RESUMEN
Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample.
Asunto(s)
Técnicas de Laboratorio Clínico , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Animales , Técnicas de Laboratorio Clínico/métodos , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/metabolismoRESUMEN
A depressed autophagy has previously been reported in cystic fibrosis patients with the common F508del-CFTR mutation. This report describes the synthesis and preliminary biological characterization of a novel series of autophagy activators involving fatty acid cysteamine conjugates. These molecular entities were synthesized by first covalently linking cysteamine to docosahexaenoic acid. The resulting conjugate 1 synergistically activated autophagy in primary homozygous F508del-CFTR human bronchial epithelial (hBE) cells at submicromolar concentrations. When conjugate 1 was used in combination with the corrector lumacaftor and the potentiator ivacaftor, it showed an additive effect, as measured by the increase in the chloride current in a functional assay. In order to obtain a more stable form for oral dosing, the sulfhydryl group in conjugate 1 was converted into a functionalized disulfide moiety. The resulting conjugate 5 is orally bioavailable in the mouse, rat, and dog and allows a sustained delivery of the biologically active conjugate 1.
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Autofagia/efectos de los fármacos , Cisteamina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Ácidos Grasos/química , Cisteamina/química , Pliegue de ProteínaRESUMEN
This report describes the synthesis and preliminary biological characterization of novel fatty acid niacin conjugates and fatty acid salicylate conjugates. These molecular entities were created by covalently linking two bioactive molecules, either niacin or salicylic acid, to an omega-3 fatty acid. This methodology allows the simultaneous intracellular delivery of two bioactives in order to elicit a pharmacological response that could not be replicated by administering the bioactives individually or in combination. The fatty acid niacin conjugate 5 has been shown to be an inhibitor of the sterol regulatory element binding protein (SREBP), a key regulator of cholesterol metabolism proteins such as PCSK9, HMG-CoA reductase, ATP citrate lyase, and NPC1L1. On the other hand, the fatty acid salicylate conjugate 11 has been shown to have a unique anti-inflammatory profile based on its ability to modulate the NF-κB pathway through the intracellular release of the two bioactives.
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Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Ácidos Grasos/química , Niacina/química , Niacina/farmacología , Ácido Salicílico/química , Ácido Salicílico/farmacología , Administración Oral , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Hidrólisis , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Estructura Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Niacina/administración & dosificación , Ratas , Ratas Sprague-Dawley , Ácido Salicílico/administración & dosificación , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Relación Estructura-Actividad , Distribución TisularRESUMEN
One possible mechanism linking inflammation with cancer involves the generation of reactive oxygen, nitrogen, and halogen species by activated macrophages and neutrophils infiltrating sites of infection or tissue damage, with these chemical mediators causing damage that ultimately leads to cell death and mutation. To determine the most biologically deleterious chemistries of inflammation, we previously assessed products across the spectrum of DNA damage arising in inflamed tissues in the SJL mouse model nitric oxide overproduction ( Pang et al. ( 2007 ) Carcinogenesis 28 , 1807 - 1813 ). Among the anticipated DNA damage chemistries, we observed significant changes only in lipid peroxidation-derived etheno adducts. We have now developed an isotope-dilution, liquid chromatography-coupled, tandem quadrupole mass spectrometric method to quantify representative species across the spectrum of RNA damage products predicted to arise at sites of inflammation, including nucleobase deamination (xanthosine and inosine), oxidation (8-oxoguanosine), and alkylation (1,N(6)-ethenoadenosine). Application of the method to the liver, spleen, and kidney from the SJL mouse model revealed generally higher levels of oxidative background RNA damage than was observed in DNA in control mice. However, compared to control mice, RcsX treatment to induce nitric oxide overproduction resulted in significant increases only in inosine and only in the spleen. Further, the nitric oxide synthase inhibitor, N-methylarginine, did not significantly affect the levels of inosine in control and RcsX-treated mice. The differences between DNA and RNA damage in the same animal model of inflammation point to possible influences from DNA repair, RcsX-induced alterations in adenosine deaminase activity, and differential accessibility of DNA and RNA to reactive oxygen and nitrogen species as determinants of nucleic acid damage during inflammation.
