RESUMO
Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
Fibroblast growth factor-21 (FGF-21) is a recently discovered metabolic regulator. Here, we investigated the effects of FGF-21 in the pancreatic beta-cell. In rat islets and INS-1E cells, FGF-21 activated extracellular signal-regulated kinase 1/2 and Akt signaling pathways. In islets isolated from healthy rats, FGF-21 increased insulin mRNA and protein levels but did not potentiate glucose-induced insulin secretion. Islets and INS-1E cells treated with FGF-21 were partially protected from glucolipotoxicity and cytokine-induced apoptosis. In islets isolated from diabetic rodents, FGF-21 treatment increased islet insulin content and glucose-induced insulin secretion. Short-term treatment of normal or db/db mice with FGF-21 lowered plasma levels of insulin and improved glucose clearance compared with vehicle after oral glucose tolerance testing. Constant infusion of FGF-21 for 8 weeks in db/db mice nearly normalized fed blood glucose levels and increased plasma insulin levels. Immunohistochemistry of pancreata from db/db mice showed a substantial increase in the intensity of insulin staining in islets from FGF-21-treated animals as well as a higher number of islets per pancreas section and of insulin-positive cells per islet compared with control. No effect of FGF-21 was observed on islet cell proliferation. In conclusion, preservation of beta-cell function and survival by FGF-21 may contribute to the beneficial effects of this protein on glucose homeostasis observed in diabetic animals.
Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Células Secretoras de Insulina/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 7 , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Teste de Tolerância a Glucose , Insulina/biossíntese , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic beta-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane.
Assuntos
Proteínas de Transporte/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/química , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/análise , Somatostatina/farmacologiaRESUMO
Most imidazolines inhibit ATP-sensitive K(+) (K(ATP)) channels. Since these drugs are potentially clinically relevant insulin secretagogues, it is important to know whether extrapancreatic K(ATP) channels are targeted. We examined the effects of three imidazoline-derived antidiabetic drugs on the cloned K(ATP) channel, expressed in Xenopus laevis oocytes, and their specificity for interaction with the pore-forming Kir6.2 or the sulphonylurea receptor (SUR) 1 subunit. Midaglizole, LY397364 and LY389382 blocked Kir6.2deltaC currents with IC(50) of 3.8, 6.1 and 0.7 microM, respectively. The block of Kir6.2/SUR1 currents by LY397364 and LY389382 was best fit by a two-site model, suggesting that these drugs also interact with SUR1. However, since all three drugs interact with the Kir6.2 subunit, and Kir6.2 forms the pore of extrapancreatic K(ATP) channels, these drugs are unlikely to be specific for the beta-cell.
Assuntos
Hipoglicemiantes/farmacologia , Imidazóis/farmacologia , Naftalenos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP , Animais , Células Cultivadas , Eletrofisiologia , Glucose/metabolismo , Insulina/metabolismo , Canais KATP , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização , Proteínas Recombinantes/metabolismoRESUMO
Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the ß-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the ß-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.