Identification of Determinants of Glucose-Dependent Insulinotropic Polypeptide Receptor That Interact with N-Terminal Biologically Active Region of the Natural Ligand

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.

Insights into the binding and activation sites of the receptors for cholecystokinin and gastrin

CCK receptors represent potential targets in a number of diseases. Knowledge of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for their ligand recognition, partial agonism, ligand-induced trafficking of signalling. In the current paper, we report studies from our laboratory and others which have provided new data on the molecularbasis of the pharmacology and functioning of CCK1 and CCK2 receptors. It has been shown that: 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs in agreement with distinct phannacophores of CCK toward the two receptors and receptor sequence variations; 2) Binding sites of most of non-peptide agonists/ antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK; Aromatic amino acids within and near the binding site, especially in helix VI, are involved in receptor activation; 4) Like for other members of family A of G-protein coupled receptors, residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation. (c) 2007 Elsevier B.V. All rights reserved.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Partial agonism, neutral antagonism, and inverse agonism at the human wild-type and constitutively active cholecystokinin-2 receptors

Cholecystokinin receptor-2 (CCK2R) is a G protein receptor that regulates a number of physiological functions. Activation of CCK2R and/or expression of a constitutively active CCK2R variant may contribute to human diseases, including digestive cancers. Search for antagonists of the CCK2R has been an important challenge during the last few years, leading to discovery of a set of chemically distinct compounds. However, several early-discovered antagonists turned out to be partial agonists. In this context, we carried out pharmacological characterization of six CCK2R antagonists using COS-7 cells expressing the human CCK2R or a CCK2R mutant having a robust constitutive activity on inositol phosphates production, and we investigated the molecular mechanisms which, at a CCK2R binding site, account for these features. Results indicated that three compounds, 3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3- yl)-N'-(3-methylphenyl)urea (L365,260), 4-{[2-[[3-(lH-indol-3-yl)-2- methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2.2.1]hept-2-yl)-oxy]carbonyl]amino] propyl]amino]-1-phenylethyl]amino-4-oxo-[lS-la.2[S*(S*)]4a]} -butanoate N-methyl-D-glucamine (PD135, 158), and (R)-1-naphthalenepropanoic acid, b-[2-[[2-(8-azaspiro-[4.5]dec-8-ylcarbonyl)-4,6-dimethylphenyl]amino]-2- oxoethyl] (CR2945), were partial agonists; one molecule, 1-[(R)-2,3-dihydro-1- (2,3-dihydro-1-(2-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl] -3-(3-methylphenyl)urea (YM022), was a neutral antagonist; and two compounds, N-(+)-[1-(adamant-1-ylmethyl)-2,4-dioxo-5-phenyl2,3,4,5-tetrahydro-1H-1, 5-benzodiazepin-3-yl]-N'-phenylurea (GV150,013X) and ([(N-[methoxy-3 phenyl] N-[N-methyl N-phenyl carbamoylmethyl], carbomoylmethyl)-3 ureido]-3-phenyl)2-propionic acid (RPR101,048), were inverse agonists. Furthermore, target- and pharmacophore-based docking of ligands followed by molecular dynamic simulation experiments resulted in consistent motion of aromatic residues belonging to a network presumably important for activation, thus providing the first structural explanations for the different pharmacological profiles of tested compounds. This study confirms that several referenced so-called antagonists are in fact partial agonists, and because of this undesired activity, we suggest that newly generated molecules should be preferred to efficiently block CCK2R-related physiological effects. Furthermore, data on the structural basis for the different pharmacological features of CCK2R ligands will serve to further clarify CCK2R mechanism of activation. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.

Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin

The cholecystokinin (CCK) receptor-2 exerts very important central and peripheral functions by binding the neuropeptides cholecystokinin or gastrin. Because this receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human cholecystokinin receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human cholecystokinin receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human cholecystokinin receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat cholecystokinin receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human cholecystokinin-2 receptors.

Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Near 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signalling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2RG) or recruiting ß-arrestin2 (CCK2Rß) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150,013X), acted as a high affinity competitive antagonist on CCK2RG but was nearly inefficient as inhibitor of CCK2Rß. Several structural elements on both GV150,013X and in CCK2R binding cavity, which hinder binding of GV150,013X only to the CCK2Rß were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulphur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2Rß state. These data establish structural evidences for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Tiotropium and olodaterol fixed-dose combination versus mono-components in COPD (GOLD 2-4)

Efficacy and safety of tiotropium+olodaterol fixed-dose combination (FDC) compared with the mono-components was evaluated in patients with moderate to very severe chronic obstructive pulmonary disease (COPD) in two replicate, randomised, double-blind, parallel-group, multicentre, phase III trials. Patients received tiotropium+olodaterol FDC 2.5/5 μg or 5/5 μg, tiotropium 2.5 μg or 5 μg, or olodaterol 5 μg delivered once-daily via Respimat inhaler over 52 weeks. Primary end points were forced expiratory volume in 1 s (FEV1) area under the curve from 0 to 3 h (AUC0-3) response, trough FEV1 response and St George's Respiratory Questionnaire (SGRQ) total score at 24 weeks. In total, 5162 patients (2624 in Study 1237.5 and 2538 in Study 1237.6) received treatment. Both FDCs significantly improved FEV1 AUC0-3 and trough FEV1 response versus the mono-components in both studies. Statistically significant improvements in SGRQ total score versus the mono-components were only seen for tiotropium+olodaterol FDC 5/5 μg. Incidence of adverse events was comparable between the FDCs and the mono-components. These studies demonstrated significant improvements in lung function and health-related quality of life with once-daily tiotropium+olodaterol FDC versus mono-components over 1 year in patients with moderate to very severe COPD.

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species' threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project - and avert - future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups - including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems - http://www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015.

After the Critique of Rights: For a Radical Democratic Theory and Practice of Human Rights

The critique of human rights has proliferated in critical legal thinking over recent years, making it clear that we can no longer uncritically approach human rights in their liberal form. In this article I assert that after the critique of rights one way human rights may be productively re-engaged in radical politics is by drawing from the radical democratic tradition. Radical democratic thought provides plausible resources to rework the shortcomings of liberal human rights, and allows human rights to be brought within the purview of a wider political project adopting a critical approach to current relations of power. Building upon previous re-engagements with rights using radical democratic thought, I return to the work of Ernesto Laclau and Chantal Mouffe to explore how human rights may be thought as an antagonistic hegemonic activity within a critical relation to power, a concept which is fundamentally futural, and may emerge as one site for work towards radical and plural democracy. I also assert, via Judith Butler's model of cultural translation, that a radical democratic practice of human rights may be advanced which resonates with and builds upon already existing activism, thereby holding possibilities to persuade those who remain sceptical as to radical re-engagements with rights.

The perihelion activity of comet 67P/Churyumov-Gerasimenko as seen by robotic telescopes

Around the time of its perihelion passage the observability of 67P/Churyumov-Gerasimenko from Earth was limited to very short windows each morning from any given site, due to the low solar elongation of the comet. The peak in the comet's activity was therefore difficult to observe with conventionally scheduled telescopes, but was possible where service/queue scheduled mode was possible, and with robotic telescopes. We describe the robotic observations that allowed us to measure the total activity of the comet around perihelion, via photometry (dust) and spectroscopy (gas), and compare these results with the measurements at this time by Rosetta's instruments. The peak of activity occurred approximately two weeks after perihelion. The total brightness (dust) largely followed the predictions from Snodgrass et al. 2013, with no significant change in total activity levels from previous apparitions. The CN gas production rate matched previous orbits near perihelion, but appeared to be relatively low later in the year.