VEGFR2 (IC50 = 20 nM); FGFR1 (IC50 = 30 nM); PDGFRβ (IC50 = 510 nM)

SU5416 (25 mg/kg, i.p.) prevents the growth of a panel of tumor cell lines under the skin in mice by blocking the angiogenic process that is linked to tumor growth.[2]

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In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.[2]

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Reversal of MCT-induced PH with the FGFR1 inhibitor SU5402. [5]
To confirm that the decreases in pulmonary vascular alterations and PH associated with FGF2-siRNA treatment were related to FGF2 knockdown, indicating a key role for FGF2 overproduction in PH, we investigated to determine whether the selective FGFR1 inhibitor SU5402 prevented and/or reversed PH induced by MCT in rats. In rats treated with SU5402 on days 21 to 42 after the MCT injection, evaluations on day 42 showed marked decreases in PAP, RV/(LV + S), and distal artery muscularization compared with rats treated with the vehicle (saline) (Figure 8).

The catalytic domain of FGF-R1 and Flk-1/KDR is expressed as GST fusion proteins after baculoviruses with altered genomes infect Spodoptera frugiperda (sf9) cells. Using glutathione sepharose chromatography, infected sf9 cell lysates are purified to homogeneity for GST-FGFR1 and GST-Flk1. In 96-well microtiter plates, 2.0 μg of a polyGlu-Tyr peptide (4:1) in 0.1 mL of PBS per well is coated overnight before the assays are carried out. The diluted purified kinases are introduced to each test well at a rate of 5 ng of GST fusion protein per 0.05 mL volume buffer using kinase assay buffer (100 mM Hepes pH 7.5, 100 mM NaCl, and 0.1 mM sodium orthovanadate). Test compounds are added to test wells (0.025 mL/well) after being diluted in 4% DMSO. To start the kinase reaction, add 0.025 mL of 40 μM ATP/40 mM MnCl2. Shake the plates for 10 minutes, and then add 0.025 mL of 0.5 M EDTA to stop the reaction. The final concentration of ATP was 10 μM, which is twice the Km value of ATP as determined experimentally. MnCl2 and no ATP are added to the negative control wells. Three rounds of washing are performed on the plates using 10 mM Tris pH 7.4, 150 mM NaCl, and 0.05% Tween-20 (TBST). For one hour, the wells are filled with a 1:10000 dilution of rabbit polyclonal anti-phosphotyrosine antiserum in TBST. After that, TBST is used to wash the plates three times. Then, for one hour, each well received a conjugate of goat anti-rabbit antiserum and horseradish peroxidase. After three TBST washes of the plates, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) is added to detect the peroxidase reaction. After allowing the assay's color readout to develop for 20 to 30 minutes, it is read using a 410 nM test filter on a Dynatech MR5000 ELISA plate reader.

The tumor cell lines that are utilized for the in vitro growth are grown in medium with 5–10% CO₂ at 37°C. One day after the start of the culture, SU5416 is serially diluted in media containing DMSO (<0.5%) and added to tumor cell cultures. The sulforhodamine B method is used to measure the growth of the cells after 96 hours. Using four-parameter analysis and curve fitting, IC50s are determined.

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Western blotting analysis: Total cell lysates (5–50 µg of protein) were separated by 10% or 4–12% SDS-PAGE and transferred to a PVDF membrane prior to immunoblotting.

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Immunofluorescence and immunohistochemical staining: Immunofluorescence staining and immunohistochemical staining were performed as previously described [3].

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Cell proliferation assay: Cell proliferation assay was performed with the cell proliferation reagent CCK-8 [3].

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Soft agar cloning assay: Soft agar colony formation assays were performed as previously described [3].

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Migration and invasion assays: Cell migration assays and Boyden chamber invasion assays were performed using the CytoSelect 24-Well Wound Healing Assay Kit and the CytoSelect 24-Well Cell Invasion Assay Kit, respectively. For collagen gel invasion assays, a collagen mixture was prepared with type I collagen solution [3].

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SMC proliferation assessed by [3H]thymidine incorporation.[5]
PA-SMCs in DMEM supplemented with 15% FCS were seeded in 24-well plates at a density of 5 × 104 cells/well and allowed to adhere. The cells were subjected to 48 hours of growth arrest in serum-free medium, then treated with 1 ml of conditioned P-EC medium. We also tested the effect of exogenous PDGF (10 ng/ml) and FGF2 (10 ng/ml) on PA-SMC proliferation with or without imatinib (10–5 M), EGF antagonist (10-5 M), and SU5402 (10-5 M). Under each condition, [3H]thymidine (1 μCi/ml) was added to each well. After incubation for 24 hours, the cells were washed twice with PBS, treated with ice-cold 10% trichloroacetic acid, and dissolved in 0.1 N NaOH (0.5 ml/well). The incorporated radioactivity was counted and reported as cpm/well.

