EGFR tyrosine kinase (IC50=0.7 μM).

AG 494 suppresses EGF-dependent DNA synthesis and Cdk2 activation in DHER-14 cells [2]. AG-494 both dramatically decreases and prevents NF-kB activation in H2O2-treated cells as well as silica-stimulated cells [4]. ALP activity produced by BMP9 is inhibited in a dose-dependent manner by AG-494 (3-9 μM; 5-7 days) [5].

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We have previously shown that the EGFR kinase selective tyrphostin AG494 fails to inhibit EGFR kinase in intact cells. Yet, AG494proved to inhibit EGF- or serum-induced cell proliferation (Osherov et al., J. Biol. Chem. 268 (1993) 11134–11142). In this preliminary communication we show that AG 494 as well as its close analogs AG 490 and AG 555 block Cdk2 activation. In contrast, AG 1478, a more selective EGFR kinase blocker which is also active as EGFR kinase blocker in intact cells, fails to do so. AG 494 exerts its full inhibitory activity on Cdk2 activation even when added 20 h subsequent to EGF addition when Cdk2 activation is maximal. The inhibitory activity on Cdk2 activation parallels its DNA synthesis inhibitory activity, strongly suggesting that its target is one of the molecular mechanisms involved in Cdk2 activation. AG 494 and its analogs may become useful lead compounds for the development of drugs aimed at the cell cycle machinery. [2]

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BMP9-induced osteogenic differentiation can be effectively blocked by EGFR inhibitors [4]
Epidermal growth factor initiates downstream events by activating its tyrosine kinase receptor EGFR (aka HER1). We sought to determine if commonly EGFR inhibitors would exert any effect on BMP9-induced osteogenic differentiation of MSCs. We tested four EGFR inhibitors, AG494, AG1478, Erlotinib and Gefitinib, and found all of them inhibited BMP9-induced ALP activity in a dose-dependent manner (Fig. 3A). Among the four tested inhibitors, Erlotinib and Gefitinib are clinically used as anticancer drugs, which were shown to be slightly more effective than AG494 and AG1478 in terms of inhibiting BMP9-induced ALP activity (Fig. 3A and B). Thus, these in vitro findings suggest that EGFR signalling may play an important role in BMP9-regulated osteogenic differentiation, although these inhibitors may also target other tyrosine kinase.

Inhibition of autophosphorylation [2]
Membranes (2 μg/assay) were pre-incubated with EGF (50 nM) in 50 mM HEPES (pH 7.4), 125 mM NaCl, for 15 min at 4°C. The assay was initiated by addition of 8 μl membranes to 12 μl reaction mixture containing 50 mM HEPES (pH 7.4), 125 mM NaCl, 2 μM ATP (=2 K m final), 1 μCi [32P]γ-ATP, 4 mM MnCl2, 24 mM MgCl2, and 4 μl tyrphostin dissolved in 10% Me2SO/45% ethanol/45% DDW (double-distilled water). The assay was conducted at 4°C and terminated after 30 s by the addition of 8 μl boiling SDS-PAGE sample buffer. Proteins were separated on 6% SDS-PAGE, and autoradiography performed. A single EGF inducible 32P-labeled band comigrating in the molecular weight of the receptor could then be detected. This band was scanned and quantified by densitometry. IC50 values were calculated using the E-Z fit program.
All reactions were performed rapidly in V-shaped 96-well plates using a multipipette. Reactions were all linear for 30 s. The exposure times of the autoradiograms were controlled so that film exposure was linear. Basal EGF-independent activity did not exceed 5% of the EGF-activated autophosphorylation of HER-1/EGF receptors. Inhibition of pp60c-src activity was essentially performed as described by using the DHER-14 cells as described in detail previously, using denatured enolase as a substrate. Inhibition of autophosphorylation of wheat-germ agglutinin (WGA) purified insulin-receptor was performed as described by Schechter et al. Inhibition of autophosphorylation of HER-2/Neu receptor was performed using membranes prepared from NIH-3T3 cells using published procedures ransfected with the human HER-2/Neu receptor as described for HER-1.

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Immunoprecipitation and Cdk2 kinase assays [2]
DHER-14 cells were grown to confluence for 48 h in Costar 6-well dishes in DMEM +10% calf serum as described. Cells were starved for 3 days. During the last 24 h, cells were activated by various mitogens (20 nM EGF; 50 ng/ml TPA phorbol ester) or 50 μM lysophosphatidic acid (LPA) for various time periods. Cells were incubated with tyrphostins as described for each experiment. Cells were washed once in PBS, then lysed on ice using 1 ml/well cold IP buffer (50 mM HEPES, 250 mM NaCl, 5 mM DTT, 0.25% NP-40, 10 mM NaF, and protease inhibitors). Lysates were incubated on ice for 30 min, then centrifuged 14 000×g for 15 min in a microfuge. Supernatants were transferred, and incubated for 2 h in the presence of a saturating concentration of 1 μg/eppendorf anti-Cdk2 antibodies. The immunocomplex was collected on protein A agarose beads (7.5 μl packed volume per eppendorf) and washed 3 times in IP buffer. The pellet was then washed a fourth time in kinase buffer (50 mM HEPES, 10 mM MgCl, 1 mM DTT, 1 μM cold ATP). All buffer was aspirated from the beads with a fine bore Hamilton syringe, and the kinase reaction initiated by the addition of 40 μl/eppendorf reaction mixture containing kinase buffer and 1 μCi/assay [γ-32P]ATP and 2.5 μg Histone H1 (freshly prepared) per assay. The reaction was performed for 20 min at 30°C, then stopped by addition of 12.5 μl/assay SDS-PAGE sample buffer ×4. Samples were separated on 10% SDS PAGE gels and exposed to film for ≈2 h at −70°C. Quantitation was performed by densitometry.

