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Purity: ≥98%
Gedatolisib (PF-05212384, PKI-587) is a brand-new, extremely powerful dual inhibitor of PI3Kα, PI3Kγ and mTOR that may have anticancer properties. In cell-free assays, it inhibits PI3Kα, PI3Kγ and mTOR with IC50 values of 0.4 nM, 5.4 nM, and 1.6 nM, respectively. By concentrating on phosphatidylinositol 3 kinase (PI3K) and mammalian target of rapamycin (mTOR) in the PI3K/mTOR signaling pathway, it may have anti-cancer effects. The cellular phosphorylation of PI3K/mTOR signaling pathway proteins is inhibited by PF-05212384. It prevents Akt from being phosphorylated along with the Akt effector proteins GSK3 kinase, ENOS, and PRAS 40. Additionally, in a number of xenograft models, including H1975, BT474, HCT116, H1975, and U87MG, PF-05212384 exhibits strong anti-tumor activity.
| Targets |
PI3Kα (IC50 = 0.4 nM); mTOR (IC50 = 1.6 nM); PI3Kγ (IC50 = 5.4 nM)
PI3Kα (IC50=0.4 nM) [1] PI3Kβ (IC50=68 nM) [1] PI3Kγ (IC50=5.4 nM in one assay; 60 nM in another assay) [1] PI3Kδ (IC50=0.6 nM) [1] mTOR (IC50=1.4 nM) [1] PI3Kα mutant H1047R (IC50=0.6 nM) [1] PI3Kα mutant E545K (IC50=0.6 nM) [1] |
|---|---|
| ln Vitro |
PKI-587 shows potent inhibitory activity against PI3K-α, PI3K-γ and mTOR with IC50 of 0.4 nM, 5.4 nM and 1.6 nM, respectively. Furthermore, PKI-587 also exhibits its potency against the most frequently occurring mutant forms of PI3Kα, notably the H1047R and E545K with IC50 of 0.6 nM and 0.6 nM, respectively.[1] In MDA-361 and PC3-MM2 cell lines, PKI-587 inhibits tumor cell growth with an IC50 of 4 nM and 13.1 nM, respectively. This effect is correlated with the suppression of phosphorylation of PI3K/mTOR signaling pathway proteins.[1]
PKI-587 exhibited potent inhibition of cell proliferation in MDA-361 breast cancer cells (IC50=4.0 nM) and PC3-MM2 prostate cancer cells (IC50=13.1 nM) [1]. It suppressed phosphorylation of Akt at T308 (IC50=8 nM) and S473 (IC50<10 nM), induced cleaved PARP at 30 nM, and suppressed phosphorylation of GSK3, eNOS, and PRAS40 at <30 nM in MDA-361 cells [1]. It also inhibited mTORC1 activity as shown by suppression of p70S6K and 4EBP1 phosphorylation at <30 nM [1]. PKI-587 was highly selective for PI3K and mTOR when tested against a panel of 236 other human protein kinases at 10 μM [1]. |
| ln Vivo |
PKI-587 treatment at 25 mg/kg intravenously results in low plasma clearance (7 (mL/min)/kg), high volume of distribution (7.2 L/kg), and a lengthy (14.4 hours) half-life in nude mice. With a minimum effective dose (MED) of 3 mg/kg and a maximum tolerated single dose (MTD) of 30 mg/kg against MDA-361 tumors, PKI-587 exhibits strong antitumor efficacy in the MDA-361 xenograft model. While PKI-587 at 25 mg/kg for 7 weeks results in 90% survival of the treated group in the H1975 (non-small-cell lung carcinoma, mutant EGFR [L858R, T790M]) xenograft model. [1]
In MDA-361 subcutaneous xenograft model in nude mice, PKI-587 administered intravenously at 20 mg/kg on days 1, 5, and 9 caused regression of large (~900 mm^3) tumors, more effective than paclitaxel (60 mg/kg, single ip dose) [1]. The minimum efficacious dose (MED) was 3 mg/kg and maximum tolerated single dose (MTD) was 30 mg/kg in the MDA-361 model [1]. In an orthotopic H1975 non-small-cell lung carcinoma model (EGFR L858R/T790M mutant), PKI-587 given once weekly at 25 mg/kg iv for 7 weeks resulted in 90% survival, whereas all untreated controls died by day 50 [1]. A single iv dose of 25 mg/kg suppressed pAkt (T308 and S473) for up to 36 h and induced cleaved PARP for up to 18 h in MDA-361 tumors [1]. |
| Enzyme Assay |
