JP2011097950A - Endogenous and non-endogenous versions of human g protein-coupled receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a receptor used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists. <P>SOLUTION: The invention relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is unknown (orphan GPCR receptors), and most particularly to mutated (non-endogenous) versions of the human GPCRs for evidence of constitutive activity. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The invention disclosed in this patent specification relates to transmembrane receptors, and more specifically to human G protein coupled receptors, and specifically to establish or enhance constitutive activity of the receptor. It relates to endogenous human GPCRs with particular emphasis on non-endogenous versions of modified GPCRs. Preferably, the modified GPCR is used for direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists with potential applicability as therapeutic agents.

  Although many receptor types exist in humans, the most numerous and therapeutically relevant ones are represented by the type of G protein-coupled receptor (GPCR or GPCRs). It is estimated that there are about 100,000 genes in the human genome, of which about 2%, or 2,000 genes, are estimated to encode GPCRs. Receptors that contain GPCRs for which endogenous ligands have been identified are termed “known” receptors, whereas receptors for which no endogenous ligand has been identified are termed “orphan” receptors. GPCRs are an important field for the development of pharmaceutical products, with about 60% being developed with prescription drugs from about 20 out of 100 known GPCRs.

  GPCRs share a common structural motif. All these receptors have 7 sequences of 22-24 hydrophobic amino acids that form 7 alpha helices, each of which spans the membrane (each span is numbered) (Ie, transmembrane part-1 (TM-1), transmembrane part-1 (TM-1), etc.) transmembrane spirals, transmembrane part-2 and transmembrane part-3, transmembrane Are bound to the outside of the cell membrane, that is, on the “extracellular” side by amino acid chains between the part-4 and the transmembrane part-5 and between the transmembrane part-6 and the transmembrane part-7 (these are “extracellular” "Regions 1, 2 and 3 (referred to as EC-1, EC-2 and EC-3, respectively)). The transmembrane helix consists of a transmembrane part-1 and a transmembrane part-2, a transmembrane part-3 and a transmembrane part-4, and a transmembrane part-5 and a transmembrane part-6. Are also bound inside, i.e. "intracellular" (these are called "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and IC-3, respectively)). The “carboxy” (“C”) terminus of the receptor is in the intracellular space inside the cell, and the “amino” (“N”) terminus of the receptor is in the extracellular space outside the cell.

  In general, when an endogenous ligand binds to a receptor (often referred to as “activation” of the receptor), a concomitant region of the intracellular region that allows coupling between the intracellular region and the intracellular “G protein”. There is a change in formation. It has been reported that GPCRs are “multiple relationships” with respect to G proteins, ie GPCRs can interact with more than one G protein. See Non-Patent Document 1. Although other G proteins exist, Gq, Gs, Gi, Gz and Go are currently identified G proteins. Endogenous ligand-activated GPCR coupling with the G protein initiates a signal transduction cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately leads to cell activation or cell inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.

  Under physiological conditions, GPCRs are present in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. Receptors that are in an inactive state cannot link to intracellular signaling pathways to generate biological responses. Changing the receptor conformation to the active state allows linkage to signal transduction pathways (via G proteins) and generates biological responses.

  A receptor may be stabilized in an active state by a compound such as an endogenous ligand or drug. Recent discoveries, including but not limited to alterations in the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in its active state by mimicking the action of an endogenous ligand that binds to the receptor. Such stabilization by ligand-dependent means is called “constitutive receptor activation”.

Kenakin, T., 43 Life Science 1095 (1988)

