Republication: Targeting PI3KC2ß Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

Novel GPR119 agonist AS1669058 potentiates insulin secretion from rat islets and has potent anti-diabetic effects in ICR and diabetic db/db mice.

AIMS: G-protein-coupled receptor 119 (GPR119), mainly expressed in pancreatic ß-cells, represents a new target for treating type 2 diabetes. GPR119 agonist is known to induce insulin secretion in a glucose-dependent manner by elevating intracellular cAMP concentrations. This study mainly examined the anti-hyperglycemic effect of a novel candidate small-molecule GPR119 agonist AS1669058 2-(4-bromo-2,5-difluorophenyl)-6-methyl-N-[2-(1-oxidopyridin-3-yl)ethyl]pyrimidin-4-amine ethanedioate on ICR mice and diabetic db/db mice. MAIN METHODS: We measured blood glucose, plasma insulin, and insulin content in the pancreas after repeated administration of AS1669058 to db/db mice twice daily for one week. KEY FINDINGS: Under high-concentration glucose conditions, AS1669058 induced insulin secretion in a dose-dependent manner in the hamster pancreatic ß-cell line HIT-T15 and in rat pancreatic islets. In addition, AS1669058 increased human insulin promoter activity in NIT-1 cells. In in vivo studies, a single administration of AS1669058 (1 mg/kg) in ICR mice improved oral glucose tolerance based on insulin secretion. Further, 1-week repeated treatment (3 mg/kg, twice daily) in diabetic db/db mice significantly reduced blood glucose levels and tended to increase insulin content in the pancreas. SIGNIFICANCE: These results suggest that AS1669058 has promising potential as an extremely more effective anti-hyperglycemic agent than other compounds we previously reported as GPR119 agonists.

Targeting PI3KC2ß impairs proliferation and survival in acute leukemia, brain tumours and neuroendocrine tumours.

BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

The therapeutic potential of GPR119 agonists for type 2 diabetes.

INTRODUCTION: Patients with type 2 diabetes mellitus (T2DM) are reaching an explosive number. Pancreatic ß cell dysfunction is the characteristic feature of the progression of T2DM and there is an increasing need for agents to improve its function. GPR119 is a G protein-coupled receptor (GPCR) expressed both in pancreatic ß cells and enteroendocrine cells and has garnered significant interest as a promising target for the next generation of T2DM drug. In vitro studies indicate that GPR119 agonists increase intracellular cAMP levels leading to enhanced glucose-induced insulin release and enhanced incretin hormone glucagon-like peptide 1 (GLP-1) secretion. In T2DM rodent models, GPR119 agonists are shown to decrease blood glucose level and preserve pancreatic ß cell function. AREAS COVERED: This review summarizes the function of GPR119 and the progresses made in the discovery of GPR119 agonists reported since 2002 in literatures. The importance of GPR119 agonists in glycemic control is discussed. EXPERT OPINION: GPR119 agonists with glucose-dependent insulin release and increased insulin promoter activity is expected to preserve pancreatic ß cell function, thereby providing great clinical benefits for T2DM patients. Both the preclinical and clinical data suggest that GPR119 agonist will be a promising anti-diabetic drug.

Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice.

G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic ß-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC50 value of 4.8 µM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 µM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes.

AS1907417, a novel GPR119 agonist, as an insulinotropic and ß-cell preservative agent for the treatment of type 2 diabetes.

