RESUMO
Androgen receptor (AR) and its splice variants (AR-SVs) promote prostate cancer (PCa) growth by orchestrating transcriptional reprogramming. Mechanisms by which the low complexity and intrinsically disordered primary transactivation domain (AF-1) of AR and AR-SVs regulate transcriptional programming in PCa remains poorly defined. Using omics, live and fixed fluorescent microscopy of cells, and purified AF-1 and AR-V7 recombinant proteins we show here that AF-1 and the AR-V7 splice variant form molecular condensates by liquid-liquid phase separation (LLPS) that exhibit disorder characteristics such as rapid intracellular mobility, coactivator interaction, and euchromatin induction. The LLPS and other disorder characteristics were reversed by a class of small-molecule-selective AR-irreversible covalent antagonists (SARICA) represented herein by UT-143 that covalently and selectively bind to C406 and C327 in the AF-1 region. Interfering with LLPS formation with UT-143 or mutagenesis resulted in chromatin condensation and dissociation of AR-V7 interactome, all culminating in a transcriptionally incompetent complex. Biochemical studies suggest that C327 and C406 in the AF-1 region are critical for condensate formation, AR-V7 function, and UT-143's irreversible AR inhibition. Therapeutically, UT-143 possesses drug-like pharmacokinetics and metabolism properties and inhibits PCa cell proliferation and tumor growth. Our work provides critical information suggesting that clinically important AR-V7 forms transcriptionally competent molecular condensates and covalently engaging C327 and C406 in AF-1, dissolves the condensates, and inhibits its function. The work also identifies a library of AF-1-binding AR and AR-SV-selective covalent inhibitors for the treatment of PCa.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Receptores Androgênicos/metabolismo , Cisteína , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Linhagem Celular Tumoral , Isoformas de Proteínas/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is a neuromuscular disease that predominantly affects boys as a result of mutation(s) in the dystrophin gene. DMD is characterized by musculoskeletal and cardiopulmonary complications, resulting in shorter life-span. Boys afflicted by DMD typically exhibit symptoms within 3-5 years of age and declining physical functions before attaining puberty. We hypothesized that rapidly deteriorating health of pre-pubertal boys with DMD could be due to diminished anabolic actions of androgens in muscle, and that intervention with an androgen receptor (AR) agonist will reverse musculoskeletal complications and extend survival. While castration of dystrophin and utrophin double mutant (mdx-dm) mice to mimic pre-pubertal nadir androgen condition resulted in premature death, maintenance of androgen levels extended the survival. Non-steroidal selective-AR modulator, GTx-026, which selectively builds muscle and bone was tested in X-linked muscular dystrophy mice (mdx). GTx-026 significantly increased body weight, lean mass and grip strength by 60-80% over vehicle-treated mdx mice. While vehicle-treated castrated mdx mice exhibited cardiopulmonary impairment and fibrosis of heart and lungs, GTx-026 returned cardiopulmonary function and intensity of fibrosis to healthy control levels. GTx-026 elicits its musculoskeletal effects through pathways that are distinct from dystrophin-regulated pathways, making AR agonists ideal candidates for combination approaches. While castration of mdx-dm mice resulted in weaker muscle and shorter survival, GTx-026 treatment increased the muscle mass, function and survival, indicating that androgens are important for extended survival. These preclinical results support the importance of androgens and the need for intervention with AR agonists to treat DMD-affected boys.
