RESUMO
Delivery of miniaturized dystrophin genes via adeno-associated viral vectors is one leading approach in development to treat Duchenne muscular dystrophy. Here we directly compared the functionality of five mini- and micro-dystrophins via skeletal muscle-specific transgenic expression in dystrophin-deficient mdx mice. We evaluated their ability to rescue defects in the microtubule network, passive stiffness and contractility of skeletal muscle. Transgenic mdx mice expressing the short dystrophin isoform Dp116 served as a negative control. All mini- and micro-dystrophins restored elevated detyrosinated α-tubulin and microtubule density of mdx muscle to values not different from C57BL/10, however, only mini-dystrophins restored the transverse component of the microtubule lattice back to C57BL/10. Passive stiffness values in mdx muscles expressing mini- or micro-dystrophins were not different from C57BL/10. While all mini- and micro-dystrophins conferred significant protection from eccentric contraction-induced force loss in vivo and ex vivo compared to mdx, removal of repeats two and three resulted in less protection from force drop caused by eccentric contraction ex vivo. Our data reveal subtle yet significant differences in the relative functionalities for different therapeutic constructs of miniaturized dystrophin in terms of protection from ex vivo eccentric contraction-induced force loss and restoration of an organized microtubule lattice.
Assuntos
Distrofina/genética , Microtúbulos/genética , Distrofia Muscular de Duchenne/genética , Tubulina (Proteína)/genética , Animais , Modelos Animais de Doenças , Distrofina/deficiência , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx/genética , Camundongos Transgênicos , Microtúbulos/patologia , Contração Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/terapiaRESUMO
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) describes a group of developmental disorders affecting the lungs with its pulmonary vasculature. Mutations in the FOXF1 gene have been reported in most cases, and extrapulmonary findings were described. We present two patients with ACDMPV and FOXF1 mutations that illustrate the variability in presentation and outcome of their disease. Patient 1 was a full-term infant with imperforate anus and pulmonary hypertension. He required Extracorporeal Membrane Oxygenation on day of life (DOL) 3, and passed away on DOL 13 after no clinical improvement. Postmortem findings were consistent with ACDMPV. FOXF1 testing revealed a heterozygous pathogenic frameshift de novo mutation, c.1057_1078dup, p.(Gly360Valfs*58). Patient 2 is a 6-month-old female, with a small omphalocele. She had intermittent retractions at 1 week of age. She was admitted with pulmonary hypertension at 7 weeks of age. Lung biopsy confirmed ACDMPV. FOXF1 testing revealed a de novo, heterozygous likely pathogenic missense mutation c.253T>C, p.(Phe85Leu]). Our two patients had different presentations, ages of onset, and progression of their disease. Our second patient had patchy lung involvement on biopsy, which may explain the relatively delayed onset and longer survival. ACDMPV is an important consideration for full-term infants with worsening pulmonary hypertension early in life.
Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Fatores de Transcrição Forkhead/genética , Mutação , Síndrome da Persistência do Padrão de Circulação Fetal/diagnóstico , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Alvéolos Pulmonares/anormalidades , Veias Pulmonares/anormalidades , Autopsia , Biomarcadores , Biópsia , Hibridização Genômica Comparativa , Análise Citogenética , Ecocardiografia , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.
Assuntos
Técnicas Citológicas/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/citologia , Antibacterianos/farmacologia , Composição de Bases/genética , Linhagem Celular , Clonagem Molecular , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , MutaçãoRESUMO
Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/terapia , Organoides/metabolismo , Modelos BiológicosRESUMO
Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.
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Dp116 is a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex (DGC), but lacks actin-binding domains. To examine the functional role of the DGC, we expressed the Dp116 transgene in mice lacking both dystrophin and utrophin (mdx:utrn(-/-)). Unexpectedly, expression of Dp116 prevented the most severe aspects of the mdx:utrn(-/-) phenotype. Dp116:mdx:utrn(-/-) transgenic mice had dramatic improvements in growth, mobility and lifespan compared with controls. This was associated with increased muscle mass and force generating capacity of limb muscles, although myofiber size and specific force were unchanged. Conversely, Dp116 had no effect on dystrophic injury as determined by muscle histopathology and serum creatine kinase levels. Dp116 also failed to restore normal fiber-type distribution or the post-synaptic architecture of the neuromuscular junction. These data demonstrate that the DGC is critical for growth and maintenance of muscle mass, a function that is independent of the ability to prevent dystrophic pathophysiology. Likewise, this is the first demonstration in skeletal muscle of a positive functional role for a dystrophin protein that lacks actin-binding domains. We conclude that both mechanical and non-mechanical functions of dystrophin are important for its role in skeletal muscle.
