EGFR (Ki = 6 pM); EGFR (IC50 = 25 pM)

With an IC50 of 14 nM, PD 153035 (SU 5271) inhibits EGF-stimulated receptor autophosphorylation in human epidermoid carcinoma A431 cells[1]. Even at 50 μM, PD153035 (SU 5271) has minimal effect on src tyrosine kinases, insulin receptor, PDGFR, FGFR, CSF-1 receptor, or CSF-1 receptor. In fibroblasts or human epidermoid carcinoma cells, PD153035 (SU 5271) quickly suppresses autophosphorylation of the EGF receptor at low nanomolar doses and specifically prevents EGF-mediated cellular activities such as mitogenesis, early gene expression, and oncogenic transformation[2]. EGF receptor-positive cell lines are subjected to a dose-dependent growth inhibition by PD 153035 (SU 5271), which starts at less than micromolar concentrations and typically has an IC50 of less than 1 pM[3].

After a single intraperitoneal (i.p.) injection of 80 mg/kg, PD153035 (SU 5271) levels in the tumor and plasma rise to 22 μM and 50 μM after 15 minutes as well. In tumors, PD153035 (SU 5271) remains at micromolar concentrations for at least 12 hours, even though its plasma levels drop below 1 μM after three hours. Tumors quickly decrease the tyrosine phosphorylation of the EGF receptor by 80–90%[4].

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PD 153035 is a potent (Ki = 6 pm) and specific inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase that suppresses tyrosine phosphorylation of the EGF receptor in A431 cells at nanomolar concentrations in cell culture. We have examined the pharmacokinetics of this compound and its ability to rapidly suppress phosphorylation of the EGF receptor in A431 human epidermoid tumors grown as xenografts in immunodeficient nude mice. Following a single i.p. dose of 80 mg/kg, the drug levels in the plasma and tumor rose to 50 and 22 microM within 15 minutes. While the plasma levels of PD153035 fell below 1 microM by 3 hours, in the tumors it remained at micromolar concentrations for at least 12 hours. The tyrosine phosphorylation of the EGF receptor was rapidly suppressed by 80-90% in the tumors. However receptor phosphorylation returned to control levels after 3 hours despite the continued presence of the drug at concentrations which, based on previous in vitro results, were predicted to maintain inhibition. EGF-stimulated tyrosine kinase activity in tumor extracts was decreased and recovered in parallel with the effects of PD153035 on receptor phosphorylation though the activity had reached only about half of the control activity after three hours. These results demonstrate the potential for using small molecule inhibitors to inhibit the EGF receptor tyrosine kinase in vivo, though a fair evaluation of their potential anti-cancer activity will have to wait for solutions to problems with sustained delivery which may allow us to maintain suppression of EGF receptor phosphorylation [4].

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Non-alcoholic fatty liver disease (NAFLD) is one of the main causes of chronic liver disease. NAFLD begins with excessive lipid accumulation in the liver and progresses to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is closely linked to dysregulated hepatic lipid metabolism. Although recent studies have reported that epidermal growth factor receptor (EGFR) signaling regulates lipid metabolism, the roles of EGFR and EGFR inhibitors as modulators of lipid metabolism are largely unknown. Here, we investigated whether inhibiting EGFR using the EGFR tyrosine kinase inhibitor (TKI) PD153035 improves NAFLD. Our results demonstrate that EGFR was activated in liver tissues from high fat diet (HFD)-induced NAFLD mice. Inhibiting EGFR using PD153035 significantly reduced phosphatidylinositol-3-kinase/protein kinase B signaling and sterol responsive elementary binding protein 1 and 2 expression, which prevented HFD-induced hepatic steatosis and hypercholesterolemia by reducing de novo lipogenesis and cholesterol synthesis and enhancing fatty acid oxidation. Additionally, inhibiting EGFR improved HFD-induced glucose intolerance. In conclusion, these results indicate that EGFR plays an important role in NAFLD and is a potential therapeutic target[5].

Enzyme reactions are performed in a total volume of 0.1 mL containing 25 mM Hepes (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 50 μM sodium vanadate, 0.5 to 1.0 ng of enzyme (which also contains enough EGF to make the final concentrations 2 μg/mL), 10 μM ATP containing 1 μCi of [32P]ATP, varying concentrations of PD153035, and 200 μM of a substrate peptide based on a portion of phospholipase C-γl having the sequence Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr472-Glu-Glu-Val. ATP is added to the mixture to start the reaction. The reaction is stopped after 10 minutes at room temperature by adding 2 mL of 75 mM phosphoric acid. The mixture is then run through a 2.5-cm phosphocellulose filter disk, which binds the peptide. The filter is put in a vial with 5 mL of scintillation fluid after being cleaned five times with 75 mM phosphoric acid. An estimated 100,000 cpm are produced by the uninhibited control activity.

