RESUMEN
Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.
Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Neuropilina-2/metabolismo , Receptores Virales/metabolismo , Anticuerpos Neutralizantes/química , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Conformación Proteica , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.
Asunto(s)
Antineoplásicos/farmacología , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/química , Animales , Antineoplásicos/química , Calorimetría , Línea Celular , Fibroblastos/metabolismo , Xenoinjertos , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transducción de Señal , Bibliotecas de Moléculas PequeñasRESUMEN
Brain rhythms provide the timing for recruitment of brain activity required for linking together neuronal ensembles engaged in specific tasks. The γ-oscillations (30 to 120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here, we report on a potent brain-permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a class of therapeutics for AD. We employed anatomical, in vitro and in vivo electrophysiological, and behavioral methods to examine the effects of our lead therapeutic candidate small molecule. As a novel in central nervous system pharmacotherapy, our lead molecule acts as a potent, efficacious, and selective negative allosteric modulator of the γ-aminobutyric acid type A receptors most likely assembled from α1ß2δ subunits. These receptors, identified through anatomical and pharmacological means, underlie the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. When orally administered twice daily for 2 wk, DDL-920 restored the cognitive/memory impairments of 3- to 4-mo-old AD model mice as measured by their performance in the Barnes maze. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.
Asunto(s)
Enfermedad de Alzheimer , Cognición , Modelos Animales de Enfermedad , Ritmo Gamma , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Ratones , Cognición/efectos de los fármacos , Ritmo Gamma/efectos de los fármacos , Memoria/efectos de los fármacos , Receptores de GABA-A/metabolismo , Ratones Transgénicos , Humanos , Masculino , Memoria a Corto Plazo/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Alanina/análogos & derivados , AzepinasRESUMEN
The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.
Asunto(s)
Diferenciación Celular , Células Epidérmicas/citología , Epidermis/fisiología , Factores Reguladores del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Anomalías Múltiples/genética , Animales , Labio Leporino/genética , Fisura del Paladar/genética , Quistes/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Epidérmicas/metabolismo , Epidermis/embriología , Anomalías del Ojo/genética , Femenino , Dedos/anomalías , Regulación de la Expresión Génica , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Rodilla/anomalías , Articulación de la Rodilla/anomalías , Labio/anomalías , Metabolismo de los Lípidos/genética , Deformidades Congénitas de las Extremidades Inferiores/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Sindactilia/genética , Anomalías Urogenitales/genéticaRESUMEN
Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function.SIGNIFICANCE STATEMENT Mossy cells (MCs), a major cell type in the hilus of the dentate gyrus (DG), are unique in providing extensive longitudinal and commissural projections throughout the DG. Although it has been assumed that all MCs have similar patterns of termination in the inner molecular layer of the DG, we discovered that the axonal projections of dorsal and ventral MCs differ. While ventral MC projections exhibit the classical pattern, with dense innervation in the inner molecular layer, dorsal MCs have a more diffuse distribution and expand into the middle molecular layer where they overlap and interact with innervation from the perforant path. These distinct locations and patterns of axonal projections suggest that dorsal and ventral MCs may have different functional roles.
