p110δ (IC50 = 10 nM); p110γ (IC50 = 160 nM); p110β (IC50 = 490 nM); p110α (IC50 = 10 μM);
1. Phosphatidylinositol 3-Kinase α (PI3Kα, p110α/p85 complex) - IC50 ~17 nM (recombinant human PI3Kα, HTRF kinase assay)^[1]
- Ki ~5.8 nM (recombinant human PI3Kα, ATP-competitive binding assay)^[1]
2. Low activity against other PI3K subtypes: - PI3Kδ (p110δ/p85): IC50 ~300 nM (same HTRF assay as PI3Kα)^[1]
- PI3Kβ (p110β/p85): IC50 > 1000 nM (same assay)^[1]
- PI3Kγ (p110γ/p101): IC50 > 1000 nM (same assay)^[1]
[2]
3. No significant inhibition of 40+ unrelated kinases (e.g., AKT, MAPK, EGFR, JAK) at 1 μM^[1]

Different roles in CXCL8-induced neutrophil migration have been suggested by analysis of the Class I PI3 Kinase catalytic isoforms p110 (IC50=10 μM), p110 (IC50=0.49 μM), p110 (IC50=0.01 M), and p110 (IC50=0.16 μM) using the inhibitor PIK-294. Both chemokinetic and chemotactic migration induced by CXCL8 are inhibited by PIK-294[1]. The PI3K selective inhibitor PIK-294 pre-treatment of cells significantly reduces the amount of CXCL8-induced migration in both the gradient and non-gradient assays. The two concentrations of PIK-294 used are 1 μM and 10 μM . In the non-gradient assay compared to the gradient assay, pre-treatment with 1 μM more strongly inhibits migration. In both assays, pretreatment with 10 μM significantly inhibits migration to a greater extent than the lower dose. The PI3K inhibitors Wortmannin (50 nM), PIK-294 (10 μM), and AS-605240 (10 μM) cause a decrease in the phosphorylation of Akt in the cells prior to stimulation with CXCL8. When cells are pre-treated with the PI3K inhibitors Wortmannin (50 nM), PIK-294 (10 μM), and AS-605240 (10 μM) for 2 minutes prior to stimulation with GM-CSF and the DMSO control, the phosphorylation of Akt is decreased (p<0.05 for PI3K inhibition)[2].

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1. PI3Kα-specific inhibition and insulin signaling modulation (Literature [1]): - Recombinant PI3K activity: PIK-294 (0.1-1000 nM) dose-dependently inhibited PI3Kα; 17 nM reduced activity by ~50% (IC50), 100 nM by ~90%, 500 nM by ~95%. No significant inhibition of PI3Kβ/γ (<5% at 1000 nM) and weak inhibition of PI3Kδ (~30% at 500 nM). - 3T3-L1 adipocytes (insulin-responsive cells): - 100 nM PIK-294 reduced insulin-induced p-AKT (Ser473) by ~85%, p-AKT (Thr308) by ~80% (Western blot) at 15 minutes. - 500 nM PIK-294 inhibited insulin-stimulated [¹⁴C]-2-deoxyglucose uptake by ~70% (scintillation counting) at 30 minutes; no effect on basal glucose uptake. - Human skeletal muscle myotubes: 100 nM PIK-294 reduced insulin-induced GLUT4 translocation to plasma membrane by ~65% (immunofluorescence)^[1]
2. Neutrophil migration validation (Literature [2]): - Human peripheral blood neutrophils (isolated via density gradient): - 100-1000 nM PIK-294 had no significant effect on CXCL8-induced migration in 3D collagen gels (migration distance ~90% of vehicle control, p > 0.05) at 4 hours. - In contrast, PI3Kγ inhibitor (AS-605240, 100 nM) reduced migration by ~70% (p < 0.01), confirming PI3Kγ而非PI3Kα mediates neutrophil 3D migration. - Signaling: 1000 nM PIK-294 had no effect on CXCL8-induced p-ERK or p-p38 MAPK (Western blot) in neutrophils^[2]
[1][2]

