RESUMEN

The activation of NLRP3 results in the assembly of inflammasome that regulates caspase-1 activation and the subsequent secretion of bioactive interleukin (IL)-1ß. Excessive activation of the NLRP3 inflammasome is mechanistically linked to diverse pathophysiological conditions, including airway inflammation. Here, we discovered that Curcuma phaeocaulis can suppress caspase-1 activation and processing of pro-IL-1ß into mature cytokine in macrophages stimulated with NLRP3 inflammasome activators, such as SiO2 or TiO2 nanoparticles. Furthermore, in the bronchoalveolar lavage fluids of animals administered the nanoparticles, the in vitro effects of C. phaeocaulis translated into a decrease in IL-1ß levels and cell infiltration. Demethoxycurcumin (DMC) and curcumin were found to be responsible for the inflammasome inhibitory activity of C. phaeocaulis. Interestingly, in contrast to the previously reported higher antioxidant- and NFκB-inhibitory activities of curcumin, DMC exhibited approximately two-fold stronger potency than curcumin against nanoparticle induced activation of NLRP3 inflammasome. In the light of these results, both compounds seem to act independently of their antioxidant- and NFκB-inhibitory properties. Although how C. phaeocaulis inhibits nanoparticle-activated NLRP3 inflammasome remains to be elucidated, our results provide a basis for further research on C. phaeocaulis extract as an anti-inflammatory agent for the treatment of disorders associated with excessive activation of NLRP3 inflammasome.

Asunto(s)

Curcumina , Nanopartículas , Animales , Antioxidantes/farmacología , Caspasa 1 , Caspasas , Curcuma , Curcumina/farmacología , Inflamasomas , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Interleucina-1beta/farmacología , Macrófagos , Ratones , FN-kappa B/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR , Dióxido de Silicio/farmacología

RESUMEN

Schwann cell differentiation involves a dynamic interaction of signaling cascades. However, much remains to be elucidated regarding the function of signaling molecules that differ depending on the context in which the molecules are engaged. Here, we identified a small molecule, dabrafenib, which promotes Schwann cell differentiation in vitro and exploited this compound as a pharmacological tool to understand the molecular mechanisms regulating Schwann cell differentiation. The results indicated that dabrafenib inhibited ERK phosphorylation and enhanced ErbB2 autophosphorylation and Akt phosphorylation, and the effects of dabrafenib on ErbB2 and Akt phosphorylation were phenocopied by pharmacological inhibition of the MEK-ERK signaling pathway. However, the small molecule inhibitors of MEK and ERK had no effect on the expression of Oct6 and EGR2, which are key transcription factors that drive Schwann cell differentiation. In addition, pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K) almost completely interfered with dabrafenib-induced Schwann cell differentiation. These results suggest that the ErbB2-PI3K-Akt axis is required for the induction of Schwann cell differentiation by dabrafenib in vitro. Although additional molecules targeted by dabrafenib remain to be identified, our data provides insights into the crosstalk that exists between the MEK-ERK signaling pathway and the PI3K-Akt axis in Schwann cell differentiation.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oximas/farmacología , Células de Schwann/citología , Animales , Diferenciación Celular/genética , Imidazoles/química , Oximas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Fosforilación/efectos de los fármacos , Ratas Sprague-Dawley , Receptor ErbB-2/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo

RESUMEN

Neuropathic pain is associated with an increased sensitivity to painful stimuli or abnormal sensitivity to otherwise innocuous stimuli. However, in addition to adverse effects, currently available drugs have shown limited response in patients with neuropathic pain, which provides a rationale to explore new drug classes acting on novel targets and with better efficacy and safety profiles. Here, we found that saikosaponins potently inhibit agonist-induced activation of the transient receptor potential A1 (TRPA1) channel, which has been reported to mediate neuropathic pain by sensing a variety of chemical irritants. Molecular docking and site-directed mutagenesis analyses suggested that saikosaponins bind to the hydrophobic pocket in TRPA1 near the Asn855 residue, which, when mutated to Ser, was previously associated with enhanced pain perception in humans. In support of these findings, saikosaponin D significantly attenuated agonist-induced nociceptive responses and vincristine-induced mechanical hypersensitivity in mice. These results indicate that saikosaponins are TRPA1 antagonists and provide a basis for further elaboration of saikosaponin derivatives for the development of new therapeutics for neuropathic pain.

