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%.

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%.

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GS-9820 (1 μM) induces rapid retraction of lamellipodia in isolated rat osteoclasts within 10-15 minutes, reducing the planar cell area to about 65-75% of the initial area. This effect is reversible upon washout of the inhibitor [1].
Treatment with GS-9820 (1 μM for 10 minutes) significantly disrupts the organization of peripheral F-actin belts and sealing zones in osteoclasts plated on glass and resorbable calcium phosphate substrates, respectively [1].
GS-9820 (1 μM) significantly suppresses the enhancement of osteoclast survival induced by RANKL (100 ng/mL) during an 18-hour incubation, but has no significant effect on basal survival in the absence of RANKL [1].
GS-9820 (1 μM) significantly inhibits the resorptive activity of rabbit osteoclasts cultured on elephant ivory slices over 48 hours, reducing the total resorbed area, pit number, and pit depth [1].
At concentrations up to 10 μM, GS-9820 did not show significant toxic effects on the viability of undifferentiated RAW264.7 cells after 24 hours of treatment as assessed by MTT assay [1].
Kinase selectivity profiling against 393 kinases (including mutants) at 10 μM GS-9820 showed no significant binding to kinases outside the PI3K family, confirming its high selectivity [1]

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].

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In a mouse model of obesity (20-week-old male ob/ob mice), daily oral administration of GS-9820 at doses of 5 mg/kg or 10 mg/kg for 15 days did not cause a statistically significant reduction in body weight, unlike the PI3Kα inhibitor BYL-719 or the dual α/δ inhibitor CNIO-PI3Ki [2].
A single oral dose of GS-9820 (15 mg/kg) administered to lean, wild-type mice did not significantly elevate energy expenditure over a 7-hour monitoring period, in contrast to BYL-719 and CNIO-PI3Ki [2].
In obese ob/ob mice, a single oral dose of GS-9820 (5 or 10 mg/kg) caused a transient, comparatively minor increase in blood glucose levels (significant only at 1 hour post-dose) under ad libitum feeding conditions. This hyperglycemic effect was less severe than that induced by PI3Kα inhibitors [2].
After 15 days of daily treatment, GS-9820 (at the tested doses) did not cause significant changes in serum levels of triglycerides (TG), free fatty acids (FFA), or lactate in ob/ob mice [2].
Daily treatment with GS-9820 (5 mg/kg) in ob/ob mice for over a week was associated with a modest increase in food intake compared to vehicle-treated controls [2].
No significant changes in locomotor activity were observed in wild-type mice following a single 15 mg/kg dose of GS-9820 [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.

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Biochemical in vitro lipid kinase assays were performed to determine the inhibitory activity of GS-9820 against recombinant PI3K enzymes. A stock solution of the compound was prepared in DMSO. Ten-point dose-response curves were generated by measuring kinase inhibitory activities over a concentration range (e.g., 5 nM to 10,000 nM) with ATP concentrations set at the Km for each specific enzyme. The resulting data were used to calculate IC₅₀ values [1].
To assess kinase binding selectivity, competition binding assays were conducted for a panel of 393 kinases. GS-9820 was tested at a concentration of 10 μM. The assay measured the compound's ability to compete with immobilized, non-selective kinase probes for binding to the kinase ATP sites. Activity was defined as less than 35% of control binding remaining [1]
.

The effect of inhibitors on RAW264.7 cell survival is evaluated using the MTT assay. RAW264.7 cells are seeded in Falcon flat bottom 96-well plates at a density of 2.5-3×104 cells/cm2 in 100 μL of DMEM with 10% FBS and 1% antibiotic solution. After seeding, the cells are allowed to attach for 24 h then exposed to control or Acalisib (GS-9820) (100 pM to 10 μM) for 24 h. After incubation at 37°C in 5% CO2, MTT substrate is added at a final concentration of 0.5 mg/mL for 4 h. Following a 4-h incubation, 100 μL of solubilization solution is added to each well to dissolve the formazan crystals and samples are analyzed after 24 h. Absorbance of the samples is assessed using a plate reader using a wavelength of 550 nm and a reference wavelength of 700 nm[1].

