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| 10mg |
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| 25mg |
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| Other Sizes |
Purity: ≥98%
| Targets |
Class I PI3K (p110α, IC₅₀ = 0.173 µM; p110β, more potent than LY294002; p110δ, expressed in WEHI231 cells)
CK2 (IC₅₀ = 0.149 µM for CK2(h), 1.127 µM for CK2α2(h)) GSK3β (inhibition confirmed by kinase selectivity screening) VCP (valosin-containing protein, identified as a novel target via affinity pull-down) ALDH2 (aldehyde dehydrogenase 2, identified as a major bound protein) Brd4 (bromodomain-containing protein 4, identified in pull-down assay) Type III phosphatidylinositol 4-kinase (230 kDa isoform, identified in pull-down) mTOR (detected by immunoblotting in pull-down assay) Various dehydrogenases and ATP/nucleotide-binding proteins (identified via proteomic screening)[1] |
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| ln Vitro |
On 4T1 breast cancer cells and 4306 ovarian cancer cells, PI-828 (0.01-100 μM) has cytotoxic effects [2]. Higher quantities of PI-828 alone (6.25-12.5 µM) induce apoptosis, while PI-828 (0.78-3.12 µM; 48 hours) decreases caspase 3 activation [3].
PI-828 was designed as an analog of LY294002 and immobilized onto epoxy-activated Sepharose beads to create an affinity matrix for chemical proteomic screening. It showed more potent inhibition of class I PI3Ks than LY294002, particularly against p110β, as assessed in a radiometric kinase assay. The immobilized PI-828-matrix was used to pull down direct protein targets from HeLa and WEHI231 cell lysates. Targets identified included PI3K isoforms (p110α, p110β, p110δ), mTOR, CK2, GSK3β, VCP, ALDH2, Brd4, and several dehydrogenases. Competitive elution experiments with free LY294002 or PI-828 confirmed binding specificity. PI-828 also inhibited CK2 activity with an IC₅₀ of 0.149 µM (human CK2) and 1.127 µM (CK2α2). Binding to VCP was validated using recombinant wild-type and ATP-binding mutant proteins, showing direct interaction independent of ATP-binding lysine residues. Docking studies suggested PI-828 binds to the ATP-binding pocket of VCP.[1] |
| ln Vivo |
In a syngeneic 4T1 breast cancer mouse model, treatment with PI-828-SNPs (at a dose equivalent to 5 mg/kg of PI-828, administered every 48 hours for 3 doses) exerted an inhibitory effect on Akt phosphorylation in vivo, which translated into superior tumor growth inhibition compared to treatment with free PI-828. However, consistent with its low potency, the antitumor efficacy of PI-828 or PI-828-SNPs was significantly lower than that seen with PI103-SNPs.[2]
In an insulin tolerance test using 4T1 tumor-bearing mice, a single dose of free PI-828 or PI-828-SNP (at doses equivalent to 5 mg/kg of the parent inhibitor) did not affect the glucose response to insulin, in contrast to the insulin resistance observed with free PI103.[2] |
| Enzyme Assay |
PI3K inhibition by PI-828 and LY294002 was determined in a radiometric assay using purified, recombinant class IA and class IB enzymes with 1 µM ATP. The kinase reaction was carried out for 1 hour at room temperature (24°C) and terminated by addition of PBS. IC₅₀ values were determined using a sigmoidal dose-response curve fit (variable slope).
