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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.
Assuntos
Encefalomielite Autoimune Experimental/genética , Perfilação da Expressão Gênica/métodos , Microglia/fisiologia , Esclerose Múltipla/genética , Inflamação Neurogênica/genética , Animais , Antioxidantes/uso terapêutico , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Redes Reguladoras de Genes , Ensaios de Triagem em Larga Escala , Humanos , Imunidade Inata , Isoxazóis/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Esclerose Múltipla/tratamento farmacológico , Inflamação Neurogênica/tratamento farmacológico , Estresse Oxidativo , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
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.
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Apolipoproteína E4/antagonistas & inibidores , Apolipoproteína E4/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Apolipoproteína E4/química , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Neurônios/metabolismo , Ftalazinas/química , Ftalazinas/farmacologia , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
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/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Antirreumáticos/administração & dosagem , Antirreumáticos/síntese química , Artrite Experimental/tratamento farmacológico , Asma/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Colágeno Tipo II/antagonistas & inibidores , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Fibronectinas/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Molecular , Esclerose Múltipla/tratamento farmacológico , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/síntese químicaRESUMO
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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Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Cromanos/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Sítios de Ligação , Células Cultivadas , Etilaminas/síntese química , Etilaminas/química , Etilaminas/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
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 , Polietilenoglicóis/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Ésteres , Meia-Vida , Humanos , Injeções Subcutâneas , Integrina alfa4/imunologia , Integrina alfa4/metabolismo , Células Jurkat , RatosRESUMO
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 & inibidores , Integrinas/antagonistas & inibidores , Administração Oral , Animais , Área Sob a Curva , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Modelos Animais de Doenças , Meia-Vida , Humanos , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Células Jurkat , Microssomos Hepáticos/metabolismo , RatosRESUMO
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 , Citoproteção , Doença de Huntington/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oxazinas/farmacologia , Animais , Células Cultivadas , Aprovação de Drogas , Doença de Huntington/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Oxazinas/química , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Serina-Treonina Quinases TORRESUMO
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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Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Caprolactama/análogos & derivados , Inibidores Enzimáticos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Caprolactama/síntese química , Caprolactama/química , Caprolactama/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Infusões Parenterais , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Fragmentos de Peptídeos/metabolismoRESUMO
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/tratamento farmacológico , Modelos Animais de Doenças , Integrina alfa4beta1/antagonistas & inibidores , Pirimidinas/uso terapêutico , Administração Oral , Animais , Asma/metabolismo , Disponibilidade Biológica , Descoberta de Drogas , Pirimidinas/farmacocinética , OvinosRESUMO
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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Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Animais , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Pirazóis/química , Pirazóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMO
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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Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Etilaminas/química , Etilaminas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/química , Cristalografia por Raios X , Etilaminas/síntese química , Humanos , Modelos Moleculares , Inibidores de Proteases/síntese química , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
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 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Butanóis/química , Cicloexilaminas/química , Inibidores de Proteases/química , Acetamidas/síntese química , Acetamidas/farmacocinética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Butanóis/síntese química , Butanóis/farmacocinética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Cicloexilaminas/síntese química , Cicloexilaminas/farmacocinética , Humanos , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Relação Estrutura-AtividadeRESUMO
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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Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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 Alcoólicas/tratamento farmacológico , Núcleo Central da Amígdala/enzimologia , Proteína Quinase C-épsilon/genética , Inibidores de Proteínas Quinases/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Alcoolismo/enzimologia , Alcoolismo/fisiopatologia , Amidas/farmacocinética , Amidas/farmacologia , Animais , Núcleo Central da Amígdala/efeitos dos fármacos , Núcleo Central da Amígdala/fisiopatologia , Depressores do Sistema Nervoso Central/farmacologia , Condicionamento Psicológico , Modelos Animais de Doenças , Etanol , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacocinética , Piridinas/farmacocinética , Piridinas/farmacologiaRESUMO
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/síntese química , Antagonistas de Receptor B1 da Bradicinina , Pirazóis/síntese química , Benzamidas/química , Benzamidas/farmacologia , Cristalografia por Raios X , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Pulmão/citologia , Estrutura Molecular , Pirazóis/química , Pirazóis/farmacologia , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
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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Dipeptídeos/farmacocinética , Integrina alfa4beta1/antagonistas & inibidores , Animais , Asma/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacologia , Humanos , Esclerose Múltipla/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering Aß production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aß40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aß production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aß was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aß reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aß production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aß in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aß was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.
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A pro-drug strategy to identify orally efficacious VLA-4 antagonists is described. Potential pro-drugs were evaluated for their physical chemical characteristics and in vitro properties, including solubility, stability, permeability and plasma stability. Based on this characterization, promising compounds were identified for in vivo pharmacokinetic evaluation. These studies resulted in the identification of a pro-drug that exhibited desirable blood levels in PK studies in several different species.
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Integrina alfa4beta1/antagonistas & inibidores , Pró-Fármacos/síntese química , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Integrina alfa4beta1/metabolismo , Masculino , Pró-Fármacos/metabolismo , RatosRESUMO
Potent, small molecule A beta inhibitors have been prepared that incorporate an alanine core bracketed by an N-terminal arylacetyl group and various C-terminal amino alcohols. The compounds exhibit stereospecific inhibition as demonstrated in an in vitro assay.