Assuntos
Androgênios/metabolismo , Distrofia Muscular de Duchenne/genética , Androgênios/genética , Animais , Modelos Animais de Doenças , Distrofina/genética , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos mdx , Debilidade Muscular/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/metabolismo , Receptores Androgênicos/metabolismo , Maturidade Sexual , Utrofina/genéticaRESUMO
Most satiety-inducing obesity therapeutics, despite modest efficacy, have safety concerns that underscore the need for effective peripherally acting drugs. An attractive therapeutic approach for obesity is to optimize/maximize energy expenditure by increasing energy-utilizing thermogenic brown adipose tissue. We used in vivo and in vitro models to determine the role of estrogen receptor ß (ER-ß) and its ligands on adipose biology. RNA sequencing and metabolomics were used to determine the mechanism of action of ER-ß and its ligands. Estrogen receptor ß (ER-ß) and its selective ligand reprogrammed preadipocytes and precursor stem cells into brown adipose tissue and increased mitochondrial respiration. An ER-ß-selective ligand increased markers of tricarboxylic acid-dependent and -independent energy biogenesis and oxygen consumption in mice without a concomitant increase in physical activity or food consumption, all culminating in significantly reduced weight gain and adiposity. The antiobesity effects of ER-ß ligand were not observed in ER-ß-knockout mice. Serum metabolite profiles of adult lean and juvenile mice were comparable, while that of adult obese mice was distinct, indicating a possible impact of obesity on age-dependent metabolism. This phenotype was partially reversed by ER-ß-selective ligand. These data highlight a new role for ER-ß in adipose biology and its potential to be a safer alternative peripheral therapeutic target for obesity.-Ponnusamy, S., Tran, Q. T., Harvey, I., Smallwood, H. S., Thiyagarajan, T., Banerjee, S., Johnson, D. L., Dalton, J. T., Sullivan, R. D., Miller, D. D., Bridges, D., Narayanan, R. Pharmacologic activation of estrogen receptor ß increases mitochondrial function, energy expenditure, and brown adipose tissue.
Assuntos
Tecido Adiposo Marrom/metabolismo , Metabolismo Energético/fisiologia , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/metabolismo , Isoquinolinas/farmacologia , Mitocôndrias/fisiologia , Tecido Adiposo Branco/fisiologia , Animais , Biomarcadores , Dieta Hiperlipídica , Receptor beta de Estrogênio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Resistência à Insulina , Masculino , Camundongos , Camundongos Knockout , Obesidade/sangue , Obesidade/metabolismoRESUMO
Stress urinary incontinence (SUI), a prevalent condition, is represented by an involuntary leakage of urine that results, at least in part, from weakened or damaged pelvic floor muscles and is triggered by physical stress. Current treatment options are limited with no oral therapies available. The pelvic floor is rich in androgen receptor and molecules with anabolic activity including selective androgen receptor modulators (SARMs) may serve as therapeutic options for individuals with SUI. In this study, two SARMs (GTx-024 and GTx-027) were evaluated in a post-menopausal animal model in order to determine their effect on pelvic floor muscles. Female C57BL/6 mice were ovariectomized and their pelvic muscles allowed to regress. The animals were then treated with vehicle or doses of GTx-024 or GTx-027. Animal total body weight, lean body mass, and pelvic floor muscle weights were measured along with the expression of genes associated with muscle catabolism. Treatment with the SARMs resulted in a restoration of the pelvic muscles to the sham-operated weight. Coordinately, the induction of genes associated with muscle catabolism was inhibited. Although a trend was observed towards an increase in total lean body mass in the SARM-treated groups, no significant differences were detected. Treatment of an ovariectomized mouse model with SARMs resulted in an increase in pelvic floor muscles, which may translate to an improvement of symptoms associated with SUI and serves as the basis for evaluating their clinical use. J. Cell. Biochem. 118: 640-646, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Anilidas/farmacologia , Músculo Esquelético , Ovariectomia , Receptores Androgênicos/metabolismo , Incontinência Urinária por Estresse , Animais , Feminino , Células HEK293 , Humanos , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Tamanho do Órgão/efeitos dos fármacos , Diafragma da Pelve , Incontinência Urinária por Estresse/tratamento farmacológico , Incontinência Urinária por Estresse/metabolismo , Incontinência Urinária por Estresse/patologia , Incontinência Urinária por Estresse/fisiopatologiaRESUMO
The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.
Assuntos
Glicoesfingolipídeos/metabolismo , Rim/fisiologia , Nefrite/tratamento farmacológico , Proteína Proto-Oncogênica c-fli-1/metabolismo , Receptores CXCR3/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Regulação da Expressão Gênica , Humanos , Rim/efeitos dos fármacos , Lactosilceramidas/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Nefrite/imunologia , Neuraminidase/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Receptores CXCR3/genéticaRESUMO
Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a â¼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.
