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Purity: ≥98%
Padnarsertib (KPT-9274; KPT9274; PAK4-IN-1) is a potent, selective, orally bioavailable small molecule, non-competitive dual inhibitor of PAK4/NAMPT inhibitor with anticancer activity. KPT-9274 disrupts the PAK4/NAMPT signaling pathway, which may lead to the reduction of G2-M transit and induction of apoptosis as well as decrease in cell invasion and migration in several human RCC cell lines. Inhibition of the PAK4 signaling pathway by KPT-9274 attenuates nuclear β-catenin as well as the Wnt/β-catenin targets cyclin D1 and c-Myc. KPT-9274 demonstrated the expected on-target effects in a mouse model and is under a phase I clinical trial for solid tumors and lymphomas.
| Targets |
Padnarsertib (KPT-9274) targets nicotinamide phosphoribosyltransferase (NAMPT) and p21-activated kinase 4 (PAK4) [2]
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| ln Vitro |
Padnarsertib inhibits the PAK4/β-catenin pathway, which results in NAD depletion and reduces the capacity of many RCC cell lines to invade, migrate, and remain viable. Padnarsertib has an IC50 of about 120 nM, according to a cell-free enzymatic experiment that used recombinant NAMPT to inhibit NAMPT. In RCC cell lines, padnarsertib can cause apoptosis and impair G2-M trafficking [2].
In multiple human renal cell carcinoma (RCC) cell lines, treatment with Padnarsertib (KPT-9274) reduced G2-M cell cycle transit (assessed by Muse Analyzer after 72h incubation with 5 μM KPT-9274, p<0.05 vs DMSO control), induced apoptosis (measured by annexin-V staining via Muse Analyzer after 72h treatment, p<0.05 vs DMSO control), and decreased cell invasion and migration. For 786-O RCC cells treated with 1 μM or 5 μM KPT-9274 for 24h: Matrigel invasion assay showed reduced migrated cells (p<0.05 vs DMSO), transwell migration assay demonstrated fewer cells migrating to the lower chamber (p<0.05 vs DMSO), and scratch/wound healing assay revealed decreased cell migration to the wound area at 12 and 24h post-scratching (p<0.05 vs DMSO). KPT-9274 dose-dependently decreased total NAD+NADH levels in RCC cell lines (48h incubation, p<0.05 vs DMSO) but had less effect on normal renal proximal tubular epithelial cells (RPTEC). Mechanistically, KPT-9274 inhibited the PAK4 pathway, attenuating nuclear β-catenin and downregulating Wnt/β-catenin targets cyclin D1 and c-Myc (detected by immunoblotting after 72h treatment with different concentrations of KPT-9274). NAPRT1 downregulation in RCC cells made them dependent on NAMPT for NAD synthesis, and NAMPT inhibition by KPT-9274 reduced RCC cell survival. KPT-9274 also downregulated sirt1 expression in RCC cells (72h treatment, immunoblotting) [2] |
| ln Vivo |
Padnarsertib and sunitinib demonstrated similar effectiveness in inhibiting the growth of xenografts. Padnarsertib does not appear to be toxic in vivo and has minimal effect on normal human RPTEC [2]. Padnarsertib (oral; 100 mg/kg or 200 mg/kg; twice daily for 14 days) resulted in decreased xenograft growth in animal models without significantly affecting animal body weight. The concentrations in mouse plasma and tumors were found to be 10757 ng/mL and 10647 ng/mL, respectively, at the conclusion of the 8-hour experiment [3].
