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1.
Cell ; 187(3): 521-525, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38306979

RESUMO

High-quality predicted structures enable structure-based approaches to an expanding number of drug discovery programs. We propose that by utilizing free energy perturbation (FEP), predicted structures can be confidently employed to achieve drug design goals. We use structure-based modeling of hERG inhibition to illustrate this value of FEP.


Assuntos
Desenho de Fármacos , Descoberta de Drogas , Termodinâmica , Entropia
2.
EMBO Mol Med ; 11(12): e10489, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31660701

RESUMO

Pantothenate kinase-associated neurodegeneration (PKAN) is an inborn error of CoA metabolism causing dystonia, parkinsonism, and brain iron accumulation. Lack of a good mammalian model has impeded studies of pathogenesis and development of rational therapeutics. We took a new approach to investigating an existing mouse mutant of Pank2 and found that isolating the disease-vulnerable brain revealed regional perturbations in CoA metabolism, iron homeostasis, and dopamine metabolism and functional defects in complex I and pyruvate dehydrogenase. Feeding mice a CoA pathway intermediate, 4'-phosphopantetheine, normalized levels of the CoA-, iron-, and dopamine-related biomarkers as well as activities of mitochondrial enzymes. Human cell changes also were recovered by 4'-phosphopantetheine. We can mechanistically link a defect in CoA metabolism to these secondary effects via the activation of mitochondrial acyl carrier protein, which is essential to oxidative phosphorylation, iron-sulfur cluster biogenesis, and mitochondrial fatty acid synthesis. We demonstrate the fidelity of our model in recapitulating features of the human disease. Moreover, we identify pharmacodynamic biomarkers, provide insights into disease pathogenesis, and offer evidence for 4'-phosphopantetheine as a candidate therapeutic for PKAN.


Assuntos
Coenzima A/metabolismo , Dopamina/metabolismo , Ferro/metabolismo , Panteteína/análogos & derivados , Neurodegeneração Associada a Pantotenato-Quinase/tratamento farmacológico , Neurodegeneração Associada a Pantotenato-Quinase/metabolismo , Animais , Biomarcadores/metabolismo , Genótipo , Camundongos , Panteteína/farmacologia , Panteteína/uso terapêutico , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
3.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29263267

RESUMO

The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.


Assuntos
Alphavirus/metabolismo , Antivirais/farmacologia , Fibroblastos/metabolismo , Proteínas de Membrana/agonistas , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/genética , Fibroblastos/patologia , Fibroblastos/virologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/genética , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/genética
4.
FASEB J ; 31(8): 3425-3438, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28428265

RESUMO

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.


Assuntos
Antitireóideos/farmacologia , Metimazol/farmacologia , Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Retina/metabolismo , Degeneração Retiniana/tratamento farmacológico , Animais , Antitireóideos/uso terapêutico , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Morte Celular , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Metimazol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenoxiacetatos/farmacologia , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retinoblastoma , Tri-Iodotironina , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo
5.
Bioorg Med Chem ; 25(10): 2743-2753, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28385597

RESUMO

Current therapeutic options for treating demyelinating disorders such as multiple sclerosis (MS) do not stimulate myelin repair, thus creating a clinical need for therapeutic agents that address axonal remyelination. Thyroid hormone is known to play an important role in promoting developmental myelination and repair, and CNS permeable thyromimetic agents could offer an increased therapeutic index compared to endogenous thyroid hormone. Sobetirome is a clinical stage thyromimetic that has been shown to have promising activity in preclinical models related to MS and X-linked adrenoleukodystrophy (X-ALD), a genetic disease that involves demyelination. Here we report a new series of sobetirome prodrugs containing ethanolamine-based promoieties that were found to undergo an intramolecular O,N acyl migration to form the pharmacologically relevant amide species. Several of these systemically administered prodrugs deliver more sobetirome to the brain compared to unmodified sobetirome. Pharmacokinetic properties of the parent drug sobetirome and amidoalcohol prodrug 3 are described and prodrug 3 was found to be more potent than sobetirome in target engagement in the brain from systemic dosing.


