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At least 775 records · Page 43Linked to original sources
PubMed1994

Alteration in protein-tyrosine phosphatase of rat epithelial cells by RSV-transformation: application of phospho-tyrosyl glutamine synthetase to the study of protein-tyrosine phosphatase.

We prepared phospho-tyrosyl glutamine synthetase (P-GS) with suppressed activity from a highly adenylylated glutamine synthetase and applied it to the assay of protein-tyrosine phosphatase (PTPase) present in non-malignant rat liver cells (BRL) by RSV-transformation. The maximum PTPase activity toward P-GS was observed at neutral pH (pH 7.5-8.0) in the soluble and particulate fractions prepared from both BRL and RSV-transformed (RSV-BRL) cells. At low activity levels (< about 0.3 U), the PTPase activity in each fraction was proportional to the sample protein concentration (A280) and the specific activity of PTPase in the soluble fraction of BRL cells was about twofold higher than that in the soluble fraction of BRL cells, while those in particulate fractions of BRL and RSV-BRL cells were almost the same as each other. Soluble fractions of BRL and RSV-BRL were subjected to molecular-sieve and anion-exchange chromatographies. One major PTPase activity, with an M(r) of about 40,000 (40k), was detected in the BRL soluble fraction, and two were detected in the RSV-BRL soluble fraction with M(r)s of about 40k and 60k. The 40k PTPases in BRL and RSV-BRL had the same profiles on anion-exchange chromatography, but the 60k PTPase in RSV-BRL cells showed a different profile. We suggest that the RSV-transformation of BRL cells induced the appearance of the 60k PTPase in the soluble fraction.

Animals↗
PubMed1995

c-Cbl is inducibly tyrosine-phosphorylated by epidermal growth factor stimulation in fibroblasts, and constitutively tyrosine-phosphorylated and associated with v-Src in v-src-transformed fibroblasts.

The c-cbl gene was cloned as the cellular homolog of the v-cbl oncogene that is the transforming component of a murine tumorigenic retrovirus, CAS NS-1, though the biological roles of c-Cbl remain to be elucidated. We have previously reported that c-Cbl is implicated in the signal transduction triggered by granulocyte-macrophage colony-stimulating factor or erythropoietin in hematopoietic cells. Here, we observed tyrosine phosphorylation of C-cbl in cells expressing epidermal growth factor receptor depending on EGF stimulation and in v-src transformed cells. Furthermore, c-Cbl was revealed to associate with v-Src in vivo. By means of binding experiments using glutathione S-transferase fusion proteins, we have found that the SH2 and SH3 domains of many proteins bind to c-Cbl. These findings strongly suggest that c-Cbl is implicated in a wide variety of signal transduction pathways, including those of EGF receptor and Src protein, as well as in the signaling pathways of hematopoietic cells.

3T3 Cells↗
PubMed1988

Predicted amino acid sequence of bovine tyrosine hydroxylase and its similarity to tyrosine hydroxylases from other species.

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.

Amino Acid Sequence↗
PubMed1988

Analysis of the 5' region of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms.

A single human gene has been described to encode multiple tyrosine hydroxylase (TH) mRNAs. The study of this variation has been extended by S1 mapping experiments and by analysis of the 5' region of the TH gene. Four different mRNAs were found to originate solely from alternative splicing of two exons. Comparison of the 5' flanking regions of human and rat genes discloses several highly conserved segments, likely to play an important role in the regulation of TH gene expression.

Base Sequence↗
PubMed2005

Induction of tyrosine hydroxylase in the locus coeruleus of transgenic mice in response to stress or nicotine treatment: lack of activation of tyrosine hydroxylase promoter activity.

Prolonged stress or chronic nicotine administration leads to induction of tyrosine hydroxylase (TH) in adrenal medulla and locus coeruleus (LC) of the rat. In this study we use mice that express a transgene encoding 4.5 kb of TH gene 5'-flanking region fused upstream of the reporter gene, human alkaline phosphatase (hAP) to test whether TH gene promoter activity is stimulated by immobilization stress, cold exposure or nicotine administration in adrenal medulla and LC. TH-hAP transgene expression is increased in response to all three stimuli in the adrenal medulla. In contrast, TH-hAP expression does not increase in response to either immobilization stress or nicotine administration in the LC and only a small induction of LC TH-hAP mRNA is observed in response to cold exposure. TH mRNA is induced 2-3 fold and TH activity is increased significantly by all three stimuli in both the adrenal and LC. These results support the hypothesis that TH expression is induced by stress or nicotine treatment in both the adrenal medulla and LC of the mouse. The induction in the adrenal is dependent primarily on transcriptional mechanisms, whereas that in the LC is apparently dependent primarily on post-transcriptional mechanisms.

