EGFR (IC50 = 3 nM); HCV; EMCV

AG-1478 (AG1478) is irreversible for controlling the growth of human lung (A549) and prostate (DU145) cancer cell lines that were grown in DMEM/F12 medium that had been chemically defined. Although AG-1478 is not able to totally stop the growth of A549 cells, it appears to be more effective at lower concentrations[1]. The angiotensin II-mediated synthesis of TGF-β and fibronectin by cardiac fibroblasts is significantly reduced when EGFR is inhibited by the specific tyrosine kinase inhibitor AG-1478 (AG1478). AG-1478, a small-molecule inhibitor with an IC50 of 4 nM, pharmacologically inhibits EGFR[2]. Flow cytometry shows that both Polyfect (PF) and Superfect (SF) treatments increase apoptosis in HEK 293 cells to a comparable degree. For both PF and SF, the antioxidant tempol significantly decreased dendrimer-mediated apoptosis. AG-1478 (AG1478) was used as a positive control and significantly induced apoptosis in HEK 293 cells at a 10-fold higher dose (100 μM) than used in signaling studies[3].

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We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true. [1]

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EGFR Inhibitors Prevented PA-induced Injury in H9C2 Cells [2]
To evaluate the effect of EGFR inhibitors on cardiomyocyte hypertrophy in vitro, H9C2 cells were pretreated with AG1478 (AG, 10 μM) or 542 (10 μM) for 2 h, and then exposed to PA (100 μM) for 6 h. Rhodamine-labled Phalloidin was used to assess the cell morphology and cell volume. Incubation of PA significantly increased H9C2 cell volume, while AG or 542 can reverse the cell morphology change (Fig. 6A,B). Meanwhile, the mRNA level of hypertrophic marker gene atrial natriuretic peptide (ANP) and profibrotic gene TGF-β in H9C2 cells were markedly decreased by AG or 542 pretreatment (Fig. 6C,D), suggesting these small molecule inhibitors prevent PA-induced cardiomyocyte hypertrophy and fibrosis.

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Genetic knockdown of EGFR Inhibited PA-induced Inflammatory Injury in H9C2 Cells [2]
To avoid the non-specificity of small-molecule inhibitors and validate the role of EGFR, we transfected the H9C2 cells with specific small-interfering RNA to down-regulate EGFR expression (si-EGFR). As shown in Fig. 7A, transfection of si-EGFR under 100 μM PA treatment significantly decreases EGFR protein expression in H9C2 cells, which further led to a decreased gene expression level of TNF-ɑ and ANP, and reduced caspase-3 activity in PA-stimulated H9c2 cells (Fig. 7B–D). These results, together with our observation of the intracellular effect of 542/AG1478, confirmed that EGFR plays an important role in mediating hyperlipidemia-induced cardiac injury. To mimic the clinical setting, we further evaluated the protective effects of EGFR inhibition against the deleterious actions of PA in H9c2 cells, which were already exposed to PA (as a treatment). The results in the supplementary Fig. S3 showed that post-treatment with EGFR inhibitors AG or 542 also decreased PA-increased TNF-α and ANP levels.

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Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent [4].

The administration of AG-1478 (AG1478) in both of the obese mouse models significantly reduces apoptosis, fibrosis, inflammation, and cardiac dysfunction. The ApoE-HFD protocol involves feeding ApoE^-/- mice HFD for the first 8 weeks, followed by oral gavage administration of AG-1478 (10 mg/kg/day) or 542 (10 mg/kg/day) for an additional 8 weeks. Low density lipoprotein (LDL) and total triglyceride (TG) levels in plasma are unaffected by AG-1478 or 542 treatment, which inhibits HFD-induced cardiac EGFR phosphorylation in vivo[2]. A well-known EGFR phosphorylation inhibitor called AG-1478 (AG478) can prevent the strong and consistent increase in EGFR phosphorylation that occurs when EGF (10 nM) is administered.