Assuntos
Acetatos/química , Barreira Hematoencefálica/metabolismo , Etanolamina/química , Fenóis/química , Administração Oral , Amidas/química , Animais , Área Sob a Curva , Encéfalo/metabolismo , Ésteres/química , Meia-Vida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Curva ROC
6.
J Clin Endocrinol Metab ; 102(3): 776-785, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27768856

RESUMO

Context: The sources and biological impact of 3,3',5,5' tetraiodothyroacetic acid (TA4) are uncertain. CD34+ fibrocytes express several proteins involved in the production of thyroid hormones. They infiltrate the orbit in Graves disease (GD), an autoimmune process known as thyroid-associated ophthalmopathy. It appears that the thyrotropin receptor plays an important role in the pathogenesis of thyroid-associated ophthalmopathy. Objective: To quantify levels of TA4 in healthy participants and those with GD, determine whether fibrocytes generate this thyroid hormone analogue, and determine whether TA4 influences the actions of thyroid-stimulating hormone and thyroid-stimulating immunoglobulins in orbital fibroblasts. Design/Setting/Participants: Patients with GD and healthy donors in an academic medical center clinical practice were recruited. Main Outcome Measures: Liquid chromatography-tandem mass spectrometry, autoradiography, real-time polymerase chain reaction, hyaluronan immunoassay. Results: Serum levels of TA4 are elevated in GD. TA4 levels are positively correlated with those of thyroxine and negatively correlated with serum levels of triiodothyronine. Several cell types in culture generate TA4 from ambient thyroxine, including fibrocytes, HELA cells, human Müller stem cells, and retinal pigmented epithelial cells. Propylthiouracil inhibits TA4 generation. TA4 enhances the induction by thyrotropin and thyroid-stimulating immunoglobulins of several participants in the pathogenesis of thyroid-associated ophthalmopathy, including interleukin 6, hyaluronan synthase 1, prostaglandin endoperoxide H synthase 2, and haluronan production. Conclusion: TA4 may be ubiquitously generated in many tissues and enhances the biological impact of thyrotropin and thyroid-stimulating immunoglobulins in orbital connective tissue. These findings may identify a physiologically important determinant of extrathyroidal thyroid-stimulating hormone action.


Assuntos
Doença de Graves/sangue , Oftalmopatia de Graves/sangue , Tiroxina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Autorradiografia , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida , Células Ependimogliais/metabolismo , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Glucuronosiltransferase/metabolismo , Doença de Graves/complicações , Oftalmopatia de Graves/etiologia , Células HeLa , Humanos , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Imunoensaio , Imunoglobulinas Estimuladoras da Glândula Tireoide/metabolismo , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Órbita , Prostaglandina-Endoperóxido Sintases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Tireotropina/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Fatores Sexuais , Espectrometria de Massas em Tandem , Tireotropina/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangue , Adulto Jovem
7.
Bioorg Med Chem ; 24(22): 5842-5854, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27707627

RESUMO

There is currently great interest in developing drugs that stimulate myelin repair for use in demyelinating diseases such as multiple sclerosis. Thyroid hormone plays a key role in stimulating myelination during development and also controls the expression of important genes involved in myelin repair in adults. Because endogenous thyroid hormone in excess lacks a generally useful therapeutic index, it is not used clinically for indications other than hormone replacement; however, selective thyromimetics such as sobetirome offer a therapeutic alternative. Sobetirome is the only clinical-stage thyromimetic that is known to cross the blood-brain-barrier (BBB) and we endeavored to increase the BBB permeability of sobetirome using a prodrug strategy. Ester prodrugs of sobetirome were prepared based on literature reports of improved BBB permeability with other carboxylic acid containing drugs and BBB permeability was assessed in vivo. One sobetirome prodrug, ethanolamine ester 11, was found to distribute more sobetirome to the brain compared to an equimolar peripheral dose of unmodified sobetirome. In addition to enhanced brain levels, prodrug 11 displayed lower sobetirome blood levels and a brain/serum ratio that was larger than that of unmodified sobetirome. Thus, these data indicate that an ester prodrug strategy applied to sobetirome can deliver increased concentrations of the active drug to the central nervous system (CNS), which may prove useful in the treatment of CNS disorders.


Assuntos
Acetatos/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Ésteres/farmacologia , Permeabilidade/efeitos dos fármacos , Fenóis/farmacologia , Pró-Fármacos/farmacologia , Acetatos/síntese química , Acetatos/química , Animais , Relação Dose-Resposta a Droga , Ésteres/síntese química , Ésteres/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Fenóis/síntese química , Fenóis/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-Atividade
8.
ChemMedChem ; 11(21): 2459-2465, 2016 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-27731931

RESUMO

Sobetirome is one of the most studied thyroid hormone receptorâ€…ß (TRß)-selective thyromimetics in the field due to its excellent selectivity and potency. A small structural change-replacing the 3,5-dimethyl groups of sobetirome with either chlorine or bromine-produces significantly more potent compounds, both in vitro and in vivo. These halogenated compounds induce transactivation of a TRß-mediated cell-based reporter with an EC50 value comparable to that of T3, access the central nervous system (CNS) at levels similar to their parent, and activate an endogenous TR-regulated gene in the brain with an EC50 value roughly five-fold lower than that of sobetirome. Previous studies suggest that this apparent increase in affinity can be explained by halogen bonding between the ligand and a backbone carbonyl group in the receptor. This makes the new analogues potential candidates for treating CNS disorders that may respond favorably to thyroid-hormone-stimulated pathways.

