Retinoic Acid Grafted to Hyaluronic Acid Activates Retinoid Gene Expression and Removes Cholesterol from Cellular Membranes.

All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin.

Formulation of hyaluronan grafted with dodecanoic acid as a potential ophthalmic treatment.

This work concerns the chemical modification of medium molecular weight hyaluronan for ophthalmic applications. The synthesis of amphiphilic HA with dodecanoyl moities was carried out under mild aqueous conditions. Perfect control of the degree of substitution was obtained by varying the molar ratio of activated fatty acid used in the reaction feed.

Biodegradable free-standing films from lauroyl derivatives of hyaluronan.

Hyaluronan (HA) films exhibit properties suitable for medical applications, but the solubility of HA limits their use in aqueous environments. This can be overcome by modifying HA with hydrophobic side groups that enable physical cross-linking. In this work, we present water insoluble free-standing films from lauroyl modified HA as novel biomaterials with properties tuneable by the degree of HA substitution.

The discovery of a selective and potent A2a agonist with extended lung retention.

Although the anti-inflammatory role of the A2a receptor is well established, controversy remains with regard to the therapeutic value for A2a agonists in treatment of inflammatory lung diseases, also as a result of unwanted A2a-mediated cardiovascular effects. In this paper, we describe the discovery and characterization of a new, potent and selective A2a agonist (compound 2) with prolonged lung retention and limited systemic exposure following local administration. To support the lead optimization chemistry program with compound selection and profiling, multiple in vitro and in vivo assays were used, characterizing compound properties, pharmacodynamics (PD), and drug concentrations.

Discovery of AZD6642, an inhibitor of 5-lipoxygenase activating protein (FLAP) for the treatment of inflammatory diseases.

A drug discovery program in search of novel 5-lipoxygenase activating protein (FLAP) inhibitors focused on driving a reduction in lipophilicity with maintained or increased ligand lipophilic efficiency (LLE) compared to previously reported compounds led to the discovery of AZD6642 (15b). Introduction of a hydrophilic tetrahydrofuran (THF) ring at the stereogenic central carbon atom led to a significant shift in physicochemical property space. The structure-activity relationship exploration and optimization of DMPK properties leading to this compound are described in addition to pharmacokinetic analysis and an investigation of the pharmacokinetic (PK)-pharmacodynamic (PD) relationship based on ex vivo leukotriene B4 (LTB4) levels in dog.

Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function.

Background And Purpose: Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC(2)) and the type 1 corticotrophin releasing factor receptor (CRF(1)) has been examined.

Experimental Approach: GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells.

Design of small molecule inhibitors of acetyl-CoA carboxylase 1 and 2 showing reduction of hepatic malonyl-CoA levels in vivo in obese Zucker rats.

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability.

Occurrence and pharmacological characterization of four human tachykinin NK2 receptor variants.

Tachykinin NK(2) receptor antagonists are potentially beneficial in treating various disorders including irritable bowel syndrome, urinary incontinence, depression and anxiety. The current study evaluates the frequency of single nucleotide polymorphisms (SNPs) in the human NK(2) receptor gene (TACR2). In addition, the potency of the endogenous peptide agonist neurokinin A (NKA), and the small molecule antagonists saredutant (NK(2)-selective) and ZD6021 (pan-NK antagonist) at the various NK(2) receptor protein variants were determined.

Senktide-induced gerbil foot tapping behaviour is blocked by selective tachykinin NK1 and NK3 receptor antagonists.

Intracerebroventricular (i.c.v.

The orphan receptor GPR55 is a novel cannabinoid receptor.

Background: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.

Experimental Approach: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands.

Adenosine is not a direct GHSR agonist--artificial cross-talk between GHSR and adenosine receptor pathways.

Aim: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR.

Methods: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP(3) experiments were performed using HEK cells stably expressing GHSR and/or adenosine A(2B) receptor (A(2B)R).

Results: Adenosine has been widely reported as a GHSR agonist.

Molecular cloning, mutations and effects of NK1 receptor antagonists reveal the human-like pharmacology of gerbil NK1 receptors.

The present study investigates the pharmacology of the cloned neurokinin 1 receptor from the gerbil (gNK(1)R), a species claimed to have human-like NK(1)R (hNK(1)R) pharmacology. The amino acid sequence of NK(1)R was cloned. The hNK(1)R, rat NK(1)R (rNK(1)R), gNK(1)R and mutants of the gNK(1)R were expressed in CHO cells.

Biochemistry of transmembrane signaling mediated by trimeric G proteins.

Many extracellular signals are at the cell surface received by specific receptors, which upon activation transduce information to the appropriate cellular effector molecules via trimeric G proteins. The G protein-mediated cascades ultimately lead to the highly refined regulation of systems such as sensory perception, cell growth, and hormonal regulation. Transmembrane signaling may be seriously deranged in various pathophysiological conditions.

Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and cointernalization with beta-arrestin 1-green fluorescent protein.

Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor.

Kinetic analysis of the internalization and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1.

The C-terminal tail of the long splice variant of the rat thyrotropin-releasing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in response to [(3)H]TRH was dependent upon the degree of truncation.

Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells.

Fusion proteins between the human A(1) adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein alpha subunit G(i1)alpha (A1/Gi), and between the human A(1) adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G(i1)alpha (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [(35)S]GTPgammaS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [(35)S]GTPgammaS binding was only seen at the A1/GFP/Gi fusion protein.

Overexpression of the G protein G11alpha prevents desensitization of Ca2+ response to thyrotropin-releasing hormone.

Doubly transfected human embryonal kidney cells (clone E2M11 of the HEK 293 cell line) expressing both thyrotropin-releasing hormone (TRH) receptors and G11alpha protein in high amounts were used to analyze the desensitization phenomenon of the Ca2+-mobilizing pathway. Quite unexpectedly, we did not observe any significant desensitization of the [Ca2+]i response to TRH in these cells after repeated or prolonged incubation with the hormone (up to 5 h). Under the same conditions, the TRH-induced [Ca2+]i response was completely desensitized in the parent cell line (293-E2 cels) expressing TRH receptors alone.

Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and gqalpha /G11alpha induced by agonist stimulation.

The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqalpha/G11alpha, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqalpha/G11alpha signals as the receptor was internalized.

Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor.

The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH.

Agonist-induced internalization of the G protein G11alpha and thyrotropin-releasing hormone receptors proceed on different time scales.

Using a combination of confocal immunofluorescence microscopy and subcellular fractionation, we demonstrate for the first time active internalization, trafficking, and down-regulation of a G protein alpha subunit subsequent to agonist occupation of a receptor. This proceeds on a much slower time scale than internalization of the corresponding receptor. In intact E2M11 HEK293 cells that express high levels of murine G11alpha and the rat thyrotropin-releasing hormone (TRH) receptor, the immunofluorescence signal of G11alpha was restricted almost exclusively to the plasma membrane.

Isolation and characterization of cytosolic malate dehydrogenase from Trichomonas vaginalis.

Malate dehydrogenase (EC 1.1.1.

Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis: purification and characterization.

Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.