RESUMEN
TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50<15nM) and good rodent pharmacokinetics (oral %F 97%).
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Triazinas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Benzamidas/administración & dosificación , Benzamidas/química , Disponibilidad Biológica , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Relación Estructura-Actividad , Triazinas/administración & dosificación , Triazinas/químicaRESUMEN
Previous efforts from our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we examined variations on this theme wherein 'directly-linked' aromatics, pendant from the indazole core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of 22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.
Asunto(s)
Antineoplásicos/farmacología , Indoles/química , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Área Bajo la Curva , Línea Celular Tumoral , Diseño de Fármacos , Xenoinjertos , Humanos , Indoles/administración & dosificación , Indoles/farmacocinética , Ratones , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , RatasRESUMEN
The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Asunto(s)
Antibacterianos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Benzofuranos/uso terapéutico , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Pironas/uso terapéutico , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Administración Oral , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Benzofuranos/farmacología , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Infecciones Comunitarias Adquiridas/microbiología , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Esquema de Medicación , Enoil-ACP Reductasa (NADH)/metabolismo , Femenino , Humanos , Cinética , Ratones , Pruebas de Sensibilidad Microbiana , Pironas/farmacología , Sepsis/microbiología , Sepsis/mortalidad , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/enzimología , Staphylococcus aureus/crecimiento & desarrollo , Tasa de SupervivenciaRESUMEN
Spiropiperidine naphthyridinone inhibitors of Staphylococcus aureus and Escherichia coli FabI have been prepared. Compounds 14a and 14c were identified as having sub-nanomolar E. coli FabI activity and are among the most potent FabI inhibitors yet described. The structural model of 14a bound to E. coli FabI is shown.
Asunto(s)
Antibacterianos/farmacología , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Enoil-ACP Reductasa (NADH)/metabolismo , Escherichia coli/enzimología , Naftiridinas/farmacología , Piperidinas/farmacología , Compuestos de Espiro/farmacología , Staphylococcus aureus/enzimología , Antibacterianos/química , Dominio Catalítico , Enoil-ACP Reductasa (NADH)/química , Modelos Moleculares , Naftiridinas/química , Piperidinas/química , Unión Proteica , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
In the search for new antibacterial agents, the enzyme FabI has been identified as an attractive target. Employing a structure guided approach, the previously reported ene-amide series of FabI inhibitors were expanded to include 2,3,4,5-tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines. These novel series incorporate additional H-bonding functions and can be more water soluble than their naphthyridinone progenitors; diazepine 16c is shown to be efficacious in a mouse infection model.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Azepinas/química , Azepinas/farmacología , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Enoil-ACP Reductasa (NADH)/metabolismo , Animales , Antibacterianos/uso terapéutico , Azepinas/uso terapéutico , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Infecciones por Escherichia coli/tratamiento farmacológico , Ratones , Modelos Moleculares , Unión Proteica , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimologíaRESUMEN
Defining protein complexes is a vital aspect of cell biology because cellular processes are often carried out by stable protein complexes and their characterization often provides insights into their function. Accurate identification of the interacting proteins in macromolecular complexes is easiest after purification to near homogeneity. To this end, the tandem affinity purification (TAP) system with subsequent protein identification by high-throughput mass spectrometry was developed (1, 2) to systematically characterize native protein complexes and transient protein interactions under near-physiological conditions. The TAP tag containing two adjacent affinity purification tags (calmodulin-binding peptide and Staphylococcus aureus protein A) separated by a tobacco etch virus (TEV) protease cleavage site is fused with the open reading frame of interest. Using homologous recombination, a fusion library was constructed for the yeast Saccharomyces cerevisiae (3) in which the carboxy-terminal end of each predicted open reading frame is individually tagged in the chromosome so that the resulting fusion proteins are expressed under the control of their natural promoters (3). In this chapter, an optimized protocol for systematic protein purification and subsequent mass spectrometry-based protein identification is described in detail for the protein complexes of S. cerevisiae (4-6).
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas en Tándem/métodos , Marcadores de Afinidad , Cromatografía de Afinidad , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Mapeo Peptídico , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.