Enzyme assays are done in fluorescent polarization (FP) format, adapted from the Echelon K-1100 PI3K FP assay kit protocol. E545K and H1047R human class I PI3K mutants can be generated in Sf9 or bought from Upstate Biotech. Escherichia coli produces GST-GRP1 (murine), which is then isolated using GST-Sepharose. Assay buffers are reaction buffer [20 mM HEPES (pH 7.1), 2 mM MgCl2, 0.05% CHAPS, and 0.01% β-mercaptoethanol] and stop/detection buffer [100 mM HEPES (pH 7.5), 4 mM EDTA, 0.05% CHAPS]. FP reaction is run for 30 minutes at room temperature in 20 μL of reaction buffer containing 20 μM phosphatidylinositol 4,5-bisphosphate (PIP2), 25 μM ATP, and<4% DMSO. FP reaction is stopped with 20 μL of stop/detection buffer (10 nM probe and 40 nM GST-GRP), and after 2 hours, data are collected using an Envision plate reader. The routine assays with purified FLAG-TOR (FL and 3.5) are performed in 96-well plates as follows. Enzymes are first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 μM microcystin LR, and 100 μg/mL BSA). To each well, 12 μL of the diluted enzyme is mixed briefly with 0.5 μL test inhibitor or control vehicle dimethyl sulfoxide (DMSO). The kinase reaction is initiated by adding 12.5 μL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 μL containing 800 ng/mL FLAG-TOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1–6 hours) at room temperature with gentle shaking and then terminated by adding 25 μL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA).
PI3Kα and PI3Kγ enzyme activities were measured using a fluorescence polarization format assay; IC50 values were determined by this method [1]. mTOR inhibition was determined using a DELFIA format ELISA that measured the kinase activity of mTOR [1]. For profiling against PI3K isoforms (β, γ, δ) and mutants (H1047R, E545K), IC50 values were determined using similar fluorescence polarization assays [1]. |
| Cell Assay |
Cells are plated in 96-well culture plates at about 3000 cells per well. PKI-587 is added to cells a day after plating. Viable cell densities are assessed three days after PKI-587 treatment by monitoring the metabolic conversion of the dye MTS (by viable cells), a tried-and-true cell proliferation assay. Freshly thawed and mixed MTS and PMS stocks (20:1) are used for each assay. Following this, 20 L of the MTS/PMS mixture is added to 96-well cell plates, and the plates are incubated for 1 to 2 hours in a cell culture incubator.
Cell proliferation inhibition was assessed in a 3-day growth inhibition assay using MDA-361 (breast, PI3K mutant E545K/Her2+) and PC3-MM2 (prostate, PTEN deletion) human tumor cell lines; cells were exposed to test compounds for 3 days and IC50 values were calculated [1]. For phosphoblot studies, MDA-361 tumor cells were exposed to PKI-587 for 4 hours, then Western blot analysis was performed to detect phosphorylated Akt (T308 and S473), cleaved PARP, phosphorylated GSK3, eNOS, PRAS40, p70S6K, and 4EBP1; densitometric scans of Western blots were used to determine IC50 values for pAkt suppression [1]. |
| Animal Protocol |
MDA-361 and H1975 cells are injected subcutaneously into the nude mice.
≤30 mg/kg Administered via i.v. For efficacy studies, female nude mice were implanted subcutaneously with MDA-361 tumors; when tumors reached approximately 900 mm^3, PKI-587 was administered intravenously at 20 mg/kg on days 1, 5, and 9 in a vehicle of 5% dextrose (D5/W) with 0.3% lactic acid, pH 3.5; paclitaxel was given intraperitoneally at 60 mg/kg as a single dose for comparison [1]. For the orthotopic H1975 model, H1975 cells were injected into the pleural cavity of nude mice; PKI-587 was given once weekly at 25 mg/kg intravenously for 7 weeks, and survival was plotted as Kaplan-Meier curves [1]. For in vivo biomarker studies, a single iv dose of 25 mg/kg PKI-587 was administered, and tumor tissues were collected at various time points to examine p-Akt suppression and cleaved PARP induction by Western blot [1]. |
| ADME/Pharmacokinetics |
In female nude mice, after a single intravenous dose of 25 mg/kg PKI-587 (formulated in 5% dextrose/0.3% lactic acid, pH 3.5), the following parameters were observed: C0=4852 ng/mL, half-life (T1/2)=14.4 h, AUClast=42593 h·ng/mL, AUC0-inf=57927 h·ng/mL, plasma clearance (Clp)=7 (mL/min)/kg, volume of distribution at steady state (Vss)=7.2 L/kg, mean residence time (MRT)=16.7 h [1].
