7TM Receptors

14,000,000 Leading Edge Experts on the ideXlab platform

Scan Science and Technology

Contact Leading Edge Experts & Companies

Scan Science and Technology

Contact Leading Edge Experts & Companies

The Experts below are selected from a list of 6444 Experts worldwide ranked by ideXlab platform

Mette M Rosenkilde - One of the best experts on this subject based on the ideXlab platform.

  • The future of antiviral immunotoxins.
    Journal of leukocyte biology, 2016
    Co-Authors: Katja Spiess, Mette Høy Jakobsen, Thomas N. Kledal, Mette M Rosenkilde
    Abstract:

    There is a constant need for new therapeutic interventions in a wide range of infectious diseases. Over the past few years, the immunotoxins have entered the stage as promising antiviral treatments. Immunotoxins have been extensively explored in cancer treatment and have achieved FDA approval in several cases. Indeed, the design of new anticancer immunotoxins is a rapidly developing field. However, at present, several immunotoxins have been developed targeting a variety of different viruses with high specificity and efficacy. Rather than blocking a viral or cellular pathway needed for virus replication and dissemination, immunotoxins exert their effect by killing and eradicating the pool of infected cells. By targeting a virus-encoded target molecule, it is possible to obtain superior selectivity and drastically limit the side effects, which is an immunotoxin-related challenge that has hindered the success of immunotoxins in cancer treatment. Therefore, it seems beneficial to use immunotoxins for the treatment of virus infections. One recent example showed that targeting of virus-encoded 7 transmembrane (7TM) Receptors by immunotoxins could be a future strategy for designing ultraspecific antiviral treatment, ensuring efficient internalization and hence efficient eradication of the pool of infected cells, both in vitro and in vivo. In this review, we provide an overview of the mechanisms of action of immunotoxins and highlight the advantages of immunotoxins as future anti-viral therapies.

  • EBV,the HumanHost,andthe7TM Receptors: Defense or Offense?
    2015
    Co-Authors: Kristine Niss Arfelt, Suzan Fares, Mette M Rosenkilde
    Abstract:

    Being present in around 90% of the worldwide population, Epstein–Barr virus (EBV) is an exceptionally prevalent virus. This highly successful virus establishes a latent infection in resting memory B cells and is maintained in a balance between viral homeostasis on one side and antiviral defense of the immune system on the other side. The life cycle of EBV is dependent on many viral proteins, but EBV also regulates a number of endogenous proteins. 7TM Receptors and ligands of viral and host origin are examples of such

  • EBV, the human host, and the 7TM Receptors: defense or offense?
    Progress in molecular biology and translational science, 2014
    Co-Authors: Kristine Niss Arfelt, Suzan Fares, Mette M Rosenkilde
    Abstract:

    Abstract Being present in around 90% of the worldwide population, Epstein–Barr virus (EBV) is an exceptionally prevalent virus. This highly successful virus establishes a latent infection in resting memory B cells and is maintained in a balance between viral homeostasis on one side and antiviral defense of the immune system on the other side. The life cycle of EBV is dependent on many viral proteins, but EBV also regulates a number of endogenous proteins. 7TM Receptors and ligands of viral and host origin are examples of such proteins. 7TM Receptors are highly druggable and they are among the most popular class of investigational drug targets. The 7TM receptor encoded by EBV-BILF1, is known to downregulate cell surface MHC class I expression as part of the immune evasion strategy of EBV. However, the functional impact of the relationship between EBV and the regulated endogenous 7TM Receptors and ligands is still unclear. This is for instance the case for the most upregulated 7TM receptor EBI2 (EBV-induced gene 2 or GPR183). Whereas some regulated genes have been suggested to be involved in the EBV life cycle, others could also be important for the antiviral immune defense. As many of these 7TM Receptors and ligands have been shown to be modulated in EBV-associated diseases, targeting these could provide an efficient and specific way to inhibit EBV-associated disease progression. Here, we will review current knowledge on EBV infection, the immune defense against EBV and 7TM Receptors and ligands being either encoded or manipulated by EBV.

