RESUMEN
Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.
Asunto(s)
Proteínas Hedgehog , Simulación del Acoplamiento Molecular , Piridinas , Pirimidinas , Proteína con Dedos de Zinc GLI1 , Piridinas/farmacología , Piridinas/química , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Pirimidinas/farmacología , Pirimidinas/química , Proteínas Hedgehog/metabolismo , Humanos , Animales , Ratones , Línea Celular Tumoral , Células 3T3 NIH , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Transducción de Señal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacosRESUMEN
Immune-mediated diseases are common and life-threatening disorders in dogs. Many canine immune-mediated diseases have strong breed predispositions and are believed to be inherited. However, the genetic mutations that cause these diseases are mostly unknown. As many immune-mediated diseases in humans share polymorphisms among a common set of genes, we conducted a candidate gene study of 15 of these genes across four immune-mediated diseases (immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis (IMPA), and atopic dermatitis) in 195 affected and 206 unaffected dogs to assess whether causative or predictive polymorphisms might exist in similar genes in dogs. We demonstrate a strong association (Fisher's exact p = 0.0004 for allelic association, p = 0.0035 for genotypic association) between two polymorphic positions (10 bp apart) in exon 2 of one allele in DLA-79, DLA-79*001:02, and multiple immune-mediated diseases. The frequency of this allele was significantly higher in dogs with immune-mediated disease than in control dogs (0.21 vs. 0.12) and ranged from 0.28 in dogs with IMPA to 0.15 in dogs with atopic dermatitis. This allele has two non-synonymous substitutions (compared with the reference allele, DLA-79*001:01), resulting in F33L and N37D amino acid changes. These mutations occur in the peptide-binding pocket of the protein, and based upon our computational modeling studies, are likely to affect critical interactions with the peptide N-terminus. Further studies are warranted to confirm these findings more broadly and to determine the specific mechanism by which the identified variants alter canine immune system function.
Asunto(s)
Alelos , Enfermedades de los Perros/genética , Enfermedades de los Perros/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Enfermedades del Sistema Inmune/veterinaria , Sustitución de Aminoácidos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Sitios de Unión , Perros , Exones , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Antígenos de Histocompatibilidad Clase I/química , Metaanálisis como Asunto , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Fenotipo , Polimorfismo de Nucleótido Simple , Conformación Proteica , Reproducibilidad de los Resultados , Relación Estructura-ActividadRESUMEN
OBJECTIVE: ß-Adrenergic receptor antagonists are widely utilized for the management of cardiac diseases in dogs. We have recently identified two deletion polymorphisms in the canine adrenoreceptor 1 (ADRB1) gene.We hypothesized that canine ADRB1 deletions would alter the structure of the protein, as well as the heart rate response to the ß-adrenergic receptor antagonist, atenolol. The objectives of this study were to predict the impact of these deletions on the predicted structure of the protein and on the heart rate response to atenolol in a population of healthy adult dogs. METHODS: Eighteen apparently healthy, mature dogs with (11) and without (seven) ADRB1 deletions were evaluated. The heart rate of the dogs was evaluated with a baseline ambulatory ECG before and 14-21 days after atenolol therapy (1 mg/kg orally q12 h). Minimum, average, and maximum heart rates were compared between groups of dogs (deletions, controls) using an unpaired t-test and within each group of dogs using a paired t-test. The protein structure of ADRB1 was predicted by computer modeling. RESULTS: Deletions were predicted to alter the structure of the ADRB1 protein. The heart rates of the dogs with deletions were lower than those of the control dogs (the average heart rates were significantly lower). CONCLUSION: ADRB1 deletions appear to have structural and functional consequences. Individual genome-based treatment recommendations could impact the management of dogs with heart disease.
Asunto(s)
Antagonistas de Receptores Adrenérgicos beta 1/administración & dosificación , Atenolol/administración & dosificación , Receptores Adrenérgicos beta 1/genética , Eliminación de Secuencia , Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Animales , Atenolol/farmacología , Perros , Electrocardiografía/veterinaria , Frecuencia Cardíaca/efectos de los fármacos , Modelos Moleculares , Estructura Secundaria de Proteína , Receptores Adrenérgicos beta 1/químicaRESUMEN
DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the ß-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM) cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de la Monoaminooxidasa , Neoplasias , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Carbolinas , Harmina/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Monoaminooxidasa , Inhibidores de la Monoaminooxidasa/farmacología , Quinasas DyrKRESUMEN
We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ9-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.
Asunto(s)
Cannabinoides/toxicidad , Trastornos del Espectro Alcohólico Fetal/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal/efectos de los fármacos , Teratogénesis/efectos de los fármacos , Animales , Etanol/efectos adversos , Etanol/farmacología , Femenino , Trastornos del Espectro Alcohólico Fetal/patología , Ratones , Receptor Smoothened/metabolismoRESUMEN
Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.
Asunto(s)
Compuestos Heterocíclicos con 2 Anillos/farmacología , Neoplasias Inflamatorias de la Mama/tratamiento farmacológico , Piridinas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Proteína con Dedos de Zinc GLI1/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias Inflamatorias de la Mama/metabolismo , Neoplasias Inflamatorias de la Mama/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE To identify cardiac tissue genes and gene pathways differentially expressed between dogs with and without dilated cardiomyopathy (DCM). ANIMALS 8 dogs with and 5 dogs without DCM. PROCEDURES Following euthanasia, samples of left ventricular myocardium were collected from each dog. Total RNA was extracted from tissue samples, and RNA sequencing was performed on each sample. Samples from dogs with and without DCM were grouped to identify genes that were differentially regulated between the 2 populations. Overrepresentation analysis was performed on upregulated and downregulated gene sets to identify altered molecular pathways in dogs with DCM. RESULTS Genes involved in cellular energy metabolism, especially metabolism of carbohydrates and fats, were significantly downregulated in dogs with DCM. Expression of cardiac structural proteins was also altered in affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that RNA sequencing may provide important insights into the pathogenesis of DCM in dogs and highlight pathways that should be explored to identify causative mutations and develop novel therapeutic interventions.