RESUMEN
Cancer stem cells (CSCs) are a niche of highly tumorigenic cells featuring self-renewal, activation of pluripotency genes, multidrug resistance, and ability to cause cancer relapse. Seven HDACi (1-7), showing either hydroxamate or 2'-aminoanilide function, were tested in colorectal cancer (CRC) and glioblastoma multiforme (GBM) CSCs to determine their effects on cell proliferation, H3 acetylation levels and in-cell HDAC activity. Two uracil-based hydroxamates, 5 and 6, which differ in substitution at C5 and C6 positions of the pyrimidine ring, exhibited the greatest cytotoxicity in GBM (5) and CRC (6) CSCs, followed by the pyridine-hydroxamate 2, with 2- to 6-fold higher potency than the positive control SAHA. Finally, increased H3 acetylation as well as HDAC inhibition directly in cells by selected 2'-aminoanilide 4 and hydroxamate 5 confirmed target engagement. Further investigation will be conducted into the broad-spectrum anticancer properties of the most potent derivatives and their effects in combination with approved, conventional anticancer drugs.
Asunto(s)
Antineoplásicos , Proliferación Celular , Neoplasias Colorrectales , Glioblastoma , Inhibidores de Histona Desacetilasas , Células Madre Neoplásicas , Piridinas , Uracilo , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/síntesis química , Piridinas/química , Piridinas/farmacología , Piridinas/síntesis química , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Uracilo/farmacología , Uracilo/química , Uracilo/análogos & derivados , Uracilo/síntesis química , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos Antitumorales , Línea Celular Tumoral , Estructura Molecular , Relación Dosis-Respuesta a DrogaRESUMEN
In the development of targeted oncology drugs, it is important to assess drug effectiveness in individual patients. We evaluated the possibility of reproducing in an ex-vivo system the biological effects observed in vitro and in vivo by the combined administration of two chemotherapeutic drugs, gemcitabine and a small inhibitor of Wee1. We found that modulation of both CDC2 phosphorylation and of a previously-identified gene signature was detectable in human skin equivalents obtained with primary keratinocytes from three individuals. Therefore, we suggest that human skin equivalents could represent a promising tool for the identification and validation of novel pharmacodynamic biomarkers.
Asunto(s)
Biomarcadores/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Piel/efectos de los fármacos , Piel/metabolismo , Células 3T3 , Animales , Proteína Quinasa CDC2 , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , GemcitabinaRESUMEN
A strategy to obtain a fully orthogonal estrogen-receptor-based gene switch responsive to molecules with acceptable pharmacological properties is presented. From a series of tetrahydrofluorenones active on the wild-type estrogen receptor (ER) an inactive analogue is chosen as a new lead compound. Coevolution of receptor mutants and ligands leads to an ER-based gene switch suitable for studies in animal models.
Asunto(s)
Fluorenos/síntesis química , Receptores de Estrógenos/efectos de los fármacos , Sitios de Unión , Estradiol/química , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/efectos de los fármacos , Receptor beta de Estrógeno/genética , Fluorenos/química , Fluorenos/farmacología , Células HeLa , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Mutación , Receptores de Estrógenos/genética , Relación Estructura-ActividadRESUMEN
It is highly desirable to design ligand-dependent transcription regulation systems based on transactivators unresponsive to endogenous ligands but induced by synthetic small molecules unable to activate endogenous receptors. Using molecular modeling and yeast selection, we identified an estrogen receptor ligand binding domain double mutant (L384M, M421G) with decreased affinity to estradiol and enhanced binding to compounds inactive on estrogen receptors. Nonresponsiveness to estrogen was achieved by additionally adding the G521R substitution while introducing an "antagonistic-type" side chain in the compound, as in 4-hydroxytamoxifen. The triple-substituted ligand binding domain is insensitive to physiological concentrations of estradiol and has nanomolar affinity for the ligand. In this binary system, both receptor and ligand are, therefore, reciprocally specific. The mutated variant in the context of a chimeric transcription factor provides tight, ligand-dependent regulation of reporter gene expression.
Asunto(s)
Estradiol/análogos & derivados , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Transcripción Genética , Sustitución de Aminoácidos , Sitios de Unión/genética , Diseño de Fármacos , Estradiol/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Genes Reporteros , Humanos , Cinética , Ligandos , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/genética , Levaduras/genéticaRESUMEN
Chemical changes performed on 1a (sirtinol) led to a series of SIRT1/2 inhibitors, in some cases more potent than 1a mainly against SIRT1. Tested in human leukemia U937 cells, the benzamide and anilide derivatives 1b, 1c, 2b, and 2c as well as the 4-(2-phenylpropyl)thioanalogue 4c showed huge apoptosis induction, while some sulfinyl and sulfonyl derivatives (5b, 5c, and 6a-c) were highly efficient in granulocytic differentiation. When assayed in human leukemia MOLT4 as well as in human breast MDA-MB-231 and colon RKO cancer cell lines, the anilide 2b (salermide) and the phenylpropylthio analogue 4b emerged as the most potent antiproliferative agents. Tested on colorectal carcinoma and glioblastoma multiforme cancer stem cells (CSCs) from patients, 2b was particularly potent against colorectal carcinoma CSCs, while 4b, 6a, and the SIRT2-selective inhibitor AGK-2 showed the highest effect against glioblastoma multiforme CSCs. Such compounds will be further explored for their broad-spectrum anticancer properties.
Asunto(s)
Antineoplásicos/síntesis química , Naftoles/síntesis química , Células Madre Neoplásicas/efectos de los fármacos , Fenilpropionatos/síntesis química , Sirtuina 1/antagonistas & inhibidores , Sirtuina 2/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Granulocitos/citología , Granulocitos/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Naftoles/química , Naftoles/farmacología , Células Madre Neoplásicas/citología , Fenilpropionatos/química , Fenilpropionatos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Inhibition of sirtuins has recently been proposed as a promising anticancer strategy. Some of the new benzodeazaoxaflavins (2a, 2b, and 2d) here reported as SIRT1/2 inhibitors were endowed with pro-apoptotic properties in human U937 leukemia cells and, most importantly, together with the prototype MC2141 (1) displayed antiproliferative effects in cancer stem cells from patients with colorectal carcinoma and glioblastoma multiforme, known to be highly tumorigenic, resistant to conventional cancer chemotherapy, and responsible, at least in part, for cancer relapse or recurrence.
Asunto(s)
Flavinas/química , Flavinas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Sirtuinas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Flavinas/síntesis química , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , HumanosRESUMEN
Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is the defective protein of the ichthyosiform condition Netherton syndrome (NS). Strongly expressed in the most differentiated epidermal layers, LEKTI is a serine protease inhibitor synthesized as three different high-molecular-weight precursors, which are rapidly processed into shorter fragments and secreted extracellularly. LEKTI polypeptides interact with several proteases to regulate skin barrier homeostasis as well as inflammatory and/or immunoallergic responses. Here, by combining antibody mapping, N-terminal sequencing, and site-specific mutagenesis, we defined the amino-acid sequence of most of the LEKTI polypeptides physiologically generated in human epidermis. We also identified three processing intermediates not described so far. Hence, a proteolytic cascade model for LEKTI activation is proposed. We then pinpointed the most effective fragments against the desquamation-related kallikreins (KLKs) and we proved that LEKTI is involved in stratum corneum shedding as some of its polypeptides inhibit the KLK-mediated proteolysis of desmoglein-1. Finally, we quantified the individual LEKTI fragments in the uppermost epidermis, showing that the ratios between LEKTI polypeptides and active KLK5 are compatible with a fine-tuned inhibition. These findings are relevant both to the understanding of skin homeostasis regulation and to the design of novel therapeutic strategies for NS.
Asunto(s)
Epidermis/metabolismo , Homeostasis/fisiología , Síndrome de Netherton/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteolisis , Piel/metabolismo , Secuencia de Aminoácidos , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Desmogleína 1/metabolismo , Epidermis/patología , Células HEK293 , Humanos , Calicreínas/metabolismo , Inhibidor de Serinpeptidasas Tipo Kazal-5 , Piel/patología , TransfecciónRESUMEN
Azumamide E, a cyclotetrapeptide isolated from the sponge Mycale izuensis, is the most powerful carboxylic acid containing natural histone deacetylase (HDAC) inhibitor known to date. In this paper, we describe design and synthesis of two stereochemical variants of the natural product. These compounds have allowed us to clarify the influence of side chain topology on the HDAC-inhibitory activity. The present contribution also reveals the identity of the recognition pattern between azumamides and the histone deacetylase-like protein (HDLP) model receptor and reports the azumamide E unprecedented isoform selectivity on histone deacetylases class subtypes. From the present studies, a plausible model for the interaction of azumamides with the receptor binding pocket is derived, providing a framework for the rational design of new cyclotetrapeptide-based HDAC inhibitors as antitumor agents.
Asunto(s)
Inhibidores de Histona Desacetilasas , Péptidos Cíclicos/farmacología , Animales , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos , Modelos Moleculares , Poríferos , Unión Proteica , Isoformas de ProteínasRESUMEN
Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.