RESUMEN
The synthesis and evaluation of tetrasubstituted aminopyridines, bearing novel azaindazole hinge binders, as potent AKT inhibitors are described. Compound 14c was identified as a potent AKT inhibitor that demonstrated reduced CYP450 inhibition and an improved developability profile compared to those of previously described trisubstituted pyridines. It also displayed dose-dependent inhibition of both phosphorylation of GSK3beta and tumor growth in a BT474 tumor xenograft model in mice.
Asunto(s)
Aminopiridinas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Canales de Potasio Éter-A-Go-Go/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pirazinas/química , Piridinas/química , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Animales , Línea Celular Tumoral , Perros , Canal de Potasio ERG1 , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Haplorrinos , Humanos , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazinas/síntesis química , Pirazinas/farmacocinética , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Insulin-like growth factor-I receptor and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways are among the most active areas of drug discovery in cancer research. However, due to their integral roles in insulin signaling, inhibitors targeting these pathways often lead to hyperglycemia and hyperinsulinemia. We investigated the mechanism of hyperglycemia induced by GSK690693, a pan-AKT kinase inhibitor in clinical development, as well as methods to ameliorate these side effects. EXPERIMENTAL DESIGN: The effect of GSK690693 on blood glucose, insulin, and glucagon levels was characterized in mice. We then evaluated the effects of commonly prescribed antidiabetic agents on GSK690693-induced hyperglycemia. The mechanism of blood glucose increase was evaluated using fasting and tracer uptake studies and by measuring liver glycogen levels. Finally, approaches to manage AKT inhibitor-induced hyperglycemia were designed using fasting and low carbohydrate diet. RESULTS: We report that treatment with antidiabetic agents does not significantly affect GSK690693-induced hyperglycemia in rodents. However, administration of GSK690693 in mice significantly reduces liver glycogen (approximately 90%), suggesting that GSK690693 may inhibit glycogen synthesis and/or activate glycogenolysis. Consistent with this observation, fasting before drug administration reduces baseline liver glycogen levels and attenuates hyperglycemia. Further, GSK690693 also inhibits peripheral glucose uptake and introduction of a low-carbohydrate (7%) or 0% carbohydrate diet after GSK690693 administration effectively reduces diet-induced hyperglycemia in mice. CONCLUSIONS: The mechanism of GSK690693-induced hyperglycemia is related to peripheral insulin resistance, increased gluconeogenesis, and/or hepatic glycogenolysis. A combination of fasting and low carbohydrate diet can reduce the magnitude of hyperglycemia induced by an AKT inhibitor.
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Hiperglucemia/inducido químicamente , Oxadiazoles/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Dieta Baja en Carbohidratos , Ayuno , Femenino , Hiperglucemia/prevención & control , Glucógeno Hepático/metabolismo , Masculino , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The PI3K/AKT signaling is activated in various hematologic malignancies. We evaluated the effect of a novel, pan-AKT kinase inhibitor, GSK690693, on the proliferation of 112 cell lines representing different hematologic neoplasia. Fifty-five percent of all cell lines tested were sensitive to AKT inhibitor (EC(50)<1 microM), with acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, and Burkitt lymphoma showing 89%, 73%, and 67% sensitivity to GSK690693, respectively. The antiproliferative effect was selective for the malignant cells, as GSK690693 did not inhibit the proliferation of normal human CD4(+) peripheral T lymphocytes as well as mouse thymocytes. Phosphorylation of downstream substrates of AKT was reduced in both sensitive and insensitive cell lines on treatment with GSK690693, suggesting that the cause of resistance was not related to the lack of AKT kinase inhibition. Consistent with the role of AKT in cell survival, GSK690693 also induced apoptosis in sensitive ALL cell lines. Overall, our data provide direct evidence for the role of AKT signaling in various hematologic malignancies, especially ALL and some lymphomas.
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Apoptosis/efectos de los fármacos , Oxadiazoles/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/patología , Linfoma/tratamiento farmacológico , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.
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Antineoplásicos/farmacología , Oxadiazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Femenino , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Oxadiazoles/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Proteínas Fluorescentes Verdes/genética , Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/fisiología , Transporte Biológico , Candida albicans/genética , Ciclo Celular , Evolución Molecular , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteína Fluorescente RojaRESUMEN
We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.