Mice: For one week, intraperitoneal injections of DMSO or SU 5402 (dissolved in DMSO at a concentration of 6 mg/mL) at 25 mg/kg body weight are given to male ΔF508 mice (CFTR^tm1Eur on a 129/FVB background) and their wild-type littermates, aged 9–12 weeks. Every day, the dosages are modified based on the mice's weight. Afterwards, isoflurane is breathed into the mice to induce anesthesia until the procedure is completed. To prevent potential cholinergic stimulation of the salivary gland, 50 μL of the cholinergic antagonist atropine (1 mM) is subcutaneously injected into the right cheek. For four minutes, the injected cheek is pressed up against a tiny strip of filter paper. The same location is then subinjected with isoprenaline (10 mM, 37.5 μL) to elicit an adrenergic secretion of saliva (time 0). For thirty minutes, replace the filter strips (pre-weighed in an Eppendorf tube) every five minutes. After the collection is complete, the weight of all six filter strips is measured, and the results are normalized to mg/g body weight.
Rats: Adult male Wistar rats (200–250 g) are given MCT (60 mg/kg s.c.) and left untreated for 21 dayIn order to evaluate the possible impact of the FGFR1 inhibitor SU 5402 on established PH, adult male Wistar rats weighing 200–250 g are administered MCT (60 mg/kg s.c.), allowed to go untreated for 21 days, and then split into two groups at random (10 animals per group): one group receives treatment with SU 5402 (25 mg/kg/day), while the other group receives no treatment from day 21 to day 42. Every treatment is administered intravenously (s.c.) once daily.

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Effect of treatment with SU5402 on established MCT PH. [5]
To assess the potential effects of the FGFR1 inhibitor SU5402 on established PH, adult male Wistar rats (200–250 g) were given MCT (60 mg/kg s.c.), left untreated for 21 days, then randomly divided into 2 groups (10 animals in each group), of which one was treated with SU5402 (25 mg/kg/day) and the other given the vehicle, from day 21 to day 42. All treatments were given once a day by s.c. injection

[1]. Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases. J Med Chem. 1999 Dec 16;42(25):5120-30.

[2]. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 1999 Jan 1;59(1):99-106.

[3]. Activation of the FGFR1 signalling pathway by the Epstein-Barr virus-encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells. J Pathol. 2015 Oct;237(2):238-48.

[4]. FGF23 affects the lineage fate determination of mesenchymal stem cells. Calcif Tissue Int. 2013 Dec;93(6):556-64.

[5]. Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents. J Clin Invest. 2009 Mar;119(3):512-23.

SU5402 is an indoleone compound with the structure 3-methyleneindolone, where one methylene hydrogen atom is substituted by a 3-(2-carboxyethyl)-4-methyl-1H-pyrrole-2-yl group. It is an ATP-competitive inhibitor of fibroblast growth factor receptor 1 tyrosine kinase activity, exhibiting fibroblast growth factor receptor antagonist activity. It is a monocarboxylic acid belonging to the pyrrole and indoleone classes. Its function is related to 3-methyleneindolone. Receptor tyrosine kinases (RTKs) have been considered potential targets for the treatment of various human diseases, including cancer, inflammatory diseases, cardiovascular diseases (including arterial restenosis), and fibrosis of the lung, liver, and kidneys. Three classes of 3-substituted indololin-2-one compounds with a propionic acid functional group linked at the C-3 position of the pyrrole ring have been identified as catalytic inhibitors of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) receptor tyrosine kinases (RTKs). Some compounds inhibited the activity of isolated vascular endothelial growth factor receptor 2 (VEGF-R2) [fetal liver tyrosine kinase 1 (Flk-1)/kinase insertion domain receptor (KDR)], fibroblast growth factor receptor (FGF-R), and platelet-derived growth factor receptor (PDGF-R) tyrosine kinases, with IC50 values reaching the nanomolar level. Therefore, compound 1 inhibited the activity of VEGF-R2 (Flk-1/KDR) and FGF-R1 tyrosine kinases, with IC50 values of 20 nM and 30 nM, respectively; while compound 16f inhibited the activity of PDGF-R tyrosine kinases, with an IC50 value of 10 nM. This paper discusses the structural modeling and structure-activity relationship analysis of these compounds against their target receptors. The cellular activity of these compounds was determined using the bromodeoxyuridine (BrdU) incorporation method. Some compounds exhibited specific and potent inhibitory effects on cell growth. These data suggest that these compounds can be used to inhibit the function of these target receptors. [1]

