WO2001067106A2 - Identification of g protein-coupled receptor ligands and modulators - Google Patents
Identification of g protein-coupled receptor ligands and modulators Download PDFInfo
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- WO2001067106A2 WO2001067106A2 PCT/US2001/007304 US0107304W WO0167106A2 WO 2001067106 A2 WO2001067106 A2 WO 2001067106A2 US 0107304 W US0107304 W US 0107304W WO 0167106 A2 WO0167106 A2 WO 0167106A2
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- arrestin
- gpcr
- mutant
- phosphorylation
- test compound
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
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Definitions
- a diverse array of external stimuli including neurotransmitters, hormones, phospholipids, photons, odorants, certain taste ligands, and growth factors can activate members of this receptor family and promote interaction between the receptor and a G protein located on the intracellular side of the membrane. This causes the exchange of GDP for GTP bound to the G protein ⁇ subunit and further causes the dissociation of the ⁇ heterodimers.
- GTP-bound G protein ⁇ subunits or ⁇ complexes initiate intracellular signaling responses by acting on effector molecules such as adenylate cyclases or phospholipases or directly regulating ion channel or kinase function.
- activated receptor is first phosphorylated by a G protein-coupled receptor kinase (GRK).
- GPK G protein-coupled receptor kinase
- An arrestin protein then binds to the activated phosphoreceptor, thereby blocking G protein interaction.
- Arrestin-receptor complex is subsequently internalized, whereupon receptor is either dephosphorylated and recycled back to the plasma membrane (resensitization) or sorted to lysosomes and destroyed (down-regulation).
- GPCRs are activated by an extremely wide variety of external stimuli and are now believed to play a role in regulating the activity of virtually all eukaryotic cells.
- the repertoire of receptor kinases and arrestins involved in the desensitization of these receptors is rather limited: six GRKs and four arrestins have thus far been found in mammals (reviewed in Freedman and Lefkowitz, supra).
- the present invention is based, at least in part, on a new use for phosphorylation-independent mutants of arrestin proteins as assay components in screening assays designed to identify ligands and/or modulators of G protein- coupled receptors (GPCRs).
- GPCRs G protein- coupled receptors
- Such phosphorylation-independent mutants of arrestin retain there dependence on agonist for GPCR-binding, however.
- phosphorylation-independent arrestin mutants are particularly well-suited for in vitro assays, for example, in assays that are directed to the identification of natural and surrogate agonists of orphan GPCRs (e.g., of known and/or orphan GPCRs, for example, in orphan GPCR ligand fishing assays).
- the phosphorylation- independent mutants of arrestin are also particularly well-suited for in vitro assays that are directed to the identification of GPCR antagonists and/or agonists.
- the present invention features methods of identifying a GPCR ligands which include contacting a composition containing the GPCR (e.g., a GPCR- containing membrane preparation) and a phosphorylation-independent arrestin mutant with a test compound and determining the ability of the test compound to modulate binding of the arrestin mutant to the GPCR, wherein modulation of binding indicates that the test compound is a GPCR ligand.
- the GPCRs being assayed are known GPCRs.
- the GPCRs being assayed are orphan G protein-coupled receptors.
- the compound being identified is a GPCR modulator (e.g. , an agonist or an antagonist or an inverse agonist for the GPCR being assayed).
- the compound being identified is a natural and/or surrogate ligand for an orphan GPCR being assayed.
- the present invention features methods of identifying G protein-coupled receptor (GPCR) agonists which include contacting a composition containing the GPCR (e.g., a GPCR-containing membrane preparation), a phosphorylation- independent arrestm mutant and a test compound and determining the ability of the test compound to modulate binding of the arrestin mutant to the GPCR, wherein modulation of binding indicates that the test compound is a GPCR agonist.
- GPCR G protein-coupled receptor
- Figure 2 is a graphic representation of the results of agonist affinity shift assays designed to demonstrate the ability of various phosphorylation-independent arrestin mutants to form high agonist affinity complexes (HACs) with both phosphorylated and non- phosphorylated GPCRs.
- HACs high agonist affinity complexes
- Figure 5 is a graphic depiction of the molecular architecture of arrestins and sequence homology of the phosphorylation recognition region.
- the major functional regions are designated as follows: Rl, basic N-terminal regulatory region; R2, acidic C- terminal regulatory region; A, activation-recognition region; P, phosphorylation recognition region; S, secondary binding site region.
- Rl basic N-terminal regulatory region
- R2 acidic C- terminal regulatory region
- A activation-recognition region
- P phosphorylation recognition region
- S secondary binding site region.
- the residues corresponding to the borders between the functional regions are shown below the schematic (numbering as in bovine ⁇ -arrestin 1A, GenBankTM Accession No. P17870, SEQ ID NO:l).