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The in vivo studies using both wild type (WT) and apolipoprotein E (ApoE) knockout mice fed with high fat diet (HFD) showed the beneficial effects of small-molecule EGFR inhibitors, AG1478 and 542, against obesity-induced myocardial injury. Administration of AG1478 and 542 significantly reduced myocardial inflammation, fibrosis, apoptosis, and dysfunction in both two obese mouse models. In vitro, EGFR signaling was blocked by either siRNA silencing or small-molecule EGFR inhibitors in palmitic acid (PA)-stimulated cardiomyocytes. EGFR inhibition attenuated PA-induced inflammatory response and apoptosis in H9C2 cells. Furthermore, we found that PA-induced EGFR activation was mediated by the upstream TLR4 and c-Src. This study has confirmed the detrimental effect of EGFR activation in the pathogenesis of obesity-induced cardiac inflammatory injuries in experimental mice, and has demonstrated the TLR4/c-Src-mediated mechanisms for PA-induced EGFR activation. Our data suggest that EGFR may be a therapeutic target for obesity-related cardiovascular diseases[2].

AG-1478 has an IC50 of >100 μM and is highly selective against ErbB2 and PDGFR. In U87MG cells, AG-1478 preferentially inhibits truncated EGFR expression (IC50 = 8.7 μM) over endogenous wt EGFR expression (IC50 = 34.6 μM and 48.4 μM, respectively), and inhibits DNA synthesis (IC50 = 4.6 μM, 19.67 μM, and 35.2 μM, respectively). Additionally, compared to endogenous or overexpressed exogenous wt EGFR, AG-1478 preferentially inhibits the tyrosine kinase activity and autophosphorylation of the ΔEGFR. In the VSMC, AG-1478 (0.25 μM) eliminates the MAPK activation caused by Ang II, a Ca2+ ionophore, and EGF, but not by a phorbol ester or platelet-derived growth factor-BB. With IC50 values of 0.07 μM and 0.2 μM, respectively, AG-1478 suppresses the EGF-induced mitogenesis of the BaF/ERX and LIM1215 cells. The ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 can be inhibited by AG1478, with a greater effect on ABCG2.

A549 (CCL-185) and DU145 (HTB-81) cells are seeded at a density of 4×10³ cells/well in either DMEM (A549 cells) or MEM (DU145 cells) on 96-well plates. After a 24-hour incubation period, serum-free DMEM/F12 (1:1) enhanced with albumin (0.5 mg/mL), sodium selenite (2 ng/mL), and transferrin (5 mg/mL) is substituted for the culture medium (DMEM/F12+). Day 0 of the incubation period is followed by a replacement of the medium with serum-free DMEM/F12+ containing tyrosine kinase inhibitors (AG494, AG-1478) at concentrations of 1–20 μM and 0.1–8 μM, respectively. For the next twenty-four hours, the incubation is maintained at 37°C in a humidified environment. Tyrphostins' impact on target cell proliferation is assessed using the MTT assay and the modified crystal violet staining method (CV). Tecan multiscan plate recorder is used to measure absorbance[1].

Mice: Four weight-matched groups of 28 C57BL/6 or ApoE^-/- mice are created at random. As a normal control group (ApoE-LF), seven mice are fed a standard animal low-fat diet consisting of 10 kcal% fat, 20 kcal% protein, and 70 kcal% carbohydrates. The remaining twenty-one mice are fed a high-fat diet consisting of 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates for a period of 16 weeks. AG1478 or 542 are given orally via gavage starting in the 9^th week, with a daily dose of 10 mg/kg for the following eight weeks. Only the vehicle (1% CMC-Na solution) is gavaged on mice in the Control and HFD groups. To ascertain the pathologic cardiac hypertrophy, doppler analysis is carried out the day prior to the sacrifice of ApoE^-/- mice.

