Factor Receptor Protein Tyrosine Kinase Research Articles

Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, and causes reversible fragmentation of the mitochondrial network.

Hyperosmotic stress can be encountered by the kidney and the skin, as well as during treatment of acute brain damage. It can lead to cell cycle arrest or apoptosis. Exactly how mammalian cells detect hyperosmolarity and how the cell chooses between cell cycle arrest or death remains to be established. It has been proposed that hyperosmolarity is detected directly by growth factor receptor protein tyrosine kinases. To investigate this, we tested whether growth factors and osmotic stress cooperate in the activation of signaling pathways. Receptors responded normally to the presence of growth factors, and we observed normal levels of GTP-bound Ras under hyperosmotic conditions. In contrast, activation of Raf, Akt, ERK1, ERK2, and c-Jun NH2-terminal kinase was strongly reduced. These observations suggest that hyperosmotic conditions block signaling directly downstream of active Ras. It is thought that apoptotic cell death due to environmental stress is initiated by cytochrome c release from the mitochondria. Visualization of cytochrome c using immunofluorescence showed that hypertonic conditions result in a breakup of the mitochondrial network, which is reestablished within 1 h after hypertonic medium is replaced with isotonic medium. When we carried out live imaging, we observed that the mitochondrial membrane potential disappeared immediately after the onset of hyperosmotic shock. Our observations provide new insights into the hypertonic stress response pathway. In addition, they show that signaling downstream of Ras and mitochondrial dynamics can easily be manipulated by the exposure of cells to hyperosmotic conditions.

Involvement of epidermal growth factor receptor-protein tyrosine kinase transactivation in endothelin-1-induced vascular contraction.

Endothelin-1 (ET-1) is one of the major inducers of vasospasm following subarachnoid hemorrhage (SAH). It is generally accepted that extracellular signal-regulated kinase 1 and 2 (ERK1/2) are involved in ET-1-induced vascular contraction. In addition, ET-1 transactivates epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK), which leads to ERK1/2 stimulation. Therefore, the authors examined whether EGFR-PTK transactivation contributes to ET-1-induced vascular contraction in this study. Mitogen-activated protein kinase inhibitor, PD98059, inhibited ET-1-induced ERK1/2 stimulation in rabbit basilar artery (BA) vascular smooth-muscle cells (VSMCs). Moreover, PD98059 inhibited ET-1-induced contraction of rabbit BA rings. A specific inhibitor of EGFR PTK, AG1478, inhibited ET-1-induced EGFR-PTK transactivation, ERK1/2 stimulation, and contraction of BA rings in a concentration-dependent manner. The concentration of AG1478 required for 50% inhibition of the ET-1-induced contraction of BA rings was similar to that for ET-1-induced EGFR-PTK transactivation. Furthermore, AG1478 also inhibited ET-1-induced BA vasospasm in vivo. The results indicate that EGFR-PTK transactivation pathway plays an important role in ET-1-induced vascular contraction.

Benzamides and benzamidines as specific inhibitors of epidermal growth factor receptor and v-Src protein tyrosine kinases

The benzamides 1 and the benzamidines 2 as well as the cyclic benzamidines 3 were designed and synthesized as the mimics of 4-anilinoquinazolines for an inhibitor of EGFR tyrosine kinase. The specific inhibitions of EGFR tyrosine kinase were observed in the benzamides 1c and 1d, and the benzamidine 2a, whereas the specific inhibitions of v-Src kinase were observed in the benzamide 1j and the benzamidine 2d at a 10 μg/mL concentration of compounds. The cyclic benzamidines 3a and 3b showed potent kinase inhibition of EGFR at a 1.0 μg/mL concentration. According to the docking simulation using the X-ray structure of EGFR kinase domain in complex with erlotinib, the LigScore2 scoring function value of erlotinib was calculated as 5.61, whereas that of the benzamide 1c was 5.05. In a similar manner, the LigScore2 value of the cyclic benzamidine 3a was calculated as 5.10.

