FGFR1 (IC50 = 52.4 nM)

PD166866 inhibits cell proliferation in FGFR1-amplified breast cancer cells. PD166866 induces autophagy in FGFR1-amplified breast cancer cells. Targeting autophagy improves the anti-cancer effects of PD166866. PD166866 induces autophagy by inhibiting Akt/mTOR pathway. [3]

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It appears that PD166866 treatment results in oxidative stress and a mitochondrial deficit[1]. With an IC50 value of 52.4 ± 0.1 nM, PD 166866 inhibits human full-length FGFR-1 tyrosine kinase. However, it has no effect on c-Src, platelet-derived growth factor receptor-β, epidermal growth factor receptor, or insulin receptor tyrosine kinases. Moreover, at concentrations of up to 50 μM, it has no effect on mitogen-activated protein kinase, protein kinase C, and CDK4. In NIH 3T3 cells expressing endogenous FGFR-1 and in L6 cells overexpressing the human FGFR-1 tyrosine kinase, PD 166866 is a strong inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation, confirming a tyrosine kinase-mediated mechanism. PD 166866's specificity for the FGFR-1 is further supported by the fact that it does not inhibit the autophosphorylation of insulin-stimulated receptor, platelet-derived growth factor, or A431 in vascular smooth muscle, respectively. Furthermore, it has been discovered that PD 166866 is a strong inhibitor of microvessel outgrowth, or angiogenesis, from human placenta fragments grown in culture. In L6 cells, PD 166866 inhibits phosphorylated 44- and 42-kDa MAPK isoforms with IC50 values of 4.3 and 7.9 nM, respectively[2]. PD166866 suppresses the Akt/mTOR signaling pathway to cause autophagy[3].

To extend our in vitro findings, mice tumor xenograft model was established by subcutaneously injecting MDA-MB-134 sub-line that stably expressed shAtg5, and the mice were treated with PD166866. In spite of no difference observed for the first five days, the tumors formed by Atg5-silenced cells showed an apparently lowered growth rate, compared to those tumors composed of mock vector-transfected cells (Fig. 3E-F). Further, Ki-67 immunostaining revealed that the number of proliferating cells in tumor xenograft was substantially decreased when Atg5 expression was suppressed, as shown in Fig. 3G. These results suggested that blockage of autophagy improves the anti-proliferative effects of PD166866 in FGFR1-amplified breast cancer [3].

Immunolocalization of Poly-ADP-Ribose-Polymerase (PARP) [1]
The enzyme PARP is activated in response to DNA fragmentation. The immunolocalization of PARP was performed as previously published. Briefly, HeLa cells were treated with PD166866 for 24 hours, the growth medium was removed, the cells were washed with PBS and fixed for 1 hour at 25°C adding a freshly made paraformaldheyde solution (4% in PBS). Samples were washed again with PBS and the endogenous oxidases were blocked for 2 minutes in the dark. Further washes with PBS followed and blocking the unspecific sites was done for 1 hour at 25°C. PARP was evidenced by immunolocalization utilizing a polyclonal antibody , directed against the N-terminal proteolytic fragment. Immuno-reaction was revealed by a secondary anti-rabbit antibody after incubation for 16 hours at 4°C. After exhaustive washing with PBS the samples were incubated for 30 minutes in solution ABC. Eventually, DAB (3,3'-Diaminobenzidine) was added and the samples were incubated for 10 minutes in the dark. The samples were washed again the plates were sealed and ready for microscopic observation.

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PD166866 is a novel small molecule inhibitor of FGFR (fibroblast growth factor-1 receptor) tyrosine kinase with an IC50 of 52.4 nM. It belongs to a new structural class of tyrosine kinase inhibitors, the 6-aryl-pyrido[2,3-d]pyrimidines. In L6 cells that overexpress the human FGFR-1 tyrosine kinase and NIH 3T3 cells that express endogenous FGFR-1, PD 166866 is a strong inhibitor of FGFR autophosphorylation. Furthermore, PD 166866 prevents L6 cells' bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa (ERK 1/2) mitogen-activated protein kinase isoforms. For therapeutic targets like tumor growth and atherosclerotic plaque neovascularization, PD 166866 has potential application as an antiproliferative/antiangiogenic agent.

