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1.
Angew Chem Int Ed Engl ; 59(6): 2420-2428, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31755636

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.


Assuntos
Benzoxazóis/química , Naftalenos/química , Proteômica/métodos , Receptores de Hidrocarboneto Arílico/metabolismo , Utrofina/metabolismo , Animais , Benzoxazóis/metabolismo , Benzoxazóis/farmacologia , Benzoxazóis/uso terapêutico , Reação de Cicloadição , Desenho de Fármacos , Humanos , Cinética , Camundongos , Sondas Moleculares/química , Distrofia Muscular de Duchenne/tratamento farmacológico , Mioblastos/citologia , Mioblastos/metabolismo , Naftalenos/metabolismo , Naftalenos/farmacologia , Naftalenos/uso terapêutico , Ligação Proteica , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Regulação para Cima/efeitos dos fármacos , Utrofina/agonistas , Utrofina/genética
2.
FEBS Lett ; 592(11): 1856-1869, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29772070

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal disorder caused by absence of functional dystrophin protein. Compensation in dystrophin-deficient (mdx) mice may be achieved by overexpression of its fetal paralogue, utrophin. Strategies to increase utrophin levels by stimulating promoter activity using small compounds are therefore a promising pharmacological approach. Here, we characterise similarities and differences existing within the mouse and human utrophin locus to assist in high-throughput screening for potential utrophin modulator drugs. We identified five novel 5'-utrophin isoforms (A',B',C,D and F) in adult and embryonic tissue. As the more efficient utrophin-based response in mdx skeletal muscle appears to involve independent transcriptional activation of conserved, myogenic isoforms (A' and F), elevating their paralogues in DMD patients is an encouraging therapeutic strategy.


Assuntos
Distrofina/deficiência , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Utrofina/metabolismo , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Utrofina/genética
3.
Curr Gene Ther ; 12(3): 206-44, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22571500

RESUMO

DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-ß, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder.


Assuntos
Aminoglicosídeos/uso terapêutico , Distrofina , Distrofia Muscular de Duchenne/tratamento farmacológico , Suramina , Utrofina , Antígenos CD/genética , Antígenos CD/metabolismo , Códon sem Sentido , Distrofina/genética , Distrofina/metabolismo , Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Terapia de Alvo Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Suramina/uso terapêutico , Utrofina/genética , Utrofina/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-30890885

RESUMO

Duchenne muscular dystrophy (DMD), an allelic X-linked progressive muscle-wasting disease, is one of the most common single-gene disorders in the developed world. Despite knowledge of the underlying genetic causation and resultant pathophysiology from lack of dystrophin protein at the muscle sarcolemma, clinical intervention is currently restricted to symptom management. In recent years, however, unprecedented advances in strategies devised to correct the primary defect through gene- and cell-based therapeutics hold particular promise for treating dystrophic muscle. Conventional gene replacement and endogenous modification strategies have greatly benefited from continued improvements in encapsidation capacity, transduction efficiency, and systemic delivery. In particular, RNA-based modifying approaches such as exon skipping enable expression of a shorter but functional dystrophin protein and rapid progress toward clinical application. Emerging combined gene- and cell-therapy strategies also illustrate particular promise in enabling ex vivo genetic correction and autologous transplantation to circumvent a number of immune challenges. These approaches are complemented by a vast array of pharmacological approaches, in particular the successful identification of molecules that enable functional replacement or ameliorate secondary DMD pathology. Animal models have been instrumental in providing proof of principle for many of these strategies, leading to several recent trials that have investigated their efficacy in DMD patients. Although none has reached the point of clinical use, rapid improvements in experimental technology and design draw this goal ever closer. Here, we review therapeutic approaches to DMD, with particular emphasis on recent progress in strategic development, preclinical evaluation and establishment of clinical efficacy. Further, we discuss the numerous challenges faced and synergistic approaches being devised to combat dystrophic pathology effectively.

5.
Cell Host Microbe ; 4(2): 89-91, 2008 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-18692766

RESUMO

Persistence of latently infected CD4(+) T cell "reservoirs" presents a major obstacle for current HIV-1 antiretroviral therapies. In Cell Host & Microbe, two separate studies use model systems of latent infection to dissect the influence of host genes on HIV-1 transcription, yielding intriguing, though partially contradictory, conclusions.


Assuntos
Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno , Transcrição Gênica , Latência Viral , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos
6.
Mol Cell ; 29(1): 56-68, 2008 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-18206969

RESUMO

HIV-1 provirus, either as a chromosomal integrant or as an episomal plasmid in HeLa cells, forms a transcription-dependent gene loop structure between the 5'LTR promoter and 3'LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocks 5' to 3'LTR juxtaposition, indicating that this structure is maintained during transcription. Analysis of mutant or hybrid HIV-1 plasmids demonstrates that replacement of the 5'LTR promoter with CMV or the 3'LTR poly(A) signal with a synthetic element (SPA) permits gene loop formation, suggesting that these interactions are not retroviral specific. In addition, activation of the 5'LTR poly(A) signal or inactivation of the 3'LTR poly(A) signal abolishes gene loop formation. Overall, we demonstrate that both ongoing transcription and pre-mRNA processing are essential for gene loop formation, and predict that these structures represent a defining feature of active gene transcription.