Benzamides and benzamidines as specific inhibitors of epidermal growth factor receptor and v-Src protein tyrosine kinases

Benzamides and benzamidines as specific inhibitors of epidermal growth factor receptor and v-Src protein tyrosine kinases

Elucidating inhibitory models of the inhibitors of epidermal growth factor receptor by docking and 3D-QSAR

Epidermal growth factor receptor (EGFR) protein tyrosine kinases (PTKs) are attractive targets for anti-tumor drug design. Although thousands of their ligands have been studied as potential inhibitors against PTKs, there is no QSAR study that covers different kinds of inhibitors with observable structural diversity. However, by using this approach, we could mine far more useful information. Hence in order to better understand the binding model and the relationship between the physicochemical properties and the inhibitory activities of different kind of various inhibitors, molecular docking and 3D-QSAR, viz. CoMFA and CoMSIA, were combined to study 124 reported inhibitors with different scaffolds. Based on the docked binding conformations, highly reliable and predictive 3D-QSAR models were derived, which reveal how steric, electrostatic, and hydrophobic interactions contribute to inhibitors' bioactivities. This result also demonstrates that it is possible to include different kinds of inhibitors with observable structural diversity into one 3D-QSAR study. Therefore, this study not only casts light on binding mechanism between EGFR and its inhibitors, but also provides new hints for de novo design of new EGFR inhibitors with observable structural diversity.

A novel macrocyclic tetrapeptide mimetic that exhibits low-picomolar Grb2 SH2 domain-binding affinity

The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that participates in the signaling of numerous oncogenic growth factor receptor protein-tyrosine kinases (PTKs). Presented herein is a 5-methylindolyl-containing macrocyclic tetrapeptide mimetic ( 5) that binds to Grb2 SH2 domain protein with K d=75 pM. This represents the highest affinity yet reported for a synthetic inhibitor against any SH2 domain. In whole cell assays this novel analogue is able to effectively block the association of Grb2 to cognate cytoplasmic erbB-2 at IC 50<10 nM without prodrug derivatization or the addition of carrier peptide motifs. Anti-mitogenic effects against erbB-2-dependent breast cancers are achieved at non-cytotoxic concentrations ( IC 50=0.6 μ M). Macrocycle 5 may be representative of a new class of therapeutically relevant Grb2 SH2 domain-directed agents.

Involvement of extracellular Ca2+ influx and epidermal growth factor receptor tyrosine kinase transactivation in endothelin-1-induced arachidonic acid release.

1. Endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in vascular smooth muscle cells (VSMCs). These channels can be distinguished by their sensitivity to Ca(2+)-channel blockers, SK&F 96365 and LOE 908. LOE 908 is sensitive to NSCC-1 and NSCC-2, and SK&F 96365 is sensitive to NSCC-2 and SOCC. Moreover, these channels play essential roles in ET-1-induced epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) transactivation. The main purpose of the present study was to demonstrate the involvement of EGFR PTK transactivation in ET-1-induced arachidonic acid release in VSMCs. 2. Both SK&F 96365 and LOE 908 inhibited ET-1-induced arachidonic acid release with the IC(50) values correlated to those of ET-1-induced Ca(2+) influx. Moreover, combined treatment with these blockers abolished ET-1-induced arachidonic acid release. 3. AG1478, a specific inhibitor of EGFR PTK, inhibited ET-1-induced arachidonic acid release and extracellular signal-regulated kinase 1 and 2 (ERK1/2). The IC(50) values of AG1478 for ET-1-induced arachidonic acid release and ERK1/2 correlated well with those for ET-1-induced EGFR PTK transactivation. 4. Mitogen-activated protein kinase kinase inhibitor, PD 98059, inhibited ET-1-induced arachidonic acid release. The IC(50) values of PD 98059 for ET-1-induced arachidonic acid release were similar to those for ET-1-induced ERK1/2 activity. In contrast, PD 98059 failed to inhibit ET-1-induced EGFR PTK transactivation. 5. These results indicate that (1) extracellular Ca(2+) influx through NSCCs and SOCC plays important roles for ET-1-induced arachidonic acid release, (2) EGFR PTK transactivation/ERK1/2 pathways are involved in ET-1-induced arachidonic acid release.