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Inflamación/metabolismo , ARN/metabolismo , Animales , Cromatografía Liquida , ADN/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Fenómenos Genéticos , Inosina , Riñón/metabolismo , Hígado/metabolismo , Ratones , Óxido Nítrico/metabolismo , Oxidación-Reducción , Bazo/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: In the event of a nuclear accident, people are exposed to elevated levels of continuous low dose-rate radiation. Nevertheless, most of the literature describes the biological effects of acute radiation. OBJECTIVES: DNA damage and mutations are well established for their carcinogenic effects. We assessed several key markers of DNA damage and DNA damage responses in mice exposed to low dose-rate radiation to reveal potential genotoxic effects associated with low dose-rate radiation. METHODS: We studied low dose-rate radiation using a variable low dose-rate irradiator consisting of flood phantoms filled with 125Iodine-containing buffer. Mice were exposed to 0.0002 cGy/min (~ 400-fold background radiation) continuously over 5 weeks. We assessed base lesions, micronuclei, homologous recombination (HR; using fluorescent yellow direct repeat mice), and transcript levels for several radiation-sensitive genes. RESULTS: We did not observe any changes in the levels of the DNA nucleobase damage products hypoxanthine, 8-oxo-7,8-dihydroguanine, 1,N6-ethenoadenine, or 3,N4-ethenocytosine above background levels under low dose-rate conditions. The micronucleus assay revealed no evidence that low dose-rate radiation induced DNA fragmentation, and there was no evidence of double strand break-induced HR. Furthermore, low dose-rate radiation did not induce Cdkn1a, Gadd45a, Mdm2, Atm, or Dbd2. Importantly, the same total dose, when delivered acutely, induced micronuclei and transcriptional responses. CONCLUSIONS: These results demonstrate in an in vivo animal model that lowering the dose-rate suppresses the potentially deleterious impact of radiation and calls attention to the need for a deeper understanding of the biological impact of low dose-rate radiation.
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Daño del ADN , Animales , Relación Dosis-Respuesta en la Radiación , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BLRESUMEN
Chronic inflammation has long been recognized as a risk factor for many human cancers. One mechanistic link between inflammation and cancer involves the generation of nitric oxide, superoxide and other reactive oxygen and nitrogen species by macrophages and neutrophils that infiltrate sites of inflammation. Although pathologically high levels of these reactive species cause damage to biological molecules, including DNA, nitric oxide at lower levels plays important physiological roles in cell signaling and apoptosis. This raises the question of inflammation-induced imbalances in physiological and pathological pathways mediated by chemical mediators of inflammation. At pathological levels, the damage sustained by nucleic acids represents the full spectrum of chemistries and likely plays an important role in carcinogenesis. This suggests that DNA damage products could serve as biomarkers of inflammation and oxidative stress in clinically accessible compartments such as blood and urine. However, recent studies of the biotransformation of DNA damage products before excretion point to a weakness in our understanding of the biological fates of the DNA lesions and thus to a limitation in the use of DNA lesions as biomarkers. This review will address these and other issues surrounding inflammation-mediated DNA damage on the road to cancer.
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Transformación Celular Neoplásica/genética , Daño del ADN/genética , Inflamación/metabolismo , Neoplasias/genética , Especies Reactivas de Oxígeno/metabolismo , Animales , Enfermedad Crónica , Humanos , Inflamación/genética , Especies de Nitrógeno Reactivo/efectos adversos , Factores de RiesgoRESUMEN
Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.
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Marcación de Gen/métodos , Mutación , Ácidos Nucleicos de Péptidos/química , Talasemia/genética , Globinas beta/genética , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Humanos , Plásmidos/genética , Recombinación Genética , Fase S , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.
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Globinas/genética , Ácidos Nucleicos de Péptidos/farmacología , Sitios de Empalme de ARN/genética , Animales , Secuencia de Bases , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Genoma/genética , Humanos , Datos de Secuencia Molecular , Mutación/genéticaRESUMEN
The design and synthesis of novel chiral PNA monomer based on trans-5-aminopipecolic acid is reported. The trans diequatorial disposition of the 1,4 ring substituents in six-membered 5-aminopipecolic acid derivative could be favorable over trans 1,3 axial-equatorial disposition in 4-aminopipecolic acid of PNA. Studies on the synthesis of trans-4/5-aminopipecolyl PNA-eagPNA chimeras and their binding preferences to DNA/RNA in duplex/triplex modes are described.
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ADN/metabolismo , Ácidos Nucleicos de Péptidos/química , ARN/metabolismo , Resonancia Magnética Nuclear Biomolecular , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The design and facile conversion of naturally occurring 4-hydroxyproline to all four diastereomers of thymine pyrrolidine PNA monomer, (2R,4S)-adenine, -guanine and -cytosine monomers and their incorporation into duplex forming PNA oligomers is reported. The interesting results of the hybridization studies with complementary DNA/RNA sequences in either parallel or antiparallel orientation reveal the stereochemistry-dependent DNA vs. RNA discriminations and parallel/antiparallel orientation selectivity.
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ADN/química , Ácidos Nucleicos de Péptidos/síntesis química , Pirrolidinas/síntesis química , ARN/química , Estructura Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Pirrolidinas/química , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
The design and facile synthesis of novel chiral piperidine PNA from naturally occurring 4-hydroxy-L-proline is reported. The stereospecific ring-expansion reaction to get six-membered piperidine derivative from 5-membered pyrrolidine derivative is exploited for this synthesis. The resulting conformationally constrained PNA is utilized for the synthesis of PNA mixmers and the concept is substantiated by UV-Tm studies of the resulting PNA(2):DNA complexes.
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ADN/química , Ácidos Nucleicos de Péptidos/química , Conformación Proteica , Rayos UltravioletaRESUMEN
Synthesis of a new six membered PNA analogue by introducing a methylene bridge between beta carbon atom of ethylene diamine and beta' carbon atom of linker to nucleobase.