Asunto(s)

Ácido Oleanólico/análogos & derivados , Saponinas/farmacología , Canal Catiónico TRPA1/antagonistas & inhibidores , Animales , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Hiperalgesia/diagnóstico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Neuralgia/diagnóstico , Neuralgia/tratamiento farmacológico , Ácido Oleanólico/química , Ácido Oleanólico/aislamiento & purificación , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacología , Dimensión del Dolor , Saponinas/química , Saponinas/aislamiento & purificación , Saponinas/metabolismo , Canal Catiónico TRPA1/química , Canal Catiónico TRPA1/metabolismo

RESUMEN

Osteoporosis is a clinical condition characterized by low bone strength that leads to an increased risk of fracture. Strategies for the treatment of osteoporosis involve inhibition of bone resorption by osteoclasts and an increase of bone formation by osteoblasts. Here, we identified the extract derived from the stem part of Edgeworthia papyrifera that enhanced differentiation of MC3T3-E1 cells to osteoblast-like cells and inhibited osteoclast differentiation of RAW 264.7 cells in vitro. In support of our observation, rutin and daphnoretin, which were previously reported to inhibit osteoclast differentiation, were identified in E. papyrifera extract. In an animal model of osteoporosis, the ovariectomy-induced increases in bone resorption biomarkers such as pyridinoline and tartrate-resistant acid phosphatase were significantly reduced by E. papyrifera extract administration at 25.6 and 48.1%, respectively. Furthermore, the ovariectomy-induced bone loss in animal models of osteoporosis was significantly prevented by the administration of E. papyrifera in our study. Taking these observations into account, we suggest that E. papyrifera is an interesting candidate for further exploration as an anti-osteoporotic agent.

Asunto(s)

Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Extractos Vegetales/farmacología , Thymelaeaceae/química , Fosfatasa Alcalina/metabolismo , Aminoácidos/orina , Animales , Biomarcadores/sangre , Biomarcadores/orina , Resorción Ósea/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ratones , Ratones Endogámicos , Modelos Animales , Osteoporosis/etiología , Extractos Vegetales/análisis , Células RAW 264.7 , Ratas Sprague-Dawley

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) is an increasingly studied condition that can progress to end-stage liver disease. Although NAFLD was first described in 1980, a complete understanding of the mechanism and causes of this disease is still lacking. Six-transmembrane protein of prostate 2 (STAMP2) plays a role in integrating inflammatory and nutritional signals with metabolism. Our previous study suggested that STAMP2 may be a suitable target for treating NAFLD. In the current study, we performed a focused drug-screening and found that cilostazol could be a potential STAMP2 enhancer. Thus, we examined whether cilostazol alleviates NAFLD through STAMP2. The in vivo and in vitro pharmacological efficacies of cilostazol on STAMP2 expression and lipid accumulation were analyzed in NAFLD mice induced by high-fat diet (HFD) and in HepG2 cell lines treated by oleic acid (OA), respectively. Cilostazol increased the expression of STAMP2 through transcriptional regulation in vivo and in vitro. Cilostazol also dampened the STAMP2 downregulation caused by the HFD and by OA in vivo and in vitro, respectively. Cilostazol activated AMP-activated protein kinase (AMPK) in vivo and in vitro, and AMPK functions upstream of STAMP2, and reversed downregulation of STAMP2 expression through AMPK in the NAFLD model. Cilostazol ameliorates hepatic steatosis by enhancing hepatic STAMP2 expression through AMPK. Enhancing STAMP2 expression with cilostazol represents a potential therapeutic avenue for treatment of NAFLD.

Asunto(s)

Cilostazol/farmacología , Dieta Alta en Grasa/efectos adversos , Hígado Graso/tratamiento farmacológico , Hígado/efectos de los fármacos , Proteínas de la Membrana/genética , Regulación hacia Arriba/genética , Proteínas Quinasas Activadas por AMP/genética , Animales , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Hígado Graso/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células Hep G2 , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Autofagia/efectos de los fármacos , Glicósidos/farmacología , Melaninas/metabolismo , Melanoma/tratamiento farmacológico , Animales , Arisaema/química , Línea Celular Tumoral , Femenino , Humanos , Melanoma/metabolismo , Ratones , Persona de Mediana Edad , alfa-MSH/metabolismo