- the term "contacting" i.e., contacting a composition comprising a G protein-coupled receptor with a compound or agent
- contacting is intended to include incubating the compound or agent and composition together in vitro (e.g., adding the compound or agent to the composition in a suitable vessel) such that the compound or agent has the potential, for example, to interact with and/or bind to a GPCR which exists in the composition.
- the present invention features methods of identifying a G protein-coupled receptor (GPCR) agonists which include contacting a composition containing the GPCR (e.g., a GPCR-containing membrane preparation) and a phosphorylation-independent arrestin mutant with a test compound and determining the ability of the test compound to modulate binding of the arrestin mutant to the GPCR, wherein modulation of binding indicates that the test compound is a GPCR agonist.
- GPCR G protein-coupled receptor
- the present invention features a variety of assay formats suitable for identification of GPCR ligands and/or modulators.
- assay components can be interacted or contacted in a reaction vessel suitable for reacting assay components (e.g., in a tube, for example, a test tube or microfuge tube, in a well, for example, in a microtitre-plate well, on a solid surface, for example, in a droplet or microdroplet, on a chip, or the like).
- at least one assay component can be immobilized in an assay well or on an assay surface either directly or indirectly and additional assay components interacted or contracted with said immobilized components.
- a GPCR or GPCR-containing composition e.g., a purified and/or isolated GPCR in a suitable buffer or medium
- a covalent attachment e.g., immobilization via native -NH 2 , -SH, -CHO and/or COOH groups in the GPCR.
- the composition can be immobilized to the assay surface indirectly.
- an antibody specific for the GPCR can be immobilized on the assay surface (e.g., via direct conjugation or via an anti-Ig or anti-Fc antibody to the specific GPCR antibody.
- a known ligand for a first target receptor in the membrane preparation or liposome can be immobilized on the assay surface and used to affinity capture the membrane preparation or liposome, containing another or second receptor (e.g., an orphan GPCR for which identification of a surrogate ligand is sought).
- another or second receptor e.g., an orphan GPCR for which identification of a surrogate ligand is sought.
- the above-described immobilization techniques are also applicable in instances in which it is desirable to utilize unlabeled arrestin mutants and/or unlabeled GPCRs as assay reagents or components, for example, in a BIACORETM assay format as described herein. (See e.g.
- Exemplary phosphorylation-independent arrestin mutants have a mutation in the phosphorylation recognition domain of the protein.
- a "phosphorylation recognition domain” includes an arrestin protein domain having about 25-25, 26-34, 27-33, 28-32, 29-31 or 30 amino acid residues and is involved in regognition of phosphorylated GPCR by an arrestin protein.
- a phosphorylation recognition includes basic residues which are involved in recognition of phosphorylated receptor by the arrestin protein.
- a "phosphorylation recognition domain” includes about amino acid residues 155-184 of SEQ ID NO:2.
- a "phosphorylation recognition domain” includes about amino acid residues 155-184 of SEQ ID NO:3.
- Peptidomimetics are intended to include compounds composed, in whole or in part, of structures such as D-amino acids, non- naturally-occurring L-amino acids, modified peptide backbones and the like, as well as compounds that are composed, in whole or in part, of molecular structures unrelated to naturally-occurring L-amino acid residues linked by natural peptide bonds.
- a computer program may be used to process samples, record output, and/or process data obtained by carrying out the above-described steps.
- the preferred computer program product comprises a computer readable storage medium having computer-readable program code means embodied in the medium.
- Hardware suitable for use in such automated apparatus will be apparent to those of skill in the art, and may include computer controllers, automated sample handlers, surface plasmon resonance (SPR) measurement tools (e.g., BIACORETM instruments), tools for measuring bound and/or free labeled reactants, printers and optical displays.
- the hardware may also contain a computer-controlled stepper motor so that each control and/or test sample can be arranged as an array of samples and automatically and repeatedly positioned for detection of binding of reagents.
- determining the effectiveness of a test compound is performed in the presence or absence of the test compound, in the presence of the test compound as compared to a suitable control (e.g., a buffer or solvent control, known positive or negative control compounds) or in the presence of the test compound as compared to a predetermined or known range or value for binding and/or activity.
- a suitable control e.g., a buffer or solvent control, known positive or negative control compounds
- cell-based agonist assays are preferably performed in the presence of a known ligand specific for the GPCR of interest or, optionally, a surrogate ligand for the GPCR of interest.
- Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
- the precise dosage level of GPCR modulator or ligand, as the active component(s) should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
- mutant constructs are then used for in vitro transcription and translation, as described herein.
- In vitro translated Arg 170 - Glu or Asp383 - Ter is then radiolabelled to a suitable specific activity.
- High specific activity (>500 Ci/mmol) labelled mutants are preferred for subsequent use in screening assays.