9.
Tetrahedron ; 71(35): 5946-5951, 2015 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-28316349

RESUMO

New synthetic routes for the preparation of isotopically labeled versions of thyroid hormone agonist sobetirome were developed using Knochel's iodine-magnesium exchange. A more efficient synthesis of the thyroid hormone antagonist NH-3 was developed from a common intermediate in the sobetirome route. Using the new synthetic routes, d6- and 3H-sobetirome were prepared for their use in studying biodistribution and the cellular uptake of sobetirome. The new route to NH-3 allows for a more rapid and efficient synthesis and provides access to an advanced intermediate to facilitate antagonist analog production in the final bond-forming synthetic step.

10.
Org Lett ; 14(16): 4038-41, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22857735

RESUMO

A set of synthetic approaches were developed and applied to the synthesis of eight frame-shifted farnesyl diphosphate (FPP) analogs. These analogs bear increased or decreased methylene units between the double bonds and/or diphosphate moieties of the isoprenoid structure. Evaluation versus mammalian FTase revealed that small structural changes can lead to dramatic changes in substrate ability.


Assuntos
Alquil e Aril Transferases/metabolismo , Fosfatos de Poli-Isoprenil/síntese química , Sesquiterpenos/síntese química , Estrutura Molecular , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/química , Relação Estrutura-Atividade
11.
Org Lett ; 13(14): 3576-9, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21699139

RESUMO

Through the use of a 1,2-metalate rearrangement, six 7-substituted farnesol analogs were generated in a concise manner. This new synthetic route allowed us to quickly prepare several diverse farnesyl diphosphate analogs with interesting biological activities against mammalian protein-farnesyl transferase.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/síntese química , Sesquiterpenos/química , Sesquiterpenos/síntese química , Alquil e Aril Transferases/metabolismo , Animais , Mamíferos , Estrutura Molecular , Fosfatos de Poli-Isoprenil/farmacologia , Sesquiterpenos/farmacologia
12.
J Pharm Biomed Anal ; 53(3): 567-75, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-20417049

RESUMO

Elucidation of pathways involved with lipid metabolism has been limited by analytical challenges associated with detection and structure identification. A discovery-based mass spectrometry lipidomic approach has been applied to identify metabolites of the endogenous cannabinoid anandamide (N-arachidonylethanolamide). Previously, a model system was established to show that anandamide can be recycled by cells to form new endocannabinoids suggesting recycling of the arachidonate carbon chain. We hypothesized that distinct cellular pathways exist to direct the anandamide-derived arachidonate chain into a specific set of metabolites, different from the metabolite pool that is comprised of non-anandamide-derived arachidonic acid. Using stable isotope encoding and liquid chromatography-mass spectrometry, we identified a distinct pool of lipid metabolites derived from exogenous anandamide or arachidonic acid in RBL-2H3 cells. We discovered that arachidonic acid-derived metabolites were primarily comprised of the eicosanoid lipid class, whereas anandamide-derived arachidonic acid, in addition to eicosanoids, was metabolized into diradylglycerols, fatty acid amides, sterols, and glycerophospholipids. From the list of anandamide metabolites of particular interest was 1-O-arachidonyl-sn-glycero-3-phosphocholine. Furthermore, we determined that while 1-O-arachidonyl-sn-glycero-3-phosphocholine may be a metabolite of anandamide, the sn-2 compound was more abundant in mouse brain tissue. Overall, our results provide a novel approach to study the metabolic fate of endocannabinoids and fatty acid-derived signaling molecules.


Assuntos
Ácidos Araquidônicos/metabolismo , Metabolismo dos Lipídeos , Alcamidas Poli-Insaturadas/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Endocanabinoides , Glicerídeos/metabolismo , Camundongos , Ratos , Espectrometria de Massas por Ionização por Electrospray
13.
Anal Biochem ; 383(1): 18-24, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18782552

RESUMO

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C(18) HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0mumol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Terpenos/análise , Linhagem Celular Tumoral , Humanos , Reprodutibilidade dos Testes
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