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Nonkeratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. EBV-encoded latent membrane protein 1 (LMP1) is considered to play a crucial role in the pathogenesis of NPC because it activates multiple cell signaling pathways that collectively promote cell proliferation, transformation, angiogenesis, and invasion, and regulate energy metabolism. This study found that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but decreases pyruvate kinase activity and pyruvate concentration. Furthermore, regardless of oxygen supply, LMP1 increases the phosphorylation levels of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c-Myc, and HIF-1α. In summary, these results indicate that LMP1 promotes aerobic glycolysis. Regarding the FGFR1 signaling pathway, LMP1 not only increases FGFR1 expression but also upregulates FGF2 expression, leading to constitutive activation of the FGFR1 signaling pathway. Furthermore, two FGFR1 inhibitors (PD161570 and SU5402) attenuated LMP1-mediated aerobic glycolysis, cell transformation (proliferation and anchorage-independent growth), cell migration, and invasion of nasopharyngeal epithelial cells, indicating that the FGFR1 signaling pathway is a key pathway in LMP1-mediated cell growth and transformation. Immunohistochemical staining showed that high levels of phosphorylated FGFR1 were prevalent in primary nasopharyngeal carcinoma specimens, and its expression was positively correlated with LMP1 expression. In addition, FGFR1 inhibitors inhibited the proliferation and anchorage-independent growth of nasopharyngeal carcinoma cells. Our current results suggest that LMP1-mediated FGFR1 activation promotes aerobic glycolysis and epithelial cell transformation, implying that FGF2/FGFR1 signaling pathway activation is involved in the EBV-driven pathogenesis of nasopharyngeal carcinoma. [2]

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FGF23 is a bone-derived hormone that regulates mineral metabolism by inhibiting renal tubular phosphate reabsorption and reducing circulating 1,25(OH)2D and PTH levels. These effects are mediated by the binding and activation of the FGF receptor and its co-receptor Klotho, which is expressed in the distal convoluted tubule of the kidney. Klotho has recently been reported to be expressed in bone tissue, suggesting that FGF23 has a direct extrarenal effect on cells involved in bone development and remodeling, but the mechanism remains unclear. In this study, we found that bone marrow stromal cells isolated from Klotho knockout mice formed fewer osteoblast colonies than wild-type mice, while adipocyte colonies were more numerous. We explored the underlying mechanism using mouse C3H10T1/2 cells. We found that Klotho expression was weaker and that FGF23 affected cell lineage fate determination in a dose-dependent manner. The effect of FGF23 on cell differentiation was attenuated by the FGF receptor-specific tyrosine kinase inhibitor SU5402. Our results indicate that FGF23 directly influences the differentiation of bone marrow stromal cells. [4] Pulmonary hypertension (PH) is a progressive, fatal lung disease characterized by proliferation of pulmonary artery smooth muscle cells (PA-SMCs) that ultimately leads to right heart failure. Molecular events originating from pulmonary endothelial cells (P-ECs) may be involved in the proliferation of PA-SMCs in PH. Therefore, we exposed cultured human pulmonary artery smooth muscle cells (PA-SMCs) to conditioned medium of pulmonary artery endothelial cells (P-ECs) from patients with idiopathic pulmonary hypertension (IPH) or healthy controls and found that IPH P-EC conditioned medium promoted PA-SMC proliferation more than control P-EC conditioned medium. FGF2 levels in IPH P-EC medium were higher than in the control group, while TGF-β1, PDGF-BB, or EGF levels were not significantly different. There was no difference in FGF2-induced proliferation or FGF receptor type 1 (FGFR1) mRNA levels between IPH and control PA-SMCs. After knocking down FGF2 in P-EC with siRNA, the stimulatory effect of IPH P-EC medium on PA-SMC growth was reduced by 60%, compared to 10% in control P-EC medium. In situ hybridization showed that FGF2 was mainly overexpressed after pulmonary vascular endothelial cell remodeling in IPH patients. Repeated intravenous injection of FGF2-siRNA eliminated the production of FGF2 in the lungs, thereby preventing and almost reversing the rat pulmonary hypertension model. Similarly, pharmacological inhibition of FGFR1 using SU5402 also reversed the established pulmonary hypertension in the same model. Therefore, the overproduction of endothelial cell FGF2 in idiopathic pulmonary hypertension and the promotion of smooth muscle cell proliferation in idiopathic pulmonary hypertension suggest that FGF2 is a promising new target for the treatment of pulmonary hypertension. [5]

C17H16N2O3

296.32

296.116

C, 68.91; H, 5.44; N, 9.45; O, 16.20

215543-92-3

SU 5402;215543-92-3

5289418

Orange solid powder

1.4±0.1 g/cm3

592.6±50.0 °C at 760 mmHg

>222ºC (dec.)

312.2±30.1 °C

0.0±1.8 mmHg at 25°C

1.688

2.03

3

3

4

22

488

0

O=C(CCC1=C(NC=C1C)/C=C2C(NC3=C\2C=CC=C3)=O)O

JNDVEAXZWJIOKB-JYRVWZFOSA-N

InChI=1S/C17H16N2O3/c1-10-9-18-15(11(10)6-7-16(20)21)8-13-12-4-2-3-5-14(12)19-17(13)22/h2-5,8-9,18H,6-7H2,1H3,(H,19,22)(H,20,21)/b13-8-

3-[4-methyl-2-[(Z)-(2-oxo-1H-indol-3-ylidene)methyl]-1H-pyrrol-3-yl]propanoic acid

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)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)