Characterization of Ca2+ channels involved in ET-1-induced transactivation of EGF receptors.

The purpose of this study was to demonstrate the involvement of Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) in endothelin-1 (ET-1)-induced transactivation of epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) using the Ca(2+) channel blockers LOE-908 and SK&F-96365 in rabbit internal carotid artery vascular smooth muscle cells. ET-1-induced EGFR PTK transactivation was completely inhibited by AG-1478, which is a specific inhibitor of EGFR PTK. In the absence of extracellular Ca(2+), the magnitude of EGFR PTK transactivation was near the basal level. Based on sensitivity to nifedipine, which is a specific blocker of voltage-operated Ca(2+) channels (VOCCs), VOCCs have minor roles in EGFR PTK transactivation. In contrast, Ca(2+) influx through VICCs plays an important role in EGFR PTK transactivation. Moreover, based on the sensitivity of VICCs to SK&F-96365 and LOE-908, VICCs were shown to consist of two types of Ca(2+)-permeable nonselective cation channels (NSCCs), which are designated NSCC-1 and NSCC-2, and a store-operated Ca(2+) channel. In summary, Ca(2+) influx through VICCs plays an essential role in ET-1-induced EGFR PTK transactivation in rabbit internal carotid artery vascular smooth muscle cells.

Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression.

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol. 72:1593-1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol. 72:9835-9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused approximately 40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

Design, synthesis, and biological evaluation of a series of lavendustin A analogues that inhibit EGFR and Syk tyrosine kinases, as well as tubulin polymerization.

A series of N-alkylamide analogues of the lavendustin A pharmacophore were synthesized and tested for inhibition of the epidermal growth factor receptor (EGFR) protein tyrosine kinase and the nonreceptor protein tyrosine kinase Syk. Although several compounds in the series were effective inhibitors of both kinases, it seemed questionable whether their inhibitory effects on these kinases were responsible for the cytotoxic properties observed in a variety of human cancer cell cultures. Accordingly, a COMPARE analysis of the cytotoxicity profile of the most cytotoxic member of the series was performed, and the results indicated that its cytotoxicity profile was similar to that of antitubulin agents. This mechanism of action was supported by demonstrating that most compounds in the series were moderately effective as inhibitors of tubulin polymerization. This suggests that the lavendustin A analogues reported here, as well as some of the previously reported lavendustin A analogues, may be acting as cytotoxic agents by a mechanism involving the inhibition of tubulin polymerization.

Inhibition of obliterative bronchiolitis by platelet-derived growth factor receptor protein-tyrosine kinase inhibitor

Inhibition of obliterative bronchiolitis by platelet-derived growth factor receptor protein-tyrosine kinase inhibitor

Adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression.

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.

ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

A potent protein-tyrosine kinase inhibitor which selectively blocks proliferation of epidermal growth factor receptor-expressing tumor cells in vitro and in vivo.

A calculated 3-D model of the kinase domain of the epidermal growth factor receptor (EGF-R) protein-tyrosine kinase (PTK) was used to develop a pharmacophore model for ATP-competitive inhibitors and, subsequently, a new class of selective EGF-R kinase inhibitors. CGP 59326A, a highly selective and potent inhibitor of the EGF-R in vitro, inhibited the proliferation of EGF-R-expressing epithelial lines, while having little anti-proliferative activity against EGF-R-negative lines. In contrast to previously described inhibitors, CGP 59326A had potent and selective in vivo anti-tumor activity at well-tolerated doses against EGF-R-expressing tumors (e.g., ED50 of 0.78 to 1.5 mg/kg for inhibition of A431 tumor growth). CGP 59326A inhibited growth of human tumor xenografts expressing the EGF-R but showed little activity against EGF-R-negative xenografts. Combination of CGP 59326A with cytotoxic agents resulted in tumor regression and cures. The high selectivity and attractive biological profile of CGP 59326A suggest that it could have therapeutic value in the treatment of proliferative diseases which involve mitogenic signaling from the EGF-R.

Inhibitors of the epidermal growth factor receptor protein tyrosine kinase: a quantitative structure-activity relationship analysis.