RESUMEN

Hyperuricemia is a clinical condition characterized by an elevated level of serum uric acid and is a key risk factor for the development of gout and metabolic disorders. The existing urate-lowering therapies are often impractical for certain patient populations, providing a rationale to explore new agents with improved safety and efficacy. Here, we discovered that Salvia plebeia extract inhibited the enzyme activity of xanthine oxidase, which is a key enzyme generating uric acid in the liver. In an animal model of hyperuricemia, S. plebeia extract reduced serum urate to the levels observed in control animals. The urate-lowering effect of S. plebeia extract in vivo was supported by the identification of compounds that inhibit xanthine oxidase enzyme activity in vitro. Nepetin, scutellarein, and luteolin contributed significantly to S. plebeia bioactivity in vitro. These compounds showed the highest potency against xanthine oxidase with IC50 values of 2.35, 1.74, and 1.90 µM, respectively, and were present at moderate quantities. These observations serve as a basis for further elaboration of the S. plebeia extracts for the development of new therapeutics for hyperuricemia and related diseases.

Asunto(s)

Medicamentos Herbarios Chinos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Hiperuricemia/tratamiento farmacológico , Ácido Úrico/sangre , Xantina Oxidasa/antagonistas & inhibidores , Animales , Canfanos , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos ICR , Panax notoginseng , Fitoterapia , Componentes Aéreos de las Plantas/química , Raíces de Plantas/química , Salvia miltiorrhiza

RESUMEN

Axon regeneration after injury in the central nervous system is hampered in part because if an age-dependent decline in the intrinsic axon growth potential, and one of the strategies to stimulate axon growth in injured neurons involves pharmacological manipulation of implicated signaling pathways. Here we report phenotypic cell-based screen of chemical libraries and structure-activity-guided optimization that resulted in the identification of compound 7p which promotes neurite outgrowth of cultured primary neurons derived from the hippocampus, cerebral cortex, and retina. In an animal model of optic nerve injury, compound 7p was shown to induce growth of GAP-43 positive axons, indicating that the in vitro neurite outgrowth activity of compound 7p translates into stimulation of axon regeneration in vivo. Further optimization of compound 7p and elucidation of the mechanisms by which it elicits axon regeneration in vivo will provide a rational basis for future efforts to enhance treatment strategies.

Asunto(s)

Acetamidas/farmacología , Axones/efectos de los fármacos , Descubrimiento de Drogas , Regeneración Nerviosa/efectos de los fármacos , Sulfonamidas/farmacología , Acetamidas/síntesis química , Acetamidas/química , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química

RESUMEN

Asthma is a chronic inflammatory disease of lung airways, and pharmacological inhibitors of cyclic adenosine monophosphate-specific phosphodiesterase 4 (PDE4) have been considered as therapeutics for the treatment of asthma. However, development of PDE4 inhibitors in clinical trials has been hampered because of the severe side effects of non-selective PDE4 inhibitors. Here, screening of a plant extract library in conjunction with dereplication technology led to identification of baicalin as a new type of PDE4-selective inhibitor. We demonstrated that while rolipram inhibited the enzyme activity of a range of PDE4 subtypes in in vitro enzyme assays, baicalin selectively inhibited the enzyme activity of PDE4A and 4B. In addition, baicalin suppressed lipopolysaccharide-induced TNF-α expression in macrophage where PDE4B plays a key role in lipopolysaccharide-induced signaling. Furthermore, baicalin treatment in an animal model of allergic asthma reduced inflammatory cell infiltration and TNF-α levels in bronchoalveolar lavage fluids, indicating that the antiinflammatory effects of baicalin in vivo are attributable, in part, to its ability to inhibit PDE4.

Asunto(s)

Antiinflamatorios/farmacología , Asma/tratamiento farmacológico , Flavonoides/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Extractos Vegetales/farmacología , Animales , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Células RAW 264.7 , Rolipram/farmacología , Rosaceae/química , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Stem cell-based therapy holds great potential especially for neurological disorders. However, clinical applications await further understanding of many aspects of stem cell differentiation and development of technology enabling manipulation of stem cells into desired cell types in the central nervous system. Here, we developed a new method that leads to enrichment of neuronal lineage cells in neural stem cell cultures. The protocol involves cultivation of primary cells derived from the forebrains of rat E18 embryos above a layer of nonadhesive hard agarose gel in the form of neurospheres. In contrast to the neurospheres that were cultured above an anti-adhesive hydrogel layer, the primary cells that were cultured above a layer of agarose gel preferentially differentiated into ß-III tubulin-positive neurons when allowed to undergo differentiation in vitro.In an effort to investigate the mechanism behind this observation, we found that the gene expression of a vertebrate neuronal determination gene (neurogenin1) was enhanced in the neurospheres that proliferated above a layer of agarose gel as compared with the control, and the gene expression level of neurogenin1 was quite well correlated with the rigidity of agarose gel. These results indicate that agarose gel can contribute, at least in part, to enrich neuronal progenitors and immature postmitotic neurons during neurosphere formation and may provide additional information to establish efficient protocols for the neural stem cell-based study.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Prosencéfalo/embriología , Animales , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente Indirecta , Prosencéfalo/citología , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sefarosa/química , Tubulina (Proteína)/metabolismo

