RESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/efectos de los fármacos , Carbazoles/farmacología , Membrana Dobles de Lípidos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Carbazoles/metabolismo , Humanos , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica MolecularRESUMEN
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.
Asunto(s)
Carbolinas/química , Carbolinas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Carbolinas/síntesis química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Programas Informáticos , Relación Estructura-Actividad , Quinasas DyrKRESUMEN
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.
RESUMEN
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.
Asunto(s)
Traumatismos de las Arterias Carótidas/enzimología , Traumatismos de las Arterias Carótidas/patología , Proliferación Celular , Metaloproteinasa 8 de la Matriz/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Neointima , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Cadherinas/metabolismo , Traumatismos de las Arterias Carótidas/genética , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Metaloproteinasa 8 de la Matriz/deficiencia , Metaloproteinasa 8 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Proteómica/métodos , Interferencia de ARN , Factores de Tiempo , Transfección , Vía de Señalización Wnt , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Evolución Biológica , Cadherinas/metabolismo , Adhesión Celular/fisiología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Neuroepiteliales/fisiología , Neuronas/fisiología , Proteínas Nucleares/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Apoptosis/genética , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Encéfalo/metabolismo , Cadherinas/genética , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Ventrículos Cerebrales/citología , Ventrículos Cerebrales/embriología , Dictyostelium , Dipéptidos/farmacología , Homólogo 1 de la Proteína Discs Large , Drosophila melanogaster , Embrión de Mamíferos , Embrión no Mamífero , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína Huntingtina , Ácidos Hidroxámicos/farmacología , Inmunoprecipitación , Proteínas de Filamentos Intermediarios/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Morfolinas/farmacología , Mutación/genética , Factores de Transcripción NFI/metabolismo , Proteínas del Tejido Nervioso/genéticaRESUMEN
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.