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1.
J Med Chem ; 67(13): 11168-11181, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38932616

RESUMO

ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.


Assuntos
Sítio Alostérico , Descoberta de Drogas , Glucosilceramidase , Glucosilceramidase/metabolismo , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/química , Humanos , Cristalografia por Raios X , Relação Estrutura-Atividade , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/metabolismo
2.
ACS Med Chem Lett ; 13(10): 1591-1597, 2022 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-36262388

RESUMO

Fragment-based ligand discovery was successfully applied to histone deacetylase HDAC2. In addition to the anticipated hydroxamic acid- and benzamide-based fragment screening hits, a low affinity (∼1 mM) α-amino-amide zinc binding fragment was identified, as well as fragments binding to other regions of the catalytic site. This alternative zinc-binding fragment was further optimized, guided by the structural information from protein-ligand complex X-ray structures, into a sub-µM, brain penetrant, HDAC2 inhibitor (17) capable of modulating histone acetylation levels in vivo.

3.
Sci Rep ; 11(1): 10309, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986302

RESUMO

Intracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.


Assuntos
Hipocampo/patologia , Tauopatias/patologia , Doença de Alzheimer/patologia , Animais , Hipocampo/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Fosforilação
4.
Stem Cell Reports ; 16(5): 1276-1289, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33891871

RESUMO

Sequestosome-1 (SQSTM1/p62) is involved in cellular processes such as autophagy and metabolic reprogramming. Mutations resulting in the loss of function of SQSTM1 lead to neurodegenerative diseases including frontotemporal dementia. The pathogenic mechanism that contributes to SQSTM1-related neurodegeneration has been linked to its role as an autophagy adaptor, but this is poorly understood, and its precise role in mitochondrial function and clearance remains to be clarified. Here, we assessed the importance of SQSTM1 in human induced pluripotent stem cell (iPSC)-derived cortical neurons through the knockout of SQSTM1. We show that SQSTM1 depletion causes altered mitochondrial gene expression and functionality, as well as autophagy flux, in iPSC-derived neurons. However, SQSTM1 is not essential for mitophagy despite having a significant impact on early PINK1-dependent mitophagy processes including PINK1 recruitment and phosphorylation of ubiquitin on depolarized mitochondria. These findings suggest that SQSTM1 is important for mitochondrial function rather than clearance.


Assuntos
Córtex Cerebral/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteína Sequestossoma-1/metabolismo , Diferenciação Celular , Respiração Celular , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/patologia , Mitofagia , Fosforilação Oxidativa , Proteínas Quinases/metabolismo , Reprodutibilidade dos Testes
5.
J Air Waste Manag Assoc ; 70(5): 504-521, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32186474

RESUMO

On January 22, 2010, the U.S. Environmental Protection Agency (EPA) established a new one-hour probabilistic National Ambient Air Quality Standard (NAAQS) for nitrogen dioxide (NO2) at 100 parts per billion (ppb) or approximately 188 micrograms per cubic meter (µg/m3). The 1-hr NO2 NAAQS is considerably more stringent than the longstanding annual standard of 53 ppb or approximately 100 µg/m3. AERMOD is the EPA recommended dispersion modeling tool for predicting impacts from emission sources, including compressor station reciprocating engines. Existing datasets were previously reviewed to evaluate and improve AERMOD 1-hr NO2 model performance. However, these datasets were not representative due to either an absence of accurate concurrent hourly emission inventories, being associated with larger electric generating units, shorter duration tracer studies, or were too site-specific (e.g., external influences such as terrain). To address this data gap, an intensive 13-month field data program was undertaken by Pipeline Research Council International (PRCI) to collect a comprehensive dataset including concurrent emissions, meteorological, and ambient ozone and nitrogen oxide measurements. This paper describes the Balko, OK natural gas compressor station host site and the design of the meteorological, ambient NOx and O3 monitors and continuous Parametric Emissions Monitoring System (PEMS). Two comparisons are made between the monitored data and AERMOD results. First, the measured onsite data (ambient air, meteorological, and emissions) were used as input to AERMOD to make comparisons between measured and modeled NOx and NO2. These comparisons showed that NOx was both over and under predicted, sometimes beyond a factor-of-two. The second comparison uses the typical permitting approach where onsite data are not available to contrast how conservative input assumptions for emissions, background O3, NO2, and offsite meteorology cause significant over predictions especially for this site where engines have a low annual utilization rate and operate infrequently throughout the year.Implications: This project resulted following the promulgation of the new 1-hr NO2 NAAQS and the challenges that natural gas pipeline compressor stations have in modeling intermittent emissions using AERMOD to demonstrate compliance with this standard. AERMOD was developed and validated with a primary focus on large sources with tall stacks, such as electric utility boilers. A recently collected dataset at a gas compressor station in Balko, Oklahoma shows that the use of permitted allowable emission values compared to actual hourly emission values causes over predictions using typical regulatory methods. Comparison of model results using actual hourly emission values to coincident ambient air monitoring values at the site shows that AERMOD over predicts near-field impacts due to simplifying assumptions including building downwash, plume dispersion, chemistry, and 1-hr invariant meteorological conditions.


