RESUMEN
Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.
Asunto(s)
Antivirales/farmacología , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Ensayos Analíticos de Alto Rendimiento , Hidrodinámica , Ratones , Modelos Moleculares , Nucleoproteínas/ultraestructura , Orthomyxoviridae/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Bibliotecas de Moléculas Pequeñas/uso terapéutico , SolucionesRESUMEN
Human TAG-1 is a neural cell adhesion molecule that is crucial for the development of the nervous system during embryogenesis. It consists of six immunoglobulin-like and four fibronectin III-like domains and is anchored to the membrane by glycosylphosphatidylinositol. Herein we present the crystal structure of the four N-terminal immunoglobulin-like domains of TAG-1 (TAG-1(Ig1-4)), known to be important in heterophilic and homophilic macromolecular interactions. The contacts of neighboring molecules within the crystal were investigated. A comparison with the structure of the chicken ortholog resulted in an alternative mode for the molecular mechanism of homophilic TAG-1 interaction. This mode of TAG-1 homophilic interaction is based on dimer formation rather than formation of a molecular zipper as proposed for the chicken ortholog.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/química , Modelos Moleculares , Animales , Proteínas Aviares/química , Sitios de Unión , Moléculas de Adhesión Celular Neuronal/aislamiento & purificación , Moléculas de Adhesión Celular Neuronal/metabolismo , Pollos , Contactina 2 , Cristalografía por Rayos X , Dimerización , Humanos , Ligandos , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kß relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).
Asunto(s)
Antineoplásicos/farmacología , Benzoxepinas/farmacología , Diseño de Fármacos , Imidazoles/farmacología , Oxazepinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Benzoxepinas/química , Benzoxepinas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazoles/química , Imidazoles/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Macaca fascicularis , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Oxazepinas/química , Oxazepinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC) can cause severe and potentially life-threatening infections. Their pathogenicity is mediated by at least 40 effector proteins which they inject into their host cells by a type-III secretion system leading to the subversion of several cellular pathways. However, the molecular function of several effectors remains unknown, even though they contribute to virulence. Here we show that one of them, NleF, binds to caspase-4, -8, and -9 in yeast two-hybrid, LUMIER, and direct interaction assays. NleF inhibits the catalytic activity of the caspases in vitro and in cell lysate and prevents apoptosis in HeLa and Caco-2 cells. We have solved the crystal structure of the caspase-9/NleF complex which shows that NleF uses a novel mode of caspase inhibition, involving the insertion of the carboxy-terminus of NleF into the active site of the protease. In conformance with our structural model, mutagenized NleF with truncated or elongated carboxy-termini revealed a complete loss in caspase binding and apoptosis inhibition. Evasion of apoptosis helps pathogenic E. coli and other pathogens to take over the host cell by counteracting the cell's ability to self-destruct upon infection. Recently, two other effector proteins, namely NleD and NleH, were shown to interfere with apoptosis. Even though NleF is not the only effector protein capable of apoptosis inhibition, direct inhibition of caspases by bacterial effectors has not been reported to date. Also unique so far is its mode of inhibition that resembles the one obtained for synthetic peptide-type inhibitors and as such deviates substantially from previously reported caspase-9 inhibitors such as the BIR3 domain of XIAP.
Asunto(s)
Inhibidores de Caspasas/metabolismo , Caspasas/metabolismo , Escherichia coli O157 , Proteínas de Escherichia coli/metabolismo , Factores de Virulencia/metabolismo , Inhibidores de Caspasas/química , Inhibidores de Caspasas/farmacología , Caspasas/química , Línea Celular , Escherichia coli O157/fisiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacología , Humanos , Modelos Moleculares , Conformación Proteica , Reproducibilidad de los Resultados , Factores de Virulencia/química , Factores de Virulencia/farmacologíaRESUMEN
B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Quinazolinas/síntesis química , Animales , Humanos , Masculino , Melanoma/tratamiento farmacológico , Ratones , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Quinazolinas/farmacocinética , Quinazolinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 A, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the "amine oxidase" class of flavoproteins, such as d-amino acid oxidase and monomeric sarcosine oxidase. With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR(2) subfamily of flavooxidases. On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior. The results do not clarify the metabolic role of this enzyme in B. subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis. However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or d-alanine as carbon or nitrogen sources.