  • Interaction of chemokines with their Receptors--from initial chemokine binding to receptor activating steps.
    Current medicinal chemistry, 2014
    Co-Authors: Stefanie Thiele, Mette M Rosenkilde
    Abstract:

    Abstract The human chemokine system comprises 19 seven-transmembrane helix (7TM) Receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM Receptors. Affinityand activity-contributing interactions are attributed to different domains and known to occur in two steps. Here, knowledge on chemokine and receptor domains involved in the first binding-step and the second activation-step is reviewed. A mechanism comprising at least two steps seems consistent; however, several intermediate interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM Receptors. Overall, the N-terminus of chemokine Receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly interact with or rely on transmembrane receptor residues, while CXC-chemokines use residues located further exterior. Moreover, different chemokines for the same receptor often bind at different sites, uncovering the existence of several orthosteric sites thereby adding another level of complexity. This gives rise to a probe-dependency of small molecule "drug-like" ligands, which, depending on the chemokine interaction, may bind allosteric for some, and orthosteric for other chemokines targeting the same receptor, thereby resulting in probedependent pharmacodynamics.

  • Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5).
    British journal of pharmacology, 2014
    Co-Authors: Anne Steen, Thomas M Frimurer, Alexander Hovard Sparre-ulrich, Stefanie Thiele, Dong Guo, Mette M Rosenkilde
    Abstract:

    Background and Purpose A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM Receptors. However, in 37% of chemokine Receptors – a subgroup of 7TM Receptors – it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. Experimental Approach The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation. Key Results [L203F]-CCR5 increased the basal level of G-protein coupling (20–70% of Emax) and β-arrestin recruitment (50% of Emax) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr244 in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist. Conclusions and Implications The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile116 acts as a gate for the movement of Tyr244 towards TM-5 in the active state, a mechanism proposed previously for the β2-adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.

Thue W. Schwartz - One of the best experts on this subject based on the ideXlab platform.

  • In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.
    Molecular informatics, 2015
    Co-Authors: Michael Lückmann, Birgitte Holst, Thue W. Schwartz, Thomas M Frimurer
    Abstract:

    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 neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM Receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds 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 observed, despite their distinct chemical nature and inverse pharmacological 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 molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer.

  • The minor binding pocket: a major player in 7TM receptor activation.
    Trends in pharmacological sciences, 2010
    Co-Authors: Mette M Rosenkilde, Thomas M Frimurer, Tau Benned-jensen, Thue W. Schwartz
    Abstract:

    From the deep part of the main ligand-binding crevice, a minor, often shallower pocket extends between the extracellular ends of transmembrane domains (TM)-I, II, III and VII of 7TM Receptors. This minor binding pocket is defined by a highly conserved kink in TM-II that is induced by a proline residue located in one of two adjacent positions. Here we argue that this minor binding pocket is important for receptor activation. Functional coupling of the Receptors seems to be mediated through the hydrogen bond network located between the intracellular segments of these TMs, with the allosteric interface between TM-II and TM-VII being of particular significance. Importantly, the minor binding pocket, especially the proline-kink in TM-II, is involved in G protein versus arrestin pathway-biased signaling, for example in the angiotensin AT1 system. Consequently, this pocket could be specifically targeted in the development of functionally biased drugs.

  • Conserved Water-mediated Hydrogen Bond Network between TM-I, -II, -VI, and -VII in 7TM Receptor Activation
    The Journal of biological chemistry, 2010
    Co-Authors: Rie Nygaard, Thomas M Frimurer, Louise Valentin-hansen, Jacek Mokrosinski, Thue W. Schwartz
    Abstract:

