Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
Add more filters










Database
Language
Publication year range
1.
ACS Med Chem Lett ; 6(8): 913-8, 2015 Aug 13.
Article in English | MEDLINE | ID: mdl-26288693

ABSTRACT

Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.

2.
Nature ; 519(7544): 425-30, 2015 Mar 26.
Article in English | MEDLINE | ID: mdl-25799996

ABSTRACT

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.


Subject(s)
Cell Movement , Endothelial Cells/cytology , Endothelial Cells/metabolism , Integrins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Motifs , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Shape/drug effects , Endothelial Cells/drug effects , Epistasis, Genetic , Focal Adhesions/metabolism , Humans , Integrin alpha1/drug effects , Integrin alpha1/metabolism , Integrin beta1/chemistry , Integrin beta1/drug effects , Integrin beta1/metabolism , Integrins/drug effects , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Neovascularization, Pathologic , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Talin/chemistry , Talin/metabolism
3.
J Med Chem ; 57(8): 3484-93, 2014 Apr 24.
Article in English | MEDLINE | ID: mdl-24673130

ABSTRACT

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.


Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemical synthesis , Animals , Drug Discovery , Female , Intracellular Signaling Peptides and Proteins/chemistry , Mice , Models, Molecular , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/chemistry , Pyrimidines/pharmacology , Structure-Activity Relationship
4.
J Clin Invest ; 123(9): 3997-4009, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23945239

ABSTRACT

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies/pharmacology , Apoptosis , Endothelial Growth Factors/immunology , Human Umbilical Vein Endothelial Cells/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Bevacizumab , Calcium-Binding Proteins , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Clinical Trials, Phase I as Topic , EGF Family of Proteins , Human Umbilical Vein Endothelial Cells/physiology , Humans , Insulinoma/blood supply , Insulinoma/drug therapy , Insulinoma/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice , Mice, Nude , Mice, Transgenic , Neoplastic Cells, Circulating/drug effects , Neoplastic Cells, Circulating/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Tumor Burden/drug effects , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/physiology , Xenograft Model Antitumor Assays
5.
Blood ; 122(22): 3678-90, 2013 Nov 21.
Article in English | MEDLINE | ID: mdl-23886837

ABSTRACT

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.


Subject(s)
Carrier Proteins/physiology , Endothelium, Vascular/embryology , Endothelium, Vascular/physiology , Guanine Nucleotide Exchange Factors/metabolism , rap1 GTP-Binding Proteins/metabolism , Actins/metabolism , Animals , Animals, Genetically Modified , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Female , Human Umbilical Vein Endothelial Cells , Humans , Intercellular Junctions/physiology , Intracellular Signaling Peptides and Proteins , Mice , Mice, Knockout , Monomeric GTP-Binding Proteins/metabolism , Neovascularization, Physiologic , Pregnancy , RNA Interference , Signal Transduction , Zebrafish , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish Proteins/physiology
6.
Development ; 134(16): 2913-23, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17626061

ABSTRACT

During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.


Subject(s)
Blood Vessels/embryology , Body Patterning , Cell Movement/genetics , Endothelial Cells/cytology , Proteins/physiology , Animals , Blood Vessels/abnormalities , Calcium-Binding Proteins , Cells, Cultured , Chick Embryo , EGF Family of Proteins , Endothelium, Vascular/abnormalities , Fetal Viability/genetics , Humans , Mice , Mice, Knockout , Models, Biological , Proteins/genetics
7.
Novartis Found Symp ; 283: 18-28; discussion 28-36, 238-41, 2007.
Article in English | MEDLINE | ID: mdl-18300411

ABSTRACT

EGFL7 was identified by a number of groups as a putative secreted factor produced by the vascular endothelial cells (ECs). In a recent publication, we showed that EGFL7 regulates midline angioblast migration in zebrafish embryos-a key step in vascular tubulogenesis. In this study, we further characterized the zebrafish vasculature in the Egfl7 knockdown embryos at the ultrastructural level, and found that malformation of axial vessels is indeed due to the accumulation of angioblasts and aberrant connection among themselves, but not abnormal interaction between ECs and other cell types. Using in vitro biochemical assays, we demonstrated that EGFL7 is tightly associated with the extracellular matrix (ECM), and it supports EC migration either as a single factor or in combination with other ECM molecules. In order to evaluate if the biological function of EGFL7 is evolutionarily conserved, we generated Egfl7 knockout mice and analysed vascular development in a number of tissues. We found that vascular coverage of a given tissue is reduced or delayed, and vascular morphogenesis is defective in the Egfl7 mutant mice. Taken together, we conclude that EGFL7 provides a proper microenvironment for endothelial cell migration, thereby enabling accurate patterning. Our study indicates that the molecular composition of the ECM influences vascular morphogenesis.


Subject(s)
Blood Vessels/embryology , Embryo, Mammalian/blood supply , Embryo, Mammalian/metabolism , Morphogenesis , Neovascularization, Physiologic , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Blood Vessels/ultrastructure , Body Patterning , Cell Movement , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/ultrastructure , Endothelial Cells/cytology , Endothelial Cells/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Mice , Neoplasms/blood supply , Neovascularization, Pathologic , Zebrafish Proteins/genetics
8.
Cell Signal ; 18(1): 40-9, 2006 Jan.
Article in English | MEDLINE | ID: mdl-15927449

ABSTRACT

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif.


Subject(s)
Cytokine Receptor gp130/metabolism , Interleukin-6/pharmacology , STAT3 Transcription Factor/metabolism , Amino Acid Motifs/drug effects , Amino Acid Motifs/physiology , Amino Acid Sequence , Animals , Cell Line , Cytokine Receptor gp130/drug effects , Cytokine Receptor gp130/genetics , Gene Expression Regulation , Mice , Molecular Sequence Data , Protein Structure, Secondary , Rats , STAT3 Transcription Factor/drug effects , STAT3 Transcription Factor/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Tyrosine/drug effects , Tyrosine/metabolism
9.
J Biol Chem ; 280(27): 25760-8, 2005 Jul 08.
Article in English | MEDLINE | ID: mdl-15894543

ABSTRACT

The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue Arg466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutation does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a yellow fluorescent protein (YFP)-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR.


Subject(s)
Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction/physiology , src Homology Domains/physiology , Amino Acid Sequence , Animals , COS Cells , Cell Line, Tumor , Chickens , Chlorocebus aethiops , Drosophila , Fibrosarcoma , Fishes , Humans , Interferons/metabolism , Interleukin-6/metabolism , Janus Kinase 1 , Macaca mulatta , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Rats , Swine , Up-Regulation/physiology
SELECTION OF CITATIONS
SEARCH DETAIL
...