Design, synthesis and biological evaluation of indane derived GPR40 agoPAMs.

GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet β-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet β-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM).

Structure-Activity Relationship of Novel and Selective Biaryl-Chroman GPR40 AgoPAMs.

A series of biaryl chromans exhibiting potent and selective agonism for the GPR40 receptor with positive allosteric modulation of endogenous ligands (AgoPAM) were discovered as potential therapeutics for the treatment of type II diabetes. Optimization of physicochemical properties through modification of the pendant aryl rings resulted in the identification of compound , which possesses an improved metabolic profile while demonstrating sustained glucose lowering.

GPR40 partial agonists and AgoPAMs: Differentiating effects on glucose and hormonal secretions in the rodent.

GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut.

GPR40 reduces food intake and body weight through GLP-1.

G protein-coupled receptor 40 (GPR40) partial agonists lower glucose through the potentiation of glucose-stimulated insulin secretion, which is believed to provide significant glucose lowering without the weight gain or hypoglycemic risk associated with exogenous insulin or glucose-independent insulin secretagogues. The class of small-molecule GPR40 modulators, known as AgoPAMs (agonist also capable of acting as positive allosteric modulators), differentiate from partial agonists, binding to a distinct site and functioning as full agonists to stimulate the secretion of both insulin and glucagon-like peptide-1 (GLP-1). Here we show that GPR40 AgoPAMs significantly increase active GLP-1 levels and reduce acute and chronic food intake and body weight in diet-induced obese (DIO) mice.

Design and Synthesis of Novel, Selective GPR40 AgoPAMs.

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT).

MLi-2, a Potent, Selective, and Centrally Active Compound for Exploring the Therapeutic Potential and Safety of LRRK2 Kinase Inhibition.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of familial and sporadic Parkinson's disease (PD). That the most prevalent mutation, G2019S, leads to increased kinase activity has led to a concerted effort to identify LRRK2 kinase inhibitors as a potential disease-modifying therapy for PD. An internal medicinal chemistry effort identified several potent and highly selective compounds with favorable drug-like properties.

Discovery of SCH 900271, a Potent Nicotinic Acid Receptor Agonist for the Treatment of Dyslipidemia.

Structure-guided optimization of a series of C-5 alkyl substituents led to the discovery of a potent nicotinic acid receptor agonist SCH 900271 (33) with an EC50 of 2 nM in the hu-GPR109a assay. Compound 33 demonstrated good oral bioavailability in all species. Compound 33 exhibited dose-dependent inhibition of plasma free fatty acid (FFA) with 50% FFA reduction at 1.

Discovery of a potent nicotinic Acid receptor agonist for the treatment of dyslipidemia.

Nicotinic acid has been used clinically for decades to control serum lipoproteins. Nicotinic acid lowers very low-density lipoprotein (VLDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and lipoprotein-a (LPa), and it is also effective in raising high-density lipoprotein (HDL)-cholesterol. However, nicotinic acid has several side effects in clinical use.

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors.

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species.

Identification and characterization of splice variants of the human P2X7 ATP channel.

The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing.

Human cord blood-derived mast cells synthesize and release I-309 in response to IgE.

Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated.

The neurokinin-1 and neurokinin-2 receptor binding sites of MDL103,392 differ.

Several small molecule non-peptide antagonists of the NK-1 and NK-2 receptors have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these non-peptide antagonists lies within the bundle created by transmembrane domains IV-VII of the receptor and differs from the binding sites of peptide agonists and antagonists. The current investigation uses site-directed mutagenesis of the NK-1 and NK-2 receptors to elucidate the amino acids that are important for binding and functional activity of the first potent dual NK-1/NK-2 antagonist MDL103,392.

Selective inhibition of IL-5 receptor alpha-chain gene transcription by IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor in human blood eosinophils.

High affinity receptor for IL-5 (IL-5R), a predominant eosinophil maturation factor, is composed of an IL-5-binding alpha-chain (IL-5R alpha) and a signal-transducing beta-chain that is shared by IL-3 and granulocyte-macrophage CSF (GM-CSF) receptors (IL-3R and GM-CSFR). By Northern blot analysis of mRNAs obtained from normal human blood eosinophils, we show in this report that the hematopoietic cytokines IL-5, IL-3, and GM-CSF down-regulate IL-5R alpha mRNA while up-regulating alpha-chain mRNAs for both IL-3R and GM-CSFR as well as the beta-chain mRNA. More detailed characterization reveals that the down-regulation of IL-5R alpha mRNA is specific to IL-3, IL-5, and GM-CSF; occurs very rapidly (reaching maximum inhibition within 2 h); is cytokine dose dependent; and does not require protein synthesis.

Evidence for multiple promoters of the human IL-5 receptor alpha subunit gene: a novel 6-base pair element determines cell-specific promoter function.