    Five highly conserved polar residues connected by a number of structural water molecules together with two rotamer micro-switches, TrpVI:13 and TyrVII:20, constitute an extended hydrogen bond network between the intracellular segments of TM-I, -II, -VI, and -VII of 7TM Receptors. Molecular dynamics simulations showed that, although the fewer water molecules in rhodopsin were relatively movable, the hydrogen bond network of the β2-adrenergic receptor was fully loaded with water molecules that were surprisingly immobilized between the two rotamer switches, both apparently being in their closed conformation. Manipulations of the rotamer state of TyrVII:20 and TrpVI:13 demonstrated that these residues served as gates for the water molecules at the intracellular and extracellular ends of the hydrogen bond network, respectively. TrpVI:13 at the bottom of the main ligand-binding pocket was shown to apparently function as a catching trap for water molecules. Mutational analysis of the β2-adrenergic receptor demonstrated that the highly conserved polar residues of the hydrogen bond network were all important for receptor signaling but served different functions, some dampening constitutive activity (AsnI:18, AspII:10, and AsnVII:13), whereas others (AsnVII:12 and AsnVII:16) located one helical turn apart and sharing a water molecule were shown to be essential for agonist-induced signaling. It is concluded that the conserved water hydrogen bond network of 7TM Receptors constitutes an extended allosteric interface between the transmembrane segments being of crucial importance for receptor signaling and that part of the function of the rotamer micro-switches, TyrVII:20 and TrpVI:13, is to gate or trap the water molecules.

  • molecular basis for agonism in the bb3 receptor an epitope located on the interface of transmembrane iii vi and vii
    Journal of Pharmacology and Experimental Therapeutics, 2010
    Co-Authors: Florence Gbahou, Birgitte Holst, Thue W. Schwartz
    Abstract:

    Epitopes determining the agonist property of two structurally distinct selective ligands for the human bombesin receptor subtype 3 (BB3), [D-Tyr6,(R)-Apa11,Phe13, Nle14]-bombesin(6-14) (Pep-1) and Ac-Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2 (Pep-2), were mapped through systematic mutagenesis of the main ligand-binding pocket of the receptor. The mutational map for the smaller Pep-2 spanned the entire binding pocket of the BB3 receptor. In contrast, the much fewer mutational hits for the larger Pep-1 were confined to the center of the pocket, i.e., the opposing faces of the extracellular segments of transmembrane (TM)-III, TM-VI, and TM-VII. All the residues, which upon mutation affected Pep-1, were also hits for Pep-2 and included those that were most essential for the function of Pep-2: LeuIII:04 (Leu(123)), TyrVI:16 (Tyr(291)), and ArgVII:06 (Arg(316)). The BB3 receptor was found to signal with 12% ligand-independent activity that was strongly influenced both positively and negatively by several mutations in the binding pocket. The substitutions, which decreased the constitutive signaling, included not only the major mutational hits for the peptide agonists but also mutations more superficially located in the receptor. It is concluded that activation of the BB3 receptor is dependent upon an epitope in the main ligand-binding pocket at the interface between TM-III, TM-VI, and TM-VII that corresponds to the site where, for example, activating metal ion sites have been constructed previously in 7TM Receptors.

  • Construction of covalently coupled, concatameric dimers of 7TM Receptors.
    Journal of receptor and signal transduction research, 2009
    Co-Authors: Marie Terpager, Christian E. Elling, D. Jason Scholl, Valentina Kubale, Lene Martini, Thue W. Schwartz
    Abstract:

    7TM Receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM Receptors. Such 15 TM concatameric homo- and heterodimers of the β2-adrenergic and the NK1 Receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type Receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two Receptors. It is concluded that covalently...

Thomas M Frimurer - One of the best experts on this subject based on the ideXlab platform.

  • In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.
    Molecular informatics, 2015
    Co-Authors: Michael Lückmann, Birgitte Holst, Thue W. Schwartz, Thomas M Frimurer
    Abstract:

    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 neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM Receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds 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 observed, despite their distinct chemical nature and inverse pharmacological 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 molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer.

  • Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5).
    British journal of pharmacology, 2014
    Co-Authors: Anne Steen, Thomas M Frimurer, Alexander Hovard Sparre-ulrich, Stefanie Thiele, Dong Guo, Mette M Rosenkilde
    Abstract:

    Background and Purpose A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM Receptors. However, in 37% of chemokine Receptors – a subgroup of 7TM Receptors – it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. Experimental Approach The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation. Key Results [L203F]-CCR5 increased the basal level of G-protein coupling (20–70% of Emax) and β-arrestin recruitment (50% of Emax) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr244 in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist. Conclusions and Implications The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile116 acts as a gate for the movement of Tyr244 towards TM-5 in the active state, a mechanism proposed previously for the β2-adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.