Assuntos
Cromatina/ultraestrutura , Regulação Viral da Expressão Gênica , HIV-1/genética , Conformação de Ácido Nucleico , Provírus/ultraestrutura , Precursores de RNA/genética , Transcrição Gênica , Cromatina/genética , Imunoprecipitação da Cromatina , Citomegalovirus/genética , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Flavonoides/farmacologia , Genes tat , Repetição Terminal Longa de HIV , HIV-1/fisiologia , Células HeLa/virologia , Humanos , Fosfosserina/análise , Piperidinas/farmacologia , Regiões Promotoras Genéticas , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Precursores de RNA/química , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Transcrição Gênica/genética , Transfecção , Células U937/virologia
7.
Mol Biol Cell ; 18(8): 2864-72, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17507653

RESUMO

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPalpha/beta to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.


Assuntos
Pareamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismo , Utrofina/genética , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/deficiência , Drosophila melanogaster , Extremidades , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Humanos , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Utrofina/metabolismo
8.
FASEB J ; 19(6): 543-9, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15791004

RESUMO

Mutations in myostatin (GDF8) cause marked increases in muscle mass, suggesting that this transforming growth factor-beta (TGF-beta) superfamily member negatively regulates muscle growth. Myostatin blockade therefore offers a strategy for reversing muscle wasting in Duchenne's muscular dystrophy (DMD) without resorting to genetic manipulation. Here, we demonstrate that pharmacological blockade using a myostatin propeptide stabilized by fusion to IgG-Fc improved pathophysiology of the mdx mouse model of DMD. Functional benefits evidenced by specific force improvement, exceeded those reported previously using myostatin antibody-mediated blockade. More importantly, use of a propeptide blockade strategy obviates possibilities of anti-idiotypic responses that could potentially limit the effectiveness of antibody-mediated myostatin blockade strategies over time. This study provides a novel pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD and since it uses an endogenous inhibitor of myostatin should help circumvent technical hurdles and toxicity associated with conventional gene or cell based therapies.


Assuntos
Distrofia Muscular Animal/terapia , Precursores de Proteínas/administração & dosagem , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Anticorpos Monoclonais , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/química , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Miostatina , Precursores de Proteínas/química , Precursores de Proteínas/imunologia , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão , Fator de Crescimento Transformador beta/imunologia , Utrofina/genética
10.
J Mol Med (Berl) ; 82(2): 102-15, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14673527

RESUMO

Duchenne muscular dystrophy (DMD) is the most common X-linked neuromuscular disorder. The devastating nature of DMD has led to an intense effort toward finding a cure for this disease, dating back to the time when Duchenne first initiated clinical trials using faradic stimulation for DMD patients. Unfortunately despite the passage of some 150 years the disease remains incurable, and its medical management is largely supportive. However, the discovery of the DMD gene about 20 years ago has allowed a change in the focus of therapeutic strategy dramatically toward delivery of the missing gene/protein. Indeed, some degree of success has been achieved in preclinical animal studies using such strategies, and gene therapy trials are currently underway in humans. Pharmacological approaches for DMD are also being developed since they can circumvent some of the technical problems associated with gene and cell based therapy. This review explores developments in therapeutic approaches for DMD.


Assuntos
Distrofia Muscular de Duchenne/terapia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Distrofina/genética , Distrofina/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Distrofias Musculares/classificação , Distrofias Musculares/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Neuregulina-1/metabolismo , Regiões Promotoras Genéticas/genética , Utrofina
11.
FEBS Lett ; 538(1-3): 168-72, 2003 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-12633873

RESUMO

Duchenne muscular dystrophy is caused by dystrophin deficiency, which can be prevented in the mdx mouse model by over-expression of an autosomal homologue, utrophin. Utrophin has two characterised full-length promoters, A and B. No data are available on the transcriptional regulation of B utrophin, which has been recently localised to the endothelium. Similar to characterised endothelial promoters, Ets and Ap-1 individually trans-activate the human B core promoter. Synergistic activation by GATA-2 and c-jun to the order of 20-fold was observed.


Assuntos
Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Endotélio/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/efeitos dos fármacos , Utrofina
12.
Neuromuscul Disord ; 12 Suppl 1: S78-89, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12206801

RESUMO

Duchenne muscular dystrophy is an X-linked recessive muscle wasting disease caused by the absence of the muscle cytoskeletal protein, dystrophin. Dystrophin is a member of the spectrin superfamily of proteins and is closely related in sequence similarity and functional motifs to three proteins that constitute the dystrophin related protein family, including the autosomal homologue, utrophin. An alternative strategy circumventing many problems associated with somatic gene therapies for Duchenne muscular dystrophy has arisen from the demonstration that utrophin can functionally substitute for dystrophin and its over-expression in muscles of dystrophin-null transgenic mice completely prevents the phenotype arising from dystrophin deficiency. One potential approach to increase utrophin levels in muscle for possible therapeutic purpose in humans is to increase expression of the utrophin gene at a transcriptional level via promoter activation. This has lead to an interest in the identification and manipulation of important regulatory regions and/or molecules that increase the expression of utrophin and their delivery to dystrophin-deficient tissue. As pre-existing cellular mechanisms are utilized, this approach would avoid many problems associated with conventional gene therapies.


Assuntos
Proteínas do Citoesqueleto/genética , Terapia Genética/métodos , Proteínas de Membrana/genética , Distrofia Muscular de Duchenne/terapia , Animais , Proteínas do Citoesqueleto/uso terapêutico , Distrofina/genética , Humanos , Proteínas de Membrana/uso terapêutico , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Regiões Promotoras Genéticas , Regulação para Cima , Utrofina
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