RESUMEN
Molecular classification of glioblastoma has enabled a deeper understanding of the disease. The four-subtype model (including Proneural, Classical, Mesenchymal and Neural) has been replaced by a model that discards the Neural subtype, found to be associated with samples with a high content of normal tissue. These samples can be misclassified preventing biological and clinical insights into the different tumor subtypes from coming to light. In this work, we present a model that tackles both the molecular classification of samples and discrimination of those with a high content of normal cells. We performed a transcriptomic in silico analysis on glioblastoma (GBM) samples (n = 810) and tested different criteria to optimize the number of genes needed for molecular classification. We used gene expression of normal brain samples (n = 555) to design an additional gene signature to detect samples with a high normal tissue content. Microdissection samples of different structures within GBM (n = 122) have been used to validate the final model. Finally, the model was tested in a cohort of 43 patients and confirmed by histology. Based on the expression of 20 genes, our model is able to discriminate samples with a high content of normal tissue and to classify the remaining ones. We have shown that taking into consideration normal cells can prevent errors in the classification and the subsequent misinterpretation of the results. Moreover, considering only samples with a low content of normal cells, we found an association between the complexity of the samples and survival for the three molecular subtypes.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Encefálicas , Encéfalo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Femenino , Glioblastoma/clasificación , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , MicrodisecciónRESUMEN
Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disease caused by lowered activity of the enzyme alpha-L-iduronidase (IDUA). Current therapeutic options show limited efficacy and do not treat some important aspects of the disease. Therefore, it may be advantageous to identify strategies that could improve the efficacy of existing treatments. Pharmacological chaperones are small molecules that protect proteins from degradation, and their use in combination with enzyme replacement therapy (ERT) has been proposed as an alternative therapeutic strategy. Using the SEE-Tx® proprietary computational drug discovery platform, a new allosteric ligand binding cavity in IDUA was identified distal from the active site. Virtual high-throughput screening of approximately 5 million compounds using the SEE-Tx® docking platform identified a subset of small molecules that bound to the druggable cavity and functioned as novel allosteric chaperones of IDUA. Experimental validation by differential scanning fluorimetry showed an overall hit rate of 11.4%. Biophysical studies showed that one exemplary hit molecule GT-01803 bound to (Kd = 22 µM) and stabilized recombinant human IDUA (rhIDUA) in a dose-dependent manner. Co-administration of rhIDUA and GT-01803 increased IDUA activity in patient-derived fibroblasts. Preliminary in vivo studies have shown that GT-01803 improved the pharmacokinetic (PK) profile of rhIDUA, increasing plasma levels in a dose-dependent manner. Furthermore, GT-01803 also increased IDUA enzymatic activity in bone marrow tissue, which benefits least from standard ERT. Oral bioavailability of GT-01803 was found to be good (50%). Overall, the discovery and validation of a novel allosteric chaperone for rhIDUA presents a promising strategy to enhance the efficacy of existing treatments for MPS I. The compound's ability to increase rhIDUA activity in patient-derived fibroblasts and its good oral bioavailability underscore its potential as a potent adjunct to ERT, particularly for addressing aspects of the disease less responsive to standard treatment.
Asunto(s)
Iduronidasa , Mucopolisacaridosis I , Iduronidasa/metabolismo , Iduronidasa/genética , Mucopolisacaridosis I/tratamiento farmacológico , Humanos , Regulación Alostérica/efectos de los fármacos , Animales , Ratones , Terapia de Reemplazo Enzimático/métodos , Descubrimiento de Drogas , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Estabilidad de Enzimas , Simulación del Acoplamiento MolecularRESUMEN
Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology is a disease-agnostic drug discovery tool that can be applied to any protein target of interest with a known three-dimensional structure. We used this proprietary technology to identify and characterize the therapeutic potential of structurally targeted allosteric regulators (STARs) of the lysosomal hydrolase ß-galactosidase (ß-Gal), which is deficient due to gene mutations in galactosidase beta 1 (GLB1)-related lysosomal storage disorders (LSDs). The biochemical HaloTag cleavage assay was used to monitor the delivery of wildtype (WT) ß-Gal and four disease-related ß-Gal variants (p.Ile51Thr, p.Arg59His, p.Arg201Cys and p.Trp273Leu) in the presence and absence of two identified STAR compounds. In addition, the ability of STARs to reduce toxic substrate was assessed in a canine fibroblast cell model. In contrast to the competitive pharmacological chaperone N-nonyl-deoxygalactonojirimycin (NN-DGJ), the two identified STAR compounds stabilized and substantially enhanced the lysosomal transport of wildtype enzyme and disease-causing ß-Gal variants. In addition, the two STAR compounds reduced the intracellular accumulation of exogenous GM1 ganglioside, an effect not observed with the competitive chaperone NN-DGJ. This proof-of-concept study demonstrates that the SEE-Tx® platform is a rapid and cost-effective drug discovery tool for identifying STARs for the treatment of LSDs. In addition, the HaloTag assay developed in our lab has proved valuable in investigating the effect of STARs in promoting enzyme transport and lysosomal delivery. Automatization and upscaling of this assay would be beneficial for screening STARs as part of the drug discovery process.