Assuntos
Poluentes Atmosféricos/análise , Monitoramento Ambiental/métodos , Modelos Teóricos , Óxidos de Nitrogênio/análise , Ozônio/análise , Gás Natural , Estados Unidos , United States Environmental Protection Agency
6.
Prion ; 7(3): 198-202, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23764833

RESUMO

Despite their devastating impact, no effective therapeutic yet exists for prion diseases at the symptomatic stage in humans or animals. Progress is hampered by the difficulty in identifying compounds that affect PrP (Sc) and the necessity of any potential therapeutic to gain access to the CNS. Synthetic polymers known as dendrimers are a particularly promising candidate in this area. Studies with cell culture models of prion disease and prion infected brain homogenate have demonstrated that numerous species of dendrimers eliminate PrP (Sc) in a dose and time dependent fashion and specific glycodendrimers are capable of crossing the CNS. However, despite their potential a number of important questions remained unanswered such as what makes an effective dendrimer and how dendrimers eliminate prions intracellularly. In a number of recent studies we have tackled these questions and revealed for the first time that a specific dendrimer can inhibit the intracellular conversion of PrP (C) to PrP (Sc) and that a high density of surface reactive groups is a necessity for dendrimers in vitro anti-prion activity. Understanding how a therapeutic works is a vital component in maximising its activity and these studies therefore represent a significant development in the race to find effective treatments for prion diseases.


Assuntos
Dendrímeros/uso terapêutico , Doenças Priônicas/tratamento farmacológico , Príons/antagonistas & inibidores , Príons/metabolismo , Animais , Dendrímeros/química , Dendrímeros/farmacologia , Humanos , Nanomedicina , Doenças Priônicas/metabolismo , Príons/química , Conformação Proteica/efeitos dos fármacos
7.
PLoS One ; 8(1): e55282, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383136

RESUMO

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future.


Assuntos
Dendrímeros/farmacologia , Polipropilenos/farmacologia , Proteínas PrPC/antagonistas & inibidores , Proteínas PrPC/metabolismo , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/metabolismo , Animais , Benzamidas/farmacologia , Bioensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Mesilato de Imatinib , Immunoblotting , Camundongos , Microscopia Confocal , Piperazinas/farmacologia , Proteína PrP 27-30/isolamento & purificação , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Suramina/farmacologia
8.
Biomacromolecules ; 14(1): 27-37, 2013 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-23234313

RESUMO

Prion diseases are characterized by the accumulation of PrP(Sc), an aberrantly folded isoform of the host protein PrP(C). Specific forms of synthetic molecules known as dendrimers are able to eliminate protease-resistant PrP(Sc) in both an intracellular and in vitro setting. The properties of a dendrimer which govern this ability are unknown. We addressed the issue by comparing the in vitro antiprion ability of numerous modified poly(propylene-imine) dendrimers, which varied in size, structure, charge, and surface group composition. Several of the modified dendrimers, including an anionic glycodendrimer, reduced the level of protease resistant PrP(Sc) in a prion strain-dependent manner. This led to the formulation of a new working model for dendrimer/prion interactions which proposes dendrimers eliminate PrP(Sc) by destabilizing the protein and rendering it susceptible to proteolysis. This ability is not dependent on any particular charge of dendrimer, but does require a high density of reactive surface groups.


Assuntos
Dendrímeros/química , Dendrímeros/farmacologia , Polipropilenos/química , Polipropilenos/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Dendrímeros/uso terapêutico , Camundongos , Camundongos Transgênicos , Polipropilenos/uso terapêutico , Proteínas PrPSc/metabolismo , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Propriedades de Superfície
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