- Equal aliquots of membrane prep containing the orphan GPCR are distributed in the wells of a multiwell reaction vessel as are equal aliquots of radiolabelled in vitro translated Arg 170 -_> Glu or Asp383 -_> Ter ⁇ -arrestin mutant. Multiple wells are then reacted with different aliquots of test compounds, each aliquot containing a distinct mixture of potential surrogate ligands.
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AU2001249107A AU2001249107A1 (en) | 2000-03-03 | 2001-03-05 | Methods of assaying for g protein-coupled receptor ligands and modulators |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002046763A1 (en) * | 2000-12-04 | 2002-06-13 | Novo Nordisk A/S | Method of identifying compounds capable of acting as agonists or antagonists of g-protein coupled receptors |
WO2003055914A2 (en) * | 2001-12-21 | 2003-07-10 | 7Tm Pharma A/S | Modified receptors for the discovery of therapeutic ligands |
WO2004034054A2 (en) * | 2002-10-11 | 2004-04-22 | 7Tm Pharma A/S | Method for enhancing and prolonging the bioluminescence resonance energy transfer (bret) signal in a bret assay and a substrate solution for use in a bret assay |
WO2004065963A2 (en) * | 2003-01-22 | 2004-08-05 | 7Tm Pharma A/S | Improved beta-arrestin based screening assays |
EP1599492A2 (en) * | 2003-01-17 | 2005-11-30 | Virologic, Inc. | Receptor deorphanization using tagged molecular libraries |
US8999653B2 (en) | 2008-07-31 | 2015-04-07 | Cis-Bio International | Method for detecting membrane protein internalization |
-
2001
- 2001-03-05 AU AU2001249107A patent/AU2001249107A1/en not_active Abandoned
- 2001-03-05 WO PCT/US2001/007304 patent/WO2001067106A2/en active Application Filing
Non-Patent Citations (4)
Title |
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BARAK L S ET AL: "A beta-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 44, no. 272, 31 October 1997 (1997-10-31), pages 27497-27500, XP002076354 ISSN: 0021-9258 * |
BIERI CHRISTOPH ET AL: "Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation" NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, vol. 17, no. 11, November 1999 (1999-11), pages 1105-1108, XP002183971 ISSN: 1087-0156 * |
GUREVICH VSEVOLOD V ET AL: "Visual arrestin binding to rhodopsin: Diverse functional roles of positively charged residues within the phosphorylation-recognition region of arrestin." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 11, 1995, pages 6010-6016, XP002187873 ISSN: 0021-9258 cited in the application * |
KOVOOR ABRAHAM ET AL: "Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 11, 12 March 1999 (1999-03-12), pages 6831-6834, XP002187872 ISSN: 0021-9258 cited in the application * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002046763A1 (en) * | 2000-12-04 | 2002-06-13 | Novo Nordisk A/S | Method of identifying compounds capable of acting as agonists or antagonists of g-protein coupled receptors |
WO2003055914A2 (en) * | 2001-12-21 | 2003-07-10 | 7Tm Pharma A/S | Modified receptors for the discovery of therapeutic ligands |
WO2003055914A3 (en) * | 2001-12-21 | 2003-10-23 | 7Tm Pharma As | Modified receptors for the discovery of therapeutic ligands |
WO2004034054A2 (en) * | 2002-10-11 | 2004-04-22 | 7Tm Pharma A/S | Method for enhancing and prolonging the bioluminescence resonance energy transfer (bret) signal in a bret assay and a substrate solution for use in a bret assay |
WO2004034054A3 (en) * | 2002-10-11 | 2004-07-29 | 7Tm Pharma As | Method for enhancing and prolonging the bioluminescence resonance energy transfer (bret) signal in a bret assay and a substrate solution for use in a bret assay |
EP1599492A2 (en) * | 2003-01-17 | 2005-11-30 | Virologic, Inc. | Receptor deorphanization using tagged molecular libraries |
EP1599492A4 (en) * | 2003-01-17 | 2008-09-24 | Virologic Inc | Receptor deorphanization using tagged molecular libraries |
WO2004065963A2 (en) * | 2003-01-22 | 2004-08-05 | 7Tm Pharma A/S | Improved beta-arrestin based screening assays |
WO2004065963A3 (en) * | 2003-01-22 | 2004-09-10 | 7Tm Pharma As | Improved beta-arrestin based screening assays |
US8999653B2 (en) | 2008-07-31 | 2015-04-07 | Cis-Bio International | Method for detecting membrane protein internalization |
Also Published As
Publication number | Publication date |
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AU2001249107A1 (en) | 2001-09-17 |
WO2001067106A3 (en) | 2002-04-11 |
WO2001067106A8 (en) | 2002-02-14 |
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