After HeLa cells are exposed to PD166866 for a full day, the growth medium is removed, PBS is used to wash the cells, and they are fixed for one hour at 25°C using a recently prepared paraformaldheyde solution (4% in PBS). After giving the samples another PBS wash, the endogenous oxidases were blocked for two minutes in the absence of light. After that, more PBS washes are performed, and the unspecified sites are blocked for an hour at 25°C. By using a polyclonal antibody to target the N-terminal proteolytic fragment for immunolocalization, PARP is demonstrated. After incubation at 4°C for 16 hours, the immunoreaction is exposed by a secondary anti-rabbit antibody. The samples are thoroughly cleaned in PBS and then incubated in solution ABC for half an hour. The samples are then incubated for 10 minutes in the dark after the addition of DAB (3,3'-Diaminobenzidine). After another wash, the plates are sealed and prepared for microscopic inspection.

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Growth and maintenance of HeLa cells [1]
Cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium - high glucose), supplemented with newborn bovine serum [final concentration (f.c.) 10%], penicillin-streptomycin (10000 U/ml) and glutamine (2 mM); the pH of the medium was 7.2 and incubation was at 37°C in a 5% CO2 atmosphere. Cells were routinely passaged when confluent. Assessment of cell viability and lipoperoxidation assay [1]
Cell viability was evaluated by the colorimetric Mosmann assay which is a quantitative method measuring the level of mitochondrial damage. The MTT [3-(4,5-dimetiltiazol-2-yl)-2,5-difenil tetrazolium-bromide] is a yellow water soluble salt which is converted into insoluble purple salts formed by the active dehydrogenases present in the mitochondria of vital cells. Absorbance values measured at 570 nm provide the number of vital cells. The cell survival data were validated by vital staining with trypan blue performed by a standard laboratory protocol.
A commercial kit was used to assess the oxidative stress at membrane level. Briefly, the assay is based on a quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA) which derives from the decomposition of poly-unsaturated fatty acids. The MDA molecule reacts with a chromogenic compound (N-methyl-2-phenylindole) thus forming a stable chromophore. Absorbance at 586 nm is directly transformed in intracellular concentration of MDA.

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TUNEL assay and analysis of the DNA fragmentation [1]
The activation of the endogenous DNases is one of the consequences of cell death causing the formation of single strand nicks and eventually fragmentation of DNA. The DNA ruptures may be evidenced by in situ labelling. Cell nuclei are permeabilized, fluorescent dUTP is added and terminal-deoxynucleotide-transferase conjugates the nucleotide where the sugar-phosphate backbone is interrupted. Fluorescence intensity provides a qualitative idea of DNA damage.

Female nude mice
20 mg/kg
i.p.

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Tumor xenograft model [3]
Healthy female nude mice (6–8 weeks, 18–20 g) were injected subcutaneously with mock vector- or shAtg5-overexpressed MDA-MB-134 cells (5 × 106 cells/mouse). When the tumors reached 100 mm3 in volume, mice were peritoneally treated with 20 mg/kg PD166866. The tumor volume was measured every 5 days and animals were sacrificed after 25 days. Tumors were dissected and frozen in liquid nitrogen or fixed in formalin immediately.

Cytotoxicity of PD166866 to cultured HeLa cells [1] We investigated the dose-response effect of HeLa cells exposed to a relatively wide range of concentrations (0.1–50 μM) of PD166866. Cell viability was assessed by MTT assay 24 hours after treatment with the drug. A significant reduction in the number of viable cells was observed at a concentration of 2.5 μM (Fig. 1, left panel). The loss of cell viability appeared to plateau at 25 μM (approximately 25% survival) and did not decrease further at a drug concentration of 50 μM. This result may indicate the presence of a cell subpopulation with intrinsic resistance to the drug. Trypan blue staining and viable cell counts confirmed this result (data obtained at a drug concentration of 2.5 μM are shown only; Fig. 1, right panel). The negative effects of PD166866 on cell growth have been observed in previous studies on 3T6 cells (a stable mouse fibroblast cell line). The results of this study confirm the published findings, and in terms of cell viability, no difference was observed between HeLa cells and 3T6 cells in the corresponding experiments conducted in this study (data not shown). Interestingly, as observed in previous studies, HeLa cells were significantly more sensitive to resveratrol (a natural product with cytotoxic and antiviral properties) than mouse cells. One explanation for this is that the two drugs may have different cellular/molecular targets.