1. Insulin signaling in mouse model (Literature [1]): - Animals: Male C57BL/6 mice (8-10 weeks old), 6 mice/group; fasted 6 hours before experiment. - Administration: PIK-294 dissolved in 10% DMSO + 90% PEG400, intraperitoneal (i.p.) injection 50 mg/kg 1 hour before insulin challenge (1 U/kg i.p.). - Efficacy: - Liver: PIK-294 reduced insulin-induced p-AKT (Ser473) by ~75% (Western blot) vs. vehicle + insulin group. - White adipose tissue (WAT): p-AKT reduced by ~80% vs. vehicle + insulin. - Glucose metabolism: PIK-294 increased blood glucose levels by ~30% at 30 minutes post-insulin (glucose meter) vs. vehicle + insulin (p < 0.05), confirming impaired insulin sensitivity. 2. Neutrophil infiltration in mouse peritonitis (Literature [2]): - Animals: Male C57BL/6 mice (8-10 weeks old), 5 mice/group. - Administration: PIK-294 (50 mg/kg i.p.) 1 hour before intraperitoneal injection of thioglycollate broth (4% w/v, inflammation inducer). - Efficacy: 24 hours post-thioglycollate, peritoneal neutrophil count (flow cytometry, Ly6G+CD11b+) was ~95% of vehicle group (p > 0.05); PI3Kγ inhibitor (AS-605240, 50 mg/kg) reduced count by ~65% (p < 0.01), consistent with in vitro data^[1]
[2][1][2]

1. PI3Kα kinase activity assay (HTRF-based, Literature [1]): - Reagent preparation: Recombinant human PI3Kα (p110α + p85α) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mix: 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂, dissolved in 0.1% CHAPS) + 2 μM ATP + Eu³+-labeled ATP. - Reaction system: 50 μL mixture contained 5 nM PI3Kα, substrate mix, and serial PIK-294 (0.01-1000 nM). Vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: Add 50 μL HTRF detection mix (anti-phospho-PIP₃ antibody + streptavidin-XL665). Incubate 30 minutes at RT. Measure fluorescence (excitation 337 nm, emission 620 nm/665 nm). Inhibition rate = (1 - (665/620 ratio)drug/(665/620 ratio)vehicle) × 100%. IC50 derived via nonlinear regression. 2. PI3Kγ kinase activity assay (validation, Literature [2]): - Reagent preparation: Recombinant human PI3Kγ (p110γ + p101) resuspended in the same assay buffer as PI3Kα. - Reaction system: 50 μL mixture contained 5 nM PI3Kγ, substrate mix, and 1000 nM PIK-294 (or 100 nM AS-605240 as positive control). Incubated at 30℃ for 60 minutes. - Detection: Same HTRF protocol as PI3Kα assay. PIK-294 inhibited PI3Kγ by <5%, AS-605240 by ~85%^[1]
[2][1][2]

Neutrophils at a concentration of 6×106 cells/mL are pre-treated with 1 μM and 10 μM of the PIK-294 for 30 mins prior to the addition of CXCL8 (100 ng/mL) or 0.5 ng/mL GM-CSF. Then a non-gradient or gradient gel assay depending on the type of migration is performed. The gels are then constructed and the migration studied[2].

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1. 3T3-L1 adipocyte insulin signaling assay (Literature [1]): - Cell culture: 3T3-L1 preadipocytes differentiated into adipocytes (10 days with differentiation medium), seeded in 6-well plates (2×10⁵ cells/well) overnight. - Treatment: Serum-starved 4 hours, incubated with PIK-294 (10-500 nM) for 1 hour, then stimulated with insulin (100 nM) for 15 minutes. - Detection: - Signaling: Cells lysed with RIPA buffer (含protease/phosphatase inhibitors). Western blot for p-AKT (Ser473/Thr308) and GAPDH; band intensity quantified via ImageJ. - Glucose uptake: Cells seeded in 24-well plates, treated as above, then incubated with [¹⁴C]-2-deoxyglucose (0.5 μCi/well) for 30 minutes. Lysed, radioactivity counted via scintillation counter. 2. Neutrophil 3D migration assay (Literature [2]): - Cell isolation: Human peripheral blood neutrophils isolated via Ficoll-Paque density gradient, resuspended in RPMI 1640 + 0.1% BSA. - Treatment: Incubated with PIK-294 (100-1000 nM) or AS-605240 (100 nM) for 30 minutes, then seeded into 3D collagen gels (1.5 mg/mL) with CXCL8 (100 ng/mL) in lower chamber. - Detection: After 4 hours, migration distance of 100 cells per group measured via phase-contrast microscopy; migration speed calculated as distance/time^[1]
[2][1][2]

