RESUMO
The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.
Assuntos
Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , RNA/genética , Fatores de Transcrição/genética , Adenosina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Elementos Reguladores de Transcrição/genética , Ativação Transcricional/genéticaRESUMO
We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.
Assuntos
Fibroblastos , Miocárdio , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Transdução de Sinais , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Apoptose/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fibrose , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Ratos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , CamundongosRESUMO
Nuclear receptors (NRs) have historically been at the forefront of cancer research, where they are known to act as critical regulators of disease. They also serve as biomarkers for tumour subclassification and targets for hormone therapy. However, most tumour types express extensive repertoires of NRs, whose interactions provide multiple paths for disease progression and offer potentially untapped mechanisms for therapeutic interventions. Recently, next-generation sequencing technologies have provided genome-wide insights into the complex interplay of NR transcriptional networks and their contribution to the development and progression of cancer. These findings have altered the traditional understanding of NR activities in oncogenesis.
Assuntos
Redes Reguladoras de Genes , Proteínas de Neoplasias , Neoplasias , Receptores Citoplasmáticos e Nucleares , Transdução de Sinais/genética , Transcrição Gênica , Animais , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.2001882.].
RESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Animais , Linhagem Celular , Sistema Livre de Células , Colesterol/sangue , Escherichia coli/genética , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Espectrometria de Massas , Terapia de Alvo Molecular , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/genética , Biossíntese de Proteínas/fisiologia , Coelhos , Ratos , Ratos Sprague-Dawley , Ribossomos/metabolismo , Ribossomos/fisiologiaRESUMO
BACKGROUND: AR-V7 is an androgen receptor (AR) splice variant that lacks the ligand-binding domain and is isolated from prostate cancer cell lines. Increased expression of AR-V7 is associated with the transition from hormone-sensitive prostate cancer to more advanced castration-resistant prostate cancer (CRPC). Due to the loss of the ligand-binding domain, AR-V7 is not responsive to traditional AR-targeted therapies, and the mechanisms that regulate AR-V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR-V7 expression and intracellular localization and their potential therapeutic role in CRPC. METHODS: We used AR high-content analysis (AR-HCA) to characterize the effects of a focused library of well-characterized clinical compounds on AR-V7 expression at the single-cell level in PC3 prostate cancer cells stably expressing green fluorescent protein (GFP)-AR-V7 (GFP-AR-V7:PC3). In parallel, an orthogonal AR-HCA screen of a small interfering (si)RNA library targeting 635 protein kinases was performed in GFP-AR-V7:PC3. The effect of the Src-Abl inhibitor PD 180970 was further characterized using cell-proliferation assays, quantitative PCR, and western blot analysis in multiple hormone-sensitive and CRPC cell lines. RESULTS: Compounds that tended to target Akt, Abl, and Src family kinases (SFKs) decreased overall AR-V7 expression, nuclear translocation, absolute nuclear level, and/or altered nuclear distribution. We identified 20 protein kinases that, when knocked down, either decreased nuclear GFP-AR-V7 levels or altered AR-V7 nuclear distribution, a set that included the SFKs Src and Fyn. The Src-Abl dual kinase inhibitor PD180970 decreased expression of AR-V7 by greater than 46% and decreased ligand-independent transcription of AR target genes in the 22RV1 human prostate carcinoma cell line. Further, PD180970 inhibited androgen-independent cell proliferation in endogenous-AR-V7-expressing prostate cancer cell lines and also overcame bicalutamide resistance observed in the 22RV1 cell line. CONCLUSIONS: SFKs, especially Src and Fyn, may be important upstream regulators of AR-V7 expression and represent promising targets in a subset of CRPCs expressing high levels of AR-V7. Prostate 77:82-93, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Androgênios/metabolismo , Proliferação de Células/fisiologia , Variação Genética/fisiologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/biossíntese , Quinases da Família src/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células MCF-7 , Masculino , Neoplasias da Próstata/patologia , Piridonas/farmacologia , Pirimidinas/farmacologia , Receptores Androgênicos/genética , Quinases da Família src/antagonistas & inibidoresRESUMO
The contribution of animal testing in drug development has been widely debated and challenged. An industry-wide nonclinical to clinical translational database was created to determine how safety assessments in animal models translate to First-In-Human clinical risk. The blinded database was composed of 182 molecules and contained animal toxicology data coupled with clinical observations from phase I human studies. Animal and clinical data were categorized by organ system and correlations determined. The 2×2 contingency table (true positive, false positive, true negative, false negative) was used for statistical analysis. Sensitivity was 48% with a 43% positive predictive value (PPV). The nonhuman primate had the strongest performance in predicting adverse effects, especially for gastrointestinal and nervous system categories. When the same target organ was identified in both the rodent and nonrodent, the PPV increased. Specificity was 84% with an 86% negative predictive value (NPV). The beagle dog had the strongest performance in predicting an absence of clinical adverse effects. If no target organ toxicity was observed in either test species, the NPV increased. While nonclinical studies can demonstrate great value in the PPV for certain species and organ categories, the NPV was the stronger predictive performance measure across test species and target organs indicating that an absence of toxicity in animal studies strongly predicts a similar outcome in the clinic. These results support the current regulatory paradigm of animal testing in supporting safe entry to clinical trials and provide context for emerging alternate models.
