RESUMEN
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RESUMEN
Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Perfilación de la Expresión Génica/métodos , Microglía/fisiología , Esclerosis Múltiple/genética , Inflamación Neurogénica/genética , Animales , Antioxidantes/uso terapéutico , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Redes Reguladoras de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunidad Innata , Isoxazoles/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Esclerosis Múltiple/tratamiento farmacológico , Inflamación Neurogénica/tratamiento farmacológico , Estrés Oxidativo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology--cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.
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Esclerosis Amiotrófica Lateral/metabolismo , Autofagia , Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/patología , Animales , Astrocitos/metabolismo , Autofagia/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Flufenazina/farmacología , Semivida , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Metotrimeprazina/farmacología , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación , Ratas , Reproducibilidad de los Resultados , Análisis de la Célula Individual/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Células Madre/metabolismoRESUMEN
Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.
Asunto(s)
Apolipoproteína E4/antagonistas & inhibidores , Apolipoproteína E4/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Apolipoproteína E4/química , Línea Celular , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Neuronas/metabolismo , Ftalazinas/química , Ftalazinas/farmacología , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
A series of (S)-2-(2-(diethylamino)-5-(N-alkyl-N-sulfonamido)pyrimidin-4-ylamino)-3-(4-(carbamoyloxy)phenyl)propanoic acid is discovered as orally available VLA-4 antagonists. Representative compounds 11b and 11p showed efficacy in multiple in vivo animal models. The in vitro selectivity of 11p is also described.
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Antirreumáticos/farmacología , Integrina alfa4beta1/antagonistas & inhibidores , Pirimidinas/farmacología , Sulfonamidas/farmacología , Animales , Antirreumáticos/administración & dosificación , Antirreumáticos/síntesis química , Artritis Experimental/tratamiento farmacológico , Asma/tratamiento farmacológico , Adhesión Celular/efectos de los fármacos , Colágeno Tipo II/antagonistas & inhibidores , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Fibronectinas/química , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Molecular , Esclerosis Múltiple/tratamiento farmacológico , Pirimidinas/administración & dosificación , Pirimidinas/síntesis química , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/síntesis químicaRESUMEN
The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Cromanos/química , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Sitios de Unión , Células Cultivadas , Etilaminas/síntesis química , Etilaminas/química , Etilaminas/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO2H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.
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Integrina alfa4/química , Polietilenglicoles/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Ésteres , Semivida , Humanos , Inyecciones Subcutáneas , Integrina alfa4/inmunología , Integrina alfa4/metabolismo , Células Jurkat , RatasRESUMEN
A series of potent α4ß1/α4ß7 integrin inhibitors is reported, including an inhibitor 12d with remarkable oral exposure and efficacy in rat models of rheumatoid arthritis and Crohn's disease.
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Integrina alfa4beta1/antagonistas & inhibidores , Integrinas/antagonistas & inhibidores , Administración Oral , Animales , Área Bajo la Curva , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Modelos Animales de Enfermedad , Semivida , Humanos , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Células Jurkat , Microsomas Hepáticos/metabolismo , RatasRESUMEN
Autophagy is an intracellular turnover pathway. It has special relevance for neurodegenerative proteinopathies, such as Alzheimer disease, Parkinson disease, and Huntington disease (HD), which are characterized by the accumulation of misfolded proteins. Although induction of autophagy enhances clearance of misfolded protein and has therefore been suggested as a therapy for proteinopathies, neurons appear to be less responsive to classic autophagy inducers than nonneuronal cells. Searching for improved inducers of neuronal autophagy, we discovered an N(10)-substituted phenoxazine that, at proper doses, potently and safely up-regulated autophagy in neurons in an Akt- and mTOR-independent fashion. In a neuron model of HD, this compound was neuroprotective and decreased the accumulation of diffuse and aggregated misfolded protein. A structure/activity analysis with structurally similar compounds approved by the US Food and Drug Administration revealed a defined pharmacophore for inducing neuronal autophagy. This pharmacophore should prove useful in studying autophagy in neurons and in developing therapies for neurodegenerative proteinopathies.
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Autofagia , Citoprotección , Enfermedad de Huntington/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxazinas/farmacología , Animales , Células Cultivadas , Aprobación de Drogas , Enfermedad de Huntington/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Oxazinas/química , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Serina-Treonina Quinasas TORRESUMEN
Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aß(x-40) levels in FVB mice via a single 100 mpk IP dose are highlighted.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Caprolactama/análogos & derivados , Inhibidores Enzimáticos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Animales , Caprolactama/síntesis química , Caprolactama/química , Caprolactama/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Infusiones Parenterales , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Fragmentos de Péptidos/metabolismoRESUMEN
A series of N-(pyrimidin-4-yl)-phenylalanine VLA-4 antagonists is described. Optimization of substituents at the 2 and 5 positions of the pyrimidine ring gave 14, a very potent VLA-4 inhibitor which is orally active in a sheep asthma model.
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Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Integrina alfa4beta1/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Administración Oral , Animales , Asma/metabolismo , Disponibilidad Biológica , Descubrimiento de Drogas , Pirimidinas/farmacocinética , OvinosRESUMEN
Utilizing a pharmacophore hypothesis, previously described gamma-secretase inhibiting HTS hits were evolved into novel tricyclic sulfonamide-pyrazoles, with high in vitro potency, good brain penetration, low metabolic stability, and high clearance.