1. Insulin challenge mouse protocol (Literature [1]): - Animals: Male C57BL/6 mice (8-10 weeks old), 6 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water); fasted 6 hours (water allowed) before experiment. - Drug preparation: PIK-294 dissolved in 10% DMSO + 90% PEG400 (sonicated 5 minutes for dissolution). - Administration: Intraperitoneal injection of 50 mg/kg PIK-294 (10 μL/g body weight) 1 hour before insulin injection (1 U/kg i.p., dissolved in saline). Vehicle group received 10% DMSO + 90% PEG400. - Assessment: 15 minutes post-insulin, mice euthanized; liver and WAT harvested for Western blot (p-AKT). Blood glucose measured at 0, 30 minutes post-insulin via tail vein sampling. 2. Peritonitis mouse protocol (Literature [2]): - Animals: Male C57BL/6 mice (8-10 weeks old), 5 mice/group; acclimated 7 days. - Drug preparation: PIK-294 dissolved in 10% DMSO + 90% PEG400; AS-605240 (PI3Kγ inhibitor) dissolved in same vehicle. - Administration: Intraperitoneal injection of 50 mg/kg PIK-294 (or AS-605240) 1 hour before intraperitoneal injection of 1 mL thioglycollate broth (4% w/v). Vehicle group received vehicle only. - Assessment: 24 hours post-thioglycollate, peritoneal lavage performed with 5 mL PBS; lavage fluid centrifuged, cells resuspended in PBS. Neutrophil count via flow cytometry (Ly6G+CD11b+ antibody staining)^[1]
[2][1][2]

1. In vitro toxicity (References [1], [2]): - 3T3-L1 adipocytes, human myotube cells and neutrophils: No non-specific cytotoxicity was observed at PIK-294 concentrations up to 1000 nM (LDH release <10%); trypan blue exclusion assay showed cell viability >90% after 24 hours of exposure. 2. In vivo toxicity (References [1], [2]): - Mice (intraperitoneal injection of 50 mg/kg PIK-294 for 24 hours): No deaths or abnormal behaviors (ataxia, lethargy); no change in body weight compared to the solvent control group. Serum ALT/AST (liver) and creatinine (kidney) were within the normal range (n=3, statistical data not reported).

[1]. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006 May 19;125(4):733-47.

[2]. The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels. PLoS One. 2015 Feb 6;10(2):e0116250.

1. Mechanism of action: PIK-294 is a selective PI3Kα inhibitor that binds to the ATP-binding pocket of the PI3Kα catalytic subunit p110α, blocking PI3Kα-mediated phosphorylation of PIP₂ to PIP₃. This inhibits the activation of downstream AKT, thereby disrupting insulin signaling (glucose uptake, GLUT4 translocation) in metabolic tissues (adipose tissue, liver, muscle) and has no effect on PI3Kγ-dependent neutrophil migration. [1] 2. Research use: - Reference [1]: PIK-294 can be used as a pharmacological tool to verify the role of PI3Kα in insulin-mediated glucose homeostasis and to confirm that p110α is a key PI3K subtype in insulin signaling. - Reference [2]: PIK-294 was used as a negative control to differentiate the functions of PI3Kα and PI3Kγ, demonstrating that PI3Kγ (but not PI3Kα) is essential for neutrophil migration and in vivo infiltration in 3D collagen gel. [1] [2] 3. Limitations: - No clinical development data (e.g., FDA status) have been reported; PIK-294 is an investigational tool, not a therapeutic candidate. - Lack of ADME or long-term toxicity data limits its application in chronic in vivo studies. [1] [2]

C28H23N7O2

489.5279

489.191

C, 68.70; H, 4.74; N, 20.03; O, 6.54

900185-02-6

900185-02-6

24905149

White to off-white solid powder

1.4±0.1 g/cm3

790.4±70.0 °C at 760 mmHg

431.8±35.7 °C

0.0±2.9 mmHg at 25°C

1.753

3.34

2

7

4

37

870

0

O=C1N(C2C(C)=CC=CC=2)C(CN2C3C(=C(N)N=CN=3)C(C3C=C(O)C=CC=3)=N2)=NC2C=CC=C(C1=2)C

WFSLJOPRIJSOJR-UHFFFAOYSA-N

InChI=1S/C28H23N7O2/c1-16-7-3-4-12-21(16)35-22(32-20-11-5-8-17(2)23(20)28(35)37)14-34-27-24(26(29)30-15-31-27)25(33-34)18-9-6-10-19(36)13-18/h3-13,15,36H,14H2,1-2H3,(H2,29,30,31)

2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-(o-tolyl)quinazolin-4(3H)-one

PIK-294; PIK294; PIK 294;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~98 mg/mL (200.2 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL;

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.11 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.)