Adrenal Medulla↗
PubMed1994

Skeletal muscle protein tyrosine phosphatase activity and tyrosine phosphatase 1B protein content are associated with insulin action and resistance.

Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent diabetes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was reduced 21% (P < 0.05) and 22% (P < 0.005) in obese nondiabetic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose disposal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of approximately 10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPase1B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased enzyme activity may have a role in the insulin resistance of obesity and NIDDM.

Adult↗
PubMed2000

Tyrosine kinase signalling in breast cancer: modulation of tyrosine kinase signalling in human breast cancer through altered expression of signalling intermediates.

The past decade has seen the definition of key signalling pathways downstream of receptor tyrosine kinases (RTKs) in terms of their components and the protein-protein interactions that facilitate signal transduction. Given the strong evidence that links signalling by certain families of RTKs to the progression of breast cancer, it is not surprising that the expression profile of key downstream signalling intermediates in this disease has also come under scrutiny, particularly because some exhibit transforming potential or amplify mitogenic signalling pathways when they are overexpressed. Reflecting the diverse cellular processes regulated by RTKs, it is now clear that altered expression of such signalling proteins in breast cancer may influence not only cellular proliferation (eg Grb2) but also the invasive properties of the cancer cells (eg EMS1/cortactin).

Adaptor Proteins, Signal Transducing↗
PubMed2005

Dual requirement for the Ig alpha immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Ig alpha ITAM tyrosine in supporting Ig alpha beta-mediated B cell development.

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Antitumor effects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase.

PURPOSE: Vascular endothelial growth factor (VEGF) is a major mitogen for endothelial cells and enhances vascular permeability. Enhanced VEGF secretion is found in human cancers and correlates with increased tumor neovascularization. ZD6474 is a p.o. bioavailable, VEGF flk-1/KDR receptor (VEGFR-2) tyrosine kinase inhibitor with antitumor activity in many human cancer xenografts and is currently in Phase I clinical development. EXPERIMENTAL DESIGN: We tested the effects of ZD6474 on EGFR phosphorylation in cell expressing functional epidermal growth factor receptor (EGFR) and the antiproliferative and the proapoptotic activity of ZD6474 alone or in combination taxanes in human cancer cell lines with functional EGFR but lacking VEGFR-2. The antitumor activity of this drug was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: ZD6474 causes a dose-dependent inhibition of EGFR phosphorylation in mouse NIH-EGFR fibroblasts and human MCF-10A ras breast cancer cells, two cell lines that overexpress the human EGFR. ZD6474 treatment resulted in a dose-dependent inhibition of soft agar growth in seven human cell lines (breast, colon, gastric, and ovarian) with functional EGFR but lacking VEGFR-2. A dose-dependent supra-additive effect in growth inhibition and in apoptosis in vitro was observed by the combined treatment with ZD6474 and paclitaxel or docetaxel. ZD6474 treatment of nude mice bearing palpable GEO colon cancer xenografts (which are sensitive to inhibition of EGFR signaling) induced dose-dependent tumor growth inhibition. Immunohistochemical analysis revealed a significant dose-dependent reduction of neoangiogenesis. The antitumor activity of ZD6474 in GEO tumor xenografts was also found to be enhanced when combined with paclitaxel. Tumor regression was observed in all mice after treatment with ZD6474 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of angiogenesis. Six of 20 mice had no histological evidence of tumors after treatment with ZD6474 plus paclitaxel. CONCLUSIONS: This study suggests that in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling, ZD6474 may also be able to inhibit cancer cell growth by blocking EGFR autocrine signaling. These results provide also a rationale for the clinical evaluation of ZD6474 combined with taxanes in cancer patients.

Agar↗
PubMed2004

ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor with additional activity against epidermal growth factor receptor tyrosine kinase, inhibits orthotopic growth and angiogenesis of gastric cancer.

