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

PD 153035 has a 14 nM IC50 and prevents EGF-stimulated receptor autophosphorylation in human epidermoid carcinoma A431 cells[1]. Even at 50 μM, PD153035 has minimal impact on src tyrosine kinases, insulin receptor, PDGFR, FGFR, CSF-1 receptor, or CSF-1 receptor. When applied to fibroblasts or human epidermoid carcinoma cells, PD153035 quickly suppresses the autophosphorylation of the EGF receptor at low nanomolar concentrations. It also specifically prevents EGF-mediated cellular processes such as mitogenesis, early gene expression, and oncogenic transformation[2]. Starting at less than micromolar concentrations, PD 153035 inhibits the growth of EGF receptor-positive cell lines in a dose-dependent manner; in most cases, the IC50 is less than 1 pM[3].

PD 153035 levels after a single intraperitoneal (i.p.) dose of 80 mg/kg rise to 50 and 22 μM in the tumor and plasma within 15 minutes. While PD 153035's plasma levels drop below 1 μM after three hours, the drug stays at micromolar concentrations in tumors for at least twelve hours. Tumors quickly reduce 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.

Mice: PD153035 (80 mg/kg) or a vehicle is injected into mice, and after 20 and 180 minutes, the rumours are removed and extracts are made. Four iterations of the experiment are conducted, using two mice at each time point. ANOVA is used in each of the four experiments to compare the inhibition of EGF-stimulation by PD 153035[3].

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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]

[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 hydrochloride is obtained by conjugating PD-153035 with an equivalent amount of hydrochloric acid. It is 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 analogues of compound 32 is very steep. The IC50 values of some derivatives are 80 times higher than those predicted by the theory of simple addition binding energy. However, analogs with similar combinations of phenyl and quinazoline substituents do not show this “hyperadditivity” effect. Since some substituents that have a slight inactivating effect when used alone can produce a strong activating effect when used in the right combination, we speculate that some substituted analogs can induce a conformational change of the receptor upon binding. The substitutions at the 6 and 7 positions of quinazoline have a certain tolerance, so compound 32 is not the best inhibitor for inducing conformational change. The diethoxy derivative 56 [4-(3-bromoanilino)-6,7-diethoxyquinazoline] has an IC50 of 0.006 nM and is the most effective 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. The inhibitor is specific for EGF receptor tyrosine kinase and can only inhibit other purified tyrosine kinases at micromolar or higher concentrations. PD 153035 can rapidly inhibit the autophosphorylation of EGF receptors at low nanomolar concentrations in fibroblasts or human epidermal-like cancer cells and selectively block EGF-mediated cellular processes, including mitosis, early gene expression and oncogenic transformation. PD 153035 has a four to five order of magnitude higher inhibitory potency against isolated EGF receptor tyrosine kinases than other tyrosine kinase inhibitors and a three to four order of magnitude higher inhibitory potency against cellular phosphorylation. [2] PD153035 has been 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 PD153035 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, exogenous EGF-induced EGF receptor autophosphorylation was completely inhibited at PD153035 concentrations >75 nM. In contrast, PD153035 only reduced noreglin-dependent tyrosine 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, and its IC50 value was 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 achieved. In colony formation assays, the growth-inhibiting activity of PD153035 in endogenous (autocrine) ligand-driven cultures was correlated with the number of EGF receptors: the activity was higher in cells with higher EGF receptor expression, while the activity was very low in cells with normal EGF receptor expression but high HER2/neu levels. PD153035 also eliminated all growth effects mediated by exogenous EGF addition; this effect was reversible upon removal of the compound. Combination therapy with the anti-EGF receptor blocking monoclonal antibody C225 further enhanced the antitumor 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]

C₁₆H₁₅BRCLN₃O₂

396.67

395.003

C, 48.45; H, 3.81; Br, 20.14; Cl, 8.94; N, 10.59; O, 8.07

183322-45-4

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

11246488

Light yellow to yellow solid powder

472.1ºC at 760 mmHg

265°C(lit.)

239.3ºC

5.028

2

5

4

23

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].Cl[H]

ZJOKWAWPAPMNIM-UHFFFAOYSA-N

InChI=1S/C16H14BrN3O2.ClH/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);1H

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

SU-5271 HCl; PD 153035; PD-153035; SU 5271 HCl; SU5271 HCl; ZM-252868 HCl; ZM 252868 HCl; ZM252868 HCl; 183322-45-4; PD153035 hydrochloride; PD-153035 hydrochloride; N-(3-Bromophenyl)-6,7-dimethoxyquinazolin-4-amine hydrochloride; PD 153035 HYDROCHLORIDE; PD153,035 HCl; SU-5271 hydrochloride; AG 1517 hydrochloride; PD153,035; PD153,035 HCl; PD 153,035 HCl; PD-153035 HCl; PD 153,035 hydrochloride; PD-153,035 hydrochloride; PD153035 hydrochloride; ZM 252868; Tyrphostin AG 1517; AG 1517; 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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~0.5 mg/mL (~1.3 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.)