Low clearance (7 mL/min/kg) compared to liver blood flow (90 mL/min/kg) indicated minimal metabolism, and high volume of distribution suggested extensive tissue distribution [1]. Oral exposure was not detectable for PKI-587 in nude mice when administered orally [1]. |
| Toxicity/Toxicokinetics |
The maximum tolerated single dose (MTD) of PKI-587 was determined to be 30 mg/kg in nude mice [1].
PKI-587 showed good microsomal stability in all three species tested and lacked CYP inhibition [1]. |
| References | |
| Additional Infomation |
Gadaprizib has been used in basic scientific research and therapeutic trials for various cancers, including ovarian cancer, breast cancer, advanced cancer, and endometrial cancer. Gadaprizib is a drug that targets phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) in the PI3K/mTOR signaling pathway, possessing potential antitumor activity. Intravenous injection of galdaprizib inhibits PI3K and mTOR kinases, leading to apoptosis and growth inhibition in PI3K/mTOR-overexpressing cancer cells. Activation of the PI3K/mTOR pathway promotes cell growth, survival, and resistance to chemotherapy and radiotherapy; mTOR is a serine/threonine kinase downstream of PI3K, and its activation may also be independent of PI3K.
The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, and metabolism; selective mTOR inhibition paradoxically activates PI3K, providing rationale for dual PI3K/mTOR inhibitors [1]. PKI-587 is an ATP-competitive inhibitor that blocks the kinase activity of both PI3K and mTOR; it is a pan-PI3K inhibitor with equivalent potency against mTOR [1]. PKI-587 was entering Phase I clinical trials as a single agent for intravenous administration at the time of publication [1]. |
| Molecular Formula |
C32H41N9O4
|
|---|---|
| Molecular Weight |
615.7258
|
| Exact Mass |
615.328
|
| Elemental Analysis |
C, 62.42; H, 6.71; N, 20.47; O, 10.39
|
| CAS # |
1197160-78-3
|
| Related CAS # |
1197160-78-3
|
| PubChem CID |
44516953
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| Appearance |
white solid powder
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| Density |
1.4±0.1 g/cm3
|
| Index of Refraction |
1.670
|
| LogP |
-0.76
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
10
|
| Rotatable Bond Count |
7
|
| Heavy Atom Count |
45
|
| Complexity |
913
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O=C(C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])C1=NC(=NC(=N1)N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])=O)N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N(C([H])([H])[H])C([H])([H])[H]
|
| InChi Key |
DWZAEMINVBZMHQ-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C32H41N9O4/c1-38(2)27-11-13-39(14-12-27)29(42)24-5-9-26(10-6-24)34-32(43)33-25-7-3-23(4-8-25)28-35-30(40-15-19-44-20-16-40)37-31(36-28)41-17-21-45-22-18-41/h3-10,27H,11-22H2,1-2H3,(H2,33,34,43)
|
| Chemical Name |
-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea
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| Synonyms |
Gedatolisib; PKI-587; PKI587; PKI 587; PF-05212384; PF05212384; PF 05212384; PF5212384; PF 5212384; PF-5212384
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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: ~2 mg/mL (~3.2 mM)
Water: <1 mg/mL Ethanol: <1 mg/mL |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6241 mL | 8.1204 mL | 16.2409 mL | |
| 5 mM | 0.3248 mL | 1.6241 mL | 3.2482 mL | |
| 10 mM | 0.1624 mL | 0.8120 mL | 1.6241 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.
| NCT Number | Status | Interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03911973 | Active Recruiting |
Drug: Gedatolisib Drug: Talazoparib |
TNBC - Triple-Negative Breast Cancer |
Kari Wisinski | April 17, 2019 | Phase 1 Phase 2 |
| NCT02626507 | Active Recruiting |
Drug: Gedatolisib Drug: Faslodex |
Breast Cancer | Hoffman Oncology | January 2016 | Phase 1 |
| NCT05501886 | Recruiting | Drug: Gedatolisib Drug: Alpelisib |
Breast Cancer | Celcuity, Inc. | September 30, 2022 | Phase 3 |
| NCT03243331 | Completed | Drug: Gedatolisib Drug: PTK7-ADC |
Metastatic Breast Cancer Triple Negative Breast Cancer |
Kathy Miller | January 19, 2018 | Phase 1 |
| NCT02684032 | Completed | Drug: Gedatolisib Drug: Palbociclib |
Breast Cancer | Celcuity, Inc. | June 14, 2016 | Phase 1 |
a,bWe treated cell lines with different concentrations of everolimus (RAD001), PKI-587, or vehicle control for 0, 24, 48, and 96 h and assessed cell viability with the WST-1 assay (Roche).Neuroendocrinology.2017;105(1):90-104. |
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