Assuntos
Escherichia coli/genética , RNA/genética , Telomerase/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Plasmídeos/genética , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Telomerase/química , Telomerase/isolamento & purificação , Telomerase/metabolismo , Transformação GenéticaRESUMO
Nearly one half of patients with lupus develop glomerulonephritis (GN), which often leads to renal failure. Although nephritis is diagnosed by the presence of proteinuria, the pathology of nephritis can fall into one of five classes defined by different forms of tissue injury, and the mechanisms involved in pathogenesis are not completely understood. Glycosphingolipids are abundant in the kidney, have roles in many cellular functions, and were shown to be involved in other renal diseases. Here, we show dysfunctional glycosphingolipid metabolism in patients with lupus nephritis and MRL/lpr lupus mice. Specifically, we found that glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels are significantly higher in the kidneys of nephritic MRL/lpr lupus mice than the kidneys of non-nephritic lupus mice or healthy controls. This elevation may be, in part, caused by altered transcriptional regulation and/or activity of LacCer synthase (GalT5) and neuraminidase 1, enzymes that mediate glycosphingolipid metabolism. We show increased neuraminidase 1 activity early during the progression of nephritis (before significant elevation of GlcCer and LacCer in the kidney). Elevated levels of urinary LacCer were detected before proteinuria in lupus mice. Notably, LacCer levels were higher in the urine and kidneys of patients with lupus and nephritis than patients with lupus without nephritis or healthy controls. Together, these results show early and significant dysfunction of the glycosphingolipid metabolic pathway in the kidneys of lupus mice and patients with lupus nephritis and suggest that molecules in this pathway may serve as early markers in lupus nephritis.
Assuntos
Glicoesfingolipídeos/metabolismo , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Neuraminidase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Análise de Variância , Animais , Biomarcadores/análise , Biópsia por Agulha , Modelos Animais de Doenças , Progressão da Doença , Seguimentos , Humanos , Immunoblotting , Imuno-Histoquímica , Testes de Função Renal , Nefrite Lúpica/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Neuraminidase/genética , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , UrináliseRESUMO
A major challenge for new drug discovery in the area of androgen receptor (AR) antagonists lies in predicting the druggable properties that will enable small molecules to retain their potency and stability during further studies in vitro and in vivo. Indole (compound 8) is a first-in-class AR antagonist with very high potency (IC50 = 0.085 µM) but is metabolically unstable. During the metabolic studies described herein, we synthesized new small molecules that exhibit significantly improved stability while retaining potent antagonistic activity for an AR. This structure-activity relationship (SAR) study of more than 50 compounds classified with three classes (Class I, II, and III) and discovered two compounds (32c and 35i) that are potent AR antagonists (e.g., IC50 = 0.021 µM, T1/2 = 120 min for compound 35i). The new antagonists exhibited improved in vivo pharmacokinetics (PK) with high efficacy antiandrogen activity in Hershberger and antiandrogen Enz-Res tumor xenograft models that overexpress AR (LNCaP-AR).
Assuntos
Antagonistas de Receptores de Andrógenos , Neoplasias da Próstata , Masculino , Humanos , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/uso terapêutico , Receptores Androgênicos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Antagonistas de Androgênios , Nitrilas/farmacologia , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (â¼80%) but also expresses AR splice variants (AR-SVs) (â¼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias de Mama Triplo Negativas/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão GênicaRESUMO
A series of propanamide derivatives were designed, synthesized, and pharmacologically characterized as selective androgen receptor degraders (SARDs) and pan-antagonists that exert a broad-scope androgen receptor (AR) antagonism. Incorporating different basic heteromonocyclic B-ring structural elements in the common A-ring-linkage-B-ring nonsteroidal antiandrogen general pharmacophore contributed to a novel scaffold of small molecules with SARD and pan-antagonist activities even compared to our recently published AF-1 binding SARDs such as UT-69 (11), UT-155 (12), and UT-34 (13). Compound 26f exhibited inhibitory and degradation effects in vitro in a wide array of wtAR, point mutant, and truncation mutant-driven prostate cancers (PCs). Further, 26f inhibited tumor cell growth in a xenograft model composed of enzalutamide-resistant (EnzR) LNCaP cells. These results demonstrate an advancement toward the development of novel SARDs and pan-antagonists with efficacy against EnzR prostate cancers.
