Lapatinib (GW572016) is a selective inhibitor of both epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases. Here, we explore the therapeutic potential of lapatinib by testing its effect on tumor cell growth in a panel of 31 characterized human breast cancer cell lines, including trastuzumab-conditioned HER-2-positive cell lines. We further characterize its activity in combination with trastuzumab and analyze whether EGFR and HER-2 expression or changes induced in the activation of EGFR, HER-2, Raf, AKT, or extracellular signal-regulated kinase (ERK) are markers of drug activity. We report that concentration-dependent antiproliferative effects of lapatinib were seen in all breast cancer cell lines tested but varied significantly between individual cell lines with up to 1,000-fold difference in the IC 50 s (range, 0.010-18.6 Mmol/L). Response to lapatinib was significantly correlated with HER-2 expression and its ability to inhibit HER-2, Raf, AKT, and ERK phosphorylation. Long-term in vivo lapatinib studies were conducted with human breast cancer xenografts in athymic mice. Treatment over 77 days resulted in a sustained and significant reduction in xenograft volume compared with untreated controls. For the combination of lapatinib plus trastuzumab, synergistic drug interactions were observed in four different HER-2-overexpressing cell lines. Moreover, lapatinib retained significant in vitro activity against cell lines selected for long-term outgrowth (>9 months) in trastuzumabcontaining (100 Mg/mL) culture medium. These observations provide a clear biological rationale to test lapatinib as a single agent or in combination with trastuzumab in HER-2-overexpressing breast cancer and in patients with clinical resistance to trastuzumab.

Dual EGFR/erbB2 inhibition is an attractive therapeutic strategy for epithelial tumors, as ligand-induced erbB2/EGFR heterodimerization triggers potent proliferative and survival signals. Here we show that a small molecule, GW572016, potently inhibits both EGFR and erbB2 tyrosine kinases leading to growth arrest and/or apoptosis in EGFR and erbB2-dependent tumor cell lines. GW572016 markedly reduced tyrosine phosphorylation of EGFR and erbB2, and inhibited activation of Erk1/2 and AKT, downstream effectors of proliferation and cell survival, respectively. Complete inhibition of activated AKT in erbB2 overexpressing cells correlated with a 23-fold increase in apoptosis compared with vehicle controls. EGF, often elevated in cancer patients, did not reverse the inhibitory effects of GW572016. These observations were reproduced in vivo, where GW572016 treatment inhibited activation of EGFR, erbB2, Erk1/2 and AKT in human tumor xenografts. Erk1/2 and AKT represent potential biomarkers to assess the clinical activity of GW572016. Inhibition of activated AKT in EGFR or erbB2-dependent tumors by GW572016 may lead to tumor regressions when used as a monotherapy, or may enhance the anti-tumor activity of chemotherapeutics, since constitutive activation of AKT has been linked to chemo-resistance.

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 M and was inactive against 26 other kinases. GW2580 at 1 M completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.CSF-1 ͉ macrophage colony-stimulating factor

November 5, 2007; 10.1073͞pnas.0702843104), the authors wish to add a reference to a paper by A. Shukla et al. (35), in which an intrinsic, nonaromatic fluorescence emission with the same excitation and emission characteristics was observed in different protein crystals and aggregates, upon UV-A excitation, and was attributed to the delocalization of peptide electrons by intra-and/or intermolecular hydrogen bond formation, consistent with the intrinsic blue-green fluorescence we report in amyloid-like nanofibrils. The added reference appears below. Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678 -1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.inhibitors ͉ enzyme ͉ irreversible ͉ thiol ͉ alkylation I nhibition of the ErbB family receptor tyrosine kinases (EGFR, ErbB-2) represents a major advance in the treatment of solid tumors, as demonstrated by the promising clinical activity of gefitinib (1), erlotinib (2), and lapatinib (3) (Fig. 1) (1). These drugs are selective, reversible ATP-competitive EGFR (e.g., 1, 2) or dual EGFR/ErbB-2 inhibitors (3), respectively. An alternative approach for targeting this family of enzymes has been through irreversible alkylation of an ErbB family-conserved cysteine residue (Cys-797 in EGFR, Cys-805 in ErbB-2, and Cys-803 in ErbB-4). i This latter approach led to the discovery of the potent, irreversible agents canertinib (4) and pelitinib (5) (Fig. 1) (2, 3). Both compounds 4 and 5 and other irreversible agents are reported to be in phase II clinical trials (4).To identify potent, efficacious EGFR/ErbB-2 inhibitors structurally distinct from lapatinib, a series of 4-anilino thienopyrimidines containing the fluorobenzyl aniline subunit common to 3 was explored. Optimization of this series on enzyme and cellular assays led to the identification of 6-ethynyl-substituted thieno[3,2-d]pyrimidines and thieno [2,3-d]pyrimidines as represented by the...

