Background
Acalisib (GS-9820) is a potent and selective inhibitor of PI3Kδ with IC50 value of 12.7 nM [1].
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylating the 3’-hydroxyl group of phosphatidylinositol-4,5-bisphosphate (PIP2) to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3). PI3Ks plays important roles in cell proliferation, survival, differentiation and metabolism. PI3Kδ plays an important role in inflammation, immune function, cell migration and vesicle trafficking, and is highly expressed in cells of hematopoietic origin [1].
Acalisib (GS-9820) is a potent and selective PI3Kδ inhibitor. GS-9820 inhibited PI3Kα, β, γ and δ with IC50 values of 5441, 3377, 1389 and 12.7 nM, respectively. In fibroblasts, GS-9820 inhibited PDGF- and LPA-induced pAkt with IC50 values of 11,585 and 2,069 nM respectively, which were through PI3Kα and PI3Kβ. In basophils, GS-9820 inhibited FcεRI PI3Kδ- and fMLP PI3Kγ-mediated CD63 expression with EC50 values of 14 nM and 2,065 nM, respectively. In isolated rat osteoclasts, GS-9820 induced prompt retraction of lamellipodia by 65-75% within 10-15 min. GS-9820 also significantly reduced the incidence of F-actin belts and inhibited osteoclast survival stimulated by RANKL [1].
Reference:
[1].? Shugg RP, Thomson A, Tanabe N, et al. Effects of isoform-selective phosphatidylinositol 3-kinase inhibitors on osteoclasts: actions on cytoskeletal organization, survival, and resorption. J Biol Chem, 2013, 288(49): 35346-35357.
Protocol
Kinase experiment: | 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]. |
Cell experiment: | 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]. |
Animal experiment: | 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. |
参考文献: [1]. Shugg RP, et al. 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]. Lopez-Guadamillas E, et al. PI3Kα inhibition reduces obesity in mice. Aging (Albany NY). 2016 Nov 4;8(11):2747-2753. |
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