RESUMEN
The mechanistic target of rapamycin (mTOR) pathway is hyperactivated in cancer and neurological disorders. Rapalogs and mTOR kinase inhibitors (TORKi) have recently been applied to alleviate epileptic seizures in tuberous sclerosis complex (TSC). Herein, we describe a pharmacophore exploration to identify a highly potent, selective, brain penetrant TORKi. An extensive investigation of the morpholine ring engaging the mTOR solvent exposed region led to the discovery of PQR626 (8). 8 displayed excellent brain penetration and was well-tolerated in mice. In mice with a conditionally inactivated Tsc1 gene in glia, 8 significantly reduced the loss of Tsc1-induced mortality at 50 mg/kg p.o. twice a day. 8 overcomes the metabolic liabilities of PQR620 (52), the first-in-class brain penetrant TORKi showing efficacy in a TSC mouse model. The improved stability in human hepatocytes, excellent brain penetration, and efficacy in Tsc1GFAPCKO mice qualify 8 as a potential therapeutic candidate for the treatment of neurological disorders.
Asunto(s)
Encéfalo/metabolismo , Morfolinas/administración & dosificación , Morfolinas/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Administración Oral , Animales , Encéfalo/efectos de los fármacos , Perros , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Morfolinas/química , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Ratas , Ratas Sprague-DawleyRESUMEN
Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Mastocitos/metabolismo , Transducción de Señal/inmunología , Proteínas ras/metabolismo , Animales , Degranulación de la Célula/inmunología , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas ras/inmunologíaRESUMEN
The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is a critical regulator of cell growth and is frequently hyperactivated in cancer. Therefore, PI3K inhibitors represent a valuable asset in cancer therapy. Herein we have developed a novel anticancer agent, the potent pan-PI3K inhibitor PQR514 (4), which is a follow-up compound for the phase-II clinical compound PQR309 (1). Compound 4 has an improved potency both in vitro and in cellular assays with respect to its predecessor compounds. It shows superiority in the suppression of cancer cell proliferation and demonstrates significant antitumor activity in an OVCAR-3 xenograft model at concentrations approximately eight times lower than PQR309 (1). The favorable pharmacokinetic profile and a minimal brain penetration promote PQR514 (4) as an optimized candidate for the treatment of systemic tumors.
RESUMEN
The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a â¼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.
Asunto(s)
Oxazinas/química , Inhibidores de Proteínas Quinasas/química , Pirimidinonas/química , Pirroles/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adenosina Trifosfato/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Perros , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Cinética , Masculino , Ratones , Conformación Molecular , Neoplasias/tratamiento farmacológico , Oxazinas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinonas/farmacocinética , Pirroles/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/químicaRESUMEN
The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure-activity relationship study, which led to the discovery of a drug-like adenosine 5'-triphosphate-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530, compound 6), which qualifies as a clinical candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compound 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compound 6 prevented cell growth in a cancer cell line panel. The preclinical in vivo characterization of compound 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncology.
Asunto(s)
Aminopiridinas/farmacología , Antineoplásicos/farmacología , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Piridinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Triazinas/farmacología , Aminopiridinas/síntesis química , Aminopiridinas/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Encéfalo/metabolismo , Línea Celular Tumoral , Perros , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Morfolinas/síntesis química , Morfolinas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Inhibidores de las Quinasa Fosfoinosítidos-3/metabolismo , Unión Proteica , Piridinas/síntesis química , Piridinas/metabolismo , Ratas Wistar , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/metabolismo , Triazinas/síntesis química , Triazinas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kß. On the basis of X-ray crystallography, hydrogen-deuterium exchange-mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenosina Trifosfatasas , Sitios de Unión , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ftalimidas , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/fisiología , Inhibidores de Proteínas Quinasas , Especificidad por SustratoAsunto(s)
Modelos Animales de Enfermedad , Linfoma de Células B Grandes Difuso/etiología , Linfoma de Células B Grandes Difuso/patología , Animales , Biomarcadores de Tumor , Biología Computacional/métodos , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Perros , Perfilación de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/mortalidad , Linfoma de Células B Grandes Difuso/terapia , Modelos Biológicos , PronósticoRESUMEN
Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, maximum concentration ( Cmax) in plasma and brain was reached after 30 min, with a half-life ( t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Piridinas/farmacología , Convulsiones/tratamiento farmacológico , Triazinas/farmacología , Animales , Compuestos de Azabiciclo/metabolismo , Compuestos de Azabiciclo/uso terapéutico , Barrera Hematoencefálica/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Conformación Proteica , Piridinas/metabolismo , Piridinas/uso terapéutico , Ratas , Triazinas/metabolismo , Triazinas/uso terapéuticoRESUMEN
Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.
Asunto(s)
Aminopiridinas/uso terapéutico , Antineoplásicos/uso terapéutico , Morfolinas/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Aminopiridinas/administración & dosificación , Aminopiridinas/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proliferación Celular/efectos de los fármacos , Perros , Humanos , Ratones , Modelos Moleculares , Morfolinas/administración & dosificación , Morfolinas/farmacocinética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Desnudas , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.