Inhibition of [3H]thymidine uptake [2]
Cells were seeded at 7000 cells/well in 96-well Costar dishes precoated with 1 μg/well human fibronectin. The cells were grown to confluence for 2 days. The medium was changed to DMEM containing 0.25% calf serum for 48 h and the cells were then incubated 16 h with either 20 nM EGF, 50 μM lysophosphatidic acid or 50 ng/ml TPA (phorbol ester). After 16 h, [3H]thymidine 0.5 μCi/ml was added for 4 h. Different concentrations of tyrphostins dissolved at ×100 concentration in 10% Me2SO were added either 30 min prior to the addition of mitogens, or during the last 4 h along with [3H]thymidine addition. The trichloroacetic acid- precipitable material was quantified by scintillation counting. Basal EGF-independent activity did not exceed 5% of mitogen-dependent [3H]thymidine uptake activity of DHER-14 cells. Tyrphostins were added either 30 min prior to EGF addition or at the time of [3H]thymidine addition. Thus AG494 and AG 1478 were present either for 20 or 4 h.

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Immunoprecipitation and immunoblot analysis [2]
Cells were plated at 2×105 cells/5 ml/well in Costar 6-well dishes, grown to confluence for 2 days and then starved in 2 ml/well DMEM containing 0.25% calf serum for 48 h. Tyrphostins dissolved at ×100 concentration in 10% Me2SO were added during the last 16 h or the last 2 h of starvation. EGF (20 nM) was then added and the plates were incubated for 2 min at 4°C with EGF (20 nM), during which time receptor autophosphorylation is linear. The reaction was terminated by addition of 1 ml/well stop lysis buffer containing 20 mM HEPES (pH 7.4), 125 mM NaCl, 1% Triton X-100, 5 mM NaF, 100 μM NaVO3, 200 μM ZnCl2, 1 mM EDTA, 2 mM EGTA, 1 mM PMSF, 10 μg/ml aprotinin, 5 μg/ml leupeptin. Immunoprecipitation using monoclonal antibody 108 was performed as described above. The immunocomplex was released in boiling sample buffer, and then samples were run on SDS-PAGE 5–15% gradient gels, transferred to nitrocellulose (ABN semi-dry blot), blocked for 30 min in TBST (Tris buffer saline (pH 7.4), 1% Tween-20, 5% bovine serum albumin), then probed with monoclonal anti-phosphotyrosine antibodies PT66 for 3 h at 40°C in TBST, washed 4 times in TBS, and then reproved with [125I]goat anti-mouse antibodies (1 μCi/ml). The blots were then washed 4 times in TBS, air dried and analyzed by autoradiography.

[1]. Tyrphostins. 2. Heterocyclic and alpha-substituted benzylidenemalononitrile tyrphostins as potent inhibitors of EGF receptor and ErbB2/neu tyrosine kinases. J Med Chem. 1991;34(6):1896-1907.

[2]. Tyrphostin AG 494 blocks Cdk2 activation. FEBS Lett. 1997;410(2-3):187-190.

[3]. Selective inhibition of the epidermal growth factor and HER2/neu receptors by tyrphostins. J Biol Chem. 1993 May 25;268(15):11134-42.

[4]. Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med. 2013;17(9):1160-1172.

[5]. SILICA-INDUCED NUCLEAR FACTOR- k B ACTIVATION: INVOLVEMENT OF REACTIVE OXYGEN SPECIES AND PROTEIN TYROSINE KINASE ACTIVATION. Journal of Toxicology and Environmental Health, Part A. Journal of Toxicology and Environmental Health, Part A. Volume 60, 2000 - Issue 1