Asunto(s)
Gangliosidosis GM1 , Enfermedades por Almacenamiento Lisosomal , Animales , Perros , Gangliosidosis GM1/tratamiento farmacológico , Gangliosidosis GM1/genética , Gangliosidosis GM1/metabolismo , 1-Desoxinojirimicina/farmacología , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Artificial intelligence (AI) has contributed substantially in recent years to the resolution of different biomedical problems, including cancer. However, AI tools with significant and widespread impact in oncology remain scarce. The goal of this study is to present an AI-based solution tool for cancer patients data analysis that assists clinicians in identifying the clinical factors associated with poor prognosis, relapse and survival, and to develop a prognostic model that stratifies patients by risk. MATERIALS AND METHODS: We used clinical data from 5275 patients diagnosed with non-small cell lung cancer, breast cancer, and non-Hodgkin lymphoma at Hospital Universitario Puerta de Hierro-Majadahonda. Accessible clinical parameters measured with a wearable device and quality of life questionnaires data were also collected. RESULTS: Using an AI-tool, data from 5275 cancer patients were analyzed, integrating clinical data, questionnaires data, and data collected from wearable devices. Descriptive analyses were performed in order to explore the patients' characteristics, survival probabilities were calculated, and a prognostic model identified low and high-risk profile patients. CONCLUSION: Overall, the reconstruction of the population's risk profile for the cancer-specific predictive model was achieved and proved useful in clinical practice using artificial intelligence. It has potential application in clinical settings to improve risk stratification, early detection, and surveillance management of cancer patients.
RESUMEN
Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next-generation sequencing (NGS)-based methods, three high-sensitivity PCR-based platforms, and two FDA-approved methods were compared using 72 plasma samples, from EGFR-mutant non-small-cell lung cancer (NSCLC) patients progressing on a first-line tyrosine kinase inhibitor (TKI). NGS platforms as well as high-sensitivity PCR-based methodologies showed excellent agreement for EGFR-sensitizing mutations (K = 0.80-0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86-0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false-positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs.
Asunto(s)
Análisis Mutacional de ADN/métodos , Mutación/genética , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Estudios de Cohortes , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Receptores ErbB/genética , Exones/genética , Femenino , Frecuencia de los Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Eliminación de Secuencia/genéticaRESUMEN
The condensation of malonoaldehyde derivatives with either a 3-amino-[1,2,4]-triazole or a 3,5-diamino-[1,2,4]-triazole precursor was studied. In agreement with previous reports, two different bicycles, namely, bearing the regioisomeric [1,2,4]triazolo[1,5-a]pyrimidine (1) or [1,2,4]triazolo[4,3-a]pyrimidine (2) structural surrogates, could be obtained. We found that, depending on the triazole precursor, only one regioisomer resulted, either of the 1 or 2 series. We also observed that these two structural surrogates could be unambiguously differentiated by indirectly measuring their (15)N chemical shifts by (1)H-(15)N HMBC experiments. The occasional conversion of [1,2,4]triazolo[4,3-a]pyrimidines to the [1,2,4]triazolo[1,5-a]pyrimidine counterparts could be unequivocally determined by (15)N NMR data.
