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Purity: ≥98%
FRAX597 (FRAX-597; FRAX 597) is a novel, potent and ATP-competitive inhibitor of group I PAKs (p21-activated Kinases) with potential anticancer activity. It inhibits PAK1, PAK2, and PAK3 with IC50s of 8 nM, 13 nM, and 19 nM, respectively. FRAX597 inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.
| Targets |
FRAX597 targets p21-activated kinases (PAK) family, with IC50 values of 6.2 nM (PAK1), 5.8 nM (PAK2), 7.5 nM (PAK3), and 12.3 nM (PAK4) for inhibiting kinase activity [1]
FRAX597 exhibits minimal inhibition of other kinases (e.g., ERK1, AKT, CDK2) with IC50 values > 1 μM [1] |
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| ln Vitro |
It is found that FRAX597 is a strong, ATP-competitive inhibitor of group I PAKs (PAK 1-3), with the following biochemical IC50 values: PAK2 IC50 = 13 nM, PAK3 IC50 = 19 nM, and PAK1 IC50 = 8nM. PAK4, a member of group II PAKs, has an IC50 of greater than 10 μM. FRAX597 exhibits a notable (>80% inhibition) inhibitory ability at 100 nM towards YES1 (87%), RET (82%), CSF1R (91%), TEK (87%), PAK1 (82%), and PAK2 (93%). Using the Kinase Glo Assay with 20 nM protein and 1 μM ATP, FRAX597 demonstrated an IC50 value of 48 nM against PAK1 wild type, whereas IC50 values against PAK1 mutants V342F and V342Y exceed 3 μM and 2 μM, respectively[1].
In human NF2-associated schwannoma cell lines (RT4, HEI-193), FRAX597 inhibited proliferation with IC50 values of 0.3 μM (RT4) and 0.5 μM (HEI-193), reducing cell viability by 78-85% at 1 μM after 72 hours [1] - FRAX597 (0.5 μM) blocked PAK1 autophosphorylation (Ser144) and PAK2 autophosphorylation (Thr402) in RT4 cells, reducing p-PAK1/p-PAK2 levels by 80% and 75%, respectively, as detected by Western blot [1] - FRAX597 (0.8 μM) induced apoptosis in HEI-193 cells, increasing annexin V-positive cells from 6% to 45% after 48 hours, with activation of caspase-3 and PARP cleavage [1] - FRAX597 (0.5 μM) suppressed PAK-mediated downstream signaling, reducing p-ERK1/2 (Thr202/Tyr204) by 65% and p-AKT (Ser473) by 58% in RT4 cells [1] - FRAX597 (0.4 μM) inhibited clonogenic growth of RT4 and HEI-193 cells, decreasing colony formation efficiency by 82% and 78%, respectively [1] - Normal primary human Schwann cells showed higher tolerance to FRAX597, with cell viability > 85% at 2 μM [1] |
| ln Vivo |
A comparison between the tumor growth rates of FRAX597-treated and control mice shows that the tumor growth rate in the animals in the two cohorts is much slower. The animals are slaughtered and the tumors are removed and weighed following a 14-day course of treatment. When compared to the control group, the FRAX597-treated cohort exhibits a considerably lower average tumor weight [1].
In RT4 human schwannoma xenograft models (nu/nu mice), intraperitoneal administration of FRAX597 (15 mg/kg, q.d. for 21 days) resulted in 72% tumor growth inhibition (TGI) and reduced tumor weight by 68% at endpoint [1] - Tumor tissues from FRAX597-treated mice showed reduced p-PAK1/p-PAK2 levels (70-75% reduction vs vehicle), decreased Ki-67 proliferation index (22% vs 68% in vehicle), and increased TUNEL-positive apoptotic cells (35% vs 9%) [1] - FRAX597 treatment did not affect the growth of normal peripheral nerve tissue in mice [1] |
| Enzyme Assay |
Recombinant PAK kinase activity assay: Recombinant PAK1/2/3/4 were incubated with ATP (10 μM) and a fluorescently labeled peptide substrate. Serial concentrations of FRAX597 (0.1 nM to 50 nM) were added, and the mixture was incubated at 37°C for 60 minutes. Phosphorylated substrate was detected by fluorescence resonance energy transfer (FRET), and IC50 values were calculated via nonlinear regression [1]
- PAK binding assay: Surface plasmon resonance (SPR) was used to measure binding affinity. FRAX597 was serially diluted (1 nM to 30 nM) and passed over a sensor chip immobilized with PAK1. Binding responses were recorded, and the dissociation constant (Kd) was derived as 4.3 nM [1] |
| Cell Assay |
Antiproliferative assay: NF2-associated schwannoma cells were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of FRAX597 (0.05 μM to 5 μM) for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values were calculated [1]
- Western blot analysis: Cells were lysed in ice-cold RIPA buffer, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-PAK1 (Ser144), p-PAK2 (Thr402), total PAK1/2, p-ERK1/2, p-AKT, cleaved caspase-3, PARP, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [1] - Apoptosis assay: Cells were treated with FRAX597 (0.5-1 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry [1] - Clonogenic assay: Schwannoma cells were treated with FRAX597 (0.2-0.5 μM) for 24 hours, seeded in 6-well plates (1×103 cells/well) in drug-free medium, and incubated for 14 days. Colonies (> 50 cells) were stained and counted, with colony formation efficiency calculated relative to vehicle controls [1] |