Hansch and Free-Wilson analyses are described on a data set, 4-anilinoquinazolines [the analogues of 4-(3-bromo-anilino)-6,7-dimethoxy quinazoline: PD 153035], as inhibitors of the epidermal growth factor receptor protein tyrosine kinase. These analyses have helped to ascertain the role of different substituents in explaining the observed inhibitory activities. From both approaches, it is concluded that the combined electron-donating nature of R1- and R2-substitutions of the quinazoline ring and the electron-withdrawing nature of the X-substitution of the anilino-ring are beneficial for increasing the inhibition activity of a compound. Further, the symmetrical alkoxy substituents present at the R1- and R2-positions are also engaged in a steric interaction which was determined quantitatively through the parabolic relationship between the activity and combined molar refraction parameter, sigma MR of the substituents.

CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

AbstractCGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL-expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL-positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome-positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL-positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1.

The growth factor TGF-beta, bone morphogenetic proteins (BMPs) and related factors regulate cell proliferation, differentiation and apoptosis, controlling the development and maintenance of most tissues. Their signals are transmitted through the phosphorylation of the tumour-suppressor SMAD proteins by receptor protein serine/threonine kinases (RS/TKs), leading to the nuclear accumulation and transcriptional activity of SMAD proteins. Here we report that Smadl, which mediates BMP signals, is also a target of mitogenic growth-factor signalling through epidermal growth factor and hepatocyte growth factor receptor protein tyrosine kinases (RTKs). Phosphorylation occurs at specific serines within the region linking the inhibitory and effector domains of Smad1, and is catalysed by the Erk family of mitogen-activated protein kinases. In contrast to the BMP-stimulated phosphorylation of Smad1, which affects carboxy-terminal serines and induces nuclear accumulation of Smad1, Erk-mediated phosphorylation specifically inhibits the nuclear accumulation of Smad1. Thus, Smadl receives opposing regulatory inputs through RTKs and RS/TKs, and it is this balance that determines the level of Smad1 activity in the nucleus, and so possibly the role of Smad1 in the control of cell fate.

ChemInform Abstract: Synthesis of Modified Tripeptides and Tetrapeptides as Potential Bisubstrate Inhibitors of the Epidermal Growth Factor Receptor Protein Tyrosine Kinase.

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Synthesis of Modified Tripeptides and Tetrapeptides as potential bisubstrate inhibitors of the epidermal growth factor receptor protein tyrosine kinase

AbstractThe synthesis of a series of bisubstrate inhibitors of the epidermal growth factor receptor protein kinase (EGF‐R PTK) consisting of small pep tides linked covalently to adenosine via appropriate triphosphate substitutes is described. Boc‐Glu(OtBu)‐Tyr‐Leu‐OBzl (5) and Ac‐Glu(OtBu)‐Tyr‐Leu‐Arg(Pmc)‐NH2 (8; Pmc = 2,2,5,7,8‐pentamethylchroman‐6‐sulfonyl) were prepared by standard peptide chemistry, (Scheme 1), then modified at the OH group of tyrosine either with adipic anhydride or with 4‐(chlorosulfonyl)benzoic acid, 4‐(chlorosulfonyl)‐2‐hydroxybenzoic acid, or benzene‐1,4‐disulfonyldichloride (Scheme 2), and finally coupled with the 5′‐OH group of 2′,3′‐O‐isopropylideneadenosine (Scheme 3). In addition, N6‐[(benzyloxy)carbonyl]‐2′,3′‐O‐isopropylidene‐adenosine 5′‐(hydrogenhexanedioate) (26), an ATP substitute, was coupled with the morpholide of 5 (Scheme 4). Removal of the protecting groups gave the bisubstrate analogs 23, 24, and 28. The compounds synthesized were tested as inhibitors of the EGF‐R PTK. The most active bisubstrate‐type inhibitor was 24, composed of the tripeptide sequence H‐Glu‐Tyr‐Leu‐OBzl, the 2‐hydroxy‐4‐sulfonylbenzoyl moiety, and adenosine; it showed an IC50 value of 33 μM.