Asunto(s)
Pirimidinas/química , Triazoles/química , Espectroscopía de Resonancia Magnética/normas , Estructura Molecular , Isótopos de Nitrógeno , Protones , Estándares de Referencia , EstereoisomerismoRESUMEN
Endogenous thyroid receptor hormones 3,5,3',5'-tetraiodo-l-thyronine (T(4), 1) and 3,5,3'-triiodo-l-thyronine (T(3), 2) exert a significant effects on growth, development, and homeostasis in mammals. They regulate important genes in intestinal, skeletal, and cardiac muscles, the liver, and the central nervous system, influence overall metabolic rate, cholesterol and triglyceride levels, and heart rate, and affect mood and overall sense of well being. The literature suggests many or most effects of thyroid hormones on the heart, in particular on the heart rate and rhythm, are mediated through the TRalpha(1) isoform, while most actions of the hormones on the liver and other tissues are mediated more through the TRbeta(1) isoform of the receptor. Some effects of thyroid hormones may be therapeutically useful in nonthyroid disorders if adverse effects can be minimized or eliminated. These potentially useful features include weight reduction for the treatment of obesity, cholesterol lowering for treating hyperlipidemia, amelioration of depression, and stimulation of bone formation in osteoporosis. Prior attempts to utilize thyroid hormones pharmacologically to treat these disorders have been limited by manifestations of hyperthyroidism and, in particular, cardiovascular toxicity. Consequently, development of thyroid hormone receptor agonists that are selective for the beta-isoform could lead to safe therapies for these common disorders while avoiding cardiotoxicity. We describe here the synthesis and evaluation of a series of novel TR ligands, which are selective for TRbeta(1) over TRalpha(1). These ligands could potentially be useful for treatment of various disorders as outlined above. From a series of homologous R(1)-substituted carboxylic acid derivatives, increasing chain length was found to have a profound effect on affinity and selectivity in a radioreceptor binding assay for the human thyroid hormone receptors alpha(1) and beta(1) (TRalpha(1) and TRbeta(2)) as well as a reporter cell assay employing CHOK1-cells (Chinese hamster ovary cells) stably transfected with hTRalpha(1) or hTRbeta(1) and an alkaline phosphatase reporter-gene downstream thyroid response element (TRAFalpha(1) and TRAFbeta(1)). Affinity increases in the order formic, acetic, and propionic acid, while beta-selectivity is highest when the R(1) position is substituted with acetic acid. Within this series 3,5-dibromo-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (11a) and 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (15) were found to reveal the most promising in vitro data based on isoform selectivity and were selected for further in vivo studies. The effect of 2, 11a, and 15 in a cholesterol-fed rat model was monitored including potencies for heart rate (ED(15)), cholesterol (ED(50)), and TSH (ED(50)). Potency for tachycardia was significantly reduced for the TRbeta selective compounds 11a and 15 compared with 2, while both 11a and 15 retained the cholesterol-lowering potency of 2. This left an approximately 10-fold therapeutic window between heart rate and cholesterol, which is consistent with the action of ligands that are approximately 10-fold more selective for TRbeta(1). We also report the X-ray crystallographic structures of the ligand binding domains of TRalpha and TRbeta in complex with 15. These structures reveal that the single amino acid difference in the ligand binding pocket (Ser277 in TRalpha or Asn331 in TRbeta) results in a slightly different hydrogen bonding pattern that may explain the increased beta-selectivity of 15.
Asunto(s)
Fenilacetatos/síntesis química , Receptores beta de Hormona Tiroidea/agonistas , Fosfatasa Alcalina/genética , Animales , Sitios de Unión , Células CHO , Colesterol/administración & dosificación , Colesterol/sangre , Cricetinae , Cristalografía por Rayos X , Genes Reporteros , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Ligandos , Masculino , Fenilacetatos/química , Fenilacetatos/farmacología , Isoformas de Proteínas , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Receptores alfa de Hormona Tiroidea/agonistas , Receptores alfa de Hormona Tiroidea/genética , Receptores alfa de Hormona Tiroidea/metabolismo , Receptores beta de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea/metabolismo , Tirotropina/sangre , Triyodotironina/farmacologíaRESUMEN
Currently, there is no efficient therapy for patients with peripheral T cell lymphoma (PTCL). The Proviral Integration site of Moloney murine leukemia virus (PIM) kinases are important mediators of cell survival. We aimed to determine the therapeutic value of PIM kinases because they are overexpressed in PTCL patients, T cell lines and primary tumoral T cells. PIM kinases were inhibited genetically (using small interfering and short hairpin RNAs) and pharmacologically (mainly with the pan-PIM inhibitor (PIMi) ETP-39010) in a panel of 8 PTCL cell lines. Effects on cell viability, apoptosis, cell cycle, key proteins and gene expression were evaluated. Individual inhibition of each of the PIM genes did not affect PTCL cell survival, partially because of a compensatory mechanism among the three PIM genes. In contrast, pharmacological inhibition of all PIM kinases strongly induced apoptosis in all PTCL cell lines, without cell cycle arrest, in part through the induction of DNA damage. Therefore, pan-PIMi synergized with Cisplatin. Importantly, pharmacological inhibition of PIM reduced primary tumoral T cell viability without affecting normal T cells ex vivo. Since anaplastic large cell lymphoma (ALK+ ALCL) cell lines were the most sensitive to the pan-PIMi, we tested the simultaneous inhibition of ALK and PIM kinases and found a strong synergistic effect in ALK+ ALCL cell lines. Our findings suggest that PIM kinase inhibition could be of therapeutic value in a subset of PTCL, especially when combined with ALK inhibitors, and might be clinically beneficial in ALK+ ALCL.
