EGFR (IC50 = 0.7 μM)
AG 494 is a protein tyrosine kinase (PTK) inhibitor that selectively inhibits the EGF receptor (EGFR) kinase activity in cell-free assays. In vitro, it inhibits EGFR autophosphorylation with an IC50 of 1.2 μM. It poorly inhibits other tyrosine kinases such as the closely related HER-2/Neu receptor (IC50 > 100 μM) and the insulin receptor kinase (IC50 > 100 μM) [2].

AG 555 (100 μM) inhibits the virus's ability to integrate and synthesise viral proteins at both early and late stages of its life cycle[1].
Tyrphostins AG555 are very effective at stopping the growth of EGFR overexpressor cells because they block Cdk2 activation, which causes growth arrest of immortalized cells at G1-S and early S[2].
Tyrphostin AG 555 can bind phosphorylated Jun/ATF-2 at a novel intragenic regulatory sequence and activate the MAP kinase pathway to selectively suppress BPV-1 transcription[3].

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In a cell-free kinase assay using crude membrane extracts from DHER-14 cells (NIH-3T3 cells transfected with the human EGFR), AG 494 inhibited EGF-stimulated receptor autophosphorylation with an IC50 of 1.2 μM. It showed minimal inhibition of HER-2/Neu receptor autophosphorylation (IC50 > 100 μM) and insulin receptor kinase activity (IC50 > 100 μM) [2].
Unlike the quinazoline AG 1478, AG 494 did not inhibit EGF receptor autophosphorylation or the phosphorylation of endogenous substrates in intact DHER-14 cells, likely due to high intracellular ATP levels competing with the inhibitor for binding to the receptor's kinase site [2].
AG 494 exhibited potent antimitogenic activity in DHER-14 cells. It inhibited EGF-dependent DNA synthesis (³H-thymidine uptake) with an IC50 of approximately 10.5 μM. It also inhibited DNA synthesis stimulated by other mitogens, with IC50 values of approximately 5 μM for both TPA (phorbol ester) and lysophosphatidic acid (LPA)-dependent pathways [2].
In growth-arrested DHER-14 cells stimulated with EGF, AG 494 (at 25 μM) completely inhibited the activation of Cdk2, a key cell cycle regulator. This inhibition was dose-dependent, with an IC50 of approximately 5 μM. The compound was effective even when added 20 hours after EGF stimulation, indicating it blocks a downstream event in the cell cycle progression pathway [2].

EGFR Autophosphorylation Inhibition Assay: Crude membranes were prepared from DHER-14 cells. Membranes (2 μg/assay) were pre-incubated with EGF (50 nM) for 15 minutes at 4°C. The kinase reaction was initiated by adding the membrane mixture to a reaction buffer containing 2 μM ATP (2× Km), 1 μCi [γ-³²P] ATP, 4 mM MnCl₂, 24 mM MgCl₂, and various concentrations of AG 494 (dissolved in 10% DMSO/45% ethanol/45% water). The reaction was carried out at 4°C for 30 seconds and stopped by adding boiling SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE, and autophosphorylation of the EGFR was visualized by autoradiography. The ³²P-labeled band corresponding to the receptor was quantified by densitometry, and IC50 values were calculated [2].
HER-2/Neu and Insulin Receptor Kinase Inhibition Assays: Inhibition of HER-2/Neu receptor autophosphorylation was assessed using membranes prepared from NIH-3T3 cells transfected with the human HER-2/Neu receptor, following procedures similar to those for the EGFR assay. Inhibition of insulin receptor kinase was performed using wheatgerm agglutinin (WGA)-purified insulin receptor, with denatured enolase as a substrate, following previously described methods [2].

Cell Line: NIH/3T3 uninfected cells and NIH/3T3-Mo-MuLV chronically infected cells.
Concentration: 100 μM.
Incubation Time: 1 hour.
Result: Inhibited Mo-MuLV proviral DNA integration.

