AZD6482

Catalog No.S1462 Batch:S146202

Technical Data

Formula

C₂₂H₂₄N₄O₄

Molecular Weight

408.45

CAS No.

1173900-33-8

Solubility (25°C)*

In vitro

DMSO

82 mg/mL (200.75 mM)

Ethanol

10 mg/mL (24.48 mM)

Water

Insoluble

In vivo (Add solvents to the product individually and in order)

Clear solution

30%PEG400 1 0.5%Tween80 2 5%propylene glycol

30.0mg/ml

Taking the 1 mL working solution as an example, add 300 μL of 100 mg/ml clarified PEG400 stock solution to 5 μL of Tween80, mix evenly to clarify it; add 50 μL Propylene glycol to the above system, mix evenly to clarify it; then continue to add 645 μL ddH2O to adjust the volume. to 1 mL. The mixed solution should be used immediately for optimal results.

* <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

AZD6482 (KIN-193) is a PI3Kβ inhibitor with IC50 of 10 nM, 8-, 87- and 109-fold more selective to PI3Kβ than PI3Kδ, PI3Kα and PI3Kγ in cell-free assays. Phase 1.

Targets

PI3Kβ ^[1] (Cell-free assay)	PI3Kδ ^[1] (Cell-free assay)	DNA-PK ^[1] (Cell-free assay)	PI3Kα ^[1] (Cell-free assay)
10 nM	80 nM	420 nM	870 nM

In vitro

AZD6482 also inhibits PI3Kα, γ, and δ, with IC50 of 80 nM to 1.09 μM, which are significantly lower than its (+)-enantiomer (S-form). AZD6482 is an antiplatelet agent; it blocks platelet activation adhesion/aggregation and promotes platelet disaggregation in assay of washed platelet aggregation (WPA), with an IC50 value of 6 nM. Furthermore, by targeting PI3Kβ, AZD6482 specifically inhibits thrombosis without interfering with normal haemostasis. Therefore, AZD6482 is used as an anti-thrombotic drug for the prophylaxis of thrombotic disorders. ^[1]

Protocol (from reference)

Kinase Assay:^{^[1]}	Assay of PI3K enzyme inhibition The inhibition of PI3Kβ, PI3Kα, PI3Kγ, and PI3Kδ is evaluated in an AlphaScreen based enzyme activity assay using human recombinant enzymes. The assay measures PI3K-mediated conversion of PIP2 to PIP3. Biotinylated PIP3, a GST-tagged pleckstrin homology (PH) domain and the two AlphaScreen beads form a complex that elicits a signal upon laser excitation at 680 nm. The PIP3 formed in the enzyme reaction competes with the biotinylated PIP3 for binding to the PH domain thus reducing the signal with increasing enzyme product. The AZD6482 is dissolved in DMSO and added to 384 well plates. PBKβ, PBKα, PBKγ, or PBKδ is added in a Tris buffer (50 mM Tris pH 7.6, 0.05% CHAPS, 5 mM DTT, and 24 mM MgCl₂) and allowed to preincubate with AZD6482 for 20 minutes prior to the addition of substrate solution containing PIP2 and ATP. The enzyme reaction is stopped after 20 minutes by addition of stop solution containing EDTA and biotin-PIP3, followed by addition of detection solution containing GST-grpl PH and AlphaScreen beads. Plates are left for a minimum of 5 hours in the dark prior to analysis. The final concentration of DMSO, ATP and PIP2 in the assay are, 0.8%, 4 μM, and 40 μM, respectively. IC50 values are calculated according to the equation, y = {a+[(b-a)/(l+(x/IC50)^s)]}, where y = % inhibition; a = 0%; b = 100%; s = the slope of the concentration-response curve; x = AZD6482 concentration.
Cell Assay:^{^[1]}	Cell lines Human platelet pellet Concentrations 0–60 nM, dissolved in DMSO Incubation Time 5 min Method For assay of washed platelet aggregation (WPA), the platelet pellets are isolated from human blood and re-suspended to 2 × 10¹⁵/L in Tyrodes buffer (TB) containing 1 μM hirudin and 0.02 U/mL apyrase. Then, the platelet suspension is left to rest at room temperature for 30 min. Just prior to time for assay, CaCl₂ is added to a final concentration of 2 mM. AZD6482, dissolved in DMSO, is added to a 96-well plate prior to the addition of the washed platelet suspension. The platelet suspension is preincubated with AZD6482 for 5 min. Light absorption at 650 nm is recorded before and after a 5 min plate shake and referred to as recording 0 (R0) and Rl. A mouse anti-human CD9 antibody is added (at a donor specific concentration) to each well prior to next 10 min plate shake and light absorption recording; R2. For data analysis, light absorbance in wells with TB are subtracted from all readings before percent aggregation is calculated according the formula: [(R1-R2)/R1] × 100 = % aggregation. Spontaneous aggregation or pro-aggregatory effect of the inhibitor is evaluated by the same formula, [(R0-Rl)/R0] × 100 = % aggregation. IC50 values are calculated according to the equation, y = {a+[(b-a)/(l+(x/IC50)^s)]}, where y = % inhibition; a = 0%; b = 100%; s = the slope of the concentration-response curve; x = AZD6482 concentration.

