p110α (IC50 = 5441 nM); p110β (IC50 = 3377 nM); p110δ (IC50 = 12.7 nM); p110γ (IC50 = 1389 nM); hVps34 (IC50 = 12682 nM); DNA-PK (IC50 = 18749 nM)

Acalisib (GS-9820) to other PI3K class I enzymes (IC50: PI3K, 5,441 nM; PI3K, 3,377 nM; PI3K, 1,389 nM), PI3K is more selective for Acalisib (IC50=12.7 nM). Additionally, compared to related kinases like PI3KCII (IC50>10 nM), hVPS34 (IC50=12.7 M), DNA-PK (IC50=18.7 M), and mTOR (IC50>10 nM), acalisib is 103-fold more selective against PI3K. Both the GPCR for lysophosphatidic acid (LPA) and the PDGF receptor signal through PI3K in fibroblasts. At 11,585 nM, acalisib reduces PDGF-induced pAkt by 50%, and at 2,069 nM, it reduces LPA-induced pAkt by 50%.

Obese hyperphagic ob/ob mice are treated with either BYL-719, a selective PI3K inhibitor, or Acalisib (GS-9820), a selective PI3K inhibitor, to examine the relative roles of PI3K and PI3K inhibition in the prevention of obesity. Surprisingly, after 15 days of treatment, BYL-719 reduces body weight to a degree comparable to CNIO-PI3Ki, whereas Acalisib has no noticeable effect at the same doses as BYL-719. It should be noted that mice with multiple myeloma xenografts can grow less rapidly when given 10 mg/kg of acalisib[2].

Biochemical in vitro lipid kinase assays are performed. A stock solution of Acalisib (GS-9820) is prepared in DMSO at a concentration of 10 mM. Ten-point kinase inhibitory activities are measured over a concentration range (5 to 104 nM) with ATP at a concentration consistent with the Km of each of the enzymes[1].

In Vitro Kinase Activity Profiling [1]
Biochemical in vitro lipid kinase assays were performed by the SelectScreen® biochemical kinase assay service. A stock solution of GS-9820 was prepared in DMSO at a concentration of 10 mm. Ten-point kinase inhibitory activities were measured over a concentration range (5 to 104 nm) with ATP at a concentration consistent with the Km of each of the enzymes.

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Kinase Binding Selectivity Profiling [1]
GS-9820 was tested at 10 μm in ATP site-dependent competition binding assays for 393 kinases (358 excluding mutant kinases) by contract with Ambit Biosciences. GS-9820 was considered active if <35% of binding to immobilized probes remained compared with DMSO control.

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PI3K Isoforms in RAW264.7 Cells [1]
Whole cell lysates from RAW264.7 cells were prepared in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin), supplemented with 1× complete mini protease inhibitor, and 1× Phosphatase Inhibitor Mixture Set I, II for 10 min on ice. Lysates were cleaned by sedimentation at 14,000 × g for 10 min at 4 °C, and the soluble protein was analyzed by Western blotting using anti-PI3Kα, anti-PI3Kβ, anti-PI3Kδ, and anti-PI3Kγ. Immunoreactive bands were visualized using LI-COR Odyssey.

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PI3K Isoform-selective Cell-Based Assays [1]
Murine embryonic fibroblasts were used for the analysis of PI3Kα and PI3Kβ signaling. Cells were transferred to serum-free medium for 2 h followed by 2 h incubation in the absence or presence of increasing concentrations of GS-9820, and then stimulated with PDGF (10 ng/ml) or lysophosphatidic acid (LPA) (10 μm) for 10 min at 37 °C to activate PI3Kα and PI3Kβ, respectively. Cells were trypsinized, washed in cold PBS, and the cell pellet was lysed in lysis buffer for 10 min on ice. Whole cell lysates were cleaned by sedimentation at 14,000 × g for 15 min at 4 °C, and the soluble protein was analyzed by Western blotting for Akt and pAkt levels.

Mice[2]
Ob/ob C57BL6J mice and Wild-type C57BL6J/Ola.Hsd mice are housed under specific pathogen free (SPF) conditions, at 22°C, and with 12 hours dark/light cycles (light cycle from 8 am to 8 pm). All mice used are males of 20 weeks of age. Mice are fed with standard chow diet (18% of fat-based caloric content). PI3K inhibitors are administered daily by oral gavage during 15 or 16 days as follows, BYL-719 (5 and 10 mg/kg) and Acalisib (5 and 10 mg/kg), CNIO-PI3Ki (1 and 5 mg/kg), dissolved in PEG-300 and 10% N-methyl-2-pyrrolidone.

[1]. Effects of isoform-selective phosphatidylinositol 3-kinase inhibitors on osteoclasts: actions on cytoskeletal organization, survival, and resorption. J Biol Chem. 2013 Dec 6;288(49):35346-57.

[2]. PI3Kα inhibition reduces obesity in mice. Aging (Albany NY). 2016 Nov 4;8(11):2747-2753.

Acalisib is under investigation in clinical trial NCT01705847 (A Phase 1b Study Evaluating GS-9820 in Subjects With Lymphoid Malignancies).
Acalisib is an inhibitor of the beta and delta isoforms of the 110 kDa catalytic subunit of class IA phosphoinositide-3 kinases (PI3K) with potential immunomodulating and antineoplastic activities. Acalisib inhibits the activity of PI3K, thereby preventing the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), which decreases tumor cell proliferation and induces cell death. PI3K-mediated signaling is often dysregulated in cancer cells; the targeted inhibition of PI3K is designed to preserve PI3K signaling in normal, non-neoplastic cells.

C21H16FN7O

401.4

401.14

C, 62.84; H, 4.02; F, 4.73; N, 24.43; O, 3.99

870281-34-8

11618268

White to off-white solid powder

1.5±0.1 g/cm3

733.7±70.0 °C at 760 mmHg

397.5±35.7 °C

0.0±2.4 mmHg at 25°C

1.759

2.43

2

7

4

30

670

1

[C@@H](C1=NC2C=CC(=CC=2C(=O)N1C1C=CC=CC=1)F)(C)NC1=NC=NC2N=CNC1=2

DOCINCLJNAXZQF-LBPRGKRZSA-N

InChI=1S/C21H16FN7O/c1-12(27-19-17-18(24-10-23-17)25-11-26-19)20-28-16-8-7-13(22)9-15(16)21(30)29(20)14-5-3-2-4-6-14/h2-12H,1H3,(H2,23,24,25,26,27)/t12-/m0/s1

Acalisib; CAL120; CAL 120; CAL-120; GS-9820; 870281-34-8; Acalisib (GS-9820); Acalisib [INN]; GS-9820; CAL-120; OVW60IDW1D; CAL120; GS9820; GS 9820

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)