  • Biased and constitutive signaling in the CC-chemokine receptor CCR5 by manipulating the interface between transmembrane helices 6 and 7.
    The Journal of biological chemistry, 2013
    Co-Authors: Anne Steen, Thomas M Frimurer, Stefanie Thiele, Dong Guo, L S Hansen, Mette M Rosenkilde
    Abstract:

    The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased signaling. Thus, β-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gαi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding. Furthermore, replacing Trp-248 with a smaller aromatic amino acid (Tyr/Phe) impaired the β-arrestin recruitment, yet with maintained G protein activity (biased signaling); also, here aplaviroc switched to a full agonist. Thus, the altered positioning of Trp-248, induced by G286F, led to a constraint of G protein active, but β-arrestin inactive and thus biased, CCR5 conformation. These results provide important information on the molecular interplay and impact of TM6 and TM7 for CCR5 activity, which may be extrapolated to other chemokine Receptors and possibly to other 7TM Receptors.

  • molecular characterization of oxysterol binding to the epstein barr virus induced gene 2 gpr183
    Journal of Biological Chemistry, 2012
    Co-Authors: Tau Bennedjensen, Andreas W Sailer, Christoffer Norn, Stephane Laurent, Christian M Madsen, Hjalte M Larsen, Kristine Niss Arfelt, Romain M Wolf, Thomas M Frimurer, Mette M Rosenkilde
    Abstract:

    Abstract Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning Receptors (7TM Receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 aka GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis we here identify four key residues for 7α,25-OHC binding: R87 in TM-II (position II:20/2.60), Y112 and Y116 (III:09/3.33 and III:13/3.37) in TM-III and Y260 in TM-VI (VI:16/6.51). Substituting these residues with Ala and/or Phe, results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Y116 interacts with the 3β-OH group in the agonist, Y260 with the 7α-OH group and R87, either directly or indirectly, with the 25-OH group although alternatives may exist here. In addition, Y112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in Receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated Receptors, here exemplified by CysLTs, P2Y12 and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM Receptors.

  • Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183).
    The Journal of biological chemistry, 2012
    Co-Authors: Tau Benned-jensen, Andreas W Sailer, Christoffer Norn, Stephane Laurent, Christian M Madsen, Hjalte M Larsen, Kristine Niss Arfelt, Romain M Wolf, Thomas M Frimurer, Mette M Rosenkilde
    Abstract:

    Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning Receptors (7TM Receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in Receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated Receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM Receptors.

Birgitte Holst - One of the best experts on this subject based on the ideXlab platform.

  • In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.
    Molecular informatics, 2015
    Co-Authors: Michael Lückmann, Birgitte Holst, Thue W. Schwartz, Thomas M Frimurer
    Abstract:

    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 neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM Receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds 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 observed, despite their distinct chemical nature and inverse pharmacological 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 molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer.

  • functionally biased signalling properties of 7TM Receptors opportunities for drug development for the ghrelin receptor
    British Journal of Pharmacology, 2013
    Co-Authors: Bjørn Sivertsen, Nicholas D. Holliday, Andreas N. Madsen, Birgitte Holst
    Abstract:

    The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several different signalling pathways including Gαq, Gαi/o, Gα12/13 and arrestin recruitment. These multiple signalling pathways allow for functionally biased signalling, where one signalling pathway may be favoured over another either by selective ligands or through mutations in the receptor. In the present review, we have described how ligands and mutations in the 7TM receptor may bias the Receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G–protein-dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have been demonstrated to signal more strongly through the Gαq-coupled pathway than the Gα12/13-coupled pathway. Similarly a ligand that promotes Gαq coupling over Gαi coupling has been described and it has been suggested that several different active conformations of the receptor may exist dependent on the properties of the agonist. Importantly, ligands with such biased signalling properties may allow the development of drugs that selectively modulate only the therapeutically relevant physiological functions, thereby decreasing the risk of side effects. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7

