 96-well plates at 1 × 10⁴ cells/well and allowed to attach overnight. SU9516 is added in concentrations from 0.05 μM to 50.00 μM for 24 h, the cells are then washed twice with PBS, and cells are replenished with complete media. The cells are fixed at 0, 4, and 7 days post-drug removal and assayed for protein levels using a modified SRB cytotoxicity assay. The cells are fixed in 10% trichloroacetic acid for 1 h, washed in distilled H2O, and stained in 0.4% SRB/acetic acid for 30 min. The cells are then washed in 0.1% acetic acid, solubilized in 10 mM Tris (pH 9), and analyzed on a Bio-Rad 360 microplate reader at 595 nm. All experiments are repeated at least three times.

References

[4] Leontovich AA, et al. Oncol Rep. 2013, 29(5), 1785-1788.

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Customer Product Validation

: Data from [Data independently produced by , , Biotechnol Lett, 2017, 39(7):951-957]

Selleck's SU9516 has been cited by 3 publications

Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation [ Mol Cell, 2022, S1097-2765(22)01064-4]	PubMed: 36417913
Differences in the Conformational Energy Landscape of CDK1 and CDK2 Suggest a Mechanism for Achieving Selective CDK Inhibition [ Cell Chem Biol, 2019, 26(1):121-130]	PubMed: 30472117
CDK inhibitor SU9516 induces tetraploid blastocyst formation from parthenogenetically activated porcine embryos. [Guo Q, et al. Biotechnol Lett, 2017, 39(7):951-957]	PubMed: 28315059

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SHIPPING AND STORAGE
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