Asunto(s)
Axones/química , Axones/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Fibras Musgosas del Hipocampo/química , Fibras Musgosas del Hipocampo/fisiología , Animales , Giro Dentado/química , Giro Dentado/citología , Giro Dentado/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Optogenética/métodosRESUMEN
Endothelial lipase (EL) inhibitors have been shown to elevate HDL-C levels in pre-clinical murine models and have potential benefit in prevention and treatment of cardiovascular diseases. Modification of the 1-ethyl-3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (DHP) lead, 1, led to the discovery of a series of potent tetrahydropyrimidinedione (THP) EL inhibitors. Synthesis and SAR studies including modification of the amide group, together with changes on the pyrimidinone core led to a series of arylcycloalkyl, indanyl, and tetralinyl substituted 5-amino or 5-hydroxypyrimidinedione-4-carboxamides. Several compounds were advanced to PK evaluation. Among them, compound 4a was one of the most potent with measurable ELHDL hSerum potency and compound 3g demonstrated the best overall pharmacokinetic parameters.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Lipasa/antagonistas & inhibidores , Pirimidinonas/química , Pirimidinonas/farmacología , Animales , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/síntesis química , Humanos , Lipasa/sangre , Lipasa/metabolismo , Ratones , Modelos Moleculares , Pirimidinonas/sangre , Pirimidinonas/síntesis química , Relación Estructura-ActividadRESUMEN
3-Methylcrotonyl-CoA carboxylase (MCC), a member of the biotin-dependent carboxylase superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans. MCC has strong sequence conservation with propionyl-CoA carboxylase (PCC), and their holoenzymes are both 750-kilodalton (kDa) α(6)ß(6) dodecamers. Therefore the architecture of the MCC holoenzyme is expected to be highly similar to that of PCC. Here we report the crystal structures of the Pseudomonas aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly, the structures show that the architecture and overall shape of PaMCC are markedly different when compared to PCC. The α-subunits show trimeric association in the PaMCC holoenzyme, whereas they have no contacts with each other in PCC. Moreover, the positions of the two domains in the ß-subunit of PaMCC are swapped relative to those in PCC. This structural information establishes a foundation for understanding the disease-causing mutations of MCC and provides new insights into the catalytic mechanism and evolution of biotin-dependent carboxylases. The large structural differences between MCC and PCC also have general implications for the relationship between sequence conservation and structural similarity.
Asunto(s)
Ligasas de Carbono-Carbono/química , Pseudomonas aeruginosa/enzimología , Sitios de Unión , Biocatálisis , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Ligasas de Carbono-Carbono/ultraestructura , Coenzima A/química , Coenzima A/metabolismo , Cristalografía por Rayos X , Enfermedad/genética , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Metilmalonil-CoA Descarboxilasa/química , Modelos Moleculares , Mutación/genética , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT: In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic inhibition. In an effort to increase tonic inhibition selectively in these interneurons, we used Cre-dependent viral vectors in SOM-Cre mice to achieve interneuron-specific expression of the nonsynaptic GABAAR subunits (α6 and δ) in vivo. We show, for the first time, that such recombinant GFP-tagged GABAAR subunits are expressed robustly, assemble to form functional receptors, substantially increase tonic inhibition in SOM interneurons, and alter circuit activity within the dentate gyrus.
Asunto(s)
Giro Dentado/citología , Red Nerviosa/metabolismo , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Somatostatina/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Giro Dentado/efectos de los fármacos , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Vectores Genéticos/metabolismo , Humanos , Isoxazoles/farmacología , Masculino , Ratones , Ratones Transgénicos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/genética , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Pentilenotetrazol/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pirimidinas/farmacología , Receptores de GABA-A/genética , Somatostatina/genéticaRESUMEN
Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).
