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1.
J Nat Prod ; 86(2): 264-275, 2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36651644

RESUMO

In this study, an integrated in silico-in vitro approach was employed to discover natural products (NPs) active against SARS-CoV-2. The two SARS-CoV-2 viral proteases, i.e., main protease (Mpro) and papain-like protease (PLpro), were selected as targets for the in silico study. Virtual hits were obtained by docking more than 140,000 NPs and NP derivatives available in-house and from commercial sources, and 38 virtual hits were experimentally validated in vitro using two enzyme-based assays. Five inhibited the enzyme activity of SARS-CoV-2 Mpro by more than 60% at a concentration of 20 µM, and four of them with high potency (IC50 < 10 µM). These hit compounds were further evaluated for their antiviral activity against SARS-CoV-2 in Calu-3 cells. The results from the cell-based assay revealed three mulberry Diels-Alder-type adducts (MDAAs) from Morus alba with pronounced anti-SARS-CoV-2 activities. Sanggenons C (12), O (13), and G (15) showed IC50 values of 4.6, 8.0, and 7.6 µM and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 Mpro proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive 1H relaxation dispersion NMR spectroscopy.


Assuntos
Produtos Biológicos , COVID-19 , Humanos , Proteases Virais , SARS-CoV-2 , Peptídeo Hidrolases , Antivirais , Simulação de Acoplamento Molecular , Inibidores de Proteases
2.
Am J Pathol ; 182(2): 485-504, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23332367

RESUMO

Duchenne muscular dystrophy (DMD) is a severe disorder characterized by progressive muscle wasting,respiratory and cardiac impairments, and premature death. No treatment exists so far, and the identification of active substances to fight DMD is urgently needed. We found that tamoxifen, a drug used to treat estrogen-dependent breast cancer, caused remarkable improvements of muscle force and of diaphragm and cardiac structure in the mdx(5Cv) mouse model of DMD. Oral tamoxifen treatment from 3 weeks of age for 15 months at a dose of 10 mg/kg/day stabilized myofiber membranes, normalized whole body force, and increased force production and resistance to repeated contractions of the triceps muscle above normal values. Tamoxifen improved the structure of leg muscles and diminished cardiac fibrosis by~ 50%. Tamoxifen also reduced fibrosis in the diaphragm, while increasing its thickness,myofiber count, and myofiber diameter, thereby augmenting by 72% the amount of contractile tissue available for respiratory function. Tamoxifen conferred a markedly slower phenotype to the muscles.Tamoxifen and its metabolites were present in nanomolar concentrations in plasma and muscles,suggesting signaling through high-affinity targets. Interestingly, the estrogen receptors ERa and ERb were several times more abundant in dystrophic than in normal muscles, and tamoxifen normalized the relative abundance of ERb isoforms. Our findings suggest that tamoxifen might be a useful therapy for DMD.


Assuntos
Antineoplásicos/uso terapêutico , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/patologia , Tamoxifeno/uso terapêutico , Animais , Antineoplásicos/farmacologia , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Fenômenos Biomecânicos/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Creatina Quinase/sangue , Diafragma/patologia , Diafragma/fisiopatologia , Modelos Animais de Doenças , Comportamento Alimentar/efeitos dos fármacos , Fibrose , Camundongos , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Tamoxifeno/sangue , Tamoxifeno/farmacologia
3.
Biomed Pharmacother ; 177: 116957, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38908198

RESUMO

Duchenne muscular dystrophy (DMD) is the most common muscular disorder affecting children. It affects nearly 1 male birth over 5000. Oxidative stress is a pervasive feature in the pathogenesis of DMD. Recent work shows that the main generators of ROS are NADPH oxidases (NOX), suggesting that they are an early and promising target in DMD. In addition, skeletal muscles of mdx mice, a murine model of DMD, overexpress NOXes. We investigated the impact of diapocynin, a dimer of the NOX inhibitor apocynin, on the chronic disease phase of mdx5Cv mice. Treatment of these mice with diapocynin from 7 to 10 months of age resulted in decreased hypertrophy of several muscles, prevented force loss induced by tetanic and eccentric contractions, improved muscle and respiratory functions, decreased fibrosis of the diaphragm and positively regulated the expression of disease modifiers. These encouraging results ensure the potential role of diapocynin in future treatment strategies.


Assuntos
Acetofenonas , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne , Animais , Acetofenonas/farmacologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Modelos Animais de Doenças , Compostos de Bifenilo/farmacologia , Diafragma/efeitos dos fármacos , Diafragma/metabolismo , Contração Muscular/efeitos dos fármacos , Fibrose , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Camundongos Endogâmicos C57BL
4.
Nat Commun ; 9(1): 2032, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29795225

RESUMO

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.


Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , RNA Mensageiro/metabolismo , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Drosophila , Avaliação Pré-Clínica de Medicamentos , Éxons/genética , Células HeLa , Humanos , Imidazóis/química , Imidazóis/uso terapêutico , Indóis/química , Indóis/uso terapêutico , Terapia de Alvo Molecular/métodos , Atrofia Muscular Espinal/genética , Fenótipo , Sítios de Splice de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genética
5.
Free Radic Res ; 39(9): 913-9, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16087472

RESUMO

Cyclosporin A (CsA) generates superoxide in smooth muscle cells. Our earlier studies have demonstrated that the increase in the vasopressin type 1 receptor induced in vascular smooth muscle cells in the presence of CsA is probably due to superoxide (Krauskopf et al., J Biol Chem 278, 41685-41690, 2003). This increase in vasopressin receptor is likely at the base of increased vascular responsiveness to vasoconstrictor hormones and hypertension induced by CsA. Here, we demonstrate that CsA produces superoxide. In addition, our data show that superoxide generation does not originate from the major cellular superoxide generating systems NAD(P)H oxidase or xanthine oxidase. Our results suggest that the side effects of CsA could be diminished with the help of SOD mimetic drugs.


Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Aorta/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Etídio/análogos & derivados , Fluoresceínas , Sequestradores de Radicais Livres/farmacologia , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética
6.
J Gen Physiol ; 132(2): 199-208, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18625851

RESUMO

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.


Assuntos
Morte Celular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Canais de Sódio/metabolismo , Sódio/metabolismo , Animais , Sobrevivência Celular , Distrofina/genética , Distrofina/metabolismo , Regulação da Expressão Gênica , Ativação do Canal Iônico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/citologia , Canal de Sódio Disparado por Voltagem NAV1.4 , Sarcolema/metabolismo , Canais de Sódio/genética , Tetrodotoxina/farmacologia
7.
J Cell Sci ; 119(Pt 18): 3733-42, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16926189

RESUMO

Duchenne muscular dystrophy is caused by deficiency of dystrophin and leads to progressive weakness. It has been proposed that the muscle degeneration occurring in this disease is caused by increased Ca2+ influx due to enhanced activity of cationic channels that are activated either by stretch of the plasma membrane (stretch-activated channels) or by Ca2+-store depletion (store-operated channels). Using both cytosolic Ca2+ measurements with Fura-2 and the manganese quench method, we show here that store-operated Ca2+ entry is greatly enhanced in dystrophic skeletal flexor digitorum brevis fibers isolated from mdx(5cv) mice, a mouse model of Duchenne muscular dystrophy. Moreover, we show for the first time that store-operated Ca2+ entry in these fibers is under the control of the Ca2+-independent phospholipase A2 and that the exaggerated Ca2+ influx can be completely attenuated by inhibitors of this enzyme. Enhanced store-operated Ca2+ entry in dystrophic fibers is likely to be due to a near twofold overexpression of Ca2+-independent phospholipase A2. The Ca2+-independent phospholipase A2 pathway therefore appears as an attractive target to reduce excessive Ca2+ influx and subsequent degeneration occurring in dystrophic fibers.


Assuntos
Sinalização do Cálcio/fisiologia , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/enzimologia , Distrofia Muscular Animal/induzido quimicamente , Fosfolipases A/metabolismo , Anilidas/farmacologia , Animais , Cafeína/farmacologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Fosfolipases A2 do Grupo VI , Transporte de Íons/efeitos dos fármacos , Manganês/metabolismo , Meliteno/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Naftalenos/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Cloreto de Potássio/metabolismo , Pironas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tiadiazóis/farmacologia
8.
Lab Invest ; 82(12): 1715-24, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12480921

RESUMO

SUMMARY: Malignant tumor cell invasion is determinant for metastasis to occur. E2 and C5 colon carcinoma cells that were derived from the parental Lovo line and that differ experimentally in spontaneous metastatic ability have been monitored for gene expression by cDNA arrays. Among genes found differentially expressed, the CD63 tetraspanin, not previously recognized in colon cancer progression, and the alpha3 integrin chain were both up-regulated in low metastatic E2 cells and were analyzed for their functional role using adhesion, migration, and invasion assays. Cell surface expression of CD63 and alpha3 integrin was about 2-fold higher in E2 than in C5 cells and confocal microscopy showed that CD63 and alpha3 integrin colocalized evenly on C5 cells whereas they concentrated at elongated tips of the low-metastatic more substrate-adhesive E2 cells. Antibody-interference experiments identified laminin-5 (LN-5) as a ligand interacting with the alpha3beta1/CD63 complex. Substrate-immobilized anti-CD63 antibodies enhanced tumor cell migration and invasion and induced prominent cell surface protrusions that were repressed by the PI3-kinase LY294002 inhibitor. Our results suggest that changes in the expression of surface CD63 and alpha3beta1 integrin interacting with LN-5 could affect migratory signals and the progression of the metastatic disease.


Assuntos
Antígenos CD/genética , Carcinoma/genética , Carcinoma/metabolismo , Neoplasias do Colo/genética , DNA Complementar/análise , Integrina alfa3beta1/genética , Glicoproteínas da Membrana de Plaquetas/genética , Anticorpos Bloqueadores/farmacologia , Antígenos CD/metabolismo , Carcinoma/secundário , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Células Clonais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , DNA de Neoplasias/análise , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Humanos , Integrina alfa3beta1/metabolismo , Morfolinas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Glicoproteínas da Membrana de Plaquetas/metabolismo , RNA Neoplásico/análise , Tetraspanina 30 , Células Tumorais Cultivadas , Regulação para Cima , Cicatrização/efeitos dos fármacos , Calinina
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