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Enzyme Assay: EGFR was prepared from human A431 carcinoma cell-shed membrane vesicles by immunoaffinity chromatography as previously described, and the assays were carried out as reported previously.13 The substrate used was based on a portion of phospholipase Cγ1, having the sequence Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr472-Glu-Glu-Val. The reaction was allowed to proceed for 10 min at room temperature and then stopped by the addition of 2 mL of 75 mM phosphoric acid. The solution was then passed through a 2.5 cm phosphocellulose disk which bound the peptide. This filter was washed with 75 mM phosphoric acid (5×), and incorporated label was assessed by scintillation counting in an aqueous fluor. Control activity (no drug) gave a count of ca. 100 000 cpm. At least two independent dose−response curves were done and the IC50 values computed. The reported values are averages; variation was generally ±15% [1].

PD153035 is applied to various EGF receptor-overexpressing cell lines (A43 1, Difi, MDA-MB-468, MDA-MB-231, DU145, SiHa, C4i, and MEl 80) at escalating 0.125-2.5 p.M. concentrations. The impact of growth inhibitors in monolayer cell culture is evaluated[3].

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EGFR Autophosphorylation in Human A431 Epidermoid Carcinoma Cells. [1]
Cells were grown to confluency in 6-well plates (35 mm diameter), exposed to serum-free medium for 18 h, and then treated with 4 or PD 153035 (32) for 2 h, followed by EGF (100 ng/mL) for 5 min. The monolayers were lysed in 0.2 mL of boiling Laemlli buffer (2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol, and 50 mM Tris, pH 6.8), and the lysates were heated to 100 °C for 5 min. Proteins in the lysate were separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The membrane was washed once in 10 mM Tris, pH 7.2, 150 mM NaCl, and 0.01% azide (TNA) and blocked overnight in TNA containing 3% bovine serum albumin and 1% ovalbumin. The membrane was blotted for 2 h with antiphosphotyrosine antibody (UBI, 1 μg/mL in blocking buffer) and then washed twice in TNA, once in TNA containing 0.05% Tween-20 and 0.05% nonidet P-40, and twice in TNA. The membranes were then incubated for 2 h in blocking buffer containing 0.1 μCi/mmol [125I]protein and then washed again as above. After the blots were dry they were loaded into a film cassette and exposed to X-AR X-ray film for 1−7 days. Band intensities were determined with a Molecular Graphics laser densitometer.

Male C57BL/6J mice and high-fat diet (HFD) composed of 60% fat were used. The animals were maintained in a controlled environment (12 h light/12 h dark cycle; 50–60% humidity; ambient temperature 22 ± 2°C). Eight-week-old male mice were fed a normal chow diet (NCD) or HFD for 8 consecutive weeks and then divided randomly into three groups: the NCD group were fed an NCD without treatment, the HFD group were fed a HFD without treatment, and the HFD+PD group were fed a HFD and treated with PD 153035 (30 mg/kg/day O.G) for the final 4 weeks. [5]

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Dissolved in water.; 80 mg/kg; i.p. injection

A431 cells are injected into the outbred nude mice.

[1]. Tyrosine kinase inhibitors. 8. An unusually steep structure-activity relationship for analogues of 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (PD 153035), a potent inhibitor of the epidermal growth factor receptor. J Med Chem. 1996 Jan 5;39(1):267-76.

[2]. A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science. 1994 Aug 19;265(5175):1093-5.

[3]. PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptoractivation and inhibits growth of cancer cells in a receptor number-dependent manner. Clin Cancer Res. 1997 Nov;3(11):2099-106.

[4]. Inhibition of the epidermal growth factor receptor tyrosine kinase by PD153035 in human A431 tumors in athymic nude mice. Invest New Drugs. 1996;13(4):295-302.

[5]. Epidermal growth factor receptor inhibition attenuates non-alcoholic fatty liver disease in diet-induced obese mice. PoS One . 2019 Feb 8;14(2):e0210828.

PD-153035 belongs to the quinazoline class of compounds, with a 3-bromophenylamino substituent at position 4 and two methoxy substituents at positions 6 and 7. It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor and an epidermal growth factor receptor antagonist. It belongs to the quinazoline, aromatic amine, secondary amine, bromobenzene, and aromatic ether classes of compounds. It is the conjugate base of PD-153035(1+). AG 1517 is a quinazoline derivative that selectively inhibits EGFR kinase activity and suppresses the growth of psoriatic keratinocytes. PD-153035 hydrochloride is prepared by reacting PD-153035 with an equivalent amount of hydrochloric acid. It functions as an epidermal growth factor receptor antagonist and an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor. It contains PD-153035(1+). AG 1517 is a quinazoline derivative that selectively inhibits EGFR kinase activity and suppresses the growth of psoriatic keratinocytes. 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (32, PD 153035) is a highly potent inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity (IC50 0.025 nM) that competitively binds to the ATP site. The structure-activity relationship of closely related analogs of 32 is very steep. Some derivatives exhibit IC50 values 80 times higher than predicted based on simple addition binding energy theory; however, analogs with similar combinations of phenyl and quinazoline substituents do not show this "hyperadditivity" effect. Since some substituents, which themselves have a slight inactivating effect, can produce a strong activating effect when used in the correct combination, we hypothesize that some substituted analogs can induce a conformational change in the receptor upon binding. Substitutions at positions 6 and 7 of quinazoline exhibit some tolerance, therefore compound 32 is not the best inhibitor of induced conformation. The diethoxy derivative 56 [4-(3-bromoanilino)-6,7-diethoxyquinazoline] has an IC50 of 0.006 nM and is the most potent inhibitor of EGFR tyrosine kinase activity reported to date. [1]