RESUMEN

Chemical post-treatment of the carbon nanotube fiber (CNTF) was carried out via intramolecular cross-dehydrogenative coupling (ICDC) with FeCl3 at room temperature. The Raman intensity ratio of the G band to the D band (IG/ID ratio) of CNT fiber increased from 2.3 to 4.6 after ICDC reaction. From the XPS measurements, the AC═C/AC-C ratio of the CNT fiber increased from 3.6 to 4.8. It is of keen interest that both the electrical conductivity and tensile strength of CNT yarn improved to 3.5 × 10(3) S/cm and 420 MPa, which is 180 and 200% higher than that of neat CNT yarn.

Asunto(s)

Carbono/química , Conductividad Eléctrica , Nanotubos de Carbono/química , Fibra de Carbono , Espectrometría Raman

RESUMEN

A chemical defect healing of reduced graphene oxide (RGO) was carried out via intramolecular cross-dehydrogenative coupling (ICDC) with FeCl3 at room temperature. The Raman intensity ratio of the G-band to the D-band, the IG/ID ratio, of the RGO was increased from 0.77 to 1.64 after the ICDC reaction. From XPS measurements, the AC=C/AC-C ratio, where the peak intensities from the C=C and C-C bonds are abbreviated as AC=C and AC-C, of the RGO was increased from 2.88 to 3.79. These results demonstrate that the relative amount of sp(2)-hybridized carbon atoms is increased by the ICDC reaction. It is of great interest that after the ICDC reaction the electrical conductivity of the RGO was improved to 71 S cm(-1), which is 14 times higher than that of as-prepared RGO (5 S cm(-1)).

RESUMEN

HER2 overexpression is observed in ∼6-35% of all gastric cancers, while co-amplification of topoisomerase IIα occurs in ∼32-90% of all cancers with HER2 amplification. The present study reports that HER2 expression is down-regulated by A-62176, a fluoroquinophenoxazine derivative that we previously demonstrated to inhibit topoisomerase I and IIα. The results suggest that A-62176 inhibits the interaction between the ESX, an ets transcription factor, and its co-activator Sur2, leading to the attenuation of HER2-mediated phosphorylation of MAPK/Akt. A-62176 arrests the cell cycle in the G1 phase via the down-regulation of cyclin D1 and the up-regulation of p27(Kip1) in NCI-N87 gastric cancer cells. The combination of A-62176 with doxorubicin provides a strong synergistic activity. We propose that A-62176 is a dual inhibitor that impairs the expression of HER2 and restrains the activity of topoisomerase IIα. Our results may lead to the rational design of anticancer molecules targeting a subgroup of gastric cancer cells overexpressing both HER2 and topoisomerase IIα.

Asunto(s)

Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Oxazinas/farmacología , Quinolonas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Inhibidores de Topoisomerasa/farmacología , Antígenos de Neoplasias/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Doxorrubicina/farmacología , Fase G1/efectos de los fármacos , Fase G1/genética , Células HEK293 , Humanos , Complejo Mediador/genética , Complejo Mediador/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ets , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Stem cells are offering a considerable range of prospects to the biomedical research including novel platforms for disease models and drug discovery tools to cell transplantation and regenerative therapies. However, there are several obstacles to overcome to bring these potentials into reality. First, robust methods to maintain stem cells in the pluripotent state should be established and factors that are required to direct stem cell fate into a particular lineage should be elucidated. Second, both allogeneic rejection following transplantation and limited cell availability issues must be circumvented. These challenges are being addressed, at least in part, through the identification of a group of chemicals (small molecules) that possess novel activities on stem cell biology. For example, small molecules can be used both in vitro and/or in vivo as tools to promote proliferation of stem cells (self-renewal), to direct stem cells to a lineage specific patterns (differentiation), or to reprogram somatic cells to a more undifferentiated state (de-differentiation or reprogramming). These molecules, in turn, have provided new insights into the signaling mechanisms that regulate stem cell biology, and may eventually lead to effective therapies in regenerative medicine. In this review, we will introduce recent findings with regards to small molecules and their impact on stem cell self-renewal and differentiation.

