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 a hydrochloride obtained by combining PD-153035 with one equivalent of hydrochloric acid. It has a role as an epidermal growth factor receptor antagonist and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It contains a PD-153035(1+).
AG 1517 is a quinazoline derivative that selectively inhibits EGFR kinase activity and suppresses the growth of psoriatic keratinocytes.

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4-(3-Bromoanilino)-6,7-dimethoxyquinazoline (32, PD 153035) is a very potent inhibitor (IC50 0.025 nM) of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), binding competitively at the ATP site. Structure-activity relationships for close analogues of 32 are very steep. Some derivatives have IC50s up to 80-fold better than predicted from simple additive binding energy arguments, yet analogues possessing combinations of similar phenyl and quinazoline substituents do not show this "supra-additive" effect. Because some substituents which are mildly deactivating by themselves can be strongly activating when used in the correct combinations, it is proposed that certain substituted analogues possess the ability to induce a change in the conformation of the receptor when they bind. There is some bulk tolerance for substitution in the 6- and 7-positions of the quinazoline, so that 32 is not the optimal inhibitor for the induced conformation. The diethoxy derivative 56 [4-(3-bromoanilino)-6,7-diethoxyquinazoline] shows an IC50 of 0.006 nM, making it the most potent inhibitor of the tyrosine kinase activity of the EGFR yet reported.[1]

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A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.[2]

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PD153035 is reported to be a specific and potent inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase and, to a lesser degree, of the closely related HER2/neu receptor. We show that PD153035 inhibits EGF-dependent EGF receptor phosphorylation and suppresses the proliferation and clonogenicity of a wide panel of EGF receptor-overexpressing human cancer cell lines. EGF receptor autophosphorylation in response to exogenous EGF was completely inhibited at PD153035 concentrations of >75 nM in cells overexpressing the EGF receptor. In contrast, PD153035 only reduced heregulin-dependent tyrosine phosphorylation in HER2/neu-overexpressing cell lines at significantly higher concentrations (1400-2800 nM). PD153035 exposure did not affect the expression of either EGF receptors or HER2/neu. PD153035 caused a dose-dependent growth inhibition of EGF receptor-overexpressing cell lines at low micromolar concentrations, and the IC50 in monolayer cultures was less than 1 microM in most cell lines tested. At doses of up to 2.5 microM, the IC50 for HER2/neu-overexpressing cells was not reached. In colony-forming assays, the PD153035 growth-inhibitory activity in cultures driven by endogenous (autocrine) ligand was correlated with EGF receptor number, with higher activity in cells expressing higher numbers of EGF receptors and only minimal activity in cells expressing normal numbers of EGF receptors but high HER2/neu levels. PD153053 also abolished all growth effects mediated by the addition of exogenous EGF; this condition could be reversed upon removal of the compound. Cotreatment with C225, an anti-EGF receptor-blocking monoclonal antibody, further enhanced the antitumor activity of PD153035, suggesting mechanisms of action for C225 other than competition with ligand binding. This latter finding also suggests that combined anti-EGF receptor strategies may be of enhanced benefit against tumors with high levels of 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.)