In nude mice bearing subcutaneous 786-O (VHL-mut) human RCC xenografts, oral administration of Padnarsertib (KPT-9274) at 100 mg/kg or 200 mg/kg twice a day resulted in dose-dependent inhibition of tumor growth (p<0.05 vs vehicle control), with no apparent toxicity (mouse body weight remained stable throughout the treatment period). Immunoblotting of harvested tumors confirmed on-target effects of KPT-9274 (downregulation of PAK4/NAMPT pathway-related proteins) [2] |
| Enzyme Assay |
A cell-free assay was conducted to evaluate NAMPT activity as a function of Padnarsertib (KPT-9274) concentration; the assay measured NAMPT enzymatic activity in the presence of different concentrations of KPT-9274 to determine the inhibitory effect of the drug on NAMPT. Additionally, CRISPR-Cas9-mediated splicing out of PAK4 in U-2 osteosarcoma cells was performed, and MTT assay was used to assess cell survival, with immunoblotting confirming PAK4 knockout and a right shift in the survival curve indicating PAK4 dependency [2]
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| Cell Assay |
Cell viability assay: RCC cell lines and normal RPTEC cells were plated in 96-well plates (3000 cells/well, n=8) and incubated with DMSO or Padnarsertib (KPT-9274) for 72h; cell viability was measured by MTT assay, showing that KPT-9274 preferentially attenuated RCC cell viability (p<0.05 vs DMSO control) with less impact on RPTEC cells.
NAD+NADH assay: 300 cells/well were plated in 96-well plates (n=4) and incubated with DMSO or KPT-9274 for 48h; total NAD+NADH levels were measured in RCC cell lines and RPTEC cells, with KPT-9274 reducing NAD+NADH levels in RCC cells (p<0.05 vs DMSO). Cell cycle analysis: RCC cells and RPTEC cells were grown to 50% confluence, incubated with DMSO or 5 μM KPT-9274 for 72h, and cell cycle distribution was analyzed by Muse Analyzer, showing reduced G2-M transit in RCC cells (p<0.05 vs DMSO). Apoptosis assay: After 72h incubation with KPT-9274, annexin-V staining was used to detect apoptosis via Muse Analyzer, with increased apoptosis in RCC cells (p<0.05 vs DMSO). Immunoblotting: RCC cells were plated in 6-well plates (1000 cells/well) and incubated with different concentrations of KPT-9274 for 72h; cell lysates were subjected to immunoblotting to detect proteins related to PAK4/NAMPT pathways (e.g., β-catenin, cyclin D1, c-Myc, sirt1) and cleaved PARP (apoptosis marker). Invasion/migration assays: 786-O cells were treated with DMSO, 1 μM or 5 μM KPT-9274 for 24h; Matrigel-coated transwells were used for invasion assay (migrated cells fixed, stained, and counted after 20h), transwell chambers for migration assay (migrated cells stained, photographed, and counted), and scratch/wound healing assay (cell migration to the wound area monitored at 0, 12, 24h post-scratching). siRNA transfection: RCC cells and RPTEC cells were grown to 50% confluence, transfected with PAK4-specific siRNA or scrambled control siRNA, then subjected to immunoblotting (to confirm PAK4 knockdown) and apoptosis assay by flow cytometry [2] |
| Animal Protocol |
Animal/Disease Models: Mice[1]
Doses: 100mg/kg or 200 mg/kg Route of Administration: Oral administration; 100mg/kg or 200 mg/kg; twice a day; 14 days Experimental Results: demonstrated a significant decrease of xenograft growth in KPT-9274 treated mouse. Nude mice (n=8 per group) were subcutaneously xenografted with 786-O human RCC cells; once tumors were established, the mice were orally gavaged with Padnarsertib (KPT-9274) at doses of 100 mg/kg or 200 mg/kg twice a day (vehicle control group received oral gavage of solvent only). Tumor volumes were measured weekly with calipers, and mean tumor volumes ± SEM were calculated. Mouse body weights were recorded at the same time points to assess toxicity. At the end of the experiment, mice were sacrificed, tumors were harvested and pooled, and immunoblotting was performed on tumor lysates to detect target proteins (PAK4/NAMPT pathway-related proteins) with β-actin as a loading control [2] |
| References | |
| Additional Infomation |