In addition to a previously characterized promoter (P1), we now show the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nuclease protection assays.

Two related neurokinin-1 receptor antagonists have overlapping but different binding sites.

The neuropeptide substance P binds to the G protein-coupled neurokinin-1 (NK-1) receptor and elicits cellular responses thought to be involved in pain, neurogenic inflammation, vasodilatation, and plasma exudation. Several small molecule nonpeptide antagonists of the substance P/NK-1 receptor interaction have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these nonpeptide antagonists lies within the bundle created by transmembrane domains IV-VII of the receptor.

Expression, purification, and characterization of human cAMP-specific phosphodiesterase (PDE4) subtypes A, B, C, and D.

Although four members (A, B, C, and D) of the cAMP-specific phosphodiesterase (PDE4) family have been cloned by different groups, no study comparing the characteristics of purified human PDE4 subtypes has been published. In this study, we have expressed human PDE4 A, B, C, and D in insect (SF9) cells by using the baculovirus expression system, purified the expressed proteins, and compared their characteristics. The recombinant PDE4 subtypes all showed catalytic activity for cAMP with a K(m) of 1-5 microM.

Phospholipase C and phospholipase D are activated independently of each other in chemotactic peptide-stimulated human neutrophils.

When cytochalasin B-treated neutrophils were stimulated with fMet-Leu-Phe (fMLP) in the presence of Ca2+, phospholipase C (PLC) activity, as measured by inositol-1,4,5-triphosphate (IP3) formation, preceded phospholipase D (PLD)-catalyzed breakdown of choline-containing phosphoglycerides to form choline and diradyl-sn-glycero-3-phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2+, PLC was fully activated by fMLP with minimal activation of PLD, indicating that PLC activation alone is not sufficient for PLD activation. Full activation of PLD by fMLP required the simultaneous presence of both Ca2+ and cytochalasin B, a condition that caused no further enhancement of PLC.

Autologous immune complex glomerulonephritis induces abnormal embryonic development.

Young female random-bred Wistar rats were immunized with homologous renal brush border membranes. The immunized animals exhibited all the clinical and immunopathological characteristics of chronic autologous immune complex glomerulonephritis (Heymann nephritis) closely resembling the idiopathic membranous glomerulonephritis in humans. The animals were subsequently mated.

Teratogenic antibodies are directed against a coated-pit glycoprotein.

It has been well established that heterologous antibodies against certain tissue components may cause congenital abnormalities when injected into pregnant rats during the critical period of organogenesis. A glycoprotein antigen (gp340) of rat renal proximal tubules was isolated (C.C.

Effects of teratogenic antibodies on cultured visceral yolk-sac endodermal cells: cytotoxicity and morphological studies.

Primary cultures of visceral yolk-sac (VYS) endodermal cells were used to assess the effects of teratogenic and nonteratogenic antibodies. When assessed by cytotoxicity assay, teratogenic antibodies appeared to be lethal to the cultured cells at high concentrations (1.25-5 mg of antibodies per ml of culture medium).

Intact teratogenic immunoglobulins may reach the rat embryo.

It is known that heterologous antiserum against rat kidney homogenate may induce congenital malformations when injected into pregnant rats during the period of organogenesis. Teratogenic rabbit antibodies against a purified rat renal tubular glycoprotein were isolated, labelled with 125I and injected into pregnant rats on the 10th day of gestation. Extracts of visceral yolk-sacs (VYS) and embryos were obtained 16 h later and chromatographed separately on a Sephacryl S-300 gel filtration column.

Teratogenic antibody internalization by rat visceral yolk-sac endoderm in vitro: an ultrastructural colloidal gold tracer study.

Previous work from our laboratory has demonstrated that specific rabbit immunoglobulins G (IgG) against a glycoprotein antigen of rat kidney proximal tubule or a cross-reacting visceral yolk-sac endodermal cell antigen will induce abnormal embryonic development when they are injected into pregnant rats during the period of organogenesis. It has been proposed that these antibodies may induce embryopathy by interfering with functions of the visceral yolk-sac placenta, an important organ providing nutrients to the embryo at this stage of development. In order to gain some insight into the underlying pathogenic mechanism(s) in which specific teratogenic IgG may interfere with visceral yolk-sac functions, we examined the uptake of these teratogenic IgG by the visceral yolk-sac endodermal cells at the electron microscopic level.

Ultrastructural pathologic changes of rat extraembryonic visceral endodermal cells exposed to teratogenic antibodies in vivo.

It has been well established that certain heterologous tissue antibodies may induce abnormal embryonic development when injected into pregnant rodents during the organogenetic period. It has been postulated that these antibodies indirectly cause embryopathy by interfering with the normal functions of the yolk-sac placenta. The exact mechanism whereby these antibodies may induce placental pathology is not known.