Assuntos
Amidas/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Amidas/síntese química , Amidas/química , Antagonistas de Receptores de Andrógenos/síntese química , Antagonistas de Receptores de Andrógenos/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Masculino , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Nitrilas/farmacologia , Feniltioidantoína/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Trocador de Sódio e Cálcio/genética , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Gatos , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
It is becoming increasingly evident that histone deacetylases (HDACs) have a prominent role in the alteration of gene expression during the growth remodeling process of cardiac hypertrophy. HDACs are generally viewed as corepressors of gene expression. However, we demonstrate that class I and class II HDACs play an important role in the basal expression and up-regulation of the sodium calcium exchanger (Ncx1) gene in adult cardiomyocytes. Treatment with the HDAC inhibitor trichostatin A (TSA) prevented the pressure-overload-stimulated up-regulation of Ncx1 expression. Overexpression of HDAC5 resulted in the dose-dependent up-regulation of basal and alpha-adrenergic stimulated Ncx1 expression. We show that Nkx2.5 recruits HDAC5 to the Ncx1 promoter, where HDAC5 complexes with HDAC1. Nkx2.5 also interacts with transcriptional activator p300, which is recruited to the Ncx1 promoter. We demonstrate that when Nkx2.5 is acetylated, it is found associated with HDAC5, whereas deacetylated Nkx2.5 is in complex with p300. Notably, TSA treatment prevents p300 from being recruited to the endogenous Ncx1 promoter, resulting in the repression of Ncx1 expression. We propose a novel model for Ncx1 regulation in which deacetylation of Nkx2.5 is required for the recruitment of p300 and results in up-regulation of exchanger expression.
Assuntos
Histona Desacetilases/fisiologia , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Gatos , Proteína p300 Associada a E1A/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilases/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Ácidos Hidroxâmicos/farmacologia , Masculino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Ratos , Trocador de Sódio e Cálcio/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Glycosphingolipids (GSLs) hexosylceramides and lactosylceramides are elevated in lupus mice and human patients with nephritis. Whereas other renal diseases characterized by increased GSL levels are thought to be a result of upregulated GSL synthesis, our results suggest elevated hexosylceramides and lactosylceramides in lupus nephritis is a result of increased catabolism of ganglioside GM3 due to significantly increased neuraminidase (NEU) activity. Thus, we hypothesized GM3 would be decreased in lupus nephritis kidneys and blocking NEU activity would reduce GSLs and improve disease in lupus mice. Female MRL/lpr lupus mice were treated with water or the NEU inhibitor oseltamivir phosphate at the onset of proteinuria to block GSL catabolism. Age-matched (non-nephritic) female MRL/MpJ lupus mice served as controls. Renal GM3 levels were significantly higher in the nephritic MRL/lpr water-treated mice compared to non-nephritic MRL/MpJ mice, despite significantly increased renal NEU activity. Blocking GSL catabolism increased, rather than decreased, renal and urine GSL levels and disease was not significantly impacted. A pilot study treating MRL/lpr females with GlcCer synthase inhibitor Genz-667161 to block GSL synthesis resulted in a strong significant negative correlation between Genz-667161 dose and renal GSL hexosylceramide and GM3 levels. Splenomegaly was negatively correlated and serum IgG levels were marginally correlated with increasing Genz-667161 dose. These results suggest accumulation of renal GM3 may be due to dysregulation of one or more of the GSL ganglioside pathways and inhibiting GSL synthesis, but not catabolism, may be a therapeutic approach for treating lupus nephritis.