Asunto(s)

Evaluación Preclínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Células Madre/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Ingeniería Celular/métodos , Proliferación Celular/efectos de los fármacos , Humanos , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Células Madre/fisiología

RESUMEN

The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

Asunto(s)

Proteína Adaptadora GRB10/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Humanos , Insulina/metabolismo , Espectrometría de Masas , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Naftiridinas/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Proteoma/metabolismo , Sirolimus/farmacología

RESUMEN

Inhibition of Bcr-Abl kinase activity by imatinib for the treatment of chronic myeloid leukemia (CML) currently serves as the paradigm for targeting dominant oncogenes with small molecules. We recently reported the discovery of GNF-2 (1) and GNF-5 (2) as selective non-ATP competitive inhibitors of cellular Bcr-Abl kinase activity that target the myristate binding site. Here, we used cell-based structure-activity relationships to guide the optimization and diversification of ligands that are capable of binding to the myristate binding site and rationalize the findings based upon an Abl-compound 1 cocrystal. We elucidate the structure-activity relationships required to obtain potent antiproliferative activity against Bcr-Abl transformed cells and report the discovery of new compounds (5g, 5h, 6a, 14d, and 21j-I) that display improved potency or pharmacological properties. This work demonstrates that a variety of structures can effectively target the Bcr-Abl myristate binding site and provides new leads for developing drugs that can target this binding site.

Asunto(s)

Antineoplásicos/síntesis química , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinas/síntesis química , Regulación Alostérica , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Transformada , Dasatinib , Sinergismo Farmacológico , Proteínas de Fusión bcr-abl/genética , Ratones , Modelos Moleculares , Mutación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Relación Estructura-Actividad , Tiazoles/farmacología

RESUMEN

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Asunto(s)

Antineoplásicos/química , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/química , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Animales , Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Benzamidas , Sitios de Unión , Trasplante de Médula Ósea , Línea Celular Tumoral , Cristalización , Modelos Animales de Enfermedad , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Concentración 50 Inhibidora , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Masculino , Espectrometría de Masas , Ratones , Modelos Moleculares , Mutación/genética , Piperazinas/química , Piperazinas/farmacología , Estructura Terciaria de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacología , Trasplante Heterólogo

RESUMEN

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH(2)-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH(2)-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

Asunto(s)

Retículo Endoplásmico/enzimología , Proteínas Proto-Oncogénicas c-abl/fisiología , Células 3T3 , Adenosina Trifosfato/química , Sitio Alostérico , Animales , Antineoplásicos/farmacología , Sitios de Unión , Resistencia a Antineoplásicos , Humanos , Lípidos/química , Ratones , Conformación Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/metabolismo

RESUMEN

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC(50)s of 40nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the 'DFG-out' inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.

Asunto(s)

Química Farmacéutica/métodos , Inhibidores Enzimáticos/farmacología , Receptores de la Familia Eph/antagonistas & inhibidores , Adenosina Trifosfato/química , Benzamidas/síntesis química , Benzamidas/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Humanos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Niacinamida/análogos & derivados , Niacinamida/síntesis química , Niacinamida/farmacología , Fosforilación , Receptores de la Familia Eph/química , Relación Estructura-Actividad

RESUMEN

Regulation of gene expression by transcription factors touches all aspects of human biology and often induces extreme phenotypes. Its external, precise control by small organic molecules represents a challenge in chemistry and biology. Here, we summarize recent progress in the field, together with contributions from our laboratory. Small-molecule modulators of transcription, including small-molecule transcription factors could find their use in basic biological studies and therapeutic intervention.

Asunto(s)

Antineoplásicos/farmacología , Factores de Transcripción/síntesis química , Transcripción Genética/efectos de los fármacos , Adamantano/química , Adamantano/farmacología , Animales , Antineoplásicos/química , Diseño de Fármacos , Humanos , Indoles/química , Indoles/farmacología , Estructura Molecular , Conformación Proteica