Asunto(s)
Microscopía por Crioelectrón , Holoenzimas/química , Holoenzimas/ultraestructura , Metilmalonil-CoA Descarboxilasa/química , Metilmalonil-CoA Descarboxilasa/ultraestructura , Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/metabolismo , Acetil-CoA Carboxilasa/ultraestructura , Biocatálisis , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Dominio Catalítico , Cristalografía por Rayos X , Acido Graso Sintasa Tipo II , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Metilmalonil-CoA Descarboxilasa/genética , Metilmalonil-CoA Descarboxilasa/metabolismo , Modelos Moleculares , Mutación/genética , Acidemia Propiónica/enzimología , Acidemia Propiónica/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Rhodobacteraceae/enzimología , Relación Estructura-ActividadRESUMEN
Axonal sprouting of excitatory neurons is frequently observed in temporal lobe epilepsy, but the extent to which inhibitory interneurons undergo similar axonal reorganization remains unclear. The goal of this study was to determine whether somatostatin (SOM)-expressing neurons in stratum (s.) oriens of the hippocampus exhibit axonal sprouting beyond their normal territory and innervate granule cells of the dentate gyrus in a pilocarpine model of epilepsy. To obtain selective labeling of SOM-expressing neurons in s. oriens, a Cre recombinase-dependent construct for channelrhodopsin2 fused to enhanced yellow fluorescent protein (ChR2-eYFP) was virally delivered to this region in SOM-Cre mice. In control mice, labeled axons were restricted primarily to s. lacunosum-moleculare. However, in pilocarpine-treated animals, a rich plexus of ChR2-eYFP-labeled fibers and boutons extended into the dentate molecular layer. Electron microscopy with immunogold labeling demonstrated labeled axon terminals that formed symmetric synapses on dendritic profiles in this region, consistent with innervation of granule cells. Patterned illumination of ChR2-labeled fibers in s. lacunosum-moleculare of CA1 and the dentate molecular layer elicited GABAergic inhibitory responses in dentate granule cells in pilocarpine-treated mice but not in controls. Similar optical stimulation in the dentate hilus evoked no significant responses in granule cells of either group of mice. These findings indicate that under pathological conditions, SOM/GABAergic neurons can undergo substantial axonal reorganization beyond their normal territory and establish aberrant synaptic connections. Such reorganized circuitry could contribute to functional deficits in inhibition in epilepsy, despite the presence of numerous GABAergic terminals in the region.
Asunto(s)
Neuronas GABAérgicas/patología , Interneuronas/patología , Somatostatina/metabolismo , Estado Epiléptico/patología , Animales , Axones/ultraestructura , Dendritas/ultraestructura , Giro Dentado/patología , Neuronas GABAérgicas/fisiología , Hipocampo/patología , Interneuronas/metabolismo , Interneuronas/fisiología , Ratones , Ratones Endogámicos C57BL , Inhibición Neural , Optogenética , Estimulación Luminosa , Pilocarpina/toxicidad , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Somatostatina/genética , Estado Epiléptico/inducido químicamenteRESUMEN
Blockade of the P2Y1 receptor is important to the treatment of thrombosis with potentially improved safety margins compared with P2Y12 receptor antagonists. Investigation of a series of urea surrogates of the diaryl urea lead 3 led to the discovery of 2-amino-1,3,4-thiadiazoles in the 7-hydroxy-N-neopentyl spiropiperidine indolinyl series as potent P2Y1 receptor antagonists, among which compound 5a was the most potent and the first non-urea analog with platelet aggregation (PA) IC50 less than 0.5 µM with 10 µM ADP. Several 2-amino-1,3,4-thiadiazole analogs such as 5b and 5f had a more favorable pharmacokinetic profile, such as higher Ctrough, lower Cl, smaller Vdss, and similar bioavailability compared with 3.
Asunto(s)
Indoles/química , Piperidinas/química , Receptores Purinérgicos P2Y1/metabolismo , Tiadiazoles/farmacología , Animales , Relación Dosis-Respuesta a Droga , Humanos , Indoles/administración & dosificación , Estructura Molecular , Piperidinas/administración & dosificación , Ratas , Relación Estructura-Actividad , Tiadiazoles/administración & dosificación , Tiadiazoles/químicaRESUMEN
A series of diphenylpyridylethanamine-based inhibitors of cholesteryl ester transfer protein with aminoheterocycles appended onto the N-terminus of the chemotype were explored as urea mimetics. Potent compounds were discovered and were further optimized to improve metabolic stability and PXR transactivation profile.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Biomimética , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Estabilidad de Medicamentos , Etilaminas/síntesis química , Etilaminas/química , Etilaminas/farmacología , Compuestos Heterocíclicos/química , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacología , Relación Estructura-Actividad , Urea/químicaRESUMEN
Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.
