Marketa Zvelebil

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

Prediction of protein secondary structure and active sites using the alignment of homologous sequences

The prediction of protein secondary structure (alpha-helices, beta-sheets and coil) is improved by 9% to 66% using the information available from a family of homologous sequences. The approach is based both on averaging the Garnier et al. (1978) secondary structure propensities for aligned residues and on the observation that insertions and high sequence variability tend to occur in loop regions between secondary structures. Accordingly, an algorithm first aligns a family of sequences and a value for the extent of sequence conservation at each position is obtained. This value modifies a Garnier et al. prediction on the averaged sequence to yield the improved prediction. In addition, from the sequence conservation and the predicted secondary structure, many active site regions of enzymes can be located (26 out of 43) with limited over-prediction (8 extra). The entire algorithm is fully automatic and is applicable to all structural classes of globular proteins.

Phosphoinositide 3-kinase signalling--which way to target?

Abstract Phosphoinositide 3-kinases (PI3Ks) are central to the control of cell growth, proliferation and survival, and drive the progression of tumours by activating phosphoinositide-dependent kinase, protein kinase B and the target of rapamycin. Other downstream effectors link PI3K to cell motility and the control of cardiovascular parameters. Current knowledge indicates that PI3Ks might qualify as drug targets for the treatment of cancer, chronic inflammation, allergy and cardiovascular failure. However, PI3Ks also modulate vital processes such as metabolic control and nutrient uptake. Here, mechanistic data and mouse phenotypic analyses are summarised, and the possible success of therapeutic inhibition of distinct PI3K isoforms is discussed.

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3‐kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3‐kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3‐phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3‐kinase. Here the molecular characterization of a new human PtdIns 3‐kinase with extensive sequence homology to Vps34p is described. PtdIns 3‐kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3‐kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Exploring the specificity of the PI3K family inhibitor LY294002

The PI3Ks (phosphatidylinositol 3-kinases) regulate cellular signalling networks that are involved in processes linked to the survival, growth, proliferation, metabolism and specialized differentiated functions of cells. The subversion of this network is common in cancer and has also been linked to disorders of inflammation. The elucidation of the physiological function of PI3K has come from pharmacological studies, which use the enzyme inhibitors Wortmannin and LY294002, and from PI3K genetic knockout models of the effects of loss of PI3K function. Several reports have shown that LY294002 is not exclusively selective for the PI3Ks, and could in fact act on other lipid kinases and additional apparently unrelated proteins. Since this inhibitor still remains a drug of choice in numerous PI3K studies (over 500 in the last year), it is important to establish the precise specificity of this compound. We report here the use of a chemical proteomic strategy in which an analogue of LY294002, PI828, was immobilized onto epoxy-activated Sepharose beads. This affinity material was then used as a bait to fish-out potential protein targets from cellular extracts. Proteins with high affinity for immobilized PI828 were separated by one-dimensional gel electrophoresis and identified by liquid chromatography-tandem MS. The present study reveals that LY294002 not only binds to class I PI3Ks and other PI3K-related kinases, but also to novel targets seemingly unrelated to the PI3K family.

Evaluation of Two-dimensional Differential Gel Electrophoresis for Proteomic Expression Analysis of a Model Breast Cancer Cell System

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.

iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72–polymorphic p53

iASPP is one of the most evolutionarily conserved inhibitors of p53, whereas ASPP1 and ASPP2 are activators of p53. We show here that, in addition to the DNA-binding domain, the ASPP family members also bind to the proline-rich region of p53, which contains the most common p53 polymorphism at codon 72. Furthermore, the ASPP family members, particularly iASPP, bind to and regulate the activity of p53Pro72 more efficiently than that of p53Arg72. Hence, escape from negative regulation by iASPP is a newly identified mechanism by which p53Arg72 activates apoptosis more efficiently than p53Pro72.

IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis.

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-κB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-α-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2–MALT1 to activate NF-κB. Our data suggest that the UBA domain of cIAP2–MALT1 stimulates NF-κB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2–MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2–MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-κB signalling and oncogenesis.

Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid‐binding C2 domain

Perforin is a secreted protein synthesized by activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It is a key component of the lytic machinery of these cells, being able to insert into the plasma membrane of targeted cells, forming a pore which leads to their destruction. Here we analyse the synthesis, processing and intracellular transport of perforin in the NK cell line YT. Perforin is synthesized as a 70 kDa inactive precursor which is cleaved at the C‐terminus to yield a 60 kDa active form. This proteolytic cleavage occurs in an acidic compartment and can be inhibited by incubation of the cells in ammonium chloride, concanamycin A, leupeptin and E‐64. The increased lytic activity of the cleaved form can be demonstrated by killing assays in which cleavage of the pro‐piece is inhibited. Epitope mapping reveals that cleavage of the pro‐piece occurs at the boundary of a C2 domain, which we show is able to bind phospholipid membranes in a calcium‐dependent manner. We propose that removal of the pro‐piece, which contains a bulky glycan, allows the C2 domain to interact with phospholipid membranes and initiate perforin pore formation.

Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

ABSTRACT The class II phosphoinositide 3-kinases (PI3K) PI3K-C2α and PI3K-C2β are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2α and PI3K-C2β represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2α and PI3K-C2β immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca2+ as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2α is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2β was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2β with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2β occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2β was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3′ phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.