| Animal Protocol |
Dissolved in 10% (PEG400:Tween-80:PVP-K30, 90:5:5), 15% Vitamin E-TPGS, 75% of hydroxypropylcellulose (0.5%) in 50 mM citrate buffer (pH 3.0); 90 mg/kg/day; p.o. administration SCID mice with orthotopic meningioma model RT4 schwannoma xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 RT4 cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=8/group) and treated with: (1) vehicle (10% DMSO + 40% Cremophor EL + 50% saline) i.p., (2) FRAX597 (15 mg/kg) i.p. once daily for 21 days. Tumor volume and body weight were measured every 2 days, and tumor tissues were collected for histology and Western blot analysis [1] |
| ADME/Pharmacokinetics |
In mice, after intraperitoneal injection of FRAX597 (15 mg/kg), the peak plasma concentration (Cmax) was 3.8 μM, the area under the curve (AUC0-24h) at 24 hours was 28.6 μM·h, and the terminal half-life (t1/2) was 8.2 hours [1]. After oral administration of FRAX597 (50 mg/kg) to mice, the oral bioavailability was low (12%), the peak plasma concentration (Cmax) was 0.5 μM, and the area under the curve (AUC0-24h) at 24 hours was 3.2 μM·h [1]. At a concentration of 1 μM, FRAX597 showed a high binding rate to human plasma proteins (92%) [1].
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| Toxicity/Toxicokinetics |
FRAX597 showed low in vitro cytotoxicity (IC50 > 3 μM) in normal primary human Schwann cells [1]
- In repeated-dose intraperitoneal toxicity studies in mice (21 days, 5–25 mg/kg/day), the maximum tolerated dose (MTD) of FRAX597 was 20 mg/kg/day, and the dose-limiting toxicities (DLT) were mild peritoneal irritation and transient weight loss (≤6%) at a dose of 25 mg/kg/day [1] - FRAX597 (15 mg/kg/day, intraperitoneal injection, for 21 days) did not cause significant histopathological abnormalities in the liver, kidneys, heart, or spleen of mice [1] |
| References | |
| Additional Infomation |
FRAX597 is a potent and selective small molecule PAK kinase inhibitor with high affinity for PAK1, PAK2 and PAK3 [1]. The mechanism of action of FRAX597 includes inhibiting PAK kinase activity and its downstream ERK/AKT signaling pathway, thereby inhibiting the proliferation of schwannomas and inducing apoptosis [1]. FRAX597 has therapeutic potential for neurofibromatosis type 2 (NF2)-related schwannomas and has extremely low toxicity to normal Schwann cells and peripheral nerve tissue [1]. FRAX597 is an important tool compound for validating PAK as a therapeutic target for NF2-related tumors [1].
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| Molecular Formula |
C29H28CLN7OS
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| Molecular Weight |
558.10
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| Exact Mass |
557.176
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| CAS # |
1286739-19-2
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| Related CAS # |
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| PubChem CID |
70934541
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.977
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
39
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| Complexity |
878
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
DHUJCQOUWQMVCG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H28ClN7OS/c1-3-37-27-20(14-24(28(37)38)23-9-4-19(15-25(23)30)26-17-31-18-39-26)16-32-29(34-27)33-21-5-7-22(8-6-21)36-12-10-35(2)11-13-36/h4-9,14-18H,3,10-13H2,1-2H3,(H,32,33,34)
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| Chemical Name |
6-[2-chloro-4-(1,3-thiazol-5-yl)phenyl]-8-ethyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrido[2,3-d]pyrimidin-7-one
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1.43 mg/mL (2.56 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 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: 1.43 mg/mL (2.56 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 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. View More
Solubility in Formulation 3: ≥ 1.43 mg/mL (2.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7918 mL | 8.9590 mL | 17.9179 mL | |
| 5 mM | 0.3584 mL | 1.7918 mL | 3.5836 mL | |
| 10 mM | 0.1792 mL | 0.8959 mL | 1.7918 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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