CC and CX3C chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor
ABSTRACT Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.
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ABSTRACT: G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.British Journal of Pharmacology 06/2011; 165(6):1617-43. DOI:10.1111/j.1476-5381.2011.01551.x · 4.99 Impact Factor
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ABSTRACT: The preclinical pharmacological profile of 6-hydroxy-8-[(1R)-1-hydroxy-2-[[2-(4-methoxyphenyl)-1,1-dimethylethyl]amino]ethyl]-2H-1,4-benzoxazin-3(4H)-one monohydrochloride (olodaterol, previously known as BI 1744 CL), a novel, enantiomeric pure, inhaled human beta(2)-adrenoceptor (hbeta(2)-AR) agonist, was compared with marketed drugs, such as salmeterol and formoterol. In vitro, olodaterol showed a potent, nearly full agonistic response at the hbeta(2)-AR (EC(50) = 0.1 nM; intrinsic activity = 88% compared with isoprenaline) and a significant selectivity profile (219- and 1622-fold against the hbeta(1)- and hbeta(3)-ARs, respectively). Likewise, olodaterol was able to potently reverse contraction induced by different stimuli in isolated human bronchi. In vivo, antagonistic effects of single doses of olodaterol and formoterol were measured against acetylcholine challenges in anesthetized guinea pigs and dogs for up to 24 h by using the Respimat Soft Mist inhaler. Heart rate and metabolic parameters (serum potassium, lactate, and glucose) were monitored to evaluate systemic pharmacodynamic effects in the dog model. In both models, olodaterol provided bronchoprotection over 24 h. Formoterol applied at an equally effective dose did not retain efficacy over 24 h. In both models olodaterol showed a rapid onset of action comparable with formoterol. Taken together, the preclinical behavior of olodaterol suggests that this novel beta(2)-AR agonist has the profile for once-daily dosing in humans concomitant with a fast onset of action and a favorable systemic pharmacodynamic profile.Journal of Pharmacology and Experimental Therapeutics 04/2010; 334(1):53-62. DOI:10.1124/jpet.110.167007 · 3.86 Impact Factor
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ABSTRACT: Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of the human muscarinic M(3) receptor (hM(3)) subtype. Tiotropium bromide (Spiriva) is a well established muscarinic antagonist in the pharmacological management of COPD with a once-daily posology. The rationale behind the sustained bronchodilation obtained with tiotropium consists in its slow dissociation from hM(3) receptors. In this study, we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs) currently in clinical development, namely aclidinium bromide and glycopyrrolate. The different muscarinic antagonists were characterized for their 1) affinity toward the different human muscarinic receptor subtypes expressed in Chinese hamster ovary cells and kinetics of receptor dissociation, 2) potency in inhibiting the agonist-induced activation of muscarinic receptors through measurement of second messengers, and 3) efficacy and duration of bronchoprotection, as tested in a model of acetylcholine-induced bronchoconstriction in anesthetized dogs over a period of 24 h. All of the tested LAMAs showed high affinity and potency toward the hM(3) receptor (tiotropium, pA(2) = 10.4; aclidinium, pA(2) = 9.6; and glycopyrrolate, pA(2) = 9.7). However, dissociation half-lives of the LAMAs from the hM(3) receptor differed significantly (tiotropium, t((1/2)) = 27 h; aclidinium, t((1/2)) = 10.7 h; and glycopyrrolate, t((1/2)) = 6.1 h). In line with their kinetic properties at the hM(3), the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24 h after administration (tiotropium = 35%, aclidinium = 21%, and glycopyrrolate = 0% at 24 h) when applied at equieffective doses.Journal of Pharmacology and Experimental Therapeutics 06/2009; 330(2):660-8. DOI:10.1124/jpet.109.152470 · 3.86 Impact Factor