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Rats: In this study, five groups of male Wistar rats, weighing approximately 300g, were utilized. Animals in Group 1 that are not diabetic (Control, C) The second group received a single intraperitoneal (i.p.) injection of C+PF (10 mg/kg). Groups 4 and 3 are C+AG-1478 (1 mg/kg i.p.) and C+SF (10 mg/kg i.p.). Group 5: Rats given a single intraperitoneal injection of streptozotocin (55 mg/kg body weight) for four weeks to induce diabetes (D); Group 6: D+PF (10 mg/kg i.p.) Groups 7 and 8 consist of D+SF (10 mg/kg i.p.) and D+AG-1478 (1 mg/kg i.p.). The treatments for dendrimer and AG1478 are given as a single dose 24 hours before sacrifice. Before the animals are killed, measurements of the rats' basal glucose levels and body weight are taken both before and after the treatments. Blood glucose levels are measured using an automated blood glucose analyzer, and as in earlier research, rats with blood glucose levels greater than 250 mg/dL (roughly 14 mM) are classified as diabetic.

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High Fat Diet-fed Animal Experiments [2]
After an acclimatization period of one week, 28 C57BL/6 or ApoE−/− mice were randomly divided into four weight-matched groups. 7 mice were fed with standard animal low-fat diet containing 10 kcal.% fat, 20 kcal.% protein and 70 kcal.% carbohydrate (MediScience Diets Co. LTD, Yangzhou, China, Cat. #MD12031) served as a normal control group (Control or ApoE-LF), while the remaining 21 mice were fed with high-fat diet containing 60 kcal.% fat, 20 kcal.% protein and 20 kcal.% carbohydrate (HFD, MediScience Diets Co. LTD, Yangzhou, China, Cat. #MD12033) for 16 weeks. Since 9th week, AG1478 or 542 were given daily by oral gavage at a dose of 10 mg/kg/day for the next 8 weeks. Mice in the Control and HFD groups were gavaged with vehicle (1% CMC-Na solution) only. At the day before the sacrifice of ApoE−/− mice, doppler analysis was performed to determine the pathologic cardiac hypertrophy. Mice was anesthetized with isoflurane, echocardiography was performed by SONOS 5500 ultrasound (Philips Electronics, Amsterdam, Netherland) with a 15-MHz linear array ultrasound transducer. At the end of experimental period, all the animals were sacrificed by cervical decapitation. The body weight was recorded. Blood samples were collected and centrifuged at 4 °C at 3000 rpm for 10 min for collecting the serum. The heart was excised aseptically, blotted dry and the weight was recorded followed by immediate freezing in liquid nitrogen and then stored at −80 °C before further analyses.

[1]. The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells. Folia Histochem Cytobiol. 2012 Jul 5;50(2):186-95.

[2]. EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice. Sci Rep. 2016 Apr 18;6:24580.

[3]. Cationic Polyamidoamine Dendrimers as Modulators of EGFR Signaling In Vitro and In Vivo. PLoS One. 2015 Jul 13;10(7):e0132215.

[4]. Tyrphostin AG1478 Inhibits Encephalomyocarditis Virus and Hepatitis C Virus by Targeting Phosphatidylinositol 4-Kinase IIIα. Antimicrob Agents Chemother. 2016 Sep 23;60(10):6402-6.

