Aberrant signaling of ErbB family members human epidermal growth factor 2 (HER2) and epidermal growth factor receptor (EGFR) is implicated in many human cancers, and HER2 expression is predictive of human disease recurrence and prognosis. Small molecule kinase inhibitors of EGFR and of both HER2 and EGFR have received approval for the treatment of cancer. We present the first high resolution crystal structure of the kinase domain of HER2 in complex with a selective inhibitor to understand protein activation, inhibition, and function at the molecular level. HER2 kinase domain crystallizes as a dimer and suggests evidence for an allosteric mechanism of activation comparable with previously reported activation mechanisms for EGFR and HER4. A unique Gly-rich region in HER2 following the ␣-helix C is responsible for increased conformational flexibility within the active site and could explain the low intrinsic catalytic activity previously reported for HER2. In addition, we solved the crystal structure of the kinase domain of EGFR in complex with a HER2/EGFR dual inhibitor (TAK-285). Comparison with previously reported inactive and active EGFR kinase domain structures gave insight into the mechanism of HER2 and EGFR inhibition and may help guide the design and development of new cancer drugs with improved potency and selectivity.

Dual inhibitors of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) have been investigated for breast, lung, gastric, prostate, and other cancers; one, lapatinib, is currently approved for breast cancer. To develop novel HER2/EGFR dual kinase inhibitors, we designed and synthesized pyrrolo[3,2-d]pyrimidine derivatives capable of fitting into the receptors' ATP binding site. Among the prepared compounds, 34e showed potent HER2 and EGFR (HER1) inhibitory activities as well as tumor growth inhibitory activity. The X-ray cocrystal structures of 34e with both HER2 and EGFR demonstrated that 34e interacts with the expected residues in their respective ATP pockets. Furthermore, reflecting its good oral bioavailability, 34e exhibited potent in vivo efficacy in HER2-overexpressing tumor xenograft models. On the basis of these findings, we report 34e (TAK-285) as a promising candidate for clinical development as a novel HER2/EGFR dual kinase inhibitor.

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The epidermal growth factor receptor (EGFR) family plays a critical role in vital cellular processes and in various cancers. Known EGFR inhibitors exhibit distinct antitumor responses against the various EGFR mutants associated with nonsmall-cell lung cancer. The L858R mutation enhances clinical sensitivity to gefitinib and erlotinib as compared with wild type and reduces the relative sensitivity to lapatinib. In contrast, the T790M mutation confers drug resistance to gefitinib and erlotinib. We determined crystal structures of the wild-type and T790M/L858R double mutant EGFR kinases with reversible and irreversible pyrrolo[3,2-d]pyrimidine inhibitors based on analogues of TAK-285 and neratinib. In these structures, M790 adopts distinct conformations to accommodate different inhibitors, whereas R858 allows conformational variations of the activation loop. These results provide structural insights for understanding the structure-activity relationships that should contribute to the development of potent inhibitors against drug-sensitive or -resistant EGFR mutations.

Group I and II hepatitis C virus genotypes were determined by a newly developed serological genotyping assay. This assay detected antibodies against group-specific recombinant proteins in the putative NS4 protein region (amino acid no. 1676-1760) by an enzyme-linked immunosorbent assay. This region of the hepatitis C virus peptide has many group-specific amino acids; fewer than 50% of these amino acids are identical between groups I and II. Genotypes determined by the serological genotyping assay were compared with those determined by a method in which the polymerase chain reaction was used in 91 chronic hepatitis patients. The group-specific polymerase chain reaction was performed within the genome region corresponding to the putative NS5 protein, where the group II hepatitis C virus genome is 57 nucleotides longer than that of group I. Among 91 chronic hepatitis C patients who had positive results in the second-generation hepatitis C virus antibody (core and NS3 region) assay, hepatitis C virus RNA was detected in 80 patients by polymerase chain reaction in the 5' untranslated region and in 78 patients by this group-specific polymerase chain reaction. As a result, in 76 of 91 patients (84%) genotypes determined by the serological genotyping assay showed complete agreement with those determined by the group-specific polymerase chain reaction, and none of the patients revealed a group opposite to that of hepatitis C virus genotype. The detection rate of the serological genotyping assay (89 of 91; 98%) was even higher than that of the polymerase chain reaction assay (78 of 91; 86%).(ABSTRACT TRUNCATED AT 250 WORDS)