Asunto(s)
Rastreo Diferencial de Calorimetría/métodos , Técnicas Químicas Combinatorias , Evaluación Preclínica de Medicamentos/métodos , Ligandos , Proteínas/metabolismo , Sitios de Unión , Citrato (si)-Sintasa/metabolismo , Indicadores y Reactivos , Ácido Oxaloacético/metabolismo , Proteínas/química , TemperaturaRESUMEN
This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.
RESUMEN
Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Indazoles/farmacología , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/análisis , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Femenino , Células HCT116 , Humanos , Indazoles/química , Indazoles/farmacocinética , Indoles/química , Indoles/farmacocinética , Células MCF-7 , Masculino , Ratones Desnudos , Ratones SCID , Modelos Químicos , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved hysicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Indoles/química , Indoles/farmacocinética , Células MCF-7 , Ratones , Modelos Químicos , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Compuestos de Espiro/química , Compuestos de Espiro/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias del Colon/tratamiento farmacológico , Indazoles/química , Indazoles/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/enzimología , Colon/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Cristalografía por Rayos X , Femenino , Humanos , Indazoles/administración & dosificación , Indazoles/farmacología , Ratones Desnudos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismoRESUMEN
PLK4 was identified as a promising therapeutic target through a systematic approach that combined RNAi screening with gene expression analysis in human breast cancers and cell lines. A drug discovery program culminated in CFI-400945, a potent and selective PLK4 inhibitor. Cancer cells treated with CFI-400945 exhibit effects consistent with PLK4 kinase inhibition, including dysregulated centriole duplication, mitotic defects, and cell death. Oral administration of CFI-400945 to mice bearing human cancer xenografts results in the significant inhibition of tumor growth at doses that are well tolerated. Increased antitumor activity in vivo was observed in PTEN-deficient compared to PTEN wild-type cancer xenografts. Our findings provide a rationale for the clinical evaluation of CFI-400945 in patients with solid tumors, in particular those deficient in PTEN.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Indazoles/farmacología , Indoles/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Centriolos/efectos de los fármacos , Centriolos/metabolismo , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AFN-1252 is a novel inhibitor of FabI, an essential enzyme in fatty acid biosynthesis in Staphylococcus spp. AFN-1252 exhibits typical MIC(90) values of ≤0·015 µg/ml against diverse clinical isolates of S. aureus, oral absorption, long elimination half-live and efficacy in animal models. We now report high binding (â¼95%) to serum proteins of mouse, rat, dog and humans, associated with an eight-fold increase in minimal inhibitory concentration (MIC) and which may be responsible for the long elimination half-lives on pharmacokinetic studies. Unlike daptomycin, AFN-1252 activity is not reduced in the presence of lung surfactant. AFN-1252 exhibits a short post-antibiotic effect of 1·1 hours against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) following a 4-hour exposure period. The AFN-1252 unique spectrum of activity is not compromised by interactions with major antibiotic classes, but demonstrates synergy with low concentrations of gentamicin against MSSA and MRSA. These studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.
Asunto(s)
Antibacterianos/sangre , Antibacterianos/farmacología , Benzofuranos/sangre , Benzofuranos/farmacología , Proteínas Sanguíneas/metabolismo , Pironas/sangre , Pironas/farmacología , Animales , Daptomicina/sangre , Daptomicina/farmacología , Perros , Gentamicinas/sangre , Gentamicinas/farmacología , Semivida , Humanos , Masculino , Meticilina/sangre , Meticilina/farmacología , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Surfactantes Pulmonares/metabolismo , Ratas , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function.
Asunto(s)
Glucógeno/metabolismo , Enfermedad de Lafora/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Carbono/metabolismo , Glucógeno Sintasa/metabolismo , Enfermedad de Lafora/enzimología , Masculino , Ratones , Fosforilación , ConejosRESUMEN
The family of Polo-like kinases is important in the regulation of mitotic progression; this work keys on one member, namely Polo-like kinase 4 (PLK4). PLK4 has been identified as a candidate anticancer target which prompted a search for potent and selective inhibitors of PLK4. The body of the paper describes lead generation and optimization work which yielded nanomolar PLK4 inhibitors. Lead generation began with directed virtual screening, using a ligand-based focused library and a PLK4 homology model. Validated hits were used as starting points for the design and discovery of PLK4 inhibitors of novel structure, namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones. Computational models, based on a published X-ray structure (PLK4 kinase domain), were used to understand and optimize the in vitro activity of the series; potent antiproliferative activity was obtained. The kinase selectivity profile and cell cycle analysis of selected inhibitors are described. The results of a xenograft study with an optimized compound 50 (designated CFI-400437) support the potential of these novel PLK4 inhibitors for cancer therapy.