Padnarsertib is an orally bioavailable inhibitor of p21-activated kinase 4 (PAK4), a serine/threonine kinase, and nicotinamide adenine dinucleotide (NAD) synthase, nicotinamide phosphoribosyltransferase (NAMPT; NAMPRTase), possessing potential antitumor activity. Upon administration, Padnarsertib binds to PAK4 via allosteric reaction, destabilizing it and leading to its degradation. This inhibits PAK4-mediated signaling, inducing death in PAK4-overexpressing tumor cells and suppressing their proliferation. Furthermore, Padnarsertib also binds to and inhibits NAMPT. This depletes intracellular NAD and inhibits NAD-dependent enzymes, which are crucial for rapid cell proliferation; ultimately leading to the death of NAMPT-overexpressing cancer cells. PAK4 is a serine/threonine kinase belonging to the PAK protein family, upregulated in various cancer cell types, and regulates cell motility, proliferation, and survival. NAMPT is an enzyme responsible for maintaining the intracellular NAD pool, plays a key role in the regulation of cell metabolism, and has cytokine-like activity. NAMPT is often overexpressed in a variety of cancers and metabolic disorders, and tumor cells depend on the activity of NAMPT to obtain NAD. Padnarsertib (KPT-9274) is a dual-specific inhibitor of NAMPT and PAK4. Renal cell carcinoma (RCC) is the sixth most common malignant tumor in the United States, with a rising incidence and widespread resistance to existing therapies; PAK4 and the NAD biosynthetic pathway are crucial for the growth of RCC. Downregulation of NAPRT1 in renal cell carcinoma (RCC) cells makes the tumor dependent on NAMPT for NAD synthesis, and the inhibitory effect of KPT-9274 on NAMPT can reduce the survival rate of RCC cells. KPT-9274 is undergoing a phase I human clinical trial for the treatment of solid tumors and lymphomas, and is expected to be translated into a treatment for RCC[2].
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| Molecular Formula |
C35H29F3N4O3
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|---|---|---|
| Molecular Weight |
610.63
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| Exact Mass |
610.219
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| CAS # |
1643913-93-2
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| Related CAS # |
1643913-93-2;Padnarsertib HCl;
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| PubChem CID |
117779453
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| Appearance |
White to light yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
857.5±65.0 °C at 760 mmHg
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| Flash Point |
472.4±34.3 °C
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| Vapour Pressure |
0.0±3.2 mmHg at 25°C
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| Index of Refraction |
1.677
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| LogP |
4.74
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
45
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| Complexity |
1040
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CN(CCC1(F)F)C(=O)C2=CC=C(C=C2)C3=CC(=C4C(=C3)C=C(O4)CNC(=O)/C=C/C5=CN=C(C=C5)N)C6=CC=C(C=C6)F
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| InChi Key |
MRFOPLWJZULAQD-SWGQDTFXSA-N
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| InChi Code |
InChI=1S/C35H29F3N4O3/c36-28-9-7-24(8-10-28)30-19-26(23-3-5-25(6-4-23)34(44)42-15-13-35(37,38)14-16-42)17-27-18-29(45-33(27)30)21-41-32(43)12-2-22-1-11-31(39)40-20-22/h1-12,17-20H,13-16,21H2,(H2,39,40)(H,41,43)/b12-2+
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| Chemical Name |
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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: 2.08 mg/mL (3.41 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 20.8 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: 2.08 mg/mL (3.41 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 20.8 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: ≥ 2.08 mg/mL (3.41 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 | 1.6377 mL | 8.1883 mL | 16.3765 mL | |
| 5 mM | 0.3275 mL | 1.6377 mL | 3.2753 mL | |
| 10 mM | 0.1638 mL | 0.8188 mL | 1.6377 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.
KPT-9274 inhibits NAMPT and PAK4 and associated signaling pathways in RCC cells.Mol Cancer Ther.2016 Sep;15(9):2119-29. |
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 KPT-9274 preferentially attenuates RCC cell viability and decreases RCC invasion and migration.Mol Cancer Ther.2016 Sep;15(9):2119-29. |
KPT-9274 decreases G2/M transit and causes apoptosis preferentially in RCC cells.Mol Cancer Ther.2016 Sep;15(9):2119-29. |
 KPT-9274 shows specificity for attenuation of PAK4 targets preferentially in RCC cells.Mol Cancer Ther.2016 Sep;15(9):2119-29. |
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 KPT-9274 shows specificity for attenuation of NAD biosynthesis targets preferentially in RCC cells.Mol Cancer Ther.2016 Sep;15(9):2119-29. |
 KPT-9274 attenuates xenograft growthin vivowith minimal toxicity and shows the expected on-target effects.Mol Cancer Ther.2016 Sep;15(9):2119-29. |
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