Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
1.
J Infect Dis ; 226(4): 585-594, 2022 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-35413121

RESUMO

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.


Assuntos
Infecções por Citomegalovirus , Vacinas contra Citomegalovirus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Citomegalovirus , Humanos , Macaca mulatta , Proteínas do Envelope Viral
2.
Hum Mol Genet ; 24(21): 6013-28, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26251043

RESUMO

Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD.


Assuntos
Adenosina Trifosfatases/metabolismo , Lisossomos/enzimologia , Proteínas de Membrana/metabolismo , Mutação , Doença de Parkinson/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Astrócitos/enzimologia , Encéfalo/metabolismo , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , ATPases Translocadoras de Prótons , Regulação para Cima
3.
J Biol Chem ; 290(32): 19433-44, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26078453

RESUMO

Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.


Assuntos
Antiparkinsonianos/farmacologia , Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , alfa-Sinucleína/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Injeções Intraventriculares , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/metabolismo
5.
J Neurochem ; 128(4): 561-76, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24117733

RESUMO

Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation-dependent LRRK2 substrate pathways. To address this gap in knowledge, we transduced SH-SY5Y neuroblastoma cells with LRRK2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK2 kinase inhibition (LRRK2-IN-1 treatment) using stable isotope labeling of amino acids in culture combined with phosphopeptide enrichment and LC-MS/MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK2-IN-1 treatment, including sites on proteins previously known to associate with LRRK2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro-inflammatory responses and neurite morphology, among other pathways. In follow-up experiments, LRRK2-IN-1 inhibited lipopolysaccharide-induced tumor necrosis factor alpha (TNFα) and C-X-C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK2-IN-1 had almost identical effects in primary glial and neuronal cultures from LRRK2 knockout mice. These data suggest LRRK2-IN-1 may inhibit pathways of perceived LRRK2 pathophysiological function independently of LRRK2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK2 function.


Assuntos
Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteômica , Adenoviridae/genética , Animais , Astrócitos/metabolismo , Células Cultivadas , Quimiocina CXCL10/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lipopolissacarídeos/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Knockout , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Fosforilação , Plasmídeos/genética , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Titânio/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
6.
Bioorg Med Chem Lett ; 24(17): 4132-40, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25113930
7.
Sci Transl Med ; 12(540)2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321864

RESUMO

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.


Assuntos
Doença de Parkinson , Animais , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Pulmão , Morfolinas , Mutação , Primatas , Pirimidinas , Pirróis
8.
J Neurochem ; 108(6): 1539-49, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19183261

RESUMO

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh-regulated gene targets, Gli-1 and Patched in a cyclopamine- and forskolin-sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular-signal-regulated kinases or c-jun N-terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh-dependent up-regulation of Gli-1, inter-alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin-like growth factor 2 or a novel Shh target, membrane-associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively-active mutant we show that Shh signaling to p38 MAPK and subsequent Gli-1 transcription requires G-protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G-protein receptor kinase 2-dependent p38 MAPK activity in regulating Shh-mediated gene transcription in astrocytes.


Assuntos
Astrócitos/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos
9.
Arthritis Rheumatol ; 69(10): 1969-1977, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28845577

RESUMO

OBJECTIVE: Patients with rheumatoid arthritis (RA) are at increased risk of herpes zoster, and vaccination is recommended for patients ages 50 years and older, prior to starting treatment with biologic agents or tofacitinib. Tofacitinib is an oral JAK inhibitor for the treatment of RA. We evaluated its effect on the immune response and safety of live zoster vaccine (LZV). METHODS: In this phase II, 14-week, placebo-controlled trial, patients ages 50 years and older who had active RA and were receiving background methotrexate were given LZV and randomized to receive tofacitinib 5 mg twice daily or placebo 2-3 weeks postvaccination. We measured humoral responses (varicella zoster virus [VZV]-specific IgG level as determined by glycoprotein enzyme-linked immunosorbent assay) and cell-mediated responses (VZV-specific T cell enumeration, as determined by enzyme-linked immunospot assay) at baseline and 2 weeks, 6 weeks, and 14 weeks postvaccination. End points included the geometric mean fold rise (GMFR) in VZV-specific IgG levels (primary end point) and T cells (number of spot-forming cells/106 peripheral blood mononuclear cells) at 6 weeks postvaccination. RESULTS: One hundred twelve patients were randomized to receive tofacitinib (n = 55) or placebo (n = 57). Six weeks postvaccination, the GMFR in VZV-specific IgG levels was 2.11 in the tofacitinib group and 1.74 in the placebo group, and the VZV-specific T cell GMFR was similar in the tofacitinib group and the placebo group (1.50 and 1.29, respectively). Serious adverse events occurred in 3 patients in the tofacitinib group (5.5%) and 0 patients (0.0%) in the placebo group. One patient, who lacked preexisting VZV immunity, developed cutaneous vaccine dissemination 2 days after starting tofacitinib (16 days postvaccination). This resolved after tofacitinib was discontinued and the patient received antiviral treatment. CONCLUSION: Patients who began treatment with tofacitinib 2-3 weeks after receiving LZV had VZV-specific humoral and cell-mediated immune responses to LZV similar to those in placebo-treated patients. Vaccination appeared to be safe in all of the patients except 1 patient who lacked preexisting VZV immunity.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Vacina contra Herpes Zoster/uso terapêutico , Herpes Zoster/prevenção & controle , Inibidores de Proteínas Quinases/uso terapêutico , Idoso , Anticorpos Antivirais/imunologia , Método Duplo-Cego , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Vacina contra Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Humanos , Imunogenicidade da Vacina/imunologia , Imunoglobulina G/imunologia , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Linfócitos T/imunologia
10.
J Med Chem ; 58(1): 419-32, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-25353650

