ABSTRACT
γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-ß peptides. Amyloid-ß peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.
Subject(s)
Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/drug effects , Carbazoles/pharmacology , Lipid Bilayers , Amyloid Precursor Protein Secretases/metabolism , Carbazoles/metabolism , Humans , Magnetic Resonance Spectroscopy/methods , Molecular Dynamics SimulationABSTRACT
DYRK1A has been associated with Down's syndrome and neurodegenerative diseases, therefore it is an important target for novel pharmacological interventions. We combined a ligand-based pharmacophore design with a structure-based protein/ligand docking using the software MOE in order to evaluate the underlying structure/activity relationship. Based on this knowledge we synthesized several novel ß-carboline derivatives to validate the theoretical model. Furthermore we identified a modified lead structure as a potent DYRK1A inhibitor (IC50=130 nM) with significant selectivity against MAO-A, DYRK2, DYRK3, DYRK4 & CLK2.
Subject(s)
Carbolines/chemistry , Carbolines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Carbolines/chemical synthesis , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Ligands , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Software , Structure-Activity Relationship , Dyrk KinasesABSTRACT
The preparation of alkyne-modified ansamitocins by mutasynthetic supplementation of Actinosynnema pretiosum mutants with alkyne-substituted aminobenzoic acids is described. This modification paved the way to introduce a thiol linker by Huisgen-type cycloaddition which can principally be utilized to create tumor targeting conjugates. In bioactivity tests, only those new ansamitocin derivatives showed strong antiproliferative activity that bear an ester side chain at C-3.
ABSTRACT
OBJECTIVE: We investigated the role of matrix metalloproteinase-8 (MMP8) in neointima formation and in vascular smooth muscle cell (VSMC) migration and proliferation. APPROACH AND RESULTS: After carotid artery wire injuring, MMP8(-/-)/apoE(-/-) mice had fewer proliferating cells in neointimal lesions and smaller lesion sizes. Ex vivo assays comparing VSMCs isolated from MMP8 knockout and wild-type mice showed that MMP8 knockout decreased proliferation and migration. Proteomics analysis revealed that a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) had lower concentrations in MMP8 knockout VSMC culture media than in MMP8 wild-type VSMC culture media. Western blot, flow cytometric, and immunocytochemical analyses showed that MMP8 knockout VSMCs contained more pro-ADAM10 but less mature ADAM10, more N-cadherin, and ß-catenin in the plasma membrane but less ß-catenin in the nucleus and less cyclin D1. Treatment of MMP8 wild-type VSMCs with an ADAM10 inhibitor, GI254023X, or siRNA knockdown of ADAM10 in MMP8 wild-type VSMCs inhibited proliferation and migration, increased N-cadherin and ß-catenin in the plasma membrane, reduced ß-catenin in the nucleus, and decreased cyclin D1 expression. Incubation of MMP8 knockout VSMCs with a recombinant ADAM10 rescued the proliferative and migratory ability of MMP8 knockout VSMCs and increased cyclin D1 expression. Furthermore, immunohistochemical analyses showed colocalization of ADAM10 with VSMCs and N-cadherin, and nuclear accumulation of ß-catenin in the neointima in apoE(-/-)/MMP8(+/+) mice. CONCLUSIONS: MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and ß-catenin-mediated pathway and plays an important role in neointima formation.
Subject(s)
Carotid Artery Injuries/enzymology , Carotid Artery Injuries/pathology , Cell Proliferation , Matrix Metalloproteinase 8/metabolism , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Neointima , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Cadherins/metabolism , Carotid Artery Injuries/genetics , Cell Movement , Cell Proliferation/drug effects , Cells, Cultured , Culture Media, Conditioned/metabolism , Cyclin D1/metabolism , Disease Models, Animal , Matrix Metalloproteinase 8/deficiency , Matrix Metalloproteinase 8/genetics , Matrix Metalloproteinase Inhibitors/pharmacology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Proteomics/methods , RNA Interference , Time Factors , Transfection , Wnt Signaling Pathway , Wnt1 Protein/metabolism , beta Catenin/metabolismABSTRACT
The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt's ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.
Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Biological Evolution , Cadherins/metabolism , Cell Adhesion/physiology , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neuroepithelial Cells/physiology , Neurons/physiology , Nuclear Proteins/metabolism , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/genetics , ADAM10 Protein , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Analysis of Variance , Animals , Animals, Genetically Modified , Apoptosis/drug effects , Apoptosis/genetics , Body Patterning/drug effects , Body Patterning/genetics , Brain/cytology , Brain/drug effects , Brain/embryology , Brain/metabolism , Cadherins/genetics , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Cerebral Ventricles/cytology , Cerebral Ventricles/embryology , Dictyostelium , Dipeptides/pharmacology , Discs Large Homolog 1 Protein , Drosophila melanogaster , Embryo, Mammalian , Embryo, Nonmammalian , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanylate Kinases/genetics , Guanylate Kinases/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Huntingtin Protein , Hydroxamic Acids/pharmacology , Immunoprecipitation , Intermediate Filament Proteins/genetics , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mice , Morpholines/pharmacology , Mutation/genetics , NFI Transcription Factors/metabolism , Nerve Tissue Proteins/geneticsABSTRACT
CD23, the low-affinity receptor for IgE, exists in membrane and soluble forms. Soluble CD23 (sCD23) fragments are released from membrane (m)CD23 by the endogenous metalloprotease a disintegrin and metalloprotease 10. When purified tonsil B cells are incubated with IL-4 and anti-CD40 to induce class switching to IgE in vitro, mCD23 is upregulated, and sCD23 accumulates in the medium prior to IgE synthesis. We have uncoupled the effects of mCD23 cleavage and accumulation of sCD23 on IgE synthesis in this system. We show that small interfering RNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an a disintegrin and metalloprotease 10 inhibitor, GI254023X, suppresses IL-4 and anti-CD40-stimulated IgE synthesis. Addition of a recombinant trimeric sCD23 enhances IgE synthesis in this system. This occurs even when endogenous mCD23 is protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. We show that recombinant trimeric sCD23 binds to cells coexpressing mIgE and mCD21 and caps these proteins on the B cell membrane. Upregulation of IgE by sCD23 occurs after class-switch recombination, and its effects are isotype-specific. These results suggest that mIgE and mCD21 cooperate in the sCD23-mediated positive regulation of IgE synthesis on cells committed to IgE synthesis. Feedback regulation may occur when the concentration of secreted IgE becomes great enough to allow binding to mCD23, thus preventing further release of sCD23. We interpret these results with the aid of a model for the upregulation of IgE by sCD23.
