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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 ( VPAC2) and the type 1 corticotrophin releasing factor receptor ( CRF1) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO- K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca2+ mobilization and GTPγ S binding to Gs, Gi, G12 and Gq were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in R amp2+/- mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC2 enhanced the cell surface expression of all three RAMPs. CRF1 enhanced the cell surface expression of RAMP2; the cell surface expression of CRF1 was also increased. There was no effect on agonist-stimulated c AMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF1 : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In R amp2+/- mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC2 and CRF1 receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF1, coupling to RAMP2 may be of physiological significance. [ABSTRACT FROM AUTHOR]

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. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.

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. Moreover, the preparation of the derivatives was optimized to achieve the desired degree of substitution (DS = 9.0 ± 0.2 %). The prepared hyaluronan derivatives were water-soluble and exhibited self-associating properties (amphiphilicity). The structure of the prepared derivatives was elucidated by NMR spectroscopy, rheology, turbidity, SEC-MALLS, and gas chromatography (GC). The hydrophobic moieties increase the solution viscosity by physical crosslinking. Low concentration of HAC12 is needed to prepare highly viscous solutions with potential use for ophthalmic applications. Amphiphilic HA kept the biocompatibility of hyaluronan. The degree of substitution and Mw of the amphiphilic HA controls the sterilization by filtration. The protection against desiccation was tested using human keratinocytes (HaCaT) cells lines., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 films are homogeneous, mechanically strong, and flexible and can be sterilized by ethylenoxide. To characterize the films, we measured their thickness, dry mass, content of residual organic solvent, mechanical properties, swelling and enzymatic degradation. The safety and biodegradability of the films were tested both in-vitro and in-vivo, showing that the films are safe and that their degradation can be tailored by the degree of HA substitution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Synthetic glucocorticoids (GC) are essential for the treatment of a broad range of inflammatory diseases. However, their use is limited by target related adverse effects on, e.g., glucose homeostasis and bone metabolism. Starting from a nonsteroidal GR ligand (4) that is a full agonist in reporter gene assays, we exploited key functional triggers within the receptor, generating a range of structurally diverse partial agonists. Of these, only a narrow subset exhibited full anti-inflammatory efficacy and a significantly reduced impact on adverse effect markers in human cell assays compared to prednisolone. This led to the discovery of AZD9567 (15) with excellent in vivo efficacy when dosed orally in a rat model of joint inflammation. Compound 15 is currently being evaluated in clinical trials comparing the efficacy and side effect markers with those of prednisolone.

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. Particularly, pharmacokinetic-PD modeling was applied to quantify the effects on the cardiovascular system, and an investigative toxicology study in rats was performed to explore potential myocardial toxicities. Compound 2, in comparison to a reference A2a agonist, UK-432,097, demonstrated higher solubility, lower lipophilicity, lower plasma protein binding, high rat lung retention (28% remaining after 24 h), and was efficacious in a lung inflammatory rat model following intratracheal dosing. Despite these properties, compound 2 did not provide a sufficient therapeutic index, that is, separation of local anti-inflammatory efficacy in the lung from systemic side effects in the cardiovascular system. The plasma concentration that resulted in induction of hypotension (half maximal effective concentration; EC50 0.5 nmol/L) correlated to the in vitro A2a potency (rIC50 0.6 nmol/L). Histopathological lesions in the heart were observed at a dose level which is threefold above the efficacious dose level in the inflammatory rat lung model. In conclusion, compound 2 is a highly potent and selective A2a agonist with significant lung retention after intratracheal administration. Despite its local anti-inflammatory efficacy in rat lung, small margins to the cardiovascular effects suggested limited therapeutic value of this compound for treatment of inflammatory lung disease by the inhaled route.

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. AZD6642 shows high specific potency and low lipophilicity, resulting in a selective and metabolically stable profile. On the basis of initial PK/PD relation measured, a low dose to human was predicted.

Inhibition of AMP deaminase (AMPD) holds the potential to elevate intracellular adenosine and AMP levels and, therefore, to augment adenosine signaling and activation of AMP-activated protein kinase (AMPK). To test the latter hypothesis, novel AMPD pan inhibitors were synthesized and explored using a panel of in vitro, ex vivo, and in vivo models focusing on confirming AMPD inhibitory potency and the potential of AMPD inhibition to improve glucose control in vivo. Repeated dosing of selected inhibitors did not improve glucose control in insulin-resistant or diabetic rodent disease models. Mice with genetic deletion of the muscle-specific isoform Ampd1 did not showany favorable metabolic phenotype despite being challenged with high-fat diet feeding. Therefore, these results do not support the development of AMPD inhibitors for the treatment of type 2 diabetes.

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. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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. The TACR2 gene was sequenced from 37 individuals. Two amino acid changing SNPs encoding the NK(2) receptor variants Ile23Thr and Arg375His were found. The frequency of the four possible protein variants differed between populations. Site-directed mutagenesis was performed introducing either SNP or both SNPs into the TACR2 gene and the constructs were transfected into CHO cells. NKA-evoked increases in intracellular Ca(2+) were monitored by FLIPR. The potency of saredutant and ZD6021 was evaluated by their ability to inhibit NKA-induced increases in intracellular Ca(2+). NKA evoked increases in intracellular Ca(2+) with a potency ranging between 1 and 5nM in CHO cells expressing the different constructs. Saredutant and ZD6021 blocked NKA-evoked increases in intracellular Ca(2+) with pK(b) values ranging between 8.8-9.3 and 7.9-8.7, respectively. The current study demonstrates that polymorphisms leading to the Ile23Thr and Arg375His amino acid exchanges are highly prevalent in the human TACR2 gene. These polymorphisms however do not appear to affect the potency of the endogenous agonist NKA or the small molecule antagonists saredutant and ZD6021 with respect to intracellular Ca(2+) signalling.