Asunto(s)
Compuestos de Fenilurea/química , Inhibidores de Agregación Plaquetaria/química , Antagonistas del Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y1/química , Tiazoles/química , Urea/análogos & derivados , Administración Oral , Animales , Perros , Semivida , Macaca fascicularis , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacocinética , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Ratas , Receptores Purinérgicos P2Y1/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacocinética , Tiazoles/farmacología , Tiazoles/uso terapéutico , Trombosis/tratamiento farmacológico , Urea/farmacocinética , Urea/farmacología , Urea/uso terapéuticoRESUMEN
A number of new amine scaffolds with good inhibitory activity in the ADP-induced platelet aggregation assay have been found to be potent antagonists of the P2Y1 receptor. SAR optimization led to the identification of isoindoline 3c and piperidine 4a which showed good in vitro binding and functional activities, as well as improved aqueous solubility. Among them, the piperidine 4a showed the best overall profile with favorable PK parameters.
Asunto(s)
Aminas/química , Agonistas del Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y1/química , Urea/análogos & derivados , Adenosina Difosfato/farmacología , Aminas/síntesis química , Aminas/farmacocinética , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Semivida , Humanos , Microsomas Hepáticos/metabolismo , Piperidinas/química , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacocinética , Unión Proteica , Agonistas del Receptor Purinérgico P2Y/síntesis química , Agonistas del Receptor Purinérgico P2Y/farmacocinética , Ratas , Receptores Purinérgicos P2Y1/metabolismo , Relación Estructura-Actividad , Urea/síntesis química , Urea/farmacocinéticaRESUMEN
ADP receptors, P2Y1 and P2Y12 have been recognized as potential targets for antithrombotic drugs. A series of P2Y1 antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y1 binding (Ki=12nM), moderate antagonism of platelet aggregation (PA IC50=5.2µM) and acceptable PK in rats.
Asunto(s)
Aminopiridinas/química , Inhibidores de Agregación Plaquetaria/química , Antagonistas del Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y1/química , Tiazoles/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacocinética , Animales , Plaquetas/metabolismo , Semivida , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/farmacocinética , Unión Proteica , Antagonistas del Receptor Purinérgico P2Y/metabolismo , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Ratas , Receptores Purinérgicos P2Y1/metabolismo , Relación Estructura-Actividad , Tiazoles/metabolismo , Tiazoles/farmacocinéticaRESUMEN
Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.
Asunto(s)
Fibrinolíticos/química , Compuestos de Fenilurea/química , Antagonistas del Receptor Purinérgico P2Y/química , Piridinas/química , Receptores Purinérgicos P2Y1/química , Urea/química , Animales , Células CACO-2 , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Fibrinolíticos/síntesis química , Fibrinolíticos/farmacocinética , Semivida , Humanos , Microsomas Hepáticos/metabolismo , Tiempo de Tromboplastina Parcial , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Piridinas/farmacocinética , Piridinas/uso terapéutico , Conejos , Ratas , Receptores Purinérgicos P2Y1/metabolismo , Solubilidad , Relación Estructura-Actividad , Trombosis/tratamiento farmacológico , Urea/farmacocinética , Urea/uso terapéutico , Agua/químicaRESUMEN
Brain rhythms provide the timing and concurrence of brain activity required for linking together neuronal ensembles engaged in specific tasks. In particular, the γ-oscillations (30-120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here we report on a potent brain permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a new class of therapeutics for AD. As a first in CNS pharmacotherapy, our lead candidate acts as a potent, efficacious, and selective negative allosteric modulator (NAM) of the γ-aminobutyric acid type A receptors (GABA A Rs) assembled from α1ß2δ subunits. We identified these receptors through anatomical and pharmacological means to mediate the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.