Assuntos
Bases de Dados Factuais , Pesquisa Translacional Biomédica , Animais , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Modelos Animais , Medição de RiscoRESUMO
Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesteraseâ 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free inâ vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. Inâ vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.
Assuntos
Fígado/efeitos dos fármacos , Inibidores de PCSK9 , Pró-Proteína Convertase 9/biossíntese , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Estrutura Molecular , Pró-Proteína Convertase 9/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other's activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/química , Genes Reporter , Células HeLa , Humanos , Complexo Mediador/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Prolactina/genética , Estrutura Secundária de Proteína , Receptores de Glucocorticoides/agonistasRESUMO
Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17ß-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.
Assuntos
Arginina/metabolismo , Biocatálise , Citrulina/metabolismo , Receptor alfa de Estrogênio/metabolismo , Histonas/metabolismo , Hidrolases/metabolismo , Ativação Transcricional , Animais , Biocatálise/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/metabolismo , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/genética , Humanos , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERß), GFP-ERß:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERß binding to ER-specific DNA response elements (EREs), ERß-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERß-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERß as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERß and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERß activity, validating the GFP-ERß:PRL-HeLa assay as a screening tool for potential ERß active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERß:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERß EDCs and improve upon currently available screening tools for this understudied nuclear receptor.
RESUMO
Phenotypic profiling by high throughput microscopy has become one of the leading tools for screening large sets of perturbations in cellular models. Of the numerous methods used over the years, the flexible and economical Cell Painting (CP) assay has been central in the field, allowing for large screening campaigns leading to a vast number of data-rich images. Currently, to analyze data of this scale, available open-source software ( i.e. , CellProfiler) requires computational resources that are not available to most laboratories worldwide. In addition, the image-embedded cell-to-cell variation of responses within a population, while collected and analyzed, is usually averaged and unused. Here we introduce SPACe ( S wift P henotypic A nalysis of Ce lls), an open source, Python-based platform for the analysis of single cell image-based morphological profiles produced by CP experiments. SPACe can process a typical dataset approximately ten times faster than CellProfiler on common desktop computers without loss in mechanism of action (MOA) recognition accuracy. It also computes directional distribution-based distances (Earth Mover's Distance - EMD) of morphological features for quality control and hit calling. We highlight several advantages of SPACe analysis on CP assays, including reproducibility across multiple biological replicates, easy applicability to multiple (â¼20) cell lines, sensitivity to variable cell-to-cell responses, and biological interpretability to explain image-based features. We ultimately illustrate the advantages of SPACe in a screening campaign of cell metabolism small molecule inhibitors which we performed in seven cell lines to highlight the importance of testing perturbations across models.
RESUMO
The initial step in estrogen-regulated transcription is the binding of a ligand to its cognate receptors, named estrogen receptors (ERα and ERß). Phytochemicals present in foods and environment can compete with endogenous hormones to alter physiological responses. We screened 224 flavonoids in our engineered biosensor ERα and ERß PRL-array cell lines to characterize their activity on several steps of the estrogen signaling pathway. We identified 83 and 96 flavonoids that can activate ERα or ERß, respectively. While most act on both receptors, many appear to be subtype-selective, including potent flavonoids that activate ER at sub-micromolar concentrations. We employed an orthogonal assay using a transgenic zebrafish in vivo model that validated the estrogenic potential of these compounds. To our knowledge, this is the largest study thus far on flavonoids and the ER pathway, facilitating the identification of a new set of potential endocrine disruptors acting on both ERα and ERß.