[1]. J Exp Clin Cancer Res. 2009 Dec 11;28(1):151.

[2]. J Pharmacol Exp Ther. 1998 Jul;286(1):569-77.

[3]. Biochem Biophys Res Commun. 2016 May 20;474(1):1-7.

PD-166866 belongs to the pyridopyrimidine class of compounds. Its structure is pyrido[2,3-d]pyrimidine, with an amino group substituted at position 2, a 3,5-dimethoxyphenyl group substituted at position 6, and a (tert-butylcarbamoyl)nitrile group substituted at position 7. It is a selective ATP-competitive human fibroblast growth factor-1 receptor (FGFR1) tyrosine kinase inhibitor with an IC50 value of 52.4 nM. It exhibits apoptosis-inducing, anti-tumor, EC2.7.10.1 (receptor protein tyrosine kinase) inhibition, and angiogenesis-inhibiting effects. It is a dimethoxybenzene, pyridopyrimidine, urea, biaryl, and primary aromatic amine compound.

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Background: Extensive experimental data indicate that overexpression of various growth factors leads to disordered cell proliferation. The role of fibroblast growth factor (FGF) in growth regulation is undeniable: particularly FGF1 and its tyrosine kinase receptor (FGFR1), which exert their effects through an extremely complex network of mechanisms and pathways. This study evaluated the antiproliferative activity of PD166866 (a synthetic molecule that inhibits the activity of FGFR1 tyrosine kinase). Methods: Cells were routinely cultured in Dulbecco modified Eagle medium supplemented with neonatal serum and a penicillin-streptomycin mixture. Cell viability was assessed using the Mosmann assay and trypan blue staining. DNA damage was assessed by in situ fluorescent staining using the terminal deoxynucleotidyl transferase (dUTP) nick-end labeling method (TUNEL assay). Membrane-level oxidative stress was assessed by quantitatively analyzing intracellular malondialdehyde (MDA) production, derived from the breakdown of polyunsaturated fatty acids. Immunohistochemistry was used to detect the expression of poly(ADP-ribose) polymerase (PARP) after DNA fragmentation using an antibody targeting the N-terminal fragment of this enzyme. Results: The bioactivity of this drug was investigated in HeLa cells. Cytotoxicity was assessed using the Mosmann assay and trypan blue live-cell staining. A significant increase in intracellular malondialdehyde concentration suggests that the target of this molecule is likely the cell membrane. The increase of this compound after PD166866 treatment suggested membrane lipid peroxidation. TUNEL assay results qualitatively but clearly indicated the presence of DNA damage. In addition, we also confirmed the intracellular accumulation of poly-ADP ribose polymerase I (PARP I). This enzyme is a sensor for DNA strand breaks, which supports the idea that drug treatment induces cell death. Conclusion: The data in this study show that PD166866 has a significant anti-proliferative effect. The negative regulation of cell proliferation may be achieved by activating the apoptosis pathway. Experimental results for this specific point, such as DNA damage assessment, cell membrane lipid peroxidation and increased PARP (an enzyme directly involved in DNA repair) expression, indicate that cells exposed to PD166866 will undergo apoptosis. However, the possibility of other cell death pathways cannot be ruled out. This article explores the potential therapeutic use of this drug. [1]