(Summary of Invention)
Disclosed herein are endogenous and non-endogenous forms of human GPCRs and uses thereof.
The present invention also provides the following items.
(Item 1) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 2.
(Item 2) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 1.
(Item 3) A plasmid comprising the vector of SEQ ID NO: 1 and cDNA.
(Item 4) A host cell comprising the plasmid of item 3.
(Item 5) G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 4.
(Item 6) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 5.
(Item 7) A plasmid comprising the vector of SEQ ID NO: 3 and cDNA.
(Item 8) A host cell comprising the plasmid of item 7.
(Item 9) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 6.
(Item 10) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 9.
(Item 11) A plasmid comprising the vector of SEQ ID NO: 5 and cDNA.
(Item 12) A host cell comprising the plasmid of item 11.
(Item 13) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 8.
(Item 14) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 13.
(Item 15) A plasmid comprising the vector of SEQ ID NO: 7 and cDNA.
(Item 16) A host cell comprising the plasmid of item 15.
(Item 17) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 10.
(Item 18) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 17.
(Item 19) A plasmid comprising the vector of SEQ ID NO: 9 and cDNA.
(Item 20) A host cell comprising the plasmid of item 19.
(Item 21) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 12.
(Item 22) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 21, comprising the amino acid sequence of SEQ ID NO: 84.
(Item 23) A plasmid comprising the vector of SEQ ID NO: 11 and cDNA.
(Item 24) A host cell comprising the plasmid of item 23.
(Item 25) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 14.
(Item 26) A non-endogenous, constitutively activated form of the G protein coupled receptor of item 25 comprising the amino acid sequence of SEQ ID NO: 88.
(Item 27) A plasmid comprising the vector of SEQ ID NO: 13 and cDNA.
(Item 28) A host cell comprising the plasmid of item 27.
(Item 29) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 16.
(Item 30) A non-endogenous, constitutively activated form of the G protein coupled receptor of item 29 comprising the amino acid sequence of SEQ ID NO: 92.
(Item 31) A plasmid comprising the vector of SEQ ID NO: 15 and cDNA.
(Item 32) A host cell comprising the plasmid of item 31.
(Item 33) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 18.
(Item 34) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 33.
(Item 35) A plasmid comprising the vector of SEQ ID NO: 17 and cDNA.
(Item 36) A host cell comprising the plasmid of item 35.
(Item 37) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 20.
(Item 38) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 37.
(Item 39) A plasmid comprising the vector of SEQ ID NO: 19 and cDNA.
(Item 40) A host cell comprising the plasmid of item 39.
(Item 41) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 22.
(Item 42) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 41.
(Item 43) A plasmid comprising the vector of SEQ ID NO: 21 and cDNA.
(Item 44) A host cell comprising the plasmid of item 43.
(Item 45) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 24.
(Item 46) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 45.
(Item 47) A plasmid comprising the vector of SEQ ID NO: 23 and cDNA.
48. A host cell comprising the plasmid of item 47.
(Item 49) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 26.
(Item 50) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 49.
(Item 51) A plasmid comprising the vector of SEQ ID NO: 25 and cDNA.
(Item 52) A host cell comprising the plasmid of item 51.
(Item 53) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 28.
(Item 54) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 53.
(Item 55) A plasmid comprising the vector of SEQ ID NO: 27 and cDNA.
(Item 56) A host cell comprising the plasmid of item 55.
(Item 57) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 30.
(Item 58) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 57.
(Item 59) A plasmid comprising the vector of SEQ ID NO: 29 and cDNA.
(Item 60) A host cell comprising the plasmid of item 59.
(Item 61) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 32.
(Item 62) A non-endogenous, constitutively activated form of the G protein coupled receptor of item 61 comprising the amino acid sequence of SEQ ID NO: 96.
(Item 63) A plasmid comprising the vector of SEQ ID NO: 95 and cDNA.
(Item 64) A host cell comprising the plasmid of item 63.
(Item 65) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 34.
(Item 66) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 65.
(Item 67) A plasmid comprising the vector of SEQ ID NO: 33 and cDNA.
68. A host cell comprising the plasmid of item 67.
(Item 69) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 36.
(Item 70) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 69.
(Item 71) A plasmid comprising the vector of SEQ ID NO: 35 and cDNA.
72. A host cell comprising the plasmid of item 71.
(Item 73) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 38.
(Item 74) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 73.
(Item 75) A plasmid comprising the vector of SEQ ID NO: 37 and cDNA.
76. A host cell comprising the plasmid of item 75.
(Item 77) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 40.
(Item 78) A non-endogenous, constitutively activated form of the G protein-coupled receptor of item 77.
(Item 79) A plasmid comprising the vector of SEQ ID NO: 39 and cDNA.
(Item 80) A host cell comprising the plasmid of item 79.

