Many diverse extracellular stimuli-including growth factors, hormones, osmolar shock, stress, and elevated temperatureresult in activation of phosphorylation cascades utilizing mitogen-activated protein kinases (MAPKs) (1-8). MAPKs (sometimes called extracellular signal-regulated kinases, or ERKs) comprise a family of related protein kinases that are themselves activated by phosphorylation on threonine and tyrosine residues. The MAPK-activating enzymes (MAPK/ ERK kinases, or MEKs) are unusual in their ability to catalyze phosphorylation on both threonine and tyrosine residues (9, 10). MEKs are in turn activated by phosphorylation on serine residues by upstream kinases. These MEK kinases, which appear to require activation by the ras protooncogene product (11, 12), include members of the Raf family (13-15), a mammalian homologue of the yeast STEll gene product (16), the tpll2 prc_ oncogene product (17), and a growth-factor sensitive enz--:e derived from PC12 rat pheochromocytoma cells (18). However, the precise specificity of these kinases in vivo is unclear, since some of them may participate in cascades leading to activation of the related stress-activated protein kinases (19,20).While the MAPK pathway is activated under many circumstances in tissue culture cells, the exact role of this pathway in vivo remains undefined. Approaches using dominant negative interfering mutant constructs of MEK have indicated that this pathway is required for nerve growth factor-dependent differentiation of PC12 cells. Furthermore, expression of constitutively activated mutants has resulted in transformation (21,22). We sought a more widely applicable method to determine the physiological role of this pathway by identifying selective inhibitors of specific components of the MAPK cascade. MATERIALS AND METHODSIn Vitro Kinase Assay. Incorporation of 32p into myelin basic protein (MBP) was assayed in the presence of glutathione S-transferase (GST) fusion proteins containing the 44-kDa MAPK (GST-MAPK) or the 45-kDa MEK (GST-MEK1). For direct evaluation of MEK activity, 10 ,tg of GST-MEK1 was incubated with 5 ,ug of a GST fusion protein containing 44-kDa MAPKwith a lysine-to-alanine mutation at position 71 (GST-MAPK-KA). This mutation eliminates kinase activity of MAPK, so that only kinase activity attributed to the added MEK remains. Similar incubations were performed with 5 ,ug of a fusion protein containing artificially partially activated MEK with serine-to-glutamate mutations at positions 218 and 222 (GST-MEK-2E). These assays utilized the same buffer and incubation conditions as described above. Phosphorylated MAPK-KA was resolved by SDS/10% PAGE and detected by autoradiography.Immunoprecipitation and Imminoblot Analysis. Tyrosinephosphorylated MAPK-KA was determined by using the same incubation protocol as for phosphorylation, but without radiolabeled, ATP. After electrophoresis, proteins on the gel were transferred to a nitrocellulose membrane, and nonspecific binding sites on the membrane were blocked by incubation with 1% ova...

A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14 C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.Considerable evidence has emerged, both preclinically and clinically, over the last decade to implicate the epidermal growth factor (EGF) receptor (EGFr) and erbB2 in the development, progression, and severity of certain human cancers. More recently, however, it has become clear that these receptors can intensify the transforming signal in a synergistic manner through their ability to form both homo-and heterodimers (1-7). Coexpression of the EGFr and erbB2 to levels where either receptor alone had little effect was highly transforming (8 -10). The association between overexpression and͞or constitutive activation of members of the type 1 receptor TK family (11) as well as coexpression of their cognate ligands (EGF, the heregulin family, transforming growth factor-␣, betacellulin) and transformation has been well established in many primary tumors. In particular, high expression levels of the EGFr and erbB2 have been frequently observed in breast, prostate, ovarian, and various squamous cell carcinomas in which overexpression positively correlates with shortened survival times and increased relapse rates (12-21).Over the past decade drug discovery efforts have produced a wide variety of chemical structures, generated either by synthetic means or as fermentation products, that reportedly inhibit purified or partially purified preparations of the EGFr tyrosine ki...

4-(3-Bromoanilino)-6,7-dimethoxyquinazoline (32, PD 153035) is a very potent inhibitor (IC50 0.025 nM) of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), binding competitively at the ATP site. Structure-activity relationships for close analogues of 32 are very steep. Some derivatives have IC50s up to 80-fold better than predicted from simple additive binding energy arguments, yet analogues possessing combinations of similar phenyl and quinazoline substituents do not show this "supra-additive" effect. Because some substituents which are mildly deactivating by themselves can be strongly activating when used in the correct combinations, it is proposed that certain substituted analogues possess the ability to induce a change in the conformation of the receptor when they bind. There is some bulk tolerance for substitution in the 6- and 7-positions of the quinazoline, so that 32 is not the optimal inhibitor for the induced conformation. The diethoxy derivative 56 [4-(3-bromoanilino)-6,7-diethoxyquinazoline] shows an IC50 of 0.006 nM, making it the most potent inhibitor of the tyrosine kinase activity of the EGFR yet reported.

A series of 4-substituted quinazolines and related compounds have been prepared and evaluated for their ability to inhibit the tyrosine kinase activity of the epidermal growth factor receptor on a phospholipase C-gamma 1-derived substrate. The results show a narrow structure-activity relationship (SAR) for the basic ring system, with quinazoline being the preferred chromophore and benzylamino and anilino the preferred side chains. In the 4-anilino series, substitution on the 3-position of the phenyl ring with small lipophilic electron-withdrawing groups provided analogues with enhanced potency. Two series of compounds [4-(phenylmethyl)amino and 4-(3-bromophenyl)amino] were studied to determine SARs for quinazoline substituents. In the more active 4-(3-bromophenyl)amino series, electron-donating groups (NH2, OMe) at the 6- or 7-position increased activity, in a pattern consistent with a requirement for high electron density in the vicinity of the 8-position of the quinazoline ring. The 6,7-dimethoxy derivatives were the most effective in both series, with the 4-(3-bromophenyl)amino derivative (3) having an IC50 of 0.029 nM, making it by far the most potent reported inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor enzyme.

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Alexander J. Bridges obtained his B.A. degree in Chemistry from Oxford University in 1972 and his Ph.D. degree under the supervision of Dr. Gordon Whitham in 1974, working on trans-cyclooctenes. He was a NATO Fellow with Professor Barry Trost at Wisconsin for the following two years, making novel dienes for Diels−Alder reactions and spent a year with Professor Pierre Potier at the CNRS at Gif-sur-Yvette working on the synthesis of vinblastine. This was followed by a final postdoctoral position at the University of Toronto with Professor J. B. Jones, working on enzymes as chiral synthetic reagents. In 1978 he joined the chemistry faculty of Northern Illinois University in DeKalb, IL, taught organic chemistry for six years, and worked on sulfur-substituted allenes and dienes. In 1984 he joined Parke-Davis in Ann Arbor, MI, and worked on adenosine agonists and quinolone anti-infectives. He moved to the Eisai Research Institute of Boston in 1988 and worked on Lipid A analogues for sepsis and urokinase inhibitors for cancer. He moved back to Parke-Davis (now Pfizer Global Research and Development, Ann Arbor Laboratories) in 1992 and worked on kinase inhibitors for cancer, especially EGFr and MEK inhibitors. He then moved to metabolic diseases chemistry and is currently

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.