Tyrphostin AG1478 belongs to the quinazoline class of compounds, with its quinazoline ring substituted with methoxy groups at positions 6 and 7 and with a (3-chlorophenyl)nitroso group at position 4. It is an epidermal growth factor receptor antagonist. Tyrphostin AG1478 possesses a variety of pharmacological activities, including epidermal growth factor receptor antagonist, antitumor drug, anti-aging drug, and antiviral drug. It belongs to the quinazoline, aromatic ether, and monochlorobenzene classes of compounds. Tyrphostin AG1478 is a member of the Tyrphostin family of tyrosine kinase inhibitors, selectively inhibiting epidermal growth factor. Cationic polyamide amines (PAMAMs) are a class of branched spherical polymers currently under investigation for various applications in nanomedicine, including nucleic acid drug delivery. Emerging evidence suggests they possess intrinsic biological and toxicological effects, but little is known about their interactions with signal transduction pathways. We have previously demonstrated that activated (fragmented) generation 6 (G) PAMAM dendrimer Superfect (SF) can stimulate epidermal growth factor receptor (EGFR) tyrosine kinase signaling in cultured human embryonic kidney (HEK 293) cells—an important signaling cascade that regulates cell growth, survival, and apoptosis. This study first investigated the effects of inactivated (intact) G6 PAMAM dendrimer Polyfect (PF) on the EGFR tyrosine kinase signaling pathway in cultured HEK 293 cells (regulating kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) via extracellular signaling), and then compared the in vivo effects of a single intraperitoneal injection of PF or SF on the EGFR signaling pathway in the kidneys of normal and diabetic male Wistar rats. The results showed that Polyfect had a dose- and time-dependent inhibitory effect on phosphorylation of EGFR, ERK1/2 and p38 MAPK in HEK-293 cells, similar to the effect of the selective EGFR inhibitor AG1478. After injecting the dendritic polymer into non-diabetic or diabetic animals for 24 hours, PF inhibited EGFR phosphorylation in the kidneys of both groups of animals, while SF stimulated EGFR phosphorylation. The inhibition of EGFR phosphorylation mediated by PF and the apoptosis of HEK 293 cells mediated by SF or PF could be significantly reversed by combined treatment with antioxidants such as temozolomide, indicating that both effects involve oxidative stress-dependent mechanisms. These results are the first to show that SF and PF PAMAM dendritic polymers can differentially modulate important EGFR signaling pathways in vivo and may represent a new class of EGFR regulators. These findings may have important clinical significance for the application of PAMAM dendritic polymers in nanomedicine. [3] In summary, we have identified PI4KA as a new cellular target of the tyrosine kinase inhibitor AG1478. AG1478 was previously thought to be only an EGFR inhibitor and Golgi dispersant. We found that AG1478 has antiviral activity against EMCV and HCV and confirmed that its mechanism of action involves the inhibition of PI4KA activity. Our in vitro experimental data suggest that AG1478 is a direct inhibitor of PI4KA; however, we cannot rule out the possibility that AG1478 acts indirectly or through both pathways on PI4KA activity. The antiviral activity of AG1478 is likely unrelated to its effect on the EGFR signaling pathway, as AG1478 can inhibit EGFR at low nanomolar concentrations (31), while inhibition of viral replication (and PI4KA activity) requires micromolar concentrations. Although EGFR inhibition is unlikely to be the cause of the antiviral activity of AG1478, it would be meaningful to investigate whether AL-9 (and other structure-related inhibitors) also have anti-EGFR properties. In summary, our study reveals important cellular effects and antiviral properties of the tyrosine kinase inhibitor AG1478, which was previously considered a promising cancer chemotherapy drug. [4]

C16H14CLN3O2

315.75

315.077

C, 60.86; H, 4.47; Cl, 11.23; N, 13.31; O, 10.13

153436-53-4

AG-1478 hydrochloride;170449-18-0

2051

White to off-white solid powder

1.3±0.1 g/cm3

458.5±45.0 °C at 760 mmHg

247 °C(dec.)

231.1±28.7 °C

0.0±1.1 mmHg at 25°C

1.668

3.77

1

5

4

22

360

0

ClC1=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]

GFNNBHLJANVSQV-UHFFFAOYSA-N

InChI=1S/C16H14ClN3O2/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-chlorophenyl)-6,7-dimethoxyquinazolin-4-amine

Tyrphostin AG-1478; 153436-53-4; AG-1478; Tyrphostin AG 1478; N-(3-chlorophenyl)-6,7-dimethoxyquinazolin-4-amine; 175178-82-2; Tyrphostin AG-1478; 4-(3-Chloroanilino)-6,7-dimethoxyquinazoline; 4-Quinazolinamine, N-(3-chlorophenyl)-6,7-dimethoxy-; AG1478; Tyrphostin AG 1478; NSC 693255; NSC-693255; NSC693255

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: ≥ 2.08 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.59 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 20.8 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 3: ≥ 2.08 mg/mL (6.59 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 15% Captisol: 30mg/mL

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Solubility in Formulation 5: 5 mg/mL (15.84 mM) in 1% CMC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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