RESUMO
Introduction: Wistar Han rats are a preferred strain of rodents for general toxicology and safety pharmacology studies in drug development. In some of these studies, visual functional tests that assess for retinal toxicity are included as an additional endpoint. Although the influence of gender on human retinal function has been documented for more than 6 decades, preclinically it is still uncertain if there are differences in retinal function between naïve male and female Wistar Han rats. Methods: In this study, sex-related differences in the retinal function were quantified by analyzing electroretinography (ERG) in 7-9-week-old (n = 52 males and 51 females) and 21-23-week-old Wistar Han rats (n = 48 males and 51 females). Optokinetic tracking response, brainstem auditory evoked potential, ultrasonic vocalization and histology were tested and evaluated in a subset of animals to investigate the potential compensation mechanisms of spontaneous blindness. Results/Discussion: Absence of scotopic and photopic ERG responses was found in 13% of 7-9-week-old (7/52) and 19% of 21-23-week-old males (9/48), but none of female rats (0/51). The averaged amplitudes of rod- and cone-mediated ERG b-wave responses obtained from males were significantly smaller than the amplitudes of the same responses from age-matched females (-43% and -26%, respectively) at 7-9 weeks of age. There was no difference in the retinal and brain morphology, brainstem auditory responses, or ultrasonic vocalizations between the animals with normal and abnormal ERGs at 21-23 weeks of age. In summary, male Wistar Han rats had altered retinal responses, including a complete lack of responses to test flash stimuli (i.e., blindness), when compared with female rats at 7-9 and 21-23 weeks of age. Therefore, sex differences should be considered when using Wistar Han rats in toxicity and safety pharmacology studies with regards to data interpretation of retinal functional assessments.
RESUMO
The number of gene therapies in development continues to increase, as they represent a novel method to treat, and potentially cure, many diseases. Gene therapies can be conducted with an in vivo or ex vivo approach, to cause gene augmentation, gene suppression, or genomic editing. Adeno-associated viruses are commonly used to deliver gene therapies, but their use is associated with several manufacturing, nonclinical and clinical challenges. As these challenges emerge, regulatory agency expectations continue to evolve. Following administration of rAAV-based gene therapies, nonclinical toxicities may occur, which includes immunogenicity, hepatotoxicity, neurotoxicity, and the potential risks for insertional mutagenesis and subsequent tumorgenicity. The mechanism for these findings and translation into the clinical setting are unclear at this time but have influenced the nonclinical studies that regulatory agencies are increasingly requesting to support clinical trials and marketing authorizations. These evolving regulatory expectations and toxicities, as well as future nonclinical considerations, are discussed herein.
Assuntos
Dependovirus , Técnicas de Transferência de Genes , Terapia Genética/métodos , Terapia Genética/tendências , Vetores Genéticos , Carcinogênese , Terapia Genética/efeitos adversos , Vetores Genéticos/toxicidade , Humanos , MutagêneseRESUMO
Transcription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor.
RESUMO
A-to-I editing is the most common editing type in humans that is catalyzed by ADAR family members (ADARs), ADAR1 and ADAR2. Although millions of A-to-I editing sites have recently been discovered, the regulation mechanisms of the RNA editing process are still not clear. Herein, we developed a two-step logistic regression model to identify genes that are potentially involved in the RNA editing process in four human cancers. In the first step, we tested the association of each editing site with known enzymes. To validate the logistic regression model, we collected 10 genes with 168 editing sites from multiple published studies and obtained a nearly 100% validation rate. ADAR1 was identified as the enzyme associated with the majority of the A-to-I editing sites. Thus, ADAR1 was taken as a control gene in the second step to identify genes that have a stronger regulation effect on editing sites than ADAR1. Using our advanced method, we successfully found a set of genes that were significantly positively or negatively associated (PA or NA) with specific sets of RNA editing sites. 51 of these genes had been reported in at least one previous study. We highlighted two genes: 1), SRSF5, supported by three previous studies, and 2) MIR22HG, supported by one previous study and two of our cancer datasets. The PA and NA genes were cancer-specific but shared common pathways. Interestingly, the PA genes from kidney cancer were enriched for survival-associated genes while the NA genes were not, indicating that the PA genes may play more important roles in kidney cancer progression.