G-protein-coupled receptor (GPR) 119 is involved in glucose-stimulated insulin secretion (GSIS) and represents a promising target for the treatment of type 2 diabetes as it is highly expressed in pancreatic ß-cells. Although a number of oral GPR119 agonists have been developed, their inability to adequately directly preserve ß-cell function limits their effectiveness. Here, we evaluated the therapeutic potential of a novel small-molecule GPR119 agonist, AS1907417, which represents a modified form of a 2,4,6-tri-substituted pyrimidine core agonist, AS1269574, we previously identified. The exposure of HEK293 cells expressing human GPR119, NIT-1 cells expressing human insulin promoter, and the pancreatic ß-cell line MIN-6-B1 to AS1907417, enhanced intracellular cAMP, GSIS, and human insulin promoter activity, respectively. In in vivo experiments involving fasted normal mice, a single dose of AS1907417 improved glucose tolerance, but did not affect plasma glucose or insulin levels. Twice-daily doses of AS1907417 for 4weeks in diabetic db/db, aged db/db mice, ob/ob mice, and Zucker diabetic fatty rats reduced hemoglobin A1c levels by 1.6%, 0.8%, 1.5%, and 0.9%, respectively. In db/db mice, AS1907417 improved plasma glucose, plasma insulin, pancreatic insulin content, lipid profiles, and increased pancreatic insulin and pancreatic and duodenal homeobox 1 (PDX-1) mRNA levels. These data demonstrate that novel GPR119 agonist AS1907417 not only effectively controls glucose levels, but also preserves pancreatic ß-cell function. We therefore propose that AS1907417 represents a new type of antihyperglycemic agent with promising potential for the effective treatment of type 2 diabetes.

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion.

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic ß-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve ß-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve ß-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC(50) value of 2.5µM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic ß-cell line MIN-6 only under high-glucose (16.8mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.

A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding.

Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.

Synthesis and biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines as novel PI3 kinase p110alpha inhibitors.

We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110alpha inhibitor; however, although 4 is a potent inhibitor of p110alpha enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110alpha inhibitor 7a was obtained. Subsequent optimization of 7a afforded exceptionally potent p110alpha inhibitors, including 8c and 8h, with IC(50) values of 0.30 nM and 0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110alpha inhibitors reported to date. Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo.

Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases.

Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.

Synthesis and biological evaluation of pyrido[3',2':4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110alpha inhibitors.

4-Morpholin-4-ylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 1.4 microM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidine derivative 10e was discovered as a p110alpha inhibitor with approximately 400-fold greater potency than 2a. Evaluation of isoform selectivity showed that 10e is a potent inhibitor of p110beta. Furthermore, 10e showed anti-proliferative activity in various cell lines, including multi-drug resistant MCF7/ADR-res cells, and was effective against HeLa human cervical tumor xenografts in nude mice.

Synthesis and biological evaluation of imidazo[1,2-a]pyridine derivatives as novel PI3 kinase p110alpha inhibitors.

3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-2-methylimidazo[1,2-a]pyridine, 2a, was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 0.67microM, through screening in a scintillation proximity assay. Optimization of the substituents of 2a increased the p110alpha inhibitory activity by more than 300-fold (2g: IC(50)=0.0018microM). Further structural modification of 2g afforded thiazole derivative 12, which has potent p110alpha inhibitory activity (IC(50) of 0.0028microM) and is highly selective for p110alpha over other PI3K isoforms. Compound 12 also inhibited serum-induced cell proliferation of A375 and HeLa cells in vitro with IC(50) values of 0.14microM and 0.21microM, respectively, and suppressed tumor growth by 37% in a mouse HeLa xenograft model when dosed intraperitoneally at 25mg/kg. These results suggest that selective p110alpha inhibitors may have potential as cancer therapeutic agents.

Synthesis and biological evaluation of 4-morpholino-2-phenylquinazolines and related derivatives as novel PI3 kinase p110alpha inhibitors.

A series of 4-morpholino-2-phenylquinazolines and related derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. In this series, the thieno[3,2-d]pyrimidine derivative 15e showed the strongest inhibitory activity against p110alpha, with an IC(50) value of 2.0 nM, and inhibited proliferation of A375 melanoma cells with an IC(50) value of 0.58 microM. Moreover, 15e was found to be selective for p110alpha over other PI3K isoforms and protein kinases, making it the first example of a selective PI3K p110alpha inhibitor.

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor.

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.

Molecular identification of nicotinic acid receptor.

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.

Molecular cloning and characterization of prokineticin receptors.

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.

cDNA cloning and characterization of porcine histamine H4 receptor.

The cDNA encoding histamine H4 receptor was cloned from the porcine spleen cDNA library. Porcine H4 receptor, which shares 72% homology with its human counterpart, bound to histamine in receptor-expressing mammalian cells. Isolation of the porcine H4 receptor, which is important for understanding of the pharmacology, will aid in better interpretation of physiological role of this subtype of histamine receptor.