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The G protein-coupled receptor GPR183/EBI2, which is activated by oxysterols, is a therapeutic target for inflammatory and metabolic diseases where both antagonists and agonists are of potential interest. Using the piperazine diamide core of the known GPR183 antagonist (E)-3-(4-bromophenyl)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)prop-2-en-1-one (NIBR189) as starting point, we identified and sourced 79 structurally related compounds that were commercially available. In vitro screening of this compound collection using a Ca2+ mobilization assay resulted in the identification of 10 compounds with agonist properties. To enable establishment of initial structure-activity relationship trends, these were supplemented with five in-house compounds, two of which were also shown to be GPR183 agonists. Taken together, our findings suggest that the agonist activity of this compound series is dictated by the substitution pattern of one of the two distal phenyl rings, which functions as a molecular efficacy-switch.

The glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue metabolic hormone that is actively being targeted for its regulatory role of glycemia and energy balance. Limited structural data of its receptor has made ligand design tedious. This study investigates the structure and function of the GIP receptor (GIPR), using a homology model based on the GLP-1 receptor. Molecular dynamics combined with in vitro mutational data were used to pinpoint residues involved in ligand binding and/or receptor activation. Significant differences in binding mode were identified for the naturally occurring agonists GIP(1-30) NH2 and GIP(1-42) compared with high potency antagonists GIP(3-30)NH2 and GIP(5-30)NH2. Residues R183(2.60), R190(2.67), and R300(5.40) are shown to be key for activation of the GIPR, and evidence suggests that a disruption of the K293(ECL2)-E362(ECL3) salt bridge by GIPR antagonists strongly reduces GIPR activation. Combinatorial use of these findings can benefit rational design of ligands targeting the GIPR.

Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3(1-70) affects its activity at CCR1. Truncated CCL3(5-70) has 10-fold higher potency and enhanced efficacy in β-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu2877.39 interacts with the N-terminal amine of truncated CCL3(5-70) and with Zn2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL2, Y184 (Cys+1) and Y187 (Cys+4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Y187 in TMIV (F171) and TMV (W194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Y187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurol. disorders and the promotion of cancer cells. Recently, a high-resoln. x-ray crystal structure of NTSR1 in complex with NTS8-13 has been detd., providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small mol. antagonist has previously been used extensively as a tool compd. to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small mol. compds. to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were obsd., despite their distinct chem. nature and inverse pharmacol. profiles. The optimized models showed significantly improved ligand recognition in a large-scale virtual screening assessment compared to the crystal structure. Our models provide new insights into small mol. ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacol. potential of NTSR1 in neurol. disorders and cancer. [on SciFinder(R)]

Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR and MOR. A short SAR exploration with the objective of identifying more polar and hence less brain penetrant agonists is described herewith. Modeling studies of the recently published structures of KOR, DOR and MOR are used to explain the receptor selectivity. The synthesis, biological evaluation and SAR of novel benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nociceptive effect with a lead compound from this series is exemplified.

OBJECTIVES: GPR40 (FFAR1), a clinically proven anti-diabetes target, is a Gq-coupled receptor for long chain fatty acids (LCFA) stimulating insulin secretion directly and mediating a major part of the dietary triglyceride-induced secretion of the incretins GLP-1 and GIP. In phase-II studies the GPR40 agonist TAK-875 decreased blood glucose but surprisingly without stimulating incretins.METHODS AND RESULTS: Here we find that GPR40 can signal through not only Gq and IP3 but also Gs and cAMP when stimulated with certain agonists such as AM-1638 and AM-5262 in contrast to the endogenous LCFA ligands and agonists such as TAK-875 and AM-837, which only signal through Gq. In competition binding against [3H]AM-1638 and [3H]L358 the Gq + Gs and the Gq-only agonists either competed for or showed positive cooperativity by increasing the binding of the two different radio-ligands, in opposite ways. Nevertheless, both the Gq-only and the Gq + Gs agonists all docked surprisingly well into the binding site for TAK-875 in the X-ray structure of GPR40. In murine intestinal primary cell-cultures the endogenous LCFAs and the Gq-only agonists stimulated GLP-1 secretion with rather poor efficacy as compared with the high efficacy Gq + Gs GPR40 agonists and a prototype GPR119 agonist. Similarly, in fasting both male and female mice the Gq + Gs agonists showed significantly higher efficacy than the Gq-only agonists in respect of increasing plasma GLP-1 and plasma GIP in a GPR40-dependent manner.CONCLUSIONS: It is concluded that stimulation of GPR40 by endogenous LCFAs or by Gq-only synthetic agonists result in a rather limited incretin response, whereas Gq + Gs GPR40 agonists stimulate incretin secretion robustly.

The superfamily of G protein-coupled receptors plays a major role in conducting signals across the cell membrane and offers enormous scope for new therapeutic targets. Predicting receptor-ligand interactions based on available structural information forms the physical basis for drug discovery in the pharmaceutical industry. Among all GPCRs, members of the ghrelin receptor family have been shown to be involved in a diverse set of medical conditions, such as type-2 diabetes, obesity, Parkinson's disease and schizophrenia. The recently solved x-ray crystal structure of the agonist- bound neurotensin receptor 1 provides a unique opportunity for the exploration of the structural basis of ligand binding to other closely related members of the ghrelin receptor family. This study investigates the underlying mechanism of small molecule binding to three receptors within the ghrelin receptor family: the ghrelin receptor (GHSR) and the two neurotensin receptor subtypes (NTSR1 and NTSR2). A combined experimental and computational approach was made to systematically probe receptor interactions with a diverse set of small molecules showing distinct pharmacological profiles. In vitro mutational mapping based on inositol-phosphate accumulation assays indicated potential residues in the binding site of GHSR that are involved in binding of the small molecule compounds Bayer YIL781 and Abbott 14c. Computational modeling and docking studies yielded novel insights into structural features of the ghrelin receptor. Most importantly, a potential salt-bridge interaction between TM-III and TM-VI was proposed that suggests reinterpretation of previous studies. Docking studies in the two neurotensin receptor subtypes, NTSR1 and NTSR2, indicated a striking overlap between the binding modes of the small molecule compounds SR48692, SR142948A and the endogenous agonist neurotensin, despite their overall distinct pharmacological effects. Taken together, these results revealed structural feat