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) have been strongly implicated in the growth and metastasis of gastric cancer. The purpose of this study was to examine the effects of ZD6474, an inhibitor of inhibitor of VEGF receptor (VEGFR) tyrosine kinase with additional activity against EGF receptor (EGFR), on tumor growth and angiogenesis in an orthotopic model of gastric cancer. In vitro, ZD6474 inhibited human umbilical vascular endothelial cell and TMK-1 human gastric tumor cell proliferation in a dose-dependent fashion. EGF-mediated activation of EGFR and Erk-1/2 was decreased in tumor cells after ZD6474 treatment. In addition, VEGF-mediated activation of VEGFR2 and Erk-1/2 was decreased in human umbilical vascular endothelial cells. TMK-1 human gastric adenocarcinoma cells were injected into the gastric wall of nude mice. ZD6474 therapy was initiated on day 10. Mice (n = 14 per group) were treated p.o. with (a) 1% Tween 80 (control), (b) 50 mg/kg/d ZD6474, or (c) 100 mg/kg/d ZD6474. Mice were sacrificed on day 33. Tumors from each group were stained for markers of blood vessels, pericytes, proliferation, and apoptosis. ZD6474 at both 50 and 100 mg/kg/d led to marked inhibition of tumor growth (P < 0.05). ZD6474 reduced tumor cell proliferation by 48% in the 50 mg/kg/d group and 65% in the 100 mg/kg/d group (P < 0.03) and increased tumor cell apoptosis (P < 0.001) in vivo. ZD6474 led to a 69% decrease in microvessel density in the 50 mg/kg/d group (P < 0.001) and a 62% decrease in the 100 mg/kg/d group (P < 0.001). Although microvessel density was decreased by ZD6474, the remaining vessels showed a relatively higher percentage of pericyte coverage (3-fold increase; P < 0.001), perhaps reflecting selective loss of uncovered vessels in the ZD6474 group. In conclusion, therapies such as ZD6474 that target two distinct aspects of tumor growth, angiogenesis and tumor cell proliferation, warrant further investigation.

Angiogenesis Inhibitors↗
PubMed1995

Association between the melanoma-inducing receptor tyrosine kinase Xmrk and src family tyrosine kinases in Xiphophorus.

Melanoma formation in the fish Xiphophorus is an in vivo model for the function of receptor tyrosine kinases (RTKs) in tumor development. The overexpression and high activity of the RTK Xmrk (Xiphophorus melanoma receptor kinase) is responsible for the formation of hereditary malignant melanoma in this fish, but the mechanism by which Xmrk signals cell proliferation has not been elucidated. Remarkably, in earlier experiments an elevated level of a pp60c-src related kinase activity was found in the melanomas. In order to evaluate the significance of src family SH2 domain interactions in the intracellular signalling of Xmrk, we determined its relative binding affinity to the ubiquitous general RTK substrate, PLC gamma, and to the Xiphophorus cytoplasmic kinases Xsrc, Xfyn and Xyes. Recombinant Xmrk purified from baculovirus infected Sf9 cells bound with high affinity to the SH2 domains of PLC gamma and Xfyn in vitro. The affinity of Xmrk to Xsrc and Xyes SH2 domains was 5- to 10-fold lower. Coprecipitation experiments revealed that the Xmrk/Xfyn interaction occurred also in melanoma cells. Moreover, stimulation of the Xmrk kinase activity was paralleled by an increase in Xfyn activity. These results suggest that in malignant melanoma of Xiphophorus the highly activated Xmrk may enhance the activity of Xfyn through direct interaction and that both kinases are linked in a signal transduction pathway.

Amino Acid Sequence↗
PubMed1995

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.

Animals↗
PubMed1994

Studies on the kinetic mechanism of pig kidney D-amino acid oxidase by site-directed mutagenesis of tyrosine 224 and tyrosine 228.

Expression conditions in Escherichia coli of wild-type, Y224F, and Y228F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties similar to those of the wild-type enzyme, in all oxidation-reduction states. All enzymes were studied by steady state and rapid reaction methods. Turnover numbers determined for Y224F DAAO with different substrates were similar to those of wild-type protein, while the Y228F DAAO always showed lower turnover numbers and higher Km values for the D-amino acid. Analyses of reduction traces at 450 and 550 nm of stopped-flow experiments with wild-type DAAO showed the presence of a new phase, the conversion between two different charge-transfer complexes of the reduced enzyme and imino acid product. The substitution of Tyr-228 totally abolished the formation of the long wavelength bands while Y224F DAAO showed long wavelength absorbance only for the first intermediate. Reoxidation of the reduced flavin results from reaction of oxygen with the first charge-transfer complex. The rate of reduction with D-alanine as substrate was 1225,45 and 10 s-1 for wild-type, Y224F, and Y228F DAAOs, respectively. Comparison of the properties of these two mutant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-reaction of the enzyme.

Animals↗
PubMed1998

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated and participates in colony-stimulating factor-1/macrophage colony-stimulating factor signaling in monocyte-macrophages.