RESUMEN
The dynamic aspects of epilepsy, in which seizures occur sporadically and are interspersed with periods of relatively normal brain function, present special challenges for neuroanatomical studies. Although numerous morphologic changes can be identified during the chronic period, the relationship of many of these changes to seizure generation and propagation remains unclear. Mossy fiber sprouting is an example of a frequently observed morphologic change for which a functional role in epilepsy continues to be debated. This review focuses on neuroanatomically identified changes that would support high levels of activity in reorganized mossy fibers and potentially associated granule cell activation. Early ultrastructural studies of reorganized mossy fiber terminals in human temporal lobe epilepsy tissue have identified morphologic substrates for highly efficacious excitatory connections among granule cells. If similar connections in animal models contribute to seizure activity, activation of granule cells would be expected. Increased labeling with two activity-related markers, Fos and phosphorylated extracellular signal-regulated kinase, has suggested increased activity of dentate granule cells at the time of spontaneous seizures in a mouse model of epilepsy. However, neuroanatomical support for a direct link between activation of reorganized mossy fiber terminals and increased granule cell activity remains elusive. As novel activity-related markers are developed, it may yet be possible to demonstrate such functional links and allow mapping of seizure activity throughout the brain. Relating patterns of neuronal activity during seizures to the underlying morphologic changes could provide important new insights into the basic mechanisms of epilepsy and seizure generation.
Asunto(s)
Giro Dentado/patología , Epilepsia/patología , Neuronas/patología , Transducción de Señal/fisiología , Animales , Gránulos Citoplasmáticos/patología , Gránulos Citoplasmáticos/ultraestructura , Giro Dentado/ultraestructura , Epilepsia/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/fisiología , Genes fos/genética , Humanos , Fibras Musgosas del Hipocampo/patología , Fibras Musgosas del Hipocampo/ultraestructura , Neuronas/ultraestructura , Convulsiones/patología , Transducción de Señal/genéticaRESUMEN
A novel series of diphenylpyridylethanamine-based inhibitors of cholesteryl ester transfer protein is described. Optimization of the urea moiety, particularly by incorporation of fluorine, is explored to balance in vitro metabolic stability with CETP potency in the whole plasma assay.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Urea/análogos & derivados , Urea/farmacología , Aminas/sangre , Aminas/química , Aminas/metabolismo , Aminas/farmacología , Animales , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Halogenación , Humanos , Ratones , Piridinas/sangre , Piridinas/metabolismo , Ratas , Urea/sangre , Urea/metabolismoRESUMEN
Decreased expression of the δ subunit of the GABAA receptor (GABAAR) has been found in the dentate gyrus in several animal models of epilepsy and other disorders with increased excitability and is associated with altered modulation of tonic inhibition in dentate granule cells (GCs). In contrast, other GABAAR subunits, including α4 and γ2 subunits, are increased, but the relationship between these changes is unclear. The goals of this study were to determine if viral transfection of δ subunits in dentate GCs could increase δ subunit expression, alter expression of potentially-related GABAAR subunits, and restore more normal network excitability in the dentate gyrus in a mouse model of epilepsy. Pilocarpine-induced seizures were elicited in DOCK10-Cre mice that express Cre selectively in dentate GCs, and two weeks later the mice were injected unilaterally with a Cre-dependent δ-GABAAR viral vector. At 4-6 weeks following transfection, δ subunit immunolabeling was substantially increased in dentate GCs on the transfected side compared to the nontransfected side. Importantly, α4 and γ2 subunit labeling was downregulated on the transfected side. Electrophysiological studies revealed enhanced tonic inhibition, decreased network excitability, and increased neurosteroid sensitivity in slices from the δ subunit-transfected side compared to those from the nontransfected side of the same pilocarpine-treated animal, consistent with the formation of δ subunit-containing GABAARs. No differences were observed between sides of eYFP-transfected animals. These findings are consistent with the idea that altering expression of key subunits, such as the δ subunit, regulates GABAAR subunit assemblies, resulting in substantial effects on network excitability.