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³H-Thymidine Uptake Assay for DNA Synthesis: DHER-14 cells were seeded in 96-well plates at 7,000 cells/well and grown to confluence for 2 days. Cells were then serum-starved for 48 hours in DMEM containing 0.25% calf serum. They were stimulated with mitogens (20 nM EGF, 50 μM LPA, or 50 ng/ml TPA) for 16 hours. ³H-thymidine (0.5 μCi/ml) was added for the final 4 hours of stimulation. AG 494, dissolved in 10% DMSO, was added either 30 minutes before the mitogens (present for 20 hours) or during the last 4 hours along with the thymidine. The trichloroacetic acid-precipitable material was quantified by scintillation counting to measure DNA synthesis [2].
Cdk2 Kinase Activity Assay: DHER-14 cells were grown to confluence in 6-well plates and serum-starved for 3 days. Cells were then stimulated with 20 nM EGF for various time periods. AG 494 was added at specified times (e.g., 20-24 hours post-EGF stimulation). Cells were lysed in a cold IP buffer. Cdk2 was immunoprecipitated from the lysates using anti-Cdk2 antibodies and collected on protein A agarose beads. The immunoprecipitate was washed and then incubated in a kinase buffer containing 1 μCi [γ-³²P] ATP and 2.5 μg Histone H1 as a substrate for 20 minutes at 30°C. The reaction was stopped with SDS-PAGE sample buffer, and proteins were separated by gel electrophoresis. Histone H1 phosphorylation was visualized by autoradiography and quantified by densitometry to determine Cdk2 activity [2].
Immunoprecipitation and Immunoblot Analysis for EGFR Phosphorylation: DHER-14 cells were plated in 6-well dishes, grown to confluence, and serum-starved for 48 hours. AG 494 was added during the last 16 or 2 hours of starvation. Cells were then stimulated with 20 nM EGF for 2 minutes at 4°C. Lysis was performed, and EGFR was immunoprecipitated using a monoclonal antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-phosphotyrosine antibody (PT66) followed by [¹²⁵I]-labeled goat anti-mouse antibodies. Tyrosine phosphorylation of the EGFR was detected by autoradiography [2].

[1]. Tyrphostin AG-555 inhibits early and late stages of Moloney murine leukemia virus replication cycle. International Journal of Oncology. 1997.

[2]. Tyrphostin AG494 blocks Cdk2 activation Nir Osherov. FEBS Letters 410 (1997) 187-190.

[3]. Tyrphostin AG 555 Inhibits Bovine Papillomavirus Transcription by Changing the Ratio between E2 Transactivator/Repressor Function. Vol. 278, No. 39, Issue of September 26, pp. 37306–37313, 2003.

Tyrphostin B46 is a potent inhibitor of epidermal growth factor receptor kinase autophosphorylation. (National Cancer Institute)

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Background: AG 494 is a tyrphostin, a class of synthetic protein tyrosine kinase inhibitors. It was developed as a selective inhibitor of the EGF receptor kinase, active in cell-free assays but surprisingly inactive against the receptor in intact cells [2].
Mechanism of Action: AG 494 is an ATP-competitive inhibitor. Its failure to inhibit EGFR in intact cells is attributed to high intracellular ATP levels. Despite this, it retains potent antimitogenic activity. This study demonstrates that its primary mode of anti-proliferative action in cells is not through direct EGFR inhibition, but by blocking a downstream signaling event, specifically the activation of Cdk2. This positions AG 494 as a potential inhibitor of cell cycle progression [2].

C19H18N2O3

322.364

322.132

C, 70.79; H, 5.63; N, 8.69; O, 14.89

133550-34-2

133550-34-2

5328770

Light yellow to yellow solid powder

1.273g/cm3

621.1ºC at 760mmHg

329.4ºC

5.09E-16mmHg at 25°C

1.649

3.144

3

4

6

24

488

0

O=C(/C(/C#N)=C(\[H])/C1C([H])=C([H])C(=C(C=1[H])O[H])O[H])N([H])C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

GSQOBTOAOGXIFL-LFIBNONCSA-N

InChI=1S/C19H18N2O3/c20-13-16(11-15-8-9-17(22)18(23)12-15)19(24)21-10-4-7-14-5-2-1-3-6-14/h1-3,5-6,8-9,11-12,22-23H,4,7,10H2,(H,21,24)/b16-11+

(E)-2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-phenylpropyl)prop-2-enamide

AG555; AG 555; AG-555

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)

DMSO: ~64 mg/mL (~198.5 mM)
Ethanol: ~64 mg/mL (~198.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.76 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 25.0 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: ≥ 2.5 mg/mL (7.76 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)