Control is the second messenger IP ₃ production described from ( "CMV") compared to the endogenous type RUP12 ( "RUP12"). 2 is a graphical representation of the results of a second messenger cell-based cyclic AMP assay that provides a comparison of constitutive signaling between endogenous RUP13 (“RUP13”) and a control vector (“CMV”). FIG. 6 is a graphical representation of signals measured by comparing CMV, endogenous RUP13 (“RUP13 wt”) and non-endogenous, constitutively activated RUP13 (“RUP13 (A268K)”) using the 8XCRE-Luc reporter plasmid. RUP13: is a graphic representation of the results of the [ ³⁵ S] GTPγS assay giving the results of comparison of constitutive signaling by “Gs fusion protein” (“RUP13-Gs”) and control vector (“CMV”). FIG. 5 is a graphical representation of signals measured by comparing CMV, endogenous RUP14 (“RUP14 wt”) and non-endogenous, constitutively activated RUP13 (“RUP14 (L246K)”) using the 8XCRE-Luc reporter plasmid. FIG. 5 is a graphical representation of signals measured by comparing CMV, endogenous RUP15 (“RUP15 wt”) and non-endogenous, constitutively activated RUP15 (“RUP15 (A398K)”) using the 8XCRE-Luc reporter plasmid. Second messenger cells that give a comparison of the constitutive signaling of endogenous RUP15 (“RUP15 wt”), a non-endogenous, constitutively activated form of RUP15 (“RUP15 (A398K)”) and a control vector (“CMV”) FIG. 4 is a graphical representation of the results of a basic cyclic AMP assay. RUP15: Graphic representation of the results of the [ ³⁵ S] GTPγS assay giving comparative results of constitutive signaling by the “Gs fusion protein” (“RUP15-Gs”) and the control vector (“CMV”). Control is the second messenger IP ₃ production described from ( "CMV") compared to the endogenous type RUP17 ( "RUP17"). FIG. 2 is an illustration of second messenger IP ₃ production from endogenous RUP21 (“RUP21”) compared to a control (“CMV”). FIG. 6 is a graphical representation of signals measured by comparing CMV, endogenous RUP23 (“RUP23 wt”) and non-endogenous, constitutively activated RUP23 (“RUP23 (W275K)”) using the 8XCRE-Luc reporter plasmid. FIG. 4 is a graphical representation of results from primary screening of various candidate compounds against RUP13. The result of “Compound A” is shown in well A2 and the result of “Compound B” is shown in well G9.

(Detailed description of the invention)
The scientific literature created in connection with receptors employs a number of terms relating to ligands that have various effects on receptors. For clarity and unity, the following definitions are used throughout this patent specification. In the event that these definitions conflict with other definitions for these terms, the definitions below shall prevail.

  “Agonist” shall mean a substance (eg, ligand, candidate compound) that activates an intracellular response when the substance binds to a receptor or enhances GTP binding to the membrane.

The “amino acid abbreviations” used herein are listed in Table A.

Table A
Alanine ALA A
Arginine ARG R
Asparagine ASN N
Aspartic acid ASP D
Cysteine CYS C
Glutamic acid GLU E
Glutamine GLN Q
Glycine GLY G
Histidine HISH
Isoleucine ILE I
Leucine LEU L
Lysine LYS K
Methionine MET M
Phenylalanine PHE F
Proline PRO P
Serine SER S
Threonine THR T
Tryptophan TRP W
Tyrosine TYR Y
Valine VAL V

“Partial agonists” activate intracellular responses to a lesser extent / range than the effects of the agonist when they are bound to the receptor, or GTP to the membrane to a lesser extent / range than the action of the agonist. It shall mean a substance that enhances binding (eg, a ligand, a candidate compound).

  An “antagonist” competitively binds to a receptor at the same site as an agonist, but does not activate an intracellular response initiated by the active form of the receptor, thereby causing an intracellular response by an agonist or partial agonist. It is intended to mean a substance that can be inhibited (eg, a ligand, a candidate compound). An “antagonist” does not reduce the baseline intracellular response in the absence of an agonist or partial agonist.

  A “candidate compound” is intended to mean a molecule (for example, a chemical compound, but not limited thereto) that can be applied to a screening technique. Preferably, the phrase “candidate compound” is determined in advance by an indirect identification process (“indirectly identified compound”) and is selected from the group consisting of an inverse agonist, agonist or antagonist for the receptor Does not include compounds generally known as compounds, more preferably does not include indirectly identified compounds previously determined to have therapeutic efficacy in at least one mammal, and most preferably It does not include indirectly identified compounds previously determined to have therapeutic efficacy in humans.

  “Composition” means a substance comprising at least one component. A “pharmaceutical composition” is an example of a composition.

  “Compound potency” shall mean a measure of a compound's ability to inhibit or stimulate receptor functionality as opposed to receptor binding affinity. Exemplary means for detecting compound potency are disclosed in the Examples section herein.

  A “codon” is generally a nucleoside that binds to a phosphate group and encodes an amino acid when translated (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)). Means a group of 3 nucleotides (or nucleotide equivalents) comprising

  “Constitutively activated receptor” shall mean a receptor that undergoes constitutive receptor activation. The constitutively activated receptor can be endogenous or non-endogenous.

  “