Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.

Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg(79)-Glu(80)-Pro(81)-Arg(82). The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP(83-132) is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP(25-47) and AGRP(50-80) had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.

, Reading RG6 6AJ1 The modulatory e ects of the allosteric e ectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D 1 , D 2 , D 3 and D 4 dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2 In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D 3 subtype for acceleration of radioligand dissociation. 3 In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D 1 , D 2(short) , D 2(long) and D 3 receptors were described by Hill coe cients exceeding unity and data were ®tted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4 At the D 4 subtype, Hill coe cients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coe cient for MIA at the D 4 subtype was less than unity and data could be ®tted well by the allosteric/competitive model, but it was not possible to de®ne unambiguously the modulatory mechanism. For this e ect a better de®nition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5 MIA reduced the potency with which dopamine stimulated [ 35 S]-GTPgS binding at the D 2 receptor. The e ects of MIA could be described by the allosteric/competitive model with e ects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Excipients, considered “inactive ingredients,” are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant Kd values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

H]spiperone in the absence of sodium ions raclopride exerted noncompetitive effects, decreasing the number of sites labeled by the radioligand. These data are interpreted in terms of a model where the receptor exists as a dimer, and in the absence of sodium ions, raclopride exerts negative cooperativity across the dimer both for its own binding and the binding of spiperone. A model of the receptor has been produced that provides a good description of the experimental phenomena described here.The G-protein-coupled receptors (GPCRs) 1 constitute a large family of proteins responsible for the transduction of a wide range of signals (e.g. hormones, neurotransmitters, odorants, light, etc.) via G-proteins (1). GPCRs possess a common structural motif of seven ␣-helical membrane-spanning domains, and it is often assumed that the functional unit (i.e. the ligand binding and G-protein interaction domains) of the GPCR is wholly contained in a single polypeptide. Indeed, most models of GPCR function assume a monomeric receptor interacting with the G-protein (see, for example, Ref. 2). Several lines of evidence, however, suggest that the some GPCRs may exist in dimeric or oligomeric forms.Immunoblotting has in several cases revealed species corresponding not only to the predicted molecular weight of the receptor but also to multiples of the molecular weight. Bands corresponding to approximately twice the predicted molecular weight of the receptor have been interpreted as homodimers for several receptors including D 2 dopamine (3, 4), D 3 dopamine (5), ␤ 2 -adrenergic (6), substance P (7), opiate (8) and M 1 and M 2 muscarinic acetylcholine receptors (9). Co-immunoprecipitation has also been used to demonstrate homodimer formation for the ␤ 2 -adrenergic receptor (6), opiate receptor (8), and somatostatin SSTR5 receptor (10). In some cases, formation of heterodimers of GPCRs has been reported with differences in the pharmacological properties of the receptors in the heterodimer (e.g. GABA B receptor isoforms (11-13), ␦ and opiate receptors (14), dopamine, and somatostatin receptors (10)).Further evidence for interaction of GPCRs was provided by Maggio et al. (15), who created two chimeric receptors ␣ 2 /M 3 and M 3 /␣ 2 , in which the C-terminal regions (transmembrane domains VI and VII) were exchanged between the ␣ 2C adrenergic and M 3 muscarinic receptors. Expression of either chimera alone did not result in any detectable binding of typical radiolabeled muscarinic or adrenergic ligands. However, cotransfection of COS7 cells with both chimeras resulted in the appearance of binding activity corresponding to both native receptors. This has lead to the proposal that some GPCRs might form domain-swapped dimers (16). Evidence for GPCR interaction in cells has been obtained by expressing GPCRs fused to different chromophores. Transfer of energy between the chromophores has been shown for the ␤ 2 -adrenergic receptor (17) and somatostatin SSTR5 receptor (10) and provides good evidence for the close proximity of the two molecules...