Assuntos
Glicoesfingolipídeos/metabolismo , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/metabolismo , Animais , Ceramidas/metabolismo , Feminino , Gangliosídeo G(M3)/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Lactosilceramidas/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Neuraminidase/metabolismo , Oseltamivir/análogos & derivados , Oseltamivir/uso terapêutico , Ácidos Fosforosos/uso terapêutico , Projetos Piloto , Proteinúria/tratamento farmacológico , Proteinúria/metabolismoRESUMO
We report herein the design, synthesis, and pharmacological characterization of a library of novel aryl pyrazol-1-yl-propanamides as selective androgen receptor degraders (SARDs) and pan-antagonists that exert broad-scope AR antagonism. Pharmacological evaluation demonstrated that introducing a pyrazole moiety as the B-ring structural element in the common A-ring-linkage-B-ring nonsteroidal antiandrogens' general pharmacophore allowed the development of a new scaffold of small molecules with unique SARD and pan-antagonist activities even compared to our recently published AF-1 binding SARDs such as UT-155 (9) and UT-34 (10). Novel B-ring pyrazoles exhibited potent AR antagonist activities, including promising distribution, metabolism, and pharmacokinetic properties, and broad-spectrum AR antagonist properties, including potent in vivo antitumor activity. 26a was able to induce an 80% tumor growth inhibition of xenografts derived from the enzalutamide-resistant (Enz-R) VCaP cell line. These results represent an advancement toward the development of novel AR antagonists for the treatment of Enz-R prostate cancer.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pirazóis/química , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/metabolismo , Antagonistas de Receptores de Andrógenos/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Meia-Vida , Humanos , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In our effort to find small-molecule treatments of advanced prostate cancers (PCs), a novel series of indolyl and indolinyl propanamides (series II and III) were discovered as selective androgen receptor degraders (SARDs). Initial studies of androgen receptor (AR) antagonist (1) and agonist (2) propanamides yielded a tertiary aniline (3) with novel SARD activity but poor metabolic stability. Cyclization to II and III produced submicromolar AR antagonism and protein degradation selective to AR and AR splice variant (AR SV). II and III maintained potency against enzalutamide-resistant (Enz-R) mutant ARs and PC cells and were efficacious in Enz-R xenografts, suggesting their potential to treat advanced PCs. Design, synthesis, and biological activity of novel SARDs that could potentially be used for the treatment of a wide spectrum of PCs including castration-resistant, Enz-R, and/or AR SV-dependent advanced PCs that are often untreatable with known hormone therapies are discussed.
Assuntos
Amidas/química , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores Androgênicos/metabolismo , Amidas/farmacologia , Amidas/uso terapêutico , Antagonistas de Receptores de Andrógenos/química , Androgênios/química , Animais , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Indóis/química , Masculino , Camundongos , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteólise , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand-binding domain (LBD)-binding antagonists, are inactivated by common resistance mechanisms. It is important to develop next-generation mechanistically distinct drugs to treat castration- and drug-resistant prostate cancers. EXPERIMENTAL DESIGN: Second-generation AR pan antagonist UT-34 was selected from a library of compounds and tested in competitive AR binding and transactivation assays. UT-34 was tested using biophysical methods for binding to the AR activation function-1 (AF-1) domain. Western blot, gene expression, and proliferation assays were performed in various AR-positive enzalutamide-sensitive and -resistant prostate cancer cell lines. Pharmacokinetic and xenograft studies were performed in immunocompromised rats and mice. RESULTS: UT-34 inhibits the wild-type and LBD-mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and -resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses, when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist enzalutamide and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-protein-coupled receptor kinase and nuclear receptor family members. CONCLUSIONS: Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/metabolismo , Administração Oral , Antagonistas de Receptores de Andrógenos/administração & dosagem , Antagonistas de Receptores de Andrógenos/efeitos adversos , Antagonistas de Receptores de Andrógenos/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Benzamidas , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Mutação , Nitrilas , Feniltioidantoína/administração & dosagem , Feniltioidantoína/efeitos adversos , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Ratos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tip-enhanced Raman spectroscopy (TERS) has been established as one the most efficient analytical techniques for probing vibrational states with nanoscale resolution. While TERS may be a source of unique information about chemical structure and interactions, it has a limited use for materials with rough or sticky surfaces. Development of the TERS approach utilizing a non-contact scanning probe microscopy mode can significantly extend the number of applications. Here we demonstrate a proof of the concept and feasibility of a non-contact TERS approach and test it on various materials. Our experiments show that non-contact TERS can provide 10 nm spatial resolution and a Raman signal enhancement factor of 105, making it very promising for chemical imaging of materials with high aspect ratio surface patterns and biomaterials.