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Osteoclasts were isolated from the long bones of neonatal rats or rabbits. Bones were minced and cells were released by repeated pipetting. The cell suspension was plated on serum-coated glass coverslips, glass-bottom dishes, or calcium phosphate-coated discs. Non-adherent cells were removed after 1 hour by gentle washing [1].
To assess morphology, osteoclasts in HEPES-buffered medium on a heated stage were imaged using phase-contrast microscopy. Time-lapse recordings were taken before and after addition of vehicle or GS-9820. The planar area of individual osteoclasts was traced and quantified using image analysis software at regular intervals [1].
To assess F-actin organization, osteoclasts plated on coverslips were treated with vehicle or GS-9820, then fixed, permeabilized, and stained with fluorescent phalloidin to label F-actin. Nuclei were counterstained. Cells were examined by fluorescence microscopy, and the percentage of osteoclasts exhibiting a complete peripheral F-actin belt or sealing zone was quantified [1].
For live-cell imaging of actin dynamics, rabbit osteoclasts were transduced with adenoviruses expressing an actin-EGFP fusion protein. Transduced cells were bathed in HEPES-buffered medium on a heated stage and imaged using confocal microscopy before and after addition of GS-9820 [1].
To assess osteoclast survival, rat osteoclasts were plated on coverslips. After cell attachment, the initial number of osteoclasts was counted. Cultures were then incubated for 18 hours in medium containing vehicle or GS-9820, with or without RANKL. The number of surviving osteoclasts was counted again, and survival was expressed as a percentage of the initial count [1].
A pit-formation assay was used to assess resorptive activity. Rabbit osteoclasts were plated on elephant ivory slices. After adherence, slices were transferred to medium containing vehicle or GS-9820 and incubated for 48 hours. Cells were then removed, and the slices were stained. Resorption pits were counted, and their area and depth were measured using microscopy and image analysis software [1].
Cell viability for RAW264.7 cells was assessed using the MTT assay. Cells were seeded in 96-well plates, allowed to attach, and then treated with a range of concentrations of GS-9820 or vehicle for 24 hours. MTT reagent was added, and after incubation, formazan crystals were dissolved. Absorbance was measured, and viability was expressed as a percentage of the vehicle-treated control [1]

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.

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The study utilized 20-week-old male C57BL/6J mice, including obese (ob/ob) and wild-type strains. Mice were housed under specific pathogen-free conditions with a 12-hour light/dark cycle and fed a standard chow diet [2].
GS-9820 was dissolved in a vehicle consisting of polyethylene glycol (PEG)-300 and 10% N-methyl-2-pyrrolidone (NMP) [2].
For the obesity reduction study in ob/ob mice, GS-9820 was administered daily by oral gavage at doses of 5 mg/kg and 10 mg/kg for 15-16 days [2].
For the energy expenditure and activity study in lean wild-type mice, a single oral dose of 15 mg/kg of GS-9820 was administered by gavage, and measurements were taken for 7 hours post-dose [2].
For serum biochemical analyses (glucose, TG, FFA, lactate), ob/ob mice were treated daily with GS-9820 for 15-16 days. On the final day, a dose was administered, and blood was collected via cardiac puncture 3-4 hours later under ad libitum feeding conditions [2].
For acute glucose excursion measurements, ob/ob mice under ad libitum feeding received a single oral dose of GS-9820 (5 or 10 mg/kg). Blood glucose was monitored from the tail tip over time [2].
Indirect calorimetry was performed using metabolic chambers. Mice were acclimated to the chambers for three days prior to the experiment. After a single oral dose of 15 mg/kg GS-9820, oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were recorded every 24 minutes for 7 hours to calculate energy expenditure. Locomotor activity was simultaneously recorded in 20-minute intervals [2]

The stability of GS-9820 was evaluated under specific conditions. The compound had a half-life of 11.7 days at 50 °C and pH 2 [1]. Other ADME/PK parameters, such as absorption, distribution, metabolism, excretion, plasma half-life, or oral bioavailability, were not described in the literature provided [1].

MTT assays on undifferentiated RAW264.7 cells showed that GS-9820 at concentrations up to 10 μM did not significantly reduce cell viability after 24 hours of treatment, indicating that the compound did not have acute cytotoxicity to the cell line at the tested concentrations [1]. Other toxicity parameters, such as lethal dose in vivo, organ toxicity, drug interactions, or plasma protein binding, were not described in reference [1].

[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 being investigated in clinical trial NCT01705847 (a phase 1b study evaluating the efficacy of GS-9820 in patients with malignant lymphomas). Acalisib is an inhibitor of the β and δ subtypes of a 110 kDa phosphatidylinositol-3 kinase (PI3K) of class IA, with potential immunomodulatory and antitumor activity. Acalisib inhibits the activity of PI3K, thereby preventing the production of the second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3), which in turn reduces tumor cell proliferation and induces cell death. PI3K-mediated signaling pathways are often dysregulated in cancer cells; targeted inhibition of PI3K aims to maintain PI3K signaling in normal non-tumor cells. GS-9820 (also known as CAL-120 during development) is a novel, potent and selective inhibitor of the δ subtype of PI3K[1].
GS-9820 has the chemical name 6-fluoro-3-phenyl-2-[(1S)-1-(9H-purin-6-ylamino)ethyl]-4(3H)-quinazolinone[1].
PI3Kδ is mainly expressed in hematopoietic cells. This study concluded that PI3Kδ plays a key role in regulating osteoclast cytoskeleton organization and absorption activity, making it an ideal target for anti-absorption therapy[1].
GS-9820 has a rapid and reversible effect on osteoclast pseudopodia retraction and actin cytoskeleton disruption, which distinguishes it from some other drugs such as calcitonin[1].

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.)