Assuntos
Distrofina/metabolismo , Longevidade , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/prevenção & controle , Animais , Fenômenos Biomecânicos , Creatina Quinase/sangue , Distrofina/química , Esôfago/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Contração Muscular , Músculo Esquelético/ultraestrutura , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/fisiopatologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Junção Neuromuscular/ultraestrutura , Tamanho do Órgão , Isoformas de Proteínas/metabolismo , Análise de Sobrevida , Utrofina/deficiência , Utrofina/metabolismoRESUMO
Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophin(DeltaR4-R23/DeltaCT) with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophin(DeltaR4-R23/DeltaCT) led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophin(DeltaR4-R23/DeltaCT) with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid alpha-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.
Assuntos
Distrofina/fisiologia , Peptídeos/fisiologia , Animais , Distrofina/química , Distrofina/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Peptídeos/químicaRESUMO
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.
Assuntos
Distrofina/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Sarcolema/enzimologia , Utrofina/metabolismo , Animais , Modelos Animais de Doenças , Distrofina/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Distrofia Muscular de Duchenne/enzimologia , Distrofia Muscular de Duchenne/genética , Óxido Nítrico Sintase Tipo I/genética , Ligação Proteica , Transporte Proteico , Sarcolema/genética , Sarcolema/metabolismo , Utrofina/genéticaRESUMO
Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.
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Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.
Assuntos
Células-Tronco Pluripotentes Induzidas , Optogenética , Humanos , Músculo Esquelético , Junção Neuromuscular , Reprodutibilidade dos TestesRESUMO
The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.
Assuntos
Sistemas CRISPR-Cas , Inversão Cromossômica , DNA/genética , DNA/metabolismo , Edição de Genes , Células-Tronco Pluripotentes Induzidas/fisiologia , Reação em Cadeia da Polimerase/métodos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Edição de Genes/métodos , Proteína Homóloga a MRE11/metabolismo , Análise de Sequência de DNA/métodos , Adenoviridae , Animais , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , DNA/química , DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Proteína Homóloga a MRE11/genética , RNA Guia de CinetoplastídeosRESUMO
Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes. We identify hundreds of thousands of long-range cis-interactions between promoters and distal promoter-interacting regions, enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several promoter-interacting regions by using clustered regularly interspaced short palindromic repeats (CRISPR) techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP and DRD2 are transcriptionally regulated by physically linked enhancers.
Assuntos
Linhagem da Célula/genética , Cromatina/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Marcadores Genéticos , Transtornos Mentais/genética , Neurônios/metabolismo , Regiões Promotoras Genéticas , Mapeamento Cromossômico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Masculino , Neurônios/citologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.
RESUMO
Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (µHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within µHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. µHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the ß-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form µHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Células Cultivadas , Imunofluorescência , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Sarcômeros , Células EstromaisRESUMO
Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , HumanosRESUMO
Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.
Assuntos
Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de Vídeo/métodos , Miócitos Cardíacos/citologia , Reconhecimento Automatizado de Padrão , Algoritmos , Diferenciação Celular , Células Cultivadas/citologia , Humanos , Processamento de Imagem Assistida por Computador , Contração Miocárdica , Técnicas de Patch-Clamp , Transdução de Sinais , Razão Sinal-Ruído , SoftwareRESUMO
Reversible phosphorylation and dephosphorylation of key proteins on tyrosine residues are important parts of intracellular signaling triggered by hormones and other agents. Recent knock-out studies in mice have identified PTP1B as a potential target for the treatment of diabetes and obesity. As a consequence, a number of academic and industrial groups are aggressively pursuing the development of selective PTP1B inhibitors. In addition, other protein-tyrosine phosphatases (PTPs) appear to be critically involved in major diseases such as cancer and autoimmunity. Given the diversity of PTPs and their potential as drug targets in different diseases, we have taken a broad approach to develop active site-directed selective inhibitors of specific members of this family of enzymes. Using a high throughput screening, we have previously identified 2-(oxalylamino)benzoic acid 3a as a relatively weak but classical competitive inhibitor of several PTPs.(4) On the basis of our early studies, indicating that 3a might be used as a starting point for the synthesis of selective PTP inhibitors, we now present our efforts in expansion of this concept and provide here a number of new chemical scaffolds for the development of inhibitors of different members of the PTP family. Although the core structure of these inhibitors is charged, good oral bioavailability has been observed in rat for some compounds. Furthermore, we have observed enhancement of 2-deoxy-glucose accumulation in C2C12 cells with prodrug analogues.
Assuntos
Inibidores Enzimáticos/síntese química , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Piridinas/síntese química , Tiofenos/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular , Cristalografia por Raios X , Desoxiglucose/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Camundongos , Modelos Moleculares , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Piridinas/química , Piridinas/farmacologia , Ratos , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologiaRESUMO
Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated alpha-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.