ABSTRACT
The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet ß-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing. A clinical study with 3 administered to subjects with T2DM provided proof of concept of 3 as a potential glucose-lowering therapy. This manuscript summarizes the scientific rationale, medicinal chemistry, preclinical, and early development data of this new class of GPR40 agonists.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Drug Discovery , Hypoglycemic Agents/pharmacology , Piperidines/pharmacology , Receptors, G-Protein-Coupled/agonists , Spiro Compounds/pharmacology , Animals , Dose-Response Relationship, Drug , Glucose Tolerance Test , HEK293 Cells , Humans , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/chemistry , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Rats , Rats, Zucker , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
Extracellular ATP released from pancreatic ß-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic ß-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic ß-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.
Subject(s)
Glucose/metabolism , Insulin-Secreting Cells/enzymology , Insulin/metabolism , Pyrophosphatases/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Cells, Cultured , Enzyme Inhibitors/pharmacology , Glucose/pharmacology , Humans , Insulin Secretion , Insulin-Secreting Cells/chemistry , Insulin-Secreting Cells/drug effects , Male , Mice , Mice, Inbred C57BL , Pyrophosphatases/analysis , Pyrophosphatases/antagonists & inhibitors , RNA, Messenger/analysis , RNA, Messenger/metabolism , Tissue DistributionABSTRACT
The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.
Subject(s)
Amidohydrolases/genetics , Arginine/analogs & derivatives , DNA-Binding Proteins/agonists , Gene Expression Regulation/drug effects , Isoxazoles/pharmacology , Liver/enzymology , Receptors, Cytoplasmic and Nuclear/agonists , Transcription Factors/agonists , Amidohydrolases/biosynthesis , Amidohydrolases/physiology , Animals , Arginine/antagonists & inhibitors , Arginine/blood , Cell Line , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Dose-Response Relationship, Drug , Female , Humans , Isoxazoles/administration & dosage , Liver/drug effects , Rats , Rats, Zucker , Receptors, Cytoplasmic and Nuclear/deficiency , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
Oxytocin (OT) has been implicated in the cardiovascular responses to exercise, stress, and baroreflex adjustments. Studies were conducted to determine the effect of genetic manipulation of the OT gene on blood pressure (BP), heart rate (HR), and autonomic/baroreflex function. OT knockout (OTKO -/-) and control +/+ mice were prepared with chronic arterial catheters. OTKO -/- mice exhibited a mild hypotension (102 +/- 3 vs. 110 +/- 3 mmHg). Sympathetic and vagal tone were tested using beta(1)-adrenergic and cholinergic blockade (atenolol and atropine). Magnitude of sympathetic and vagal tone to the heart and periphery was not significantly different between groups. However, there was an upward shift of sympathetic tone to higher HR values in OTKO -/- mice. This displacement combined with unchanged basal HR led to larger responses to cholinergic blockade (+77 +/- 25 vs. +5 +/- 15 beats/min, OTKO -/- vs. control +/+ group). There was also an increase in baroreflex gain (-13.1 +/- 2.5 vs. -4.1 +/- 1.2 beats x min(-1) x mmHg(-1), OTKO -/- vs. control +/+ group) over a smaller BP range. Results show that OTKO -/- mice are characterized by 1) hypotension, suggesting that OT is involved in tonic BP maintenance; 2) enhanced baroreflex gain over a small BP range, suggesting that OT extends the functional range of arterial baroreceptor reflex; and 3) shift in autonomic balance, indicating that OT reduces the sympathetic reserve.