RESUMO
Sustained treatment of estrogen receptor (ER)-positive breast cancer with ER-targeting drugs results in ER mutations and refractory unresponsive cancers. Androgen receptor (AR), which is expressed in 80%-95% of ER-positive breast cancers, could serve as an alternate therapeutic target. Although AR agonists were used in the past to treat breast cancer, their use is currently infrequent due to virilizing side effects. Discovery of tissue-selective AR modulators (SARMs) has renewed interest in using AR agonists to treat breast cancer. Using translational models, we show that AR agonist and SARM, but not antagonist, inhibit the proliferation and growth of ER-positive breast cancer cells, patient-derived tissues, and patient-derived xenografts (PDX). Ligand-activated AR inhibits wild-type and mutant ER activity by reprogramming the ER and FOXA1 cistrome and rendering tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer.
RESUMO
Non-alcoholic steatohepatitis (NASH) affects 8-10 million people in the US and up to 75% of obese individuals. Despite this, there are no approved oral therapeutics to treat NASH and therefore the need for novel approaches exists. The estrogen receptor ß (ER-ß)-selective agonist, ß-LGND2, inhibits body weight and white adipose tissue, and increases metabolism, resulting in higher energy expenditure and thermogenesis. Due to favorable effects of ß-LGND2 on obesity, we hypothesized that ß-LGND2 will prevent NASH directly by reducing lipid accumulation in the liver or indirectly by favorably changing body composition. Male C57BL/6 mice fed with high fat diet (HFD) for 10 weeks or methionine choline-deficient diet for four weeks and treated with vehicle exhibited altered liver weights by twofold and increased serum transaminases by 2-6-folds. These changes were not observed in ß-LGND2-treated animals. Infiltration of inflammatory cells and collagen deposits, an indication of fibrosis, were observed in the liver of mice fed with HFD for 10 weeks, which were effectively blocked by ß-LGND2. Gene expression studies in the liver indicate that pregnane X receptor target genes were significantly increased by HFD, and the increase was inhibited by ß-LGND2. On the other hand, metabolomics indicate that bile acid metabolites were significantly increased by ß-LGND2. These studies demonstrate that an ER-ß agonist might provide therapeutic benefits in NASH by directly modulating the function of xenobiotic and bile acid receptors in the liver, which have important functions in the liver, and indirectly, as demonstrated before, by inhibiting adiposity. Impact statement Over 75-90% of those classified as clinically obese suffer from co-morbidities, the most common of which is non-alcoholic steatohepatitis (NASH). While there are currently no effective treatment approaches for NASH, data presented here provide preliminary evidence that an estrogen receptor ß-selective ligand could have the potential to reduce lipid accumulation and inflammation, and protect liver from NASH.
Assuntos
Ácidos e Sais Biliares/antagonistas & inibidores , Receptor beta de Estrogênio/agonistas , Isoquinolinas/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Deficiência de Colina/complicações , Dieta/efeitos adversos , Fígado/patologia , Masculino , Metabolômica , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
Androgen receptor (AR) mediates the growth of prostate cancer throughout its course of development, including in abnormal splice variants (AR-SV)-driven advanced stage castration-resistant disease. AR stabilization by androgens makes it distinct from other steroid receptors, which are typically ubiquitinated and degraded by proteasomes after ligand binding. Thus, targeting AR in advanced prostate cancer requires the development of agents that can sustainably degrade variant isoforms for effective therapy. Here we report the discovery and characterization of potent selective AR degraders (SARD) that markedly reduce the activity of wild-type and splice variant isoforms of AR at submicromolar doses. Three SARDs (UT-69, UT-155, and (R)-UT-155) bind the amino-terminal transcriptional activation domain AF-1, which has not been targeted for degradation previously, with two of these SARD (UT-69 and UT-155) also binding the carboxy-terminal ligand binding domain. Despite different mechanisms of action, all three SARDs degraded wild-type AR and inhibited AR function, exhibiting greater inhibitory potency than the approved AR antagonists. Collectively, our results introduce a new candidate class of next-generation therapeutics to manage advanced prostate cancer. Cancer Res; 77(22); 6282-98. ©2017 AACR.