Purpose: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. Experimental Design: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. Results: The IC 50 of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor^induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate^induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells.Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line.When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/ 2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. Conclusions: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.Excessive growth factor signaling leads to unregulated growth that can contribute to the pathogenesis of human cancer. The signaling cascade is initiated by the binding of peptide growth factors to their tyrosine kinase receptors at the plasma membrane. The receptor kinases are activated and through the recruitment of the growth factor receptor binding protein 2/son of sevenless complex to autophosphorylated sites on the receptors, the G protein Ras is induced to its active GTP-bound state. Ras recruits the serine/threonine kinase Raf to the plasma membrane, where it is then able to phosphorylate and activate mitogen-activated protein kinase kinases (MEK) 1 and 2, which are dual specificity protein kinases that phosphorylate serine/ threonine and tyrosine residues. The MEK kinases in turn phosphorylate and activate their only currently known substrates, extracellular signal-regulated kinases (ERK) 1 and 2. ERK1/2 proteins translocate to the nucleus where they phosphorylate and activate effector proteins and transcription factors, resulting in diverse cellular responses, including proliferation.The overexpression and/or mutation of epidermal growth factor (EGF) receptor (EGFR), erbB2, platelet-derived growth factor receptor, RET, and othe...

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires W , and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn 2؉ -dependent glutathione transferases but with a preference for Mg 2؉ and L-cysteine as cofactors.Sequencing of the Bacillus subtilis genome revealed the presence of seven new factors, all members of the extracytoplasmic function subfamily (12, 13). We have begun to investigate their functions by mutation of each gene and the identification of target operons (8)(9)(10)(11). In this work, we demonstrate that yndN encodes a fosfomycin resistance (Fos r ) protein that depends on W for expression. We have renamed yndN as fosB, based on its similarity to the fosB gene identified on a Staphylococcus epidermidis plasmid (Fig. 1B).Transcription of fosB requires W . Previously, 15 W -dependent operons were identified by searching the genome for sequences matching the W autoregulatory site, P w : TGAAAC N 16 CGTA (10). Additional candidate promoters, including one for fosB (Fig. 1A), were identified with 17-bp spacer regions (10).To confirm the role of this predicted W -dependent promoter, we generated a P fosB -cat-lacZ operon fusion inserted ectopically in the SP␤ prophage (HB8083; Table 1) and transduced the reporter fusion into wild-type, sigW, and rsiW mutant backgrounds. Promoter activity as determined in early-stationary-phase cells yielded 18.4 Miller units of ␤-galactosidase in the wild-type strain (HB0052), and this was reduced to background levels (ϳ1 unit) in the sigW mutant (HB0023). In the rsiW (anti-W ) mutant (HB0012), expression was elevated approximately twofold (30.5 units). This pattern is precisely that expected for a W -dependent promoter. We used reverse transcriptase primer extension mapping to identify the transcriptional start site for fosB as a G residue 10 bases downstream from the Ϫ10 region CGTA motif (Fig. 2). There were no other start sites visible in the primer extension experiment, which is consistent with the idea that W is largely, if not exclusively, responsible for fosB transcription. The fosB gene is apparently monocistronic, as it is flanked on either side by genes transcribed from the complementary strand of the genome (Fig. 1A).fosB and sigW mutants are sensitive to fosfomycin. Both fosB (HB0008) and sigW (HB0020) mutants are fosfomycin sensitive: an MIC of 50 g/ml for the mutants compared to 800 g/ml for the wild type in liquid culture. Similarly, the sigW and fosB mutants have a much greater zone of growth inhibition in disk diffusion assays (ϳ25-mm zone for wild type versus Ͼ50 mm for the mutants). The fosB and sigW mutant strains did not display altered sensitivity to several other antibiotics, including vancomycin, cephalosporin C, penicillin G, D-cycloserine, tunicamycin, nisin, and bacitracin. Induction of fosB from a xylose-inducible promoter completely restores Fos r to either the sigW mutant (HB0081) or, as expected, t...

The enzyme conferring resistance to the antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] originally reported by Suarez and co-workers [Area, P., Hardisson, C., & Suarez, J. E. (1990) Antimicrob. Agents Chemother. 34, 844-848] is demonstrated in this study to be a metalloglutathione transferase. The apoenzyme is a dimer of 16 kDa subunits. Electron paramagnetic resonance spectroscopy and water proton nuclear magnetic resonance longitudinal relaxation rates suggest that each subunit contains a mononuclear Mn2+ center that interacts strongly with the substrate fosfomycin (Kd = 17 microM) more weakly with the product (Kd = 1.1 mM) and very weakly or not at all with GSH. Inhomogeneous broadening of the EPR signals of enzyme-bound Mn2+ in the presence of H2(17)O indicates that three of the coordination sites on the metal are occupied by water. Sequence alignments, three-dimensional structures, and mechanistic considerations suggest that FosA is related to at least two other metalloenzymes, glyoxalase I and the Mn2+- or Fe2+-containing extradiol dioxygenases. The mechanistic imperative driving the evolution of this previously unidentified superfamily of metalloenzymes is proposed to be bidentate coordination of a substrate or intermediate to the metal center in the enzyme-catalyzed reactions.

Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs). Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2). A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors. We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites. The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured. Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two. It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in. This interface is characterized by a binding "hot-spot" consisting of a core of three residues in ECD1 and two in ECD2. There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH. This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.

The fosfomycin resistance protein FosA is a member of a distinct superfamily of metalloenzymes containing glyoxalase I, extradiol dioxygenases, and methylmalonyl-CoA epimerase. The dimeric enzyme, with the aid of a single mononuclear Mn2+ site in each subunit, catalyzes the addition of glutathione (GSH) to the oxirane ring of the antibiotic, rendering it inactive. Sequence alignments suggest that the metal binding site of FosA is composed of three residues: H7, H67, and E113. The single mutants H7A, H67A, and E113A as well as the more conservative mutants H7Q, H67Q, and E113Q exhibit marked decreases in the ability to bind Mn2+ and, in most instances, decreases in catalytic efficiency and the ability to confer resistance to the antibiotic. The enzyme also requires the monovalent cation K+ for optimal activity. The K+ ion activates the enzyme 100-fold with an activation constant of 6 mM, well below the physiologic concentration of K+ in E. coli. K+ can be replaced by other monovalent cations of similar ionic radii. Several lines of evidence suggest that the K+ ion interacts directly with the active site. Interaction of the enzyme with K+ is found to be dependent on the presence of the substrate fosfomycin. Moreover, the E113Q mutant exhibits a kcat which is 40% that of wild-type in the absence of K+. This mutant is not activated by monovalent cations. The behavior of the E113Q mutant is consistent with the proposition that the K+ ion helps balance the charge at the metal center, further lowering the activation barrier for addition of the anionic nucleophile. The fully activated, native enzyme provides a rate acceleration of >10(15) with respect to the spontaneous addition of GSH to the oxirane.