Asunto(s)
Antineoplásicos/síntesis química , Indazoles/síntesis química , Indoles/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Indazoles/química , Indazoles/farmacología , Indoles/química , Indoles/farmacología , Ratones , Ratones SCID , Modelos Moleculares , Estereoisomerismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Adhesion complexes typically assemble from clustered receptors that link to the cytoskeleton via cytoplasmic adapter proteins. However, it is unclear how phospholipid-anchored adhesion molecules, such as the Dictyostelium receptor gp80, interact with the cytoskeleton. gp80 has been found to form adhesion complexes from raftlike membrane domains, which can be isolated as a Triton X-100-insoluble floating fraction (TIFF). We report here that the actin-binding protein ponticulin mediates TIFF-cytoskeleton interactions. Analysis of gp80-null cells revealed that these interactions were minimal in the absence of gp80. During development, gp80 was required to enhance these interactions as its adhesion complexes assembled. Whereas ponticulin and gp80 could partition independently into TIFF, gp80 was shown to recruit ponticulin to cell-cell contacts and to increase its partitioning into TIFF. However, these proteins did not co-immunoprecipitate. Furthermore, sterol sequestration abrogated the association of ponticulin with TIFF without affecting gp80, suggesting that sterols may mediate the interactions between ponticulin and gp80. In ponticulin-null cells, large gp80 adhesion complexes assembled in the absence of ponticulin despite the lack of cytoskeleton association. We propose that such nascent gp80 adhesion complexes produce expanded raftlike domains that recruit ponticulin and thereby establish stable cytoskeleton interactions to complete the assembly process.
Asunto(s)
Proteínas Portadoras , Citoesqueleto/metabolismo , Dictyostelium/metabolismo , Glicoproteínas de Membrana/química , Proteínas de Microfilamentos , Actinas/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas Portadoras/farmacología , Adhesión Celular , Comunicación Celular , Detergentes/farmacología , Electroforesis en Gel de Poliacrilamida , Enterobacter aerogenes/metabolismo , Metabolismo de los Lípidos , Glicoproteínas de Membrana/farmacología , Proteínas de Microfilamentos/farmacología , Microscopía Fluorescente , Octoxinol/farmacología , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Tiazoles/farmacología , TiazolidinasRESUMEN
Transcript elongation can be interrupted by a variety of obstacles, including certain DNA sequences, DNA-binding proteins, chromatin, and DNA lesions. Bypass of many of these impediments is facilitated by elongation factor TFIIS through a mechanism that involves cleavage of the nascent transcript by the RNA polymerase II/TFIIS elongation complex. Highly purified yeast RNA polymerase II is able to perform transcript hydrolysis in the absence of TFIIS. The "intrinsic" cleavage activity is greatly stimulated at mildly basic pH and requires divalent cations. Both arrested and stalled complexes can carry out the intrinsic cleavage reaction, although not all stalled complexes are equally efficient at this reaction. Arrested complexes in which the nascent transcript was cleaved in the absence of TFIIS were reactivated to readthrough blocks to elongation. Thus, cleavage of the nascent transcript is sufficient for reactivating some arrested complexes. Small RNA products released following transcript cleavage in stalled ternary complexes differ depending upon whether the cleavage has been induced by TFIIS or has occurred in mildly alkaline conditions. In contrast, both intrinsic and TFIIS-induced small RNA cleavage products are very similar when produced from an arrested ternary complex. Although alpha-amanitin interferes with the transcript cleavage stimulated by TFIIS, it has little effect on the intrinsic cleavage reaction. A mutant RNA polymerase previously shown to be refractory to TFIIS-induced transcript cleavage is essentially identical to the wild type polymerase in all tested aspects of intrinsic cleavage.