The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC 50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (∼1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 μmol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials. Cancer Res; 70(1); 288-98. ©2010 AACR.

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Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.

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Poly(ADP-ribose) polymerase (PARP) inhibitors enhance DNA topoisomerase I (topo I) poisoninduced cytotoxicity and antitumor activity in vitro and in vivo, but the mechanism has not been defined. We investigated the role of PARP-1 in the response to topo I poisons using PARP-1 À/À and PARP-1 +/+ mouse embryonic fibroblasts and the potent PARP-1 inhibitor, AG14361 (K i < 5 nmol/L). PARP-1 À/À mouse embryonic fibroblasts were 3-fold more sensitive to topotecan than PARP-1 +/+ mouse embryonic fibroblasts (GI 50 , 21 and 65 nmol/L, respectively). AG14361caused a >3-fold sensitization of PARP-1 +/+ cells to topotecan compared with a <1.4-fold sensitization in PARP-1 À/À cells. In human leukemia K562 cells, AG14361 caused a 2-fold sensitization to camptothecin-induced cytotoxicity. AG14361 did not affect the cellular activity of topo I as determined by measurement of cleavable complexes and topo I relaxation activity, showing that sensitization was not due to topo I activation. In contrast, repair of DNA following camptothecin removal, normally very rapid, was significantly retarded by AG14361, resulting in a 62% inhibition of repair 10 minutes after camptothecin removal. This led to a 20% increase in the net accumulation of camptothecin-induced DNA strand break levels in cells coexposed to AG14361 for 16 hours. We investigated the DNA repair mechanism involved using a panel of DNA repair^deficient Chinese hamster ovary cells. AG14361 significantly potentiated camptothecin-mediated cytotoxicity in all cells, except the base excision repair^deficient EM9 cells. Therefore, the most likely mechanism for the potentiation of topo I poison-mediated cytotoxicity by AG14361 is via PARP-1-dependent base excision repair.

The nucleocapsid protein from the Rous sarcoma virus has two regions of sequence with the motif CysXaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Xaa-Cys.All retrovirus nucleocapsid proteins contain one or two of these motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleocapsid protein only in three residues near the carboxyl terminus. The hypothesized role of the conserved cysteines and histidines as zinc ligands was tested experimentally. No tightly bound metal ions were detected for avian myeloblastosis nucleocapsid protein, and the molar amount of zinc in virions was less by a factor of 50 than that of the nucleocapsid protein.Added Zn2+ did not significantly affect nucleocapsid binding to poly(ethenoadenylic acid) or its secondary structure, as determined from circular dichroism. Nevertheless, the conserved cysteine and histidine residues of the Rous sarcoma (Prague-C strain) nucleocapsid protein are essential for fully functional virus, as shown by the fact that single-site substitutions of five of the six conserved cysteines and either of the two histidine residues blocked viral replication.Retrovirus nucleocapsid (NC) proteins are members of a larger class of nucleic acid-binding proteins with "Cys-His" boxes (1). All of the NC proteins that have been sequenced contain at least one sequence with the motif Cys-Xaa-XaaCys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Xaa-Cys, where the first two cysteines and the histidine are invariant for nine Cys-His boxes from NC proteins from five viral strains infecting four different species (2). The Cys-His motif also represents the only conserved sequence noted for retroviral NC proteins, implying that it plays a key structural and/or functional role in these proteins.ppl2 is the major NC protein of the avian retroviruses, including avian myeloblastosis virus (AMV) and Rous sarcoma virus, Prague-C strain (RSV). About 2500 copies (3,4) of NC are tightly bound to genomic RNA within the viral core, resulting in approximately one NC for every seven or eight nucleotides.The NC of RSV and AMV has two of the Cys-His sequences (ref. 5 and this report). As part of our ongoing characterization of the function and structure of the NC from both AMV and RSV (6-8), we undertook an investigation of its properties due to the residues in the Cys-His sequences. We report the primary sequence ofAMV NC, the distribution of its secondary structural elements, an analysis of its metal content, and the results of site-directed mutagenesis experiments on conserved cysteine and histidine residues in RSV NC. This communication provides experimental evidence showing the conserved residues to be essential to virus replication. However, our biochemical evidence argues against the hypothesis (9) that the ppl2 protein is a metalloprotein. MATERIALS AND METHODSIsolation and Sequencing of the DNA for NC. A pBR322 clone...

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