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In the course of our study aimed at developing new types of DMARDs (disease-modifying antirheumatic drugs), we found that quinoline derivative 1a had a potent anti-inflammatory effect in an adjuvant arthritis (AA) rat model, starting from the potent bone resorption inhibitors justicidins as the lead compounds. Further modification of 1a was performed, and various quinoline and quinazoline derivatives having a heteroaryl moiety on the alkyl side chain at the 2-position of the skeleton were prepared. These compounds were evaluated for anti-inflammatory effects using the AA rat model. Most of these compounds, especially those having an imidazole or a triazole moiety on the 2-alkyl chain, exhibited a potent effect. Among the compounds synthesized, ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2, 4-triazol-1-yl-methyl)quinoline-3-carboxylate (12d), having an ED50 value of 2.6 mg/kg/day (anti-inflammatory effect in an AA rat model, po), was selected as a candidate for further investigation. In vitro, 12d inhibited mitogen-induced proliferation at 10(-7)-10(-5) M but not prostaglandin E2 production at 10(-5) M. Moreover, 12d preferentially inhibited the IFN-gamma production by Th1-type clones over the IL-4 production by Th2-type clones. This preferential suppression of Th1 cytokine production is considered the essential immunomodulating action of 12d for the present. Synthesis and structure-activity relationships for this novel series of quinoline and quinazoline derivatives are detailed.

Cells from breast cancers lacking hormone receptors (estrogen receptor[I n mammary glands, estrogen plays a major role in duct elongation and branching, and accelerates the proliferation of epithelial cells, whereas progesterone contributes to lobule formation. In non-neoplastic mammary glands, estradiol is thought to act as a paracrine mitogen because no co expression of estrogen receptor (ER)α and cell proliferation markers (Ki-67 or proliferating cell nuclear antigen [PCNA]) has been detected. A recent study by Dimitrakakis et al. demonstrated that ERα and the estrogen-induced proteins MYC, cyclin D1, and stromal cell-derived factor-1 are co-expressed within the nuclei of monkey mammary glands.(1) Responsiveness to estrogen is retained after carcinogenesis in approximately 70% of all breast cancers. Two different mechanisms have been proposed to explain the mitogenic effects of estrogens in breast cancer tissue:(2) genomic action via estrogen-responsive elements located in the promoter regions of c-fos (3) and c-myc, (4) which have mitogenic activity involving G 1 -phase progression; or indirect action on cyclin D1 gene transcription.(5) Induction of cyclin D1 is strictly regulated in a hormone-dependent manner, and cAMP response elements in the promoter region of the cyclin D1 gene require the activation function (AF)-1 and AF-2 domains of ERα. Other non-genomic actions of estrogen via mitogen-activated protein kinase (MAPK)-external signal-regulated kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)-Akt have also been proposed. (2) Human epidermal growth factor receptor (HER)2 is a tyrosine kinase receptor, the biological and clinical role of which has been investigated thoroughly in breast cancer. No specific ligands for HER2 have been found, but dimers containing HER2 (e.g. homodimers of HER2 or HER2-HER3 heterodimers) can strongly activate intracellular signaling for cell proliferation, cell survival, motility, and adhesion.(6) Two major signaling pathways for the HER family are the Raf-MEK-ERK pathway and the PI3K-phosphoinositide-dependent kinase (PDK1)-Akt pathway. These signal-transduction pathways regulate the expression of genes in many ways to promote cell-cycle progression by inhibition of p27 (8,9) and activation of cyclin D1, (10) and to inhibit apoptosis by phosphorylation of Bad and caspase 9. (11) Thus, the mechanisms of cell proliferation have been thoroughly investigated for hormone receptor-positive and HER2-positive breast cancers. In contrast, in breast cancers lacking hormone receptors and HER2, the signaling pathways responsible for cell proliferation are not well characterized. Cell proliferation in triplenegative breast cancer cells must be regulated in an ER or HER2 signaling-independent manner. The present study was conducted to examine the phosphorylation of HER2, ERK1/2, and Akt kinases, which are key kinases in two major signaling pathways, in order to obtain data to support further investigations into the molecular events contributing to cell proliferation. Materials and Me...