Asunto(s)
Aminopiridinas/farmacología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Aminopiridinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Células HCT116 , Humanos , Estructura Molecular , Morfolinas/química , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Tubulina (Proteína)/química , Moduladores de Tubulina/químicaRESUMEN
All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCß phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca²âº- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCß-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca²âº and PKCß. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Proteína Quinasa C beta/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Calcio/metabolismo , Dominio Catalítico , Degranulación de la Célula/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase Ib/química , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Mastocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Moleculares , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
BACKGROUND: Tissue mast cell numbers are dynamically regulated by recruitment of progenitors from the vasculature. It is unclear whether progenitors are recruited during allergic sensitization and whether recruitment promotes allergic responses. OBJECTIVE: We sought to (1) determine the effect of mast cell recruitment on acute allergic responses and (2) to define the role of phosphoinositide 3-kinase (PI3K) isoforms in sequential steps to allergic responses. METHODS: Gene-targeted mice for PI3Kγ or PI3Kδ or mice treated with isoform-specific PI3K inhibitors (a novel PI3Kγ-specific inhibitor [NVS-PI3-4] and the PI3Kδ inhibitor IC87114) were used to monitor IgE-mediated mast cell recruitment, migration, adhesion by means of intravital microscopy, degranulation, TNF-α release, and subsequent endothelial cell activation in vivo or in bone marrow-derived mast cells. RESULTS: Functional PI3Kγ, but not PI3Kδ, was crucial for mast cell accumulation in IgE-challenged skin, TNF-α release from IgE/antigen-stimulated mast cells, and mast cell/endothelial interactions and chemotaxis. PI3Kγ-deficient bone marrow-derived mast cells did not adhere to the endothelium in TNF-α-treated cremaster muscle, whereas PI3Kδ was not required. Depletion of TNF-α blocked IgE-induced mast cell recruitment, which links tissue mast cell-derived cytokine release to endothelial activation and mast cell recruitment. Interference with mast cell recruitment protected against anaphylaxis and was superior to blockage of tissue mast cell degranulation. CONCLUSIONS: Interference with mast cell recruitment to exacerbated tissues provides a novel strategy to alleviate allergic reactions and surpassed attenuation of tissue mast cell degranulation. This results in prolonged drug action and allows for reduction of drug doses required to block anaphylaxis, an important feature for drugs targeting inflammatory disease in general.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Hipersensibilidad/inmunología , Mastocitos/inmunología , Anafilaxia/tratamiento farmacológico , Anafilaxia/inmunología , Anafilaxia/metabolismo , Animales , Antialérgicos/uso terapéutico , Degranulación de la Célula/inmunología , Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Células Endoteliales/inmunología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Hipersensibilidad/tratamiento farmacológico , Hipersensibilidad/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR-/-) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR-/- macrophages with increasing concentrations of (125)I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on (125)I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect (125)I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR-/- mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.
Asunto(s)
Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Lipoproteínas LDL/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Pinocitosis/fisiología , Actinas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Citocalasina D/farmacología , Dinaminas/metabolismo , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Inhibidores de las Quinasa Fosfoinosítidos-3 , Multimerización de Proteína/efectos de los fármacos , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²5I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase pinocytosis, although the isoform mediating this process has not been determined. Because PI3Kγ is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3Kγ inhibitor AS605240 or PI3Kγ-null macrophages incubated with LDL showed an â¼50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase pinocytosis of LDL and identify PI3Kγ as contributing to this process.
Asunto(s)
LDL-Colesterol/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Células Espumosas/fisiología , Macrófagos/efectos de los fármacos , Pinocitosis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Células Espumosas/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Lipoproteínas LDL , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Quinoxalinas/farmacología , Tiazolidinedionas/farmacologíaRESUMEN
Recognition of bacterial constituents by mast cells (MCs) is dependent on the presence of pattern recognition receptors, such as Toll-like receptors (TLRs). The final cellular response, however, depends on the influence of multiple environmental factors. In the current study we tested the hypothesis that the PI3K-activating ligands insulin-like growth factor-1 (IGF-1), insulin, antigen, and Steel Factor (SF) are able to modulate the TLR4-mediated production of proinflammatory cytokines in murine MCs. Costimulation with any of these ligands caused increased LPS-triggered secretion of IL-6 and TNF-α, but attenuated the production of IL-1ß, though all three cytokines were produced in an NFκB-dependent manner. The pan-specific PI3K-inhibitor Wortmannin reverted the altered production of these cytokines. In agreement, MCs deficient for SHIP1, a negative regulator of the PI3K pathway, showed augmented secretion of IL-6/TNF-α and reduced production of IL-1ß in response to LPS alone. The differential effects of IGF-1 on TLR4-mediated cytokine production were also observed in the context of TLR2 and IL-33 receptor-mediated MC activation. Importantly, these effects were seen in both bone marrow-derived and peritoneal MCs, suggesting general relevance for MCs. Using pharmacological and genetic tools, we could show that the p110δ isoform of PI3K is strongly implicated in SF-triggered suppression of LPS-induced IL-1ß production. Costimulation with antigen was affected to a lesser extent. In conclusion, NFκB-dependent production of proinflammatory cytokines in MCs is differentially controlled by PI3K-activating ligand/receptor systems.
