Discovery of a potent and highly selective PDK1 inhibitor via fragment-based drug discovery.

We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation.

Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.

Growth factor induction of Cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migration.

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms.

Targeting the lymphotoxin-beta receptor with agonist antibodies as a potential cancer therapy.

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor.

An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcepsilonRIalpha.

The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcepsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood.

Expression, purification, and characterization of rat interferon-beta, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters.

To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.

Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo.

Cripto, a cell surface-associated protein belonging to the EGF-CFC family of growth factor-like molecules, is overexpressed in many human solid tumors, including 70-80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-beta ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-beta ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells.

Fucosylation of Cripto is required for its ability to facilitate nodal signaling.

O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors.

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR).

Identification of a palmitic acid-modified form of human Sonic hedgehog.

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated.

Cytotoxic activities of recombinant soluble murine lymphotoxin-alpha and lymphotoxin-alpha beta complexes.

Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized.

Characterization of lymphotoxin-alpha beta complexes on the surface of mouse lymphocytes.

The lymphotoxin-alpha beta complex (LT alpha beta) is found on the surface of activated lymphocytes and binds to a specific receptor called the LT beta receptor (LT beta R). In the mouse, signaling through this pathway is important for lymph node development and splenic organization, yet the biochemical properties of murine LT alpha and LT beta are essentially unknown. Here we have used soluble receptor-Ig forms of LT beta R and TNF-R55 and mAbs specific for murine LT alpha, LT beta, and LT beta R to characterize the appearance of surface LT alpha beta complexes and LT beta R on several common murine cell lines.

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step.

Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxins alpha and beta.

The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells.

Signaling through the lymphotoxin beta receptor induces the death of some adenocarcinoma tumor lines.

Surface lymphotoxin (LT) is a heteromeric complex of LT-alpha and LT-beta chains that binds to the LT-beta receptor (LT-beta-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.

Immunomodulation by LFA3TIP, an LFA-3/IgG1 fusion protein: cell line dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers.

LFA3TIP, a fusion protein comprised of the first extracellular domain of LFA-3 fused to the hinge, CH2, and CH3 domains of human IgG1 inhibits responses of human and non-human primate T cells in vitro. In seeking to optimize the expression efficiency to prepare large quantities of LFA3TIP for primate studies, the protein was produced in both the CHO (Chinese hamster ovary) and murine NS-0 myeloma cell lines. Although LFA3TIP derived from these cell lines performs identically in vitro in CD2 receptor binding and T cell assays, examination of a pharmacodynamic marker-the reduction in CD2+ lymphocyte numbers-following the administration of equal doses of NS-0 or CHO derived LFA3TIP to baboons, suggested that the effect of the NS-0 derived material was less sustained.

Characterization of surface lymphotoxin forms. Use of specific monoclonal antibodies and soluble receptors.

Lymphotoxin (LT) is a cytokine related to TNF, found in human systems in both secreted and membrane bound forms. The well characterized secreted form is a trimer of a single protein, LT-alpha, whereas the surface form is composed of a complex between two related molecules, LT-alpha and LT-beta. Because there is a distinct receptor for the complex, the membrane form is believed to signal via events different from those elicited by TNF and secreted LT-alpha.

The ELAM ligand fucosyltransferase, ELFT, directs E-selectin binding to a secreted scaffold protein: a method to produce and purify large quantities of specific carbohydrate structures.

Many biological processes depend on cell surface recognition of receptor-ligand pairs. Some receptors, such as the selectins, recognize specific carbohydrate structures as part of their ligands. The ability to synthesize such ligands for use in the study of cell adhesion mechanisms or as inhibitors of a variety of pathological conditions would be extremely useful.