EGFR (IC50 = 3 nM); HCV; EMCV

In chemically specified DMEM/F12 media, human lung (A549) and prostate (DU145) cancer cell lines are grown. AG-1478 (AG1478) is irreversible for growth regulation of these cell lines. Although AG-1478 is not able to totally stop the proliferation of A549 cells, it appears to be more effective at lower concentrations[1]. The angiotensin II-mediated production of TGF-β and fibronectin by cardiac fibroblasts is considerably 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 was used to determine how much both Polyfect (PF) and Superfect (SF) treatment boosted apoptosis in HEK 293 cells. Tempol, an antioxidant, dramatically decreased PF and SF dendrimer-mediated apoptosis. As a positive control, AG-1478 (AG1478) was utilized at a 10-fold higher dose (100 μM) than that of signaling studies, and it effectively caused apoptosis in HEK 293 cells[3].

---

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].

---

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]

---

---

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.

---

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.

---

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].

In both of the obese mouse models, the administration of AG-1478 (AG1478) dramatically lowers myocardial inflammation, fibrosis, apoptosis, and dysfunction. ApoE-/- mice are given oral gavage with AG -1478 (10 mg/kg/day) or 542 (10 mg/kg/day) for 8 weeks after being fed HFD for 8 weeks (ApoE-HFD). In vivo, HFD-induced cardiac EGFR phosphorylation is blocked by AG-1478 or 542 therapy, with no impact on plasma levels of total triglycerides (TG) or low density lipoprotein (LDL)[2]. EGFR phosphorylation is robustly and consistently elevated upon administration of EGF (10 nM), and this elevation can be inhibited by the known EGFR phosphorylation inhibitor AG-1478 (AG478). Although not as much as with AG1478, increasing dosages of Polyfect (PF) significantly reduce EGF-induced EGFR phosphorylation (p<0.05)[3].

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.

---

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.

---

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 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 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 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.

---

Dissolved in 100 mM Captisol; 1 mg/kg; i.p. injection

Female BALB/c nu/nu mice inoculated s.c. with A431 or U87MG.Δ2-7 tumor cells

[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 is a member of the class of quinazolines that is quinazoline substituted by methoxy groups at positions 6 and 7 and a (3-chlorophenyl)nitrilo group at position 4. It acts as an epidermal growth factor receptor antagonist. It has a role as an epidermal growth factor receptor antagonist, an antineoplastic agent, a geroprotector and an antiviral agent. It is a member of quinazolines, an aromatic ether and a member of monochlorobenzenes.
Tyrphostin AG 1478 is a member of the tyrphostin family of tyrosine kinase inhibitors, that selectively inhibits epidermal growth factor.

---

Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling-an important signaling cascade that regulates cell growth, survival and apoptosis- in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM dendrimers in nanomedicine.[3]

---

Summarizing, we here identify PI4KA as a novel cellular target of tyrphostin AG1478, a compound previously recognized only as an EGFR inhibitor and a Golgi apparatus-dispersing agent. We reveal that AG1478 exerts antiviral properties against EMCV and HCV and demonstrate that its mode of action involves inhibition of PI4KA activity. Our in vitro data suggested that AG1478 is a direct inhibitor of PI4KA; however, we cannot exclude the possibility that AG1478 targets PI4KA activity indirectly or a combination of both. The antiviral properties of AG1478 are most likely not linked to its effects on EGFR signaling, since AG1478 was shown to inhibit EGFR in the low nanomolar range (31), whereas inhibition of virus replication (and PI4KA activity) requires micromolar concentrations. Although it is unlikely that EGFR inhibition accounts for the antiviral activity of AG1478, it would be interesting to investigate in the future whether AL-9 (and other structurally related inhibitors) may also exhibit anti-EGFR properties. In conclusion, our study uncovers important cellular effects and antiviral properties of tyrphostin AG1478, a compound proposed earlier as a promising treatment in cancer chemotherapy. [4]

C16H15CL2N3O2

352.21

351.054

C, 54.56; H, 4.29; Cl, 20.13; N, 11.93; O, 9.08

170449-18-0

AG-1478;153436-53-4

3035187

Typically exists as solid at room temperature

1.337g/cm3

458.5ºC at 760mmHg

231.1ºC

1.37E-08mmHg at 25°C

4.919

2

5

4

23

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].Cl[H]

WDJDYIUSDDVWKB-UHFFFAOYSA-N

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

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)