  • Functionally biased signalling properties of 7TM Receptors – opportunities for drug development for the ghrelin receptor
    British journal of pharmacology, 2013
    Co-Authors: Bjørn Sivertsen, Nicholas D. Holliday, Andreas N. Madsen, Birgitte Holst
    Abstract:

    The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several different signalling pathways including Gαq, Gαi/o, Gα12/13 and arrestin recruitment. These multiple signalling pathways allow for functionally biased signalling, where one signalling pathway may be favoured over another either by selective ligands or through mutations in the receptor. In the present review, we have described how ligands and mutations in the 7TM receptor may bias the Receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G–protein-dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have been demonstrated to signal more strongly through the Gαq-coupled pathway than the Gα12/13-coupled pathway. Similarly a ligand that promotes Gαq coupling over Gαi coupling has been described and it has been suggested that several different active conformations of the receptor may exist dependent on the properties of the agonist. Importantly, ligands with such biased signalling properties may allow the development of drugs that selectively modulate only the therapeutically relevant physiological functions, thereby decreasing the risk of side effects. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7

  • molecular basis for agonism in the bb3 receptor an epitope located on the interface of transmembrane iii vi and vii
    Journal of Pharmacology and Experimental Therapeutics, 2010
    Co-Authors: Florence Gbahou, Birgitte Holst, Thue W. Schwartz
    Abstract:

    Epitopes determining the agonist property of two structurally distinct selective ligands for the human bombesin receptor subtype 3 (BB3), [D-Tyr6,(R)-Apa11,Phe13, Nle14]-bombesin(6-14) (Pep-1) and Ac-Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2 (Pep-2), were mapped through systematic mutagenesis of the main ligand-binding pocket of the receptor. The mutational map for the smaller Pep-2 spanned the entire binding pocket of the BB3 receptor. In contrast, the much fewer mutational hits for the larger Pep-1 were confined to the center of the pocket, i.e., the opposing faces of the extracellular segments of transmembrane (TM)-III, TM-VI, and TM-VII. All the residues, which upon mutation affected Pep-1, were also hits for Pep-2 and included those that were most essential for the function of Pep-2: LeuIII:04 (Leu(123)), TyrVI:16 (Tyr(291)), and ArgVII:06 (Arg(316)). The BB3 receptor was found to signal with 12% ligand-independent activity that was strongly influenced both positively and negatively by several mutations in the binding pocket. The substitutions, which decreased the constitutive signaling, included not only the major mutational hits for the peptide agonists but also mutations more superficially located in the receptor. It is concluded that activation of the BB3 receptor is dependent upon an epitope in the main ligand-binding pocket at the interface between TM-III, TM-VI, and TM-VII that corresponds to the site where, for example, activating metal ion sites have been constructed previously in 7TM Receptors.

  • In Vivo Characterization of High Basal Signaling from the Ghrelin Receptor
    Endocrinology, 2009
    Co-Authors: Pia Steen Petersen, Andreas N. Madsen, David P.d. Woldbye, Kristoffer L. Egerod, Chunyu Jin, Manja Lang, Maria Rasmussen, Annette G. Beck-sickinger, Birgitte Holst
    Abstract:

    The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM Receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution. Food intake and body weight were significantly decreased in the group of rats treated with the inverse agonist, as compare...

Christian E. Elling - One of the best experts on this subject based on the ideXlab platform.

  • Construction of covalently coupled, concatameric dimers of 7TM Receptors.
    Journal of receptor and signal transduction research, 2009
    Co-Authors: Marie Terpager, Christian E. Elling, D. Jason Scholl, Valentina Kubale, Lene Martini, Thue W. Schwartz
    Abstract:

    7TM Receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM Receptors. Such 15 TM concatameric homo- and heterodimers of the β2-adrenergic and the NK1 Receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type Receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two Receptors. It is concluded that covalently...