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Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Pirazoles/farmacología , Sulfonamidas/farmacología , Animales , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Pirazoles/química , Pirazoles/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMEN
Discovery of a series of pyrazolopiperidine sulfonamide based gamma-secretase inhibitors and its SAR evolution is described. Significant increases in APP potency on the pyrazolopiperidine scaffold over the original N-bicyclic sulfonamide scaffold were achieved and this potency increase translated in an improved in vivo efficacy.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Piperidinas/química , Piperidinas/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Humanos , Ratones , Modelos Moleculares , Piperidinas/uso terapéutico , Relación Estructura-Actividad , Sulfonamidas/uso terapéuticoRESUMEN
Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2' position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.
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Acetamidas/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Butanoles/química , Ciclohexilaminas/química , Inhibidores de Proteasas/química , Acetamidas/síntesis química , Acetamidas/farmacocinética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Butanoles/síntesis química , Butanoles/farmacocinética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Ciclohexilaminas/síntesis química , Ciclohexilaminas/farmacocinética , Humanos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Relación Estructura-ActividadRESUMEN
The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Etilaminas/química , Etilaminas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/química , Ácido Aspártico Endopeptidasas/química , Cristalografía por Rayos X , Etilaminas/síntesis química , Humanos , Modelos Moleculares , Inhibidores de Proteasas/síntesis química , Unión Proteica , Relación Estructura-ActividadRESUMEN
Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Abeta cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S1' residues critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity but poor permeability and high P-gp efflux.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: Despite the high cost and widespread prevalence of alcohol use disorders, treatment options are limited, underscoring the need for new, effective medications. Previous results using protein kinase C epsilon (PKCε) knockout mice, RNA interference against PKCε, and peptide inhibitors of PKCε predict that small-molecule inhibitors of PKCε should reduce alcohol consumption in humans. METHODS: We designed a new class of PKCε inhibitors based on the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In vitro kinase and binding assays were used to identify the most potent compounds. Their effects on ethanol-stimulated synaptic transmission; ethanol, sucrose, and quinine consumption; ethanol-induced loss of righting; and ethanol clearance were studied in mice. RESULTS: We identified two compounds that inhibited PKCε with Ki <20 nM, showed selectivity for PKCε over other kinases, crossed the blood-brain barrier, achieved effective concentrations in mouse brain, prevented ethanol-stimulated gamma-aminobutyric acid release in the central amygdala, and reduced ethanol consumption when administered intraperitoneally at 40 mg/kg in wild-type but not in Prkce-/- mice. One compound also reduced sucrose and saccharin consumption, while the other was selective for ethanol. Both transiently impaired locomotion through an off-target effect that did not interfere with their ability to reduce ethanol intake. One compound prolonged recovery from ethanol-induced loss of righting but this was also due to an off-target effect since it was present in Prkce-/- mice. Neither altered ethanol clearance. CONCLUSIONS: These results identify lead compounds for development of PKCε inhibitors that reduce alcohol consumption.
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Consumo de Bebidas Alcohólicas/tratamiento farmacológico , Núcleo Amigdalino Central/enzimología , Proteína Quinasa C-epsilon/genética , Inhibidores de Proteínas Quinasas/farmacología , Transmisión Sináptica/efectos de los fármacos , Alcoholismo/enzimología , Alcoholismo/fisiopatología , Amidas/farmacocinética , Amidas/farmacología , Animales , Núcleo Amigdalino Central/efectos de los fármacos , Núcleo Amigdalino Central/fisiopatología , Depresores del Sistema Nervioso Central/farmacología , Condicionamiento Psicológico , Modelos Animales de Enfermedad , Etanol , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacocinética , Piridinas/farmacocinética , Piridinas/farmacologíaRESUMEN
The B1 receptor is an attractive target for the treatment of pain and inflammation. A series of 3-carboxamido-5-phenacylamino pyrazole B1 receptor antagonists are described that exhibit good potency against B1 and high selectivity over B2. Initially, N-unsubstituted pyrazoles were studied, but these compounds suffered from extensive glucuronidation in primates. This difficulty could be surmounted by the use of N-substituted pyrazoles. Optimization efforts culminated in compound 41, which has high receptor potency and metabolic stability.
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Benzamidas/síntesis química , Antagonistas del Receptor de Bradiquinina B1 , Pirazoles/síntesis química , Benzamidas/química , Benzamidas/farmacología , Cristalografía por Rayos X , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Pulmón/citología , Estructura Molecular , Pirazoles/química , Pirazoles/farmacología , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
The identification of orally active, small molecule antagonists of the alpha4beta1 integrin, VLA-4, could lead to therapeutic agents with utility in a number of clinical settings, including asthma, multiple sclerosis and IBD. Starting from CDR3 sequences conserved among neutralizing alpha4 antibodies, peptides were identified that antagonized VLA-4 mediated adhesion in vitro. Through a series of structural modifications, these peptides evolved into small molecules that exhibited high potency and selectivity for VLA-4 in cell adhesion assays. Finally, through the optimization of physical and pharmacokinetic properties, compounds were identified that exhibited oral activity in animal models of asthma and multiple sclerosis.
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Dipéptidos/farmacocinética , Integrina alfa4beta1/antagonistas & inhibidores , Animales , Asma/tratamiento farmacológico , Adhesión Celular/efectos de los fármacos , Dipéptidos/administración & dosificación , Dipéptidos/farmacología , Humanos , Esclerosis Múltiple/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.