RAFTK, a novel nonreceptor protein kinase, has been shown to be involved in focal adhesion signal transduction pathways in neuronal PC12 cells, megakaryocytes, platelets, and T cells. Because focal adhesions may modulate cytoskeletal functions and thereby alter phagocytosis, cell migration, and adhesion in monocyte-macrophages, we investigated the role of RAFTK signaling in these cells. RAFTK was abundantly expressed in THP1 monocytic cells as well as in primary alveolar and peripheral blood-derived macrophages. Colony-stimulating factor-1 (CSF-1)/macrophage colony-stimulating factor (M-CSF) stimulation of THP1 cells increased the tyrosine phosphorylation of RAFTK; similar increases in phosphorylation were also detected after lipopolysaccharide stimulation. RAFTK was phosphorylated with similar kinetics in THP1 cells and peripheral blood-derived macrophages. Immunoprecipitation analysis showed associations between RAFTK and the signaling molecule phosphatidylinositol-3 (PI-3) kinase. PI-3 kinase enzyme activity also coprecipitated with the RAFTK antibody, further confirming this association. The CSF-1/M-CSF receptor c-fms and RAFTK appeared to associate in response to CSF-1/M-CSF treatment of THP1 cells. Inhibition of RAFTK by a dominant-negative kinase mutant reduced CSF-1/M-CSF-induced MAPK activity. These data indicate that RAFTK participates in signal transduction pathways mediated by CSF-1/M-CSF, a cytokine that regulates monocyte-macrophage growth and function.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Tyrosine kinase inhibitors. 7. 7-Amino-4-(phenylamino)- and 7-amino-4-[(phenylmethyl)amino]pyrido[4,3-d]pyrimidines: a new class of inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor.

The synthesis of 7-aminopyrido[4,3-d]pyrimidines bearing aromatic side chains at the 4-position is reported. These compounds are shown to be a new class of inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Structure-activity relationships (SARs) for substitution in both 4-(phenylamino)- and 4-[(phenylmethyl)amino] side chains were determined, using a series of substituents (NO2, Br, CF3, OMe, NH2, and NMe2) selected primarily for their wide range of electronic properties. In the phenylamino series, 3-substituted derivatives were more potent than the corresponding 2- and 4-substituted analogues. For the 3-substituted compounds, activity was favored by electron withdrawal, in a relationship which could be quantified, with the 3-Br being the most potent compound (IC50 = 0.01 microM compared with IC50 = 0.34 microM for the unsubstituted side chain derivative). No such correlation was apparent for the 2- or 4-substituent, although Br was still the best substituent. In contrast, in the 4-[(phenylmethyl)amino] series, substitution of the side chain was not beneficial. For the 4-(phenylamino) series, the substituent SARs are broadly similar to that found previously for 4-(phenylamino)quinazolines. These results suggest that side chain SARs may be broadly invariant over a range of different chromophores, with the side chain of choice for optimization of EGFR inhibitory activity being 4-[(3-bromophenyl)amino].

Carcinoma, Squamous Cell↗
PubMed2003

Discovery of 5-[5-fluoro-2-oxo-1,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase.

To improve the antitumor properties and optimize the pharmaceutical properties including solubility and protein binding of indolin-2-ones, a number of different basic and weakly basic analogues were designed and synthesized. 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (12b or SU11248) has been found to show the best overall profile in terms of potency for the VEGF-R2 and PDGF-Rbeta tyrosine kinase at biochemical and cellular levels, solubility, protein binding, and bioavailability. 12b is currently in phase I clinical trials for the treatment of cancers.

3T3 Cells↗
PubMed1996

Tyrosine kinase inhibitors. 10. Isomeric 4-[(3-bromophenyl)amino]pyrido[d]-pyrimidines are potent ATP binding site inhibitors of the tyrosine kinase function of the epidermal growth factor receptor.

Following the discovery of the very high inhibitory ability of the 4-[(3-bromophenyl)amino]-quinazolines against the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (e.g., 3, IC50 0.029 nM), four series of related pyrido[d]pyrimidines bearing electron-donating groups at the 6- or 7-positions have been synthesized and evaluated. The compounds were prepared by nucleophilic substitution of the corresponding 6- and 7-fluoro analogues. While members of all series showed potent inhibitory activity against isolated EGFR, there were important differences between the different isomeric pyrido[d]pyrimidines and the parent quinazolines. Overall, the [3,4-d] and [4,3-d] series were the most potent, followed by the [3,2-d] compounds, with the [2,3-d] analogues being least active. Whereas in the parent quinazoline series the addition of steric bulk to a 6- or 7-NH2 substituent (i.e., NHMe and NMe2 groups) dramatically decreased potency, no such trend was discernable in the [3,2-d] series. Furthermore, in the 7-substituted pyrido[4,3-d]- and 6-substituted pyrido[3,4-d]pyrimidine series, and to a limited extent in the 7-substituted pyrido[2,3-d] series, such substitution increased potency dramatically, to the extent that the 7-(methylamino)pyrido[4,3-d]pyrimidine (5f) (IC50 0.13 nM) and 6-(methylamino)pyrido[3,4-d]pyrimidine (7f) (IC50 0.008 nM) constitute important new leads. Selected compounds were evaluated for their ability to inhibit EGFR autophosphorylation in A431 cells, and a positive quantitative correlation was found between this activity and inhibitory activity against the isolated enzyme.

ErbB Receptors↗