  • Metal Ion Site Engineering Indicates a Global Toggle Switch Model for Seven-transmembrane Receptor Activation
    The Journal of biological chemistry, 2006
    Co-Authors: Christian E. Elling, Rasmus Jorgensen, Thomas M Frimurer, Birgitte Holst, Lars-ole Gerlach, Thue W. Schwartz
    Abstract:

    Much evidence indicates that, during activation of seven-transmembrane (7TM) Receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp113), the anchor point for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of the beta2-adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC50 decreased to 0.5 microm) and efficacious agonists in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest that the pivots for these vertical seesaw movements are the highly conserved proline bends of the involved helices.

  • molecular mechanism of 7TM receptor activation a global toggle switch model
    Annual Review of Pharmacology and Toxicology, 2006
    Co-Authors: Thue W. Schwartz, Thomas M Frimurer, Mette M Rosenkilde, Birgitte Holst, Christian E. Elling
    Abstract:

    The multitude of chemically highly different agonists for 7TM Receptors apparently do not share a common binding mode or active site but nevertheless act through induction of a common molecular activation mechanism. A global toggle switch model is proposed for this activation mechanism to reconcile the accumulated biophysical data supporting an outward rigid-body movement of the intracellular segments, as well as the recent data derived from activating metal ion sites and tethered ligands, which suggests an opposite, inward movement of the extracellular segments of the transmembrane helices. According to this model, a vertical see-saw movement of TM-VI-and to some degree TM-VII-around a pivot corresponding to the highly conserved prolines will occur during receptor activation, which may involve the outer segment of TM-V in an as yet unclear fashion. Small-molecule agonists can stabilize such a proposed active conformation, where the extracellular segments of TM-VI and -VII are bent inward toward TM-III, by acting as molecular glue deep in the main ligand-binding pocket between the helices, whereas larger agonists, peptides, and proteins can stabilize a similar active conformation by acting as Velcro at the extracellular ends of the helices and the connecting loops.

  • MOLECULAR MECHANISM OF 7TM RECEPTOR ACTIVATION—A GLOBAL TOGGLE SWITCH MODEL
    Annual review of pharmacology and toxicology, 2006
    Co-Authors: Thue W. Schwartz, Thomas M Frimurer, Mette M Rosenkilde, Birgitte Holst, Christian E. Elling
    Abstract:

    The multitude of chemically highly different agonists for 7TM Receptors apparently do not share a common binding mode or active site but nevertheless act through induction of a common molecular activation mechanism. A global toggle switch model is proposed for this activation mechanism to reconcile the accumulated biophysical data supporting an outward rigid-body movement of the intracellular segments, as well as the recent data derived from activating metal ion sites and tethered ligands, which suggests an opposite, inward movement of the extracellular segments of the transmembrane helices. According to this model, a vertical see-saw movement of TM-VI-and to some degree TM-VII-around a pivot corresponding to the highly conserved prolines will occur during receptor activation, which may involve the outer segment of TM-V in an as yet unclear fashion. Small-molecule agonists can stabilize such a proposed active conformation, where the extracellular segments of TM-VI and -VII are bent inward toward TM-III, by acting as molecular glue deep in the main ligand-binding pocket between the helices, whereas larger agonists, peptides, and proteins can stabilize a similar active conformation by acting as Velcro at the extracellular ends of the helices and the connecting loops.

  • A physicogenetic method to assign ligand-binding relationships between 7TM Receptors
    Bioorganic & medicinal chemistry letters, 2005
    Co-Authors: Thomas M Frimurer, Trond Ulven, Evi Kostenis, Christian E. Elling, Lars-ole Gerlach, Thomas Hogberg
    Abstract:

    A computational protocol has been devised to relate 7TM receptor proteins (GPCRs) with respect to physicochemical features of the core ligand-binding site as defined from the crystal structure of bovine rhodopsin. The identification of such Receptors that already are associated with ligand information (e.g., small molecule ligands with mutagenesis or SAR data) is used to support structure-guided drug design of novel ligands. A case targeting the newly identified prostaglandin D2 receptor CRTH2 serves as a primary example to illustrate the procedure.