Kinase Assay:^{^[1]}

Assay of PI3K enzyme inhibition
The inhibition of PI3Kβ, PI3Kα, PI3Kγ, and PI3Kδ is evaluated in an AlphaScreen based enzyme activity assay using human recombinant enzymes. The assay measures PI3K-mediated conversion of PIP2 to PIP3. Biotinylated PIP3, a GST-tagged pleckstrin homology (PH) domain and the two AlphaScreen beads form a complex that elicits a signal upon laser excitation at 680 nm. The PIP3 formed in the enzyme reaction competes with the biotinylated PIP3 for binding to the PH domain thus reducing the signal with increasing enzyme product. The AZD6482 is dissolved in DMSO and added to 384 well plates. PBKβ, PBKα, PBKγ, or PBKδ is added in a Tris buffer (50 mM Tris pH 7.6, 0.05% CHAPS, 5 mM DTT, and 24 mM MgCl₂) and allowed to preincubate with AZD6482 for 20 minutes prior to the addition of substrate solution containing PIP2 and ATP. The enzyme reaction is stopped after 20 minutes by addition of stop solution containing EDTA and biotin-PIP3, followed by addition of detection solution containing GST-grpl PH and AlphaScreen beads. Plates are left for a minimum of 5 hours in the dark prior to analysis. The final concentration of DMSO, ATP and PIP2 in the assay are, 0.8%, 4 μM, and 40 μM, respectively. IC50 values are calculated according to the equation, y = {a+[(b-a)/(l+(x/IC50)^s)]}, where y = % inhibition; a = 0%; b = 100%; s = the slope of the concentration-response curve; x = AZD6482 concentration.

Cell Assay:^{^[1]}

Cell lines
Human platelet pellet
Concentrations
0–60 nM, dissolved in DMSO
Incubation Time
5 min
Method
For assay of washed platelet aggregation (WPA), the platelet pellets are isolated from human blood and re-suspended to 2 × 10¹⁵/L in Tyrodes buffer (TB) containing 1 μM hirudin and 0.02 U/mL apyrase. Then, the platelet suspension is left to rest at room temperature for 30 min. Just prior to time for assay, CaCl₂ is added to a final concentration of 2 mM. AZD6482, dissolved in DMSO, is added to a 96-well plate prior to the addition of the washed platelet suspension. The platelet suspension is preincubated with AZD6482 for 5 min. Light absorption at 650 nm is recorded before and after a 5 min plate shake and referred to as recording 0 (R0) and Rl. A mouse anti-human CD9 antibody is added (at a donor specific concentration) to each well prior to next 10 min plate shake and light absorption recording; R2. For data analysis, light absorbance in wells with TB are subtracted from all readings before percent aggregation is calculated according the formula: [(R1-R2)/R1] × 100 = % aggregation. Spontaneous aggregation or pro-aggregatory effect of the inhibitor is evaluated by the same formula, [(R0-Rl)/R0] × 100 = % aggregation. IC50 values are calculated according to the equation, y = {a+[(b-a)/(l+(x/IC50)^s)]}, where y = % inhibition; a = 0%; b = 100%; s = the slope of the concentration-response curve; x = AZD6482 concentration.

References

[1] Fjellstrom O, et al. US Patent, WO2009093972.

Customer Product Validation

: , , Mol Cancer Res, 2017, 15(6):765-775

: , , One customer

: , , Dr. Zhang of Tianjin Medical University

: Data from [Data independently produced by , , Oncotarget, 2016, 7(34):54897-54912]

Selleck's AZD6482 has been cited by 25 publications

A Gene Co-Expression Network-Based Drug Repositioning Approach Identifies Candidates for Treatment of Hepatocellular Carcinoma [ Cancers (Basel), 2022, 14(6)1573]	PubMed: 35326724
ZAP70 Activation Compensates for Loss of Class IA PI3K Isoforms Through Activation of the JAK-STAT3 Pathway [ Cancer Diagn Progn, 2022, 2(3):391-404]	PubMed: 35530641
Synergism between the phosphatidylinositol 3-kinase p110β isoform inhibitor AZD6482 and the mixed lineage kinase 3 inhibitor URMC-099 on the blockade of glioblastoma cell motility and focal adhesion formation [ Cancer Cell Int, 2021, 21(1):24]	PubMed: 33407478
Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER+ Breast Cancer [ Cancer Cell, 2020, S1535-6108(20)30427-X]	PubMed: 32976773
PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition [ Proc Natl Acad Sci U S A, 2020, 117(39):24427-24433]	PubMed: 32929011
Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma [ Blood Adv, 2020, 4(18):4382-4392]	PubMed: 32926124
Application of a Biphasic Mathematical Model of Cancer Cell Drug Response for Formulating Potent and Synergistic Targeted Drug Combinations to Triple Negative Breast Cancer Cells. [ Cancers (Basel), 2020, 27;12(5)pii: E1087]	PubMed: 32349331
Biphasic Mathematical Model of Cell-Drug Interaction That Separates Target-Specific and Off-Target Inhibition and Suggests Potent Targeted Drug Combinations for Multi-Driver Colorectal Cancer Cells. [ Cancers (Basel), 2020, 13;12(2) pii: E436]	PubMed: 32069833
Cisplatin Resistance in Osteosarcoma: In vitro Validation of Candidate DNA Repair-Related Therapeutic Targets and Drugs for Tailored Treatments. [ Front Oncol, 2020, 10;10:331]	PubMed: 32211337
SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers. [ Nat Commun, 2019, 10(1):2532]	PubMed: 31182717

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