Intracerebroventricular (i.c.v.) administration of tachykinin NK(1) receptor agonists induces tapping of the hind legs in gerbils, so-called gerbil foot tapping, which is thought to reflect a fear-related response. The aim of the present study was to examine how ligands selective for NK(1), NK(2) and NK(3) receptors affect the gerbil foot tap response. Agonists selective for NK receptor subtypes were administered i.c.v. and the gerbil foot tap response was monitored. The effect of systemically administered antagonists was also studied. The interaction of ligands with gerbil NK(1) receptors was evaluated using autoradiography on gerbil brain slices with [(3)H]-Sar,Met(O(2))-substance P or [(3)H]GR205171 as radioligand. The effects of ligands on NK(1) and NK(3) receptor-mediated increases in intracellular calcium in vitro were studied in Chinese hamster ovary cells expressing the cloned gerbil receptors. The selective NK(1) receptor agonist ASMSP and the selective NK(3) receptor agonist senktide induced dose-dependent increases in gerbil foot tapping with similar potency. The maximal effect of senktide was approximately 40% of the maximal response evoked by ASMSP. The effects of ASMSP and senktide were blocked by administration of the selective NK(1) receptor antagonist CP99,994 (10 micromol/kg s.c.). The effects of senktide, but not ASMSP, were blocked by administration of the selective NK(3) receptor antagonist SB223412 (50 micromol/kg i.p.). Senktide did not displace NK(1) receptor radioligand binding and was >1000-fold less potent than ASMSP at activating gerbil NK(1) receptors. The selective NK(3) receptor agonist senktide evokes fear-related gerbil foot tapping, an effect which probably involves indirect enhancement of NK(1) receptor signalling.

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. The affinity and potency of NKR agonists and the NK(1)R antagonists CP99994 and RP67580 (NK(1)R-selective) and ZD6021 (NK1/2R) were assessed in vitro by monitoring [(3)H]-SarMet SP binding and substance P-evoked mobilization of intracellular Ca(2+). The gerbil foot tap (GFT) method was used to assess the potency of the antagonists in vivo. The gNK(1)R coding sequence displayed an overall 95% and 97% homology with hNK(1)R and rNK(1)R, respectively. The affinity of the NK(1)R-selective agonist (3)H-SarMet SP for human and gerbil NK(1)R was similar (2.0 and 3.1 nM) but lower for rNK(1)R (12.4 nM). The rank order potency of the agonists for NK(1)R was SP > or = ASMSP > or = NKA >>> pro7NKB in all species. The NK(1)R antagonists, ZD6021 and CP99994, had comparable affinity and potency for gerbil and human NK(1)R, but were 1000-fold less potent for rNK(1)R. In contrast, RP67580 had comparable affinity and potency for all three species. Mutations in positions 116 and 290 did not affect agonist potency at the gNK(1)R while the potency of the antagonists ZD6021 and CP99994 were markedly decreased (10-20-fold). It is concluded that gNK(1)R has similar antagonist pharmacology as the human-like orthologue and that species differences in antagonist function depend on key residues in the coding sequence and antagonist structure.

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. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.

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. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the re-ceptor, which altered both the association and dissociation rates of [(3)H]TRH, the kinetics of ligand binding were unaffected by C-terminal truncation. Equally, the rate of recycling to the plasma membrane of internalized receptors was unaffected by C-terminal truncation. Although the extent of internalization of the full-length receptor was impaired by pre-exposure of cells to TRH, this was not true of C-terminal truncation mutants, which displayed limited steady-state internalization ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length receptor.

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. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein alpha subunit.

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. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqalpha/G11alpha was also internalized, although much of the Gqalpha/G11alpha immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqalpha/G11alpha immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqalpha/G11alpha immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.

Malate dehydrogenase (EC 1.1.1.37.) (MDH) was purified to apparent homogeneity from the cytosolic fraction of the protozoan Trichomonas vaginalis Donné. The four step purification included ion-exchange chromatography (DEAE-Sephacel and Q-Sepharose, elution with NaCl) and affinity chromatography (Reactive Red Agarose, elution with NADH and NaCl). The enzyme was purified about 132-fold (30.6% yield) to a specific activity of 352 units mg-1. The Km values determined at pH 7.8 (pH optimum from 7.5 to 8.3) for oxaloacetate and NADH were 16.2 microM and 10.6 microM, respectively. The MDH activity was inhibited by the substrate, decreasing to 50% at about 1 microM concentration of oxaloacetate. The reverse reaction from malate to oxaloacetate showed a pH optimum around pH 9.5. The Km for malate and NAD+ (determined at pH 7.8) were 1220 microM and 69.9 microM, respectively. SDS-PAGE analysis of the purified MDH revealed a single band with an apparent size of 34.5 kDa. The native molecular weight was estimated by HPLC gel filtration to be 60 kDa, which indicates that the T. vaginalis MDH exists as a dimer.