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 on HeLa cells in culture [1]
We explored the dose/response effect of HeLa cells exposed to a relatively broad range of PD166866 concentrations (0.1 - 50 μM). Cells were treated for 24 hours with the drug and their vitality assessed by the MTT assay. A significant reduction of vital cells can be monitored already at 2.5 μM concentration (Figure 1, left panel). The loss of viability seems to stabilize at 25 μM (about 25% survival) with no further decrease at a 50 μM concentration of drug. This result may indicate the presence of a cell subpopulation, intrinsically resistant to the drug. This result was confirmed by vital cell count with trypan blue (only the data obtained at 2.5 μM of drug is shown; Figure 1, right panel). The negative effect of PD166866 on the cell growth was already observed in a previous works performed on 3T6 cells: a stabilized murine fibroblast line. The results presented here validate those already published and, as far as cell survival is concerning, no difference can be monitored on HeLa in comparison to 3T6 cells in matching experiments also run in this work (not shown). Interestingly, as observed in a former study, HeLa cells showed a significantly higher sensitivity than murine cells towards resveratrol, a natural product showing both cytotoxic and antiviral properties. One way to rationalize this data is that the cellular/molecular target of the two drugs could be different.

[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 is a member of the class of pyridopyrimidines that is pyrido[2,3-d]pyrimidine substituted by an amino group at position 2, 3,5-dimethoxyphenyl group at position 6, and by a (tert-butylcarbamoyl)nitrilo group at position 7. It is a selective ATP competitive inhibitor of the human fibroblast growth factor-1 receptor (FGFR1) tyrosine kinase with an IC50 of 52.4 nM. It has a role as an apoptosis inducer, an antineoplastic agent, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an angiogenesis inhibitor. It is a dimethoxybenzene, a pyridopyrimidine, a member of ureas, a biaryl and a primary arylamine.

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Background: Many experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF) in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1) act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1. Methods: Cells were routinely grown in Dulbecco Modified Eagle's medium supplemented with newborn serum and a penicillin-streptomycin mixture.Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by in situ fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay).Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA) deriving from the decomposition of poly-unsaturated fatty acids.The expression of Poly-ADP-Ribose-Polymerase (PARP), consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme. Results: The bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell death. Conclusions: Data presented in this work show that PD166866 has clear antiproliferative effects. The negative control of cell proliferation may be exerted through the activation of the apoptotic pathway. The results of experiments addressing this specific point, such as: evaluation of DNA damage, lipoperoxidation of the cell membrane and increase of expression of PARP, an enzyme directly involved in DNA repair. Results suggest that cells exposed to PD16866 undergo apoptosis. However, concomitant modes of cell death cannot be ruled out. The possible use of this drug for therapeutic purposes is discussed.[1]

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Through direct synthetic efforts, we discovered a small molecule that is a nanomolar inhibitor of the human fibroblast growth factor-1 receptor (FGFR) tyrosine kinase. PD 166866, a member of a new structural class of tyrosine kinase inhibitors, the 6-aryl-pyrido[2,3-d]pyrimidines, was identified by screening a compound library with assays that measure protein tyrosine kinase activity. PD 166866 inhibited human full-length FGFR-1 tyrosine kinase with an IC50 value of 52.4 +/- 0.1 nM and was further characterized as an ATP competitive inhibitor of the FGFR-1. In contrast, PD 166866 had no effect on c-Src, platelet-derived growth factor receptor-beta, epidermal growth factor receptor or insulin receptor tyrosine kinases or on mitogen-activated protein kinase, protein kinase C and CDK4 at concentrations as high as 50 microM. PD 166866 was a potent inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation in NIH 3T3 cells expressing endogenous FGFR-1 and in L6 cells overexpressing the human FGFR-1 tyrosine kinase, confirming a tyrosine kinase-mediated mechanism. PD 166866 also inhibited bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa (ERK 1/2) mitogen-activated protein kinase isoforms in L6 cells, presumably via inhibition of bFGF-stimulated FGFR-1 tyrosine kinase activation. PD 166866 did not inhibit platelet-derived growth factor, epidermal growth factor or insulin-stimulated receptor autophosphorylation in vascular smooth muscle, A431 or NIHIR cells, respectively, further supporting its specificity for the FGFR-1. In addition, daily exposure of PD 166866 to L6 cells at concentrations from 1 to 100 nM resulted in a concentration-related 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 growth of L6 cells or serum-stimulated vascular smooth muscle cell proliferation. Finally, PD 166866 was found to be a potent inhibitor of microvessel outgrowth (angiogenesis) from cultured artery fragments of human placenta. These results highlight the discovery of PD 166866, a new nanomolar potent and selective small molecule inhibitor of the FGFR-1 tyrosine kinase with potential use as antiproliferative/antiangiogenic agent for such therapeutic targets as tumor growth and neovascularization of atherosclerotic plaques. [2]

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Breast cancer, representing approximately 30% of all gynecological cancer cases diagnosed yearly, is a leading cause of cancer-related mortality for women. Amplification of FGFR1 is frequently observed in breast cancers and is associated with poor prognosis. Though FGFRs have long been considered as anti-cancer drug targets, and a cluster of FGFR antagonists are currently under clinical trials, the precise cellular responses under the treatment of FGFR antagonists remains unclear. Here, we show that PD166866, an FGFR1-selective inhibitor, inhibits proliferation and triggers anoikis in FGFR1-amplified breast cancer cell lines. Notably, we demonstrate that PD166866 induces autophagy in FGFR1-amplified breast cancer cell lines, while blockage of autophagy by Atg5 knockdown further enhances the anti-proliferative activities of PD166866. Moreover, mechanistic study reveals that PD166866 induces autophagy through repressing Akt/mTOR signaling pathway. Together, the present study provides new insights into the molecular mechanisms underlying the anti-tumor activities of FGFR antagonists, and may further assist the FGFRs-based drug discovery.[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.)