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A small molecule called PD 153035 inhibits epidermal growth factor (EGF) receptor tyrosine kinase with an inhibition constant of 5 pM. This inhibitor is specific for EGF receptor tyrosine kinase and only inhibits other purified tyrosine kinases at micromolar or higher concentrations. PD 153035 rapidly inhibits EGF receptor autophosphorylation at low nanomolar concentrations in fibroblasts or human epidermal-like cancer cells and selectively blocks EGF-mediated cellular processes, including mitosis, early gene expression, and oncogenic transformation. PD 153035 showed four to five orders of magnitude greater inhibitory efficacy against isolated EGF receptor tyrosine kinase than other tyrosine kinase inhibitors, and three to four orders of magnitude greater inhibitory efficacy against cellular phosphorylation. [2] PD 153035 is reported to be a specific and potent epidermal growth factor (EGF) receptor tyrosine kinase inhibitor, and also has some inhibitory effect on the closely related HER2/neu receptor. We found that PD 153035 can inhibit EGF-dependent EGF receptor phosphorylation and inhibit the proliferation and colony formation of various human cancer cell lines overexpressing EGF receptors. In EGF receptor overexpressing cells, when the concentration of PD 153035 > 75 nM, exogenous EGF-induced EGF receptor autophosphorylation was completely inhibited. In contrast, PD153035 only reduced hereinine-dependent phosphorylation in HER2/neu-overexpressing cell lines at significantly elevated concentrations (1400–2800 nM). PD153035 treatment did not affect EGF receptor or HER2/neu expression. PD153035 dose-dependently inhibited the growth of EGF receptor-overexpressing cell lines at low micromolar concentrations, with IC50 values less than 1 μM in monolayer cultures of most tested cell lines. Even at concentrations up to 2.5 μM, the IC50 value for HER2/neu-overexpressing cells was not reached. In colony formation assays, the growth-inhibiting activity of PD153035 in endogenous (autocrine) ligand-driven cultures was correlated with the number of EGF receptors: higher activity was observed in cells with higher EGF receptor expression levels, while extremely low activity was observed in cells with normal EGF receptor expression but high HER2/neu levels. PD153035 can also eliminate all growth effects mediated by exogenous EGF addition; this effect can be reversed after the compound is removed. Combination therapy with the anti-EGF receptor blocking monoclonal antibody C225 further enhanced the anti-tumor activity of PD153035, suggesting that the mechanism of action of C225 is not merely competitive binding with ligands. The latter finding also suggests that the combined anti-EGF receptor strategy may have a stronger therapeutic effect on tumors with high EGF receptor expression. [3]

C16H14BRN3O2

360.2053

359.026

C, 53.35; H, 3.92; Br, 22.18; N, 11.67; O, 8.88

153436-54-5

PD153035 Hydrochloride;183322-45-4; PD153035;153436-54-5; 205195-07-9 (xHCl); 586347-97-9 (nitrate)

4705

White to off-white solid powder

1.5±0.1 g/cm3

472.1±45.0 °C at 760 mmHg

239.3±28.7 °C

0.0±1.2 mmHg at 25°C

1.679

4.08

1

5

4

22

360

0

BrC1=C([H])C([H])=C([H])C(=C1[H])N([H])C1C2=C([H])C(=C(C([H])=C2N=C([H])N=1)OC([H])([H])[H])OC([H])([H])[H]

LSPANGZZENHZNJ-UHFFFAOYSA-N

InChI=1S/C16H14BrN3O2/c1-21-14-7-12-13(8-15(14)22-2)18-9-19-16(12)20-11-5-3-4-10(17)6-11/h3-9H,1-2H3,(H,18,19,20)

N-(3-bromophenyl)-6,7-dimethoxyquinazolin-4-amine

PD153035; PD 153035; PD-153035; 153436-54-5; N-(3-bromophenyl)-6,7-dimethoxyquinazolin-4-amine; PD153,035; PD-153,035; pd 153,035; 4-(3-BROMOANILINO)-6,7-DIMETHOXYQUINAZOLINE; WHI-P-79; NSC-669364; ZM 252868; Tyrphostin AG 1517; AG 1517; ZM 252868;ZM-252868; ZM252868; SU-5271, SU 5271;SU5271;

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: ≥ 2.5 mg/mL (6.94 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 30% propylene glycol, 5% Tween 80, 65% D5W:30mg/mL

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