Asunto(s)
Antineoplásicos/farmacología , Linfoma de Células T Periférico/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Sinergismo Farmacológico , Humanos , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patología , Fosforilación , Proteínas Proto-Oncogénicas c-pim-1/genética , ARN Interferente PequeñoRESUMEN
The serine/threonine Pim 1 kinase is an oncogene whose expression is deregulated in several human cancers. Overexpression of Pim 1 facilitates cell cycle progression and suppresses apoptosis. Hence pharmacologic inhibitors of Pim 1 are of therapeutic interest for cancer. ETP-45299 is a potent and selective inhibitor of Pim 1 that inhibits the phosphorylation of Bad and 4EBP1 in cells and suppresses the proliferation of several non-solid and solid human tumor cell lines. The combination of the PI3K inhibitor GDC-0941 with ETP-45299 was strongly synergistic in MV-4-11 AML cells, indicating that the combination of selective Pim kinase inhibitors and PI3K inhibitor could have clinical benefit.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Leucemia Mieloide Aguda , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Piridazinas/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Indazoles/farmacología , Leucemia Mieloide Aguda/metabolismo , Piridazinas/química , Sulfonamidas/farmacologíaRESUMEN
Based on the scaffold of the pharmacologically selective thyromimetic 2b, structurally a close analog to KB-141 (2a), a number of novel N-acylated-alpha-amino acid derivatives were synthesized and tested in a TR radioligand binding assay as well as in a reporter cell assay. On the basis of TRbeta(1)-isoform selectivity and affinity, as well as affinity to the reporter cell assay, 3d was selected for further studies in the cholesterol-fed rat model. In this model 3d revealed an improved therapeutic window between cholesterol and TSH lowering but decreased margins versus tachycardia compared with 2a.
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Aminoácidos/farmacología , Imitación Molecular , Éteres Fenílicos/farmacología , Fenilacetatos/farmacología , Receptores de Hormona Tiroidea/efectos de los fármacos , Aminoácidos/química , Animales , Células CHO , Colesterol en la Dieta/administración & dosificación , Cricetinae , Cricetulus , Ligandos , Ensayo de Unión Radioligante , Ratas , Receptores de Hormona Tiroidea/metabolismoRESUMEN
Based on the examination of the X-ray crystallographic structures of the LBD of TRalpha and TRbeta in complex with KB-141 (2), a number of novel 4'-hydroxy bioisosteric thyromimetics were prepared. Optimal affinity and beta-selectivity (33 times), was found with a medium-sized alkyl-substituted amido group; iso-butyl (12c). It can be concluded that bioisosteric replacements of the 4'-hydroxy position represent a new promising class of TRbeta-selective synthetic thyromimetics.
Asunto(s)
Amidas/farmacología , Receptores beta de Hormona Tiroidea/efectos de los fármacos , Amidas/síntesis química , Amidas/química , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Éteres Fenílicos/química , Fenilacetatos/química , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Receptores alfa de Hormona Tiroidea/efectos de los fármacos , Hormonas Tiroideas/química , Triyodotironina/químicaRESUMEN
Based on the examination of the crystal structure of rat TRbeta complexed with 3,5,3'-triiodo-l-thyronine (2) a novel TRbeta-selective indole derivative 6b was prepared and tested in vitro. This compound was found to be 14 times selective for TRbeta over TRalpha in binding and its beta-selectivity could be rationalized through the comparison of the X-ray crystallographic structures of 6b complexed with TRalpha and TRbeta.