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By direct synthesis, we discovered a small molecule that is a nanomolar inhibitor of human fibroblast growth factor-1 receptor (FGFR) tyrosine kinase. PD 166866 belongs to a novel class of tyrosine kinase inhibitors—6-aryl-pyrido[2,3-d]pyrimidine compounds—identified through screening a compound library and using methods to determine protein tyrosine kinase activity. PD 166866 exhibits an IC50 value of 52.4 ± 0.1 nM for inhibiting full-length human FGFR-1 tyrosine kinase and has been further confirmed as an ATP-competitive inhibitor of FGFR-1. Conversely, at concentrations up to 50 μM, PD 166866 has no effect on c-Src, platelet-derived growth factor receptor-β, epidermal growth factor receptor or insulin receptor tyrosine kinases, as well as mitogen-activated protein kinase, protein kinase C, and CDK4. PD 166866 is a potent inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation in NIH 3T3 cells expressing endogenous FGFR-1 and L6 cells overexpressing human FGFR-1 tyrosine kinase, confirming a tyrosine kinase-mediated mechanism. PD 166866 also inhibits bFGF-induced tyrosine phosphorylation of 44 kDa and 42 kDa (ERK 1/2) mitogen-activated protein kinase isoforms in L6 cells, suggesting its mechanism of action is through inhibition of bFGF-stimulated FGFR-1 tyrosine kinase activation. PD 166866 does not inhibit autophosphorylation of platelet-derived growth factor, epidermal growth factor, or insulin-stimulated receptors in vascular smooth muscle cells, A431 cells, or NIHIR cells, further confirming its specificity for FGFR-1. In addition, daily exposure of L6 cells to PD 166866 at concentrations of 1 to 100 nM resulted in concentration-dependent inhibition of bFGF-stimulated cell growth for 8 consecutive days, with an IC50 value of 24 nM. In contrast, PD 166866 had little effect on platelet-derived growth factor-BB-stimulated L6 cell growth or serum-stimulated vascular smooth muscle cell proliferation. Furthermore, PD 166866 was found to be a potent inhibitor of microangiogenesis (vascularization) in human placental artery fragment cultures. These results highlight the discovery of PD 166866, a novel, nanomolar-level potent and selective small molecule FGFR-1 tyrosine kinase inhibitor with the potential to be used as an antiproliferative/antiangiogenic drug for the treatment of diseases such as tumor growth and neovascularization of atherosclerotic plaques. [2]

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Breast cancer accounts for approximately 30% of all gynecological cancer cases each year and is one of the leading causes of cancer-related deaths in women. FGFR1 amplification is common in breast cancer and is associated with poor prognosis. Although FGFRs have long been considered targets for anticancer drugs and several FGFR antagonists are currently undergoing clinical trials, the exact response of cells to FGFR antagonist treatment remains unclear. This study shows that the selective FGFR1 inhibitor PD166866 can inhibit the proliferation of FGFR1-amplified breast cancer cell lines and induce anointing apoptosis. Notably, we found that PD166866 can induce autophagy in FGFR1-amplified breast cancer cell lines, and knocking down Atg5 to block autophagy further enhances the antiproliferative activity of PD166866. In addition, mechanistic studies show that PD166866 induces autophagy by inhibiting the Akt/mTOR signaling pathway. This study provides new insights into the molecular mechanism of the antitumor activity of FGFR antagonists and may further help in the discovery of FGFR-based drugs. [3]

C20H24N6O3

396.44

396.191

C, 60.59; H, 6.10; N, 21.20; O, 12.11

192705-79-6

5328127

white solid powder

1.277g/cm3

1.643

3.545

3

7

5

29

545

0

C1=C2C(N=C(NC(=O)NC(C)(C)C)C(C3C=C(OC)C=C(OC)C=3)=C2)=NC(N)=N1

NHJSWORVNIOXIT-UHFFFAOYSA-N

InChI=1S/C20H24N6O3/c1-20(2,3)26-19(27)25-17-15(8-12-10-22-18(21)24-16(12)23-17)11-6-13(28-4)9-14(7-11)29-5/h6-10H,1-5H3,(H4,21,22,23,24,25,26,27)

1-[2-amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-3-tert-butylurea

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)

Solubility in Formulation 1: ≥ 1 mg/mL (2.52 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 10.0 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.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5% DMSO+40% PEG 300+5% Tween 80+50% ddH2O: 0.5mg/mL

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Solubility in Formulation 4: 3.33 mg/mL (8.40 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 40°C.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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