The therapeutic efficacy of orally administered zidovudine (3'-azido-3'-deoxythymidine) was determined in animals infected with Escherichia coli and Salmonella dublin. The 50% effective dose (ED50) of zidovudine (9.6 to 11.8 mg/kg of body weight) compared favorably with that of trimethoprim (19.4 to 22.2 mg/kg) in mice with systemic E. coli infection. At 50 mg/kg, both zidovudine and ampicillin reduced the number of bacteria in the kidneys of mice and prevented lethal infection in mice with ascending pyelonephritis caused by E. coli. Zidovudine prevented a lethal S. dublin infection in calves over a wide dose range (8.0 to 31.0 mg/kg per day). Zidovudine levels in plasma of uninfected mice were 28.2 ± 4.5 and 7.9 ± 2.2 ,ug/ml at 30 and 60 min, respectively, exceeding the MICs for the bacteria used in the infections. Few zidovudine-resistant strains were observed. The in vivo data raise the possibility that zidovudine may have an antibacterial effect in patients receiving this therapy.The synthetic nucleoside zidovudine (3'-azido-3'-deoxythymidine) is an antimicrobial agent with a spectrum of in vitro activity that encompasses human immunodeficiency virus (HIV) (8) AIDS, abstr. no. 556, 1985), and gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, and Haemophilus influenzae (1). Zidovudine has shown no activity against Pseudomonas aeruginosa, grampositive or anaerobic bacteria, or common pathogenic fungi (such as Pneumocystis carinii) frequently isolated from HIV-infected patients (Nusinoff-Lehrman et al., Int. Conf. AIDS, 1985). E. coli and Salmonella dublin have been shown to be exquisitely susceptible to zidovudine, with in vitro MICs in the range of 0.0025 to 0.3 ,xg/ml (Table 1).We extended these in vitro observations to determine the in vivo efficacy of zidovudine in experimental bacterial infections that included a mouse systemic E. coli infection, acute ascending E. coli pyelonephritis in mice, and S. dublin salmonellosis in calves.MATERIALS AND METHODS Antimicrobial agents. Zidovudine and trimethoprim were laboratory reference preparations (Burroughs Wellcome Co., Research Triangle Park, N.C.). Ampicillin was purchased as the sodium salt (Sigma Chemical Co., St. Louis, Mo.). For all studies on mice, compounds were dissolved or suspended just before use in an aqueous suspending vehicle consisting of 1.0% Tween 80 and 0.5% low-viscosity sodium carboxymethylcellulose (Sigma). For studies in calves, zidovudine was administered by subcutaneous injection in an aqeuous solution of 20% dimethylformamide.Bacteria. The bacteria used for animal infections were veterinary and clinical isolates previously found to exhibit susceptibility in vitro to zidovudine and the reference antimicrobial agents used in this study. E. coli P855, used for the mouse systemic infection studies, is a clone from E. coli CN348 (isolated from a sick calf) maintained in the Well-* Corresponding author.come Research Laboratories strain collection. E. coli J96 was used for the mouse ascendin...

The role of type 1 pili in promoting bladder colonization was examined by constructing two mutant strains of a clinical Escherichia coli isolate. One mutant was isogenic to the parental strain save for a lesion in a gene required for pilus receptor binding; the other mutant was isogenic save for a lesion in the gene encoding the pilus structural subunit. Using mixed infections of the parental and mutant strains in an ascending rat cystitis model, we found that type 1-piliated mutants that lacked the receptor-binding function were as ineffective in bladder colonization as were mutants lacking the entire organelle.

No abstract

Escherichia coli K-12 mutants possessing defined lesions affecting type 1 pilus production, receptor binding, or length were examined for their ability to resist killing by mouse peritoneal macrophages in vitro. Mutants were mixed pairwise at known ratios in wells containing macrophages, and after incubation, the ratio of the survivors was assayed. The difference in phagocytic killing between type 1 piliated cells and isogenic nonpiliated cells was significant, the piliated cells being approximately threefold more resistant. Pilus length had little effect upon survival, as the long-piliated mutants were no more resistant to killing than the normal-length parents. Interestingly, the receptor-binding function of type 1 pili was mostimportant-in effecting resistance, as mutants lacking the ability to bind receptor were killed as effectively as nonpiliated mutants. These data are consistent with the notion that pili actually-impede killing by macrophages rather than serve as passive physical barriers to uptake.