CK2 and GSK3β inhibition was established by kinase selectivity screening. Inhibitor (10 µM PI-828 and LY294002) was tested against a panel of kinases in 10 µM ATP.[1] |
| Cell Assay |
Cell viability assay [2]
Cell Types: 4T1 breast cancer cells and 4306 ovarian cancer cells Tested Concentrations: 0.01, 0.1, 1, 10 and 100 μM Incubation Duration: Experimental Results: demonstrated cytotoxic effect. Apoptosis analysis[3] Cell Types: Human Embryonic Carcinoma NCCIT Cell Tested Concentrations: 0.78, 1.56, 3.12, 6.25, 12.5 μM Incubation Duration: 48 hrs (hours) Experimental Results: 0.78 to 3.12 μM Concentration range reduces caspase 3 activation; higher concentrations cause Apoptosis. For large-scale pull-down assays, HeLa cells (2.5×10⁶ cells) were lysed in lysis buffer A (20 mM Hepes pH 7.5, 150 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, with protease and phosphatase inhibitors). WEHI231 cells were grown in suspension in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, and antibiotics. Total cellular extract (15 mg) was adjusted to 1 M NaCl and incubated with 300 µl of 50% PI-828-matrix bead slurry for 4 hours. For competition assays, lysates were pre-incubated with 1 mM of LY294002 or PI-828 in lysis buffer A containing 10 mM ATP and 20 mM MgCl₂. Affinity complexes were washed extensively in high-salt lysis buffer and then in standard lysis buffer. Elution was performed either with 2× Laemmli sample buffer by boiling or with free inhibitor (LY294002, PI-828, PI103, or LY303511) in lysis buffer with ATP and MgCl₂.[1] Bound proteins were separated by one-dimensional gel electrophoresis, stained with colloidal Coomassie Blue, and identified by in-gel digestion followed by LC-ESI-MS/MS analysis. Identifications were accepted when at least two individual peptide masses matched a protein (mass error ± 100 mDa, allowing one missed cleavage), MOWSE scores exceeded the threshold (P=0.05), and predicted protein mass matched the gel-based mass.[1] |
| Animal Protocol |
In the 4T1 breast cancer model, 4-week-old BALB/c mice were implanted subcutaneously with 4T1 cells (1×10⁵ cells). Therapy began on day 9 post-implantation. Animals were randomized into treatment groups. The group treated with PI-828-SNPs received the nanoparticles at a dose equivalent to 5 mg/kg of the PI-828 inhibitor via intravenous injection, administered every 48 hours.[2]
For the insulin tolerance test, random-fed mice bearing 4T1 tumors were injected with a single dose of empty nanoparticles (control), free PI-828, or PI-828-SNPs (at doses equivalent to 5 mg/kg of the parent inhibitor) via the tail vein. At defined time points after drug administration, mice were injected with freshly prepared insulin solution (0.75 U/kg) in saline. Blood glucose levels were measured before and 45 minutes after insulin injections using a glucometer.[2] |
| Toxicity/Toxicokinetics |
Unlike PI103, treatment with free PI-828 or PI-828-SNPs did not induce insulin resistance. No significant changes in body weight were observed in the treated animals. Histological analysis (TUNEL assay) of liver, kidney, and spleen sections from animals treated with PI103-SNPs (the primary study subjects) showed no signs of increased apoptosis; Western blotting of apoptosis markers (cleaved caspase-3, PARP) showed no difference between the treatment groups, indicating that this nanoparticle formulation strategy had no acute toxicity to these organs. [2]
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| References |
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| Additional Infomation |
8-(4-aminophenyl)-2-(4-morpholino)-1-benzopyran-4-one belongs to the chromone class of compounds. PI-828 is an analogue of the widely used PI3K inhibitor LY294002. Its design features the introduction of an amine linker at the 4-position of the phenyl substituent on the outer ring, so as to immobilize it on a solid support for chemical proteomics studies. This study mainly uses it as an affinity tool to identify novel cellular targets of LY294002 and reveal its off-target interactions with proteins such as VCP, ALDH2, and Brd4, which may lead to the reported PI3K-independent effects of LY294002. This study demonstrates the practicality of immobilized PI-828 as a proteomics platform in mapping compound interaction maps and verifying inhibitor specificity. [1]
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| Molecular Formula |
C19H18N2O3
|
|---|---|
| Molecular Weight |
322.35782
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| Exact Mass |
322.132
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| CAS # |
942289-87-4
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| PubChem CID |
25181195
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| Appearance |
White to yellow solid powder
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| Density |
1.316g/cm3
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| Boiling Point |
534.2ºC at 760 mmHg
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| Flash Point |
276.9ºC
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| Index of Refraction |
1.658
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| LogP |
3.525
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
24
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| Complexity |
493
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
WUKMIBOGGXMBAC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H18N2O3/c20-14-6-4-13(5-7-14)15-2-1-3-16-17(22)12-18(24-19(15)16)21-8-10-23-11-9-21/h1-7,12H,8-11,20H2
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| Chemical Name |
8-(4-aminophenyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one
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| Synonyms |
PI-828; PI 828; PI828
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 12.5 mg/mL (~38.78 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (3.88 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 12.5 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. Solubility in Formulation 2: ≥ 1.25 mg/mL (3.88 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 12.5 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1021 mL | 15.5106 mL | 31.0212 mL | |
| 5 mM | 0.6204 mL | 3.1021 mL | 6.2042 mL | |
| 10 mM | 0.3102 mL | 1.5511 mL | 3.1021 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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