Tyrphostin B48, a member of the tyrosine kinase inhibitor family, inhibits epidermal growth factor receptor (EGFR). We previously demonstrated that the EGFR-selective tyrosine kinase inhibitor AG494 failed to inhibit EGFR kinase in intact cells. However, AG494 has been shown to inhibit EGF- or serum-induced cell proliferation (Osherov et al., Journal of Biochemistry 268 (1993) 11134-11142). In this preliminary report, we found that AG494 and its analogues AG490 and AG555 can block Cdk2 activation. Conversely, the more selective EGFR kinase inhibitor AG1478 (which also exhibits EGFR kinase inhibitory activity in intact cells) does not. Even when Cdk2 activation reached its maximum (i.e., added 20 hours after EGF addition), AG494 completely inhibited Cdk2 activation. Its inhibitory activity against Cdk2 activation is parallel to its inhibitory activity against DNA synthesis, strongly suggesting that its target is one of the molecular mechanisms involved in Cdk2 activation. AG 494 and its analogues may become useful lead compounds for developing drugs targeting cell cycle mechanisms. [2] Mesenchymal stem cells (MSCs) are pluripotent progenitor cells that can differentiate into various lineages, including bone, cartilage, and adipose tissue. Epidermal growth factor (EGF) can stimulate cell growth, proliferation, and differentiation. EGF exerts its effects by binding with a high affinity to the epidermal growth factor receptor (EGFR) on the cell surface, stimulating the intrinsic protein tyrosine kinase activity of its receptor, thereby initiating a signal transduction cascade, causing various intracellular biochemical changes, and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in mesenchymal stem cells (MSCs). This study aims to investigate whether the EGF signaling pathway interacts with BMP9 and regulates BMP9-induced osteogenic differentiation. We found that EGF enhances the expression of early and late osteogenic markers induced by BMP9 in vitro, while EGFR inhibitors gefitinib and erlotinib, or receptor tyrosine kinase inhibitors AG-1478 and AG494, effectively inhibit this enhancement in a dose- and time-dependent manner. Furthermore, EGF significantly enhances BMP9-induced bone formation in mouse fetal limb explants. In vivo stem cell transplantation experiments showed that exogenous expression of EGF in MSCs effectively enhances BMP9-induced ectopic bone formation, resulting in larger and more mature bone masses. Interestingly, we found that while EGF can induce BMP9 expression in MSCs, EGFR expression is directly upregulated by BMP9 through the Smad1/5/8 signaling pathway. Therefore, the interaction between the EGF and BMP9 signaling pathways in MSCs may highlight their important roles in regulating osteogenic differentiation. Utilizing the synergistic effect between BMP9 and EGF holds promise for promoting osteogenic processes in regenerative medicine. [4] Nuclear factor-κB (NF-κB) is a multi-protein complex that can regulate a variety of inflammatory cytokines involved in the occurrence and development of silicosis. This study recorded the ability of silica exposure to induce NF-κB DNA binding activity in a mouse peritoneal macrophage line (RAW264.7 cells) in vitro and explored the role of reactive oxygen species (ROS) and/or protein tyrosine kinases in this activation process. In in vitro experiments, exposure of mouse macrophages to silica (100 µg/ml) doubled the amount of reactive oxygen species (ROS), which was manifested as enhanced chemiluminescence (CL) and activated NF-κB. Superoxide dismutase (SOD) completely inhibited silica-induced CL, while catalase inhibited it by 75%. Various antioxidants (catalase, superoxide dismutase, α-tocopherol, pyrrolidine dithiocarbamate, or N-acetylcysteine) could inhibit NF-κB activation. Further evidence suggests that ROS are involved in NF-κB activation: 1 mM H₂O₂ enhances NF-κB binding to DNA, while catalase inhibits this activation. Specific inhibitors of protein tyrosine kinases, such as hapbin A, genistein, and AG494, prevent NF-κB activation in silica-treated cells. Genistein and AG494 also reduce NF-κB activation in H₂O₂-treated cells. Results confirmed that in silica-exposed macrophages, the tyrosine phosphorylation levels of several cellular proteins (molecular weights of approximately 39, 58-70, and 103 kDa) were elevated, and genistein inhibited this silica-induced phosphorylation. Conversely, inhibitors of protein kinases A or C, such as H89, astrocytin, calciphosphoprotein C, and H7, did not significantly inhibit silica-induced NF-κB activation. The results suggest that reactive oxygen species (ROS) may play a role in silica-induced macrophage NF-κB activation, and that tyrosine kinase-mediated phosphorylation events may be involved in this activation process. [5]

C16H12N2O3

280.2781

280.084

C, 68.56; H, 4.32; N, 9.99; O, 17.12

133550-35-3

5328771

Yellow to brown solid powder

1.4±0.1 g/cm3

586.9±50.0 °C at 760 mmHg

249 °C(dec.)

308.8±30.1 °C

0.0±1.7 mmHg at 25°C

1.736

2.4

3

4

3

21

446

0

O([H])C1=C(C([H])=C([H])C(/C(/[H])=C(\C#N)/C(N([H])C2C([H])=C([H])C([H])=C([H])C=2[H])=O)=C1[H])O[H]

HKHOVJYOELRGMV-XYOKQWHBSA-N

InChI=1S/C16H12N2O3/c17-10-12(8-11-6-7-14(19)15(20)9-11)16(21)18-13-4-2-1-3-5-13/h1-9,19-20H,(H,18,21)/b12-8+

2E-cyano-3-(3,4-dihydroxyphenyl)-N-phenyl-2-propenamide

AG-494; AG 494; 133550-35-3; Tyrphostin B48; alpha-Cyano-(3,4-dihydroxy)-N-phenylcinnamide; (E)-2-cyano-3-(3,4-dihydroxyphenyl)-N-phenylacrylamide; 139087-53-9; AG494; Tyrphostin AG-494.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~356.79 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)