Asunto(s)
Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Mastocitos/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Inositol Polifosfato 5-Fosfatasas , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteína 1 Similar al Receptor de Interleucina-1 , Lipopolisacáridos/toxicidad , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Ratones , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal , Receptor Toll-Like 2/metabolismoRESUMEN
Class I(B) phosphoinositide 3-kinase gamma (PI3Kgamma) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kgamma consists of a catalytic p110gamma and a regulatory p87(PIKAP) (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein betagamma (Gbetagamma)-mediated PI3Kgamma regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110gamma activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kgamma-complemented bone marrow-derived mast cells (BMMCs) from p110gamma(-/-) mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3Kgamma activation. Although p101 strongly interacted with Gbetagamma, thereby mediating PI3Kgamma membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gbetagamma-dependent membrane recruitment of p87/p110gamma by direct interaction with p110gamma, suggesting the indispensability of Ras for activation of p87/p110gamma. Consequently, interference with Ras signaling indeed selectively blocked p87/p110gamma, but not p101/p110gamma, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110gamma activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kgamma that could open up new possibilities for therapeutic intervention.
Asunto(s)
Activación Enzimática/fisiología , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Proteínas ras/metabolismo , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Humanos , Mastocitos/metabolismo , Ratones , Microscopía ConfocalRESUMEN
Phosphoinositide 3-kinase gamma (PI3Kgamma) plays a major role in chronic inflammation and allergy. It is a heterodimer of a catalytic p110gamma subunit and an adaptor protein, either p101 or the p101 homolog p84 (p87(PIKAP)). It is unclear whether both PI3Kgamma complexes specifically modulate responses such as chemotaxis and degranulation. In mast cells, the p84:p110gamma complex synergizes with immunoglobulin E (IgE)- and antigen-clustered FcepsilonRI receptor signaling and is required to achieve maximal degranulation. During this process, PI3Kgamma is activated by ligands of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), in particular adenosine receptors, through autocrine and paracrine pathways. Here, we show that p110gamma needs p84 to relay signals from GPCRs to formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], phosphorylation of Akt, migration of cells, and synergistic adenosine-enforced degranulation. Furthermore, the absence of adaptor subunits could not be compensated for by increased p110gamma abundance. Differentiated, p110gamma null cells also lost adaptor proteins. Complementation of p110gamma null mast cells with p101 and p110gamma restored the activation of Akt and cell migration, but failed to support degranulation. Lack of degranulation was attributed to a change in the spatiotemporal localization of PI3Kgamma-derived PtdIns(3,4,5)P(3); although both p84:p110gamma and p101:p110gamma complexes initially deposited PtdIns(3,4,5)P(3) at the plasma membrane, p101:p110gamma-derived PtdIns(3,4,5)P(3) was rapidly endocytosed to motile, microtubule-associated vesicles. In addition, p84:p110gamma, but not p101:p110gamma signaling was sensitive to disruption of lipid rafts. Our results demonstrate a nonredundant function for the p101 and p84 PI3Kgamma adaptor proteins and show that distinct pools of PtdIns(3,4,5)P(3) at the plasma membrane can elicit specific cell responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Degranulación de la Célula , Movimiento Celular , Mastocitos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Proteínas de Arabidopsis/metabolismo , Células de la Médula Ósea/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ib , Humanos , Isoenzimas/fisiología , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Canales de Potasio/metabolismo , Subunidades de Proteína/fisiología , Ratas , Transducción de SeñalRESUMEN
Phosphoinositide 3-kinase (PI3K)/protein kinase B/Akt and Ras/mitogen-activated protein kinase pathways are often constitutively activated in melanoma and have thus been considered as promising drug targets. Exposure of melanoma cells to NVP-BAG956, NVP-BBD130, and NVP-BEZ235, a series of novel, potent, and stable dual PI3K/mammalian target of rapamycin (mTOR) inhibitors, resulted in complete G1 growth arrest, reduction of cyclin D1, and increased levels of p27(KIP1), but negligible apoptosis. In contrast, treatment of melanoma with the pan-class I PI3K inhibitor ZSTK474 or the mTORC1 inhibitor rapamycin resulted only in minor reduction of cell proliferation. In a syngeneic B16 mouse melanoma tumor model, orally administered NVP-BBD130 and NVP-BEZ235 efficiently attenuated tumor growth at primary and lymph node metastatic sites with no obvious toxicity. Metastatic melanoma in inhibitor-treated mice displayed reduced numbers of proliferating and significantly smaller tumor cells. In addition, neovascularization was blocked and tumoral necrosis increased when compared with vehicle-treated mice. In conclusion, compounds targeting PI3K and mTOR simultaneously were advantageous to attenuate melanoma growth and they develop their potential by targeting tumor growth directly, and indirectly via their interference with angiogenesis. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential in metastatic melanoma therapy.