Assuntos
Adenosina Desaminase , Neoplasias , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Humanos , Neoplasias/genética , Edição de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Human health is at risk from environmental exposures to a wide range of chemical toxicants and endocrine-disrupting chemicals (EDCs). As part of understanding this risk, the U.S. Environmental Protection Agency (EPA) has been pursuing new high-throughput in vitro assays and computational models to characterize EDCs. EPA models have incorporated our high-content analysis-based green fluorescent protein estrogen receptor (GFP-ER): PRL-HeLa assay, which allows direct visualization of ER binding to DNA regulatory elements. Here, we characterize a modified functional assay based on the stable expression of a chimeric androgen receptor (ARER), wherein a region containing the native AR DNA-binding domain (DBD) was replaced with the ERα DBD (amino acids 183-254). We demonstrate that the AR agonist dihydrotestosterone induces GFP-ARER nuclear translocation, PRL promoter binding, and transcriptional activity at physiologically relevant concentrations (<1 nM). In contrast, the AR antagonist bicalutamide induces only nuclear translocation of the GFP-ARER receptor (at µM concentrations). Estradiol also fails to induce visible chromatin binding, indicating androgen specificity. In a screen of reference chemicals from the EPA and the Agency for Toxic Substances and Disease Registry, the GFP-ARER cell model identified and mechanistically grouped activity by known (anti-)androgens based on the ability to induce nuclear translocation and/or chromatin binding. Finally, the cell model was used to identify potential (anti-)androgens in environmental samples in collaboration with the Houston Ship Channel/Galveston Bay Texas A&M University EPA Superfund Research Program. Based on these data, the chromatin-binding, in vitro assay-based GFP-ARER model represents a selective tool for rapidly identifying androgenic activity associated with drugs, chemicals, and environmental samples.
Assuntos
Disruptores Endócrinos/farmacologia , Receptor alfa de Estrogênio/genética , Receptores Androgênicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas de Ligação a DNA/genética , Di-Hidrotestosterona/farmacologia , Disruptores Endócrinos/efeitos adversos , Exposição Ambiental/efeitos adversos , Proteínas de Fluorescência Verde/farmacologia , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Estados UnidosRESUMO
: A zymogen-like activated factor X variant (FXa) is being developed for treating acute bleeding conditions. Activated factor V is an essential cofactor to FXa for activating prothrombin to thrombin. Thrombi/emboli formation was observed microscopically in FXa toxicity studies in animals. The objective of this research was to evaluate candidate biomarkers for FXa-induced thrombi/emboli formation to inform safety monitoring and dose-escalation decisions in FXa clinical trials. Effects of intravenous FXa administration on platelets, fibrinogen, activated partial thromboplastin time (aPTT), prothrombin time (PT), D-dimer, tissue factor pathway inhibitor, thrombinâ:âantithrombin complex, antithrombin, and factor V, and protein C (PC) activities were evaluated in mice, rats, and monkeys. Mice had endogenous factor V activity 10× that of monkeys and were overly sensitive to FXa-induced thrombi/emboli formation. In monkeys, decreases in fibrinogen and prolongation in aPTT and PT emerged as potential biomarkers for impending FXa-induced thrombi/emboli formation, based on association of changes with microscopically observable thrombi/emboli (0-97 thrombi/emboli per monkey). PC decreases, measured by a clot-based assay, were also observed. A similar reduction in PC activity, when measured by clot-based assay, was observed in a phase 1 clinical trial. However, an in-vitro experiment with human plasma spiked with increasing concentrations of FXa indicated dose-dependent FXa-induced interference with clot-based assays and no depletion of PC or S by FXa in non-clot-based assays. Nonclinical biomarker studies identified fibrinogen, aPTT and PT as potential biomarkers for monitoring the clinical safety of FXa. Results of clot-based assays with FXa treatment should be interpreted with caution.
Assuntos
Anticoagulantes/uso terapêutico , Biomarcadores/metabolismo , Testes de Coagulação Sanguínea/métodos , Fator Xa/uso terapêutico , Trombose/tratamento farmacológico , Animais , Anticoagulantes/farmacologia , Fator Xa/farmacologia , Haplorrinos , Humanos , Camundongos , Ratos , Ratos WistarRESUMO
Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question.