RESUMO

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.


Assuntos
Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteoma/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Sequência de Aminoácidos , Animais , Área Sob a Curva , Encéfalo/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação de Sentido Incorreto , Nitrilas/química , Nitrilas/farmacocinética , Doença de Parkinson/tratamento farmacológico , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteoma/química , Proteoma/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirróis/química , Pirróis/farmacocinética , Ratos
11.
Neurobiol Aging ; 33(7): 1481.e13-23, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22227005

RESUMO

Transgenic mice are used to model increased brain amyloid-ß (Aß) and amyloid plaque formation reflecting Alzheimer's disease pathology. In our study hippocampal network oscillations, population spikes, and long-term potentiation (LTP) were recorded in APPswe/PS1dE9 (APP/PS1) and presenilin1 (PS1) transgenic and wild type mice at 2, 4, and 8 months of age under urethane anesthesia. Hippocampal theta oscillations elicited by brainstem stimulation were similar in wild type and PS1 mice at all age groups. In contrast, APP/PS1 mice showed an age-dependent decrease in hippocampal activity, characterized by a significant decline in elicited theta power and frequency at 4 and 8 months. Magnitudes of population spikes and long-term potentiation in the dentate gyrus were similar across groups at both 4 and 8 months. In APP/PS1 mice, soluble and insoluble Aß, and hippocampal and cortical plaque load increased with age, and the disruption in hippocampal theta oscillation showed a significant correlation with plaque load. Our study shows that, using in vivo electrophysiological methods, early Aß-related functional deficits can be robustly detected in the brainstem-hippocampus multisynaptic network.


Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Hipocampo/fisiologia , Ritmo Teta/fisiologia , Precursor de Proteína beta-Amiloide/genética , Animais , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1/genética
12.
Biol Reprod ; 77(1): 147-55, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17392500

RESUMO

Comprehensive understanding of the cellular mechanisms utilized by luteal cells in response to extracellular hormonal signals resulting in the normal synthesis and secretion of their steroid and peptide products has yet to be achieved. Previous studies have established that cAMP functions as a second messenger in mediating gonadotropin stimulated luteal progesterone secretion. Classically, increased intracellular concentrations of cAMP result in activation of protein kinase A (PKA), which in turn phosphorylates gene regulatory transcription factors. Recent studies demonstrate that non-PKA mediated actions of cAMP exist, yet the mechanisms are not well understood. In addition to gonadotropic hormones, such growth factors as insulin, insulin-like growth factor 1, and epidermal growth factor have been shown to modulate luteal steroid hormone synthesis and steroidogenic enzyme expression as either independent effects or via amplification or modulation of the action of gonadotropic hormones or cAMP. Thus, mechanisms independent of cAMP and also downstream to cAMP that do not involve PKA are likely to be important in steroidogenesis in mammalian cells. The present studies were performed to help define the cellular mediators involved in cAMP-stimulated progesterone expression. Our data demonstrate that, in an in vitro steroidogenic cell model, 1) cAMP-stimulated progesterone occurs in a manner that is independent of PKA, 2) neither phosphatidylinositol-3-kinase nor mitogen-activated protein kinase are involved in PKA-independent cAMP-stimulated progesterone production, 3) tyrosine kinase activity does mediate cAMP-stimulated progesterone production, and 4) cAMP directly activates the Ras protein. These data suggest novel mediators of cAMP-stimulated progesterone production.


Assuntos
Corpo Lúteo/citologia , Corpo Lúteo/metabolismo , AMP Cíclico/metabolismo , Progesterona/metabolismo , Proteínas Tirosina Quinases/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Progesterona/biossíntese , Progesterona/genética , Ratos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA