RESUMEN
Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.
Asunto(s)
Autofagia , Células Eritroides/patología , Neuroacantocitosis/patología , Adulto , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Autofagia/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Benzoquinonas/farmacología , Bortezomib/farmacología , Diferenciación Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Demografía , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/patología , Eritrocitos/ultraestructura , Células Eritroides/efectos de los fármacos , Células Eritroides/ultraestructura , Eritropoyesis/efectos de los fármacos , Femenino , Proteínas de Choque Térmico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lactamas Macrocíclicas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Peso Molecular , Cuerpos Multivesiculares/efectos de los fármacos , Cuerpos Multivesiculares/metabolismo , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, is in an inhibited form because of the synergistic contribution of 2 events, the interaction with its physiologic inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized, and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analog (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients.
Asunto(s)
Chaperonas de Histonas/metabolismo , Inmunosupresores/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Glicoles de Propileno/farmacología , Proteína Fosfatasa 2/metabolismo , Esfingosina/análogos & derivados , Factores de Transcripción/metabolismo , Familia-src Quinasas/metabolismo , Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN , Clorhidrato de Fingolimod , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Inmunosupresores/química , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Glicoles de Propileno/química , Mapas de Interacción de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Esfingosina/química , Esfingosina/farmacología , Células Tumorales CultivadasRESUMEN
Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn's action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/patología , Proteína Fosfatasa 2/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Transducción de Señal/efectos de los fármacos , Retroalimentación Fisiológica , Humanos , Indoles/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Células Tumorales CultivadasRESUMEN
Lyn, a member of the group of tyrosine kinases named the Src family kinases (SFKs), is overexpressed, associated with an aberrant multiprotein complex and constitutively active in B-cell chronic lymphocytic leukemia (B-CLL) cells, resulting in a high level of tyrosine phosphorylation and contributing to their resistance to apoptosis. By using biochemical and bioinformatics tools, we identified procaspase-8 (procasp8), the caspase-8 zymogen, as a cytosolic target for Lyn in B-CLL cells, the phosphorylation of which at Tyr380 promotes the formation of an inactive procasp8 homodimer. This complex remains segregated in the cytosol and appears to be crucial in mediating the antiapoptotic function of Lyn in this disease. The significance of the Lyn-procasp8 axis in impairing apoptosis in B-CLL cells was further confirmed by pharmacological and genetic inhibition of procasp8, which drastically reduced the apoptosis induced by the SFK inhibitors PP2 and dasatinib. Our data highlight that Lyn's dysregulated expression, activity, and localization in B-CLLs support resistance to cell demise by inhibiting an early player of apoptotic signaling, and potentially broaden the perspectives of developing new strategies for the treatment of this disease.
Asunto(s)
Apoptosis , Caspasa 8/química , Leucemia Linfocítica Crónica de Células B/patología , Familia-src Quinasas/metabolismo , Western Blotting , Caspasa 8/metabolismo , Proliferación Celular , Biología Computacional , Citosol/metabolismo , Electroforesis en Gel Bidimensional , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Fosforilación , Multimerización de Proteína , Proteoma/análisis , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-ß and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-ß activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.
Asunto(s)
Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Animales , Becaplermina , Benzamidas/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Mesilato de Imatinib , Masculino , Piperazinas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-sis/farmacología , Pirimidinas/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Triterpenos/farmacologíaRESUMEN
Mitochondria, once merely considered as the "powerhouse" of cells, as they generate more than 90 % of cellular ATP, are now known to play a central role in many metabolic processes, including oxidative stress and apoptosis. More than 40 known human diseases are the result of excessive production of reactive oxygen species (ROS), bioenergetic collapse and dysregulated apoptosis. Mitochondria are the main source of ROS in cells, due to the activity of the respiratory chain. In normal physiological conditions, ROS generation is limited by the anti-oxidant enzymatic systems in mitochondria. However, disregulation of the activity of these enzymes or interaction of respiratory complexes with mitochondriotropic agents may lead to a rise in ROS concentrations, resulting in oxidative stress, mitochondrial permeability transition (MPT) induction and triggering of the apoptotic pathway. ROS concentration is also increased by the activity of amine oxidases located inside and outside mitochondria, with oxidation of biogenic amines and polyamines. However, it should also be recalled that, depending on its concentration, the polyamine spermine can also protect against stress caused by ROS scavenging. In higher organisms, cell signaling pathways are the main regulators in energy production, since they act at the level of mitochondrial oxidative phosphorylation and participate in the induction of the MPT. Thus, respiratory complexes, ATP synthase and transition pore components are the targets of tyrosine kinases and phosphatases. Increased ROS may also regulate the tyrosine phosphorylation of target proteins by activating Src kinases or phosphatases, preventing or inducing a number of pathological states.
Asunto(s)
Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Poliaminas/metabolismo , Transducción de Señal , Tirosina/metabolismo , Apoptosis , Transporte de Electrón/genética , Regulación de la Expresión Génica , Humanos , Mitocondrias/patología , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/patología , Poro de Transición de la Permeabilidad Mitocondrial , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Moduladores de Tubulina/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Comunicación Celular/fisiología , Técnicas de Cocultivo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Microscopía Confocal , Persona de Mediana Edad , Nocodazol/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Moduladores de Tubulina/metabolismo , Células Tumorales CultivadasRESUMEN
Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.
Asunto(s)
Empalme Alternativo , Cortactina/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfocitos B/metabolismo , Linfocitos B/patología , Estudios de Casos y Controles , Cortactina/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Isoformas de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs.
Asunto(s)
Encéfalo/metabolismo , Hígado/metabolismo , Mitocondrias/metabolismo , Peróxidos/farmacología , Espermina/farmacología , Animales , Transporte Biológico , Fosforilación , Ratas , Ratas Wistar , Tirosina/metabolismoRESUMEN
The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type 1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano/química , Proteínas Mitocondriales/química , Dominios Proteicos Ricos en Prolina , Proteínas de los Retroviridae/química , Dominios Homologos src , Familia-src Quinasas/química , Secuencias de Aminoácidos , Animales , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Proteínas Mitocondriales/genética , Unión Proteica , Multimerización de Proteína/genética , Transporte de Proteínas/genética , Conejos , Ratas , Proteínas de los Retroviridae/genética , Familia-src Quinasas/genéticaRESUMEN
CK2 is a Ser/Thr protein kinase overexpressed in many cancers. It is usually present in cells as a tetrameric enzyme, composed of two catalytic (α or α') and two regulatory (ß) subunits, but it is active also in its monomeric form, and the specific role of the different isoforms is largely unknown. CK2 phosphorylates several substrates related to the uncontrolled proliferation, motility, and survival of cancer cells. As a consequence, tumor cells are addicted to CK2, relying on its activity more than healthy cells for their life, and exploiting it for developing multiple oncological hallmarks. However, little is known about CK2 contribution to the metabolic rewiring of cancer cells. With this study we aimed at shedding some light on it, especially focusing on the CK2 role in the glycolytic onco-phenotype. By analyzing neuroblastoma and osteosarcoma cell lines depleted of either one (α) or the other (α') CK2 catalytic subunit, we also aimed at disclosing possible pro-tumor functions which are specific of a CK2 isoform. Our results suggest that both CK2 α and α' contribute to cell proliferation, survival and tumorigenicity. The analyzed metabolic features disclosed a role of CK2 in tumor metabolism, and suggest prominent functions for CK2 α isoform. Results were also confirmed by CK2 pharmacological inhibition. Overall, our study provides new information on the mechanism of cancer cells addiction to CK2 and on its isoform-specific functions, with fundamental implications for improving future therapeutic strategies based on CK2 targeting.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Glucólisis , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Quinasa de la Caseína II/genética , Línea Celular Tumoral , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Protein kinase CK2 is an antiapoptotic cancer-sustaining protein. Curcumin, reported previously as a CK2 inhibitor, is too bulky to be accommodated in the CK2 active site and rapidly degrades in solution generating various ATP-mimetic inhibitors; with a detailed comparative analysis, by means of both protein crystallography and enzymatic inhibition, ferulic acid was identified as the principal curcumin degradation product responsible for CK2 inhibition. The other curcumin derivatives vanillin, feruloylmethane and coniferyl aldehyde are weaker CK2 inhibitors. The high instability of curcumin in standard buffered solutions flags this compound, which is included in many commercial libraries, as a possible source of misleading interpretations, as was the case for CK2. Ferulic acid does not show any cytotoxicity and any inhibition of cellular CK2, due to its poor cellular permeability. However, curcumin acts as a prodrug in the cellular context, by generating its degradation products inside the treated cells, thus rescuing CK2 inhibition and consequently inducing cell death. Through the intracellular release of its degradation products, curcumin is expected to affect various target families; here, we identify the first bromodomain of BRD4 as a new target for those compounds. DATABASE: Structural data are available in the PDB database under the accession numbers 6HOP (CK2α/curcumin), 6HOQ (CK2α/ferulic acid), 6HOR (CK2α/feruloylmethane), 6HOT (CK2α/ferulic aldehyde), 6HOU (CK2α/vanillin) and 6HOV (BRD4/ferulic acid).
Asunto(s)
Antineoplásicos/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Curcumina/farmacología , Profármacos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/química , Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Curcumina/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Profármacos/química , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Protein kinase CK2 sustains cancer growth, especially in hematological malignancies. Its inhibitor SRPIN803, based on a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold, showed notable specificity. Our synthesis of the initially proposed SRPIN803 resulted in its constitutional isomer SRPIN803-revised, where the 2-cyano-2-propenamide group does not cyclise and fuse to the thiadiazole ring. Its crystallographic structure in complex with CK2α identifies the structural determinants of the reported specificity. SRPIN803-revised explores the CK2 open hinge conformation, extremely rare among kinases, also interacting with side chains from this region. Its optimization lead to the more potent compound 4, which inhibits endocellular CK2, significantly affects viability of tumour cells and shows remarkable selectivity on a panel of 320 kinases.
Asunto(s)
Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/química , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinasa de la Caseína II/metabolismo , Humanos , Células Jurkat , Simulación del Acoplamiento Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/metabolismo , Tiadiazoles/farmacologíaRESUMEN
The two human monoamine oxidase isoforms (namely MAO A and MAO B) are enzymes involved in the catabolism of monoamines, including neurotransmitters, and for this reason are well-known and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases, for which novel pharmacological approaches are necessary. Benextramine is a tetraamine disulfide mainly known as irreversible α-adrenergic antagonist, but able to hit additional targets involved in neurodegeneration. As the molecular structures of monoamine oxidases contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds were found to induce irreversible inactivation of MAOs with inactivation potency depending on both the polyamine structure and the enzyme isoform. The more effective compounds generally showed preference for MAO B. Structure-activity relationships studies revealed the key role played by the disulfide core of these molecules in the inactivation mechanism. Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of this type of inhibitors thus suggesting that their covalent binding inside the MAO active site sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line. These results demonstrated for the first time that benextramine and its derivatives can inactivate human MAOs exploiting a mechanism different from that of the classical MAO inhibitors and could be a starting point for the development of pharmacological tools in neurodegenerative diseases.
Asunto(s)
Cistamina/análogos & derivados , Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Cistamina/química , Cistamina/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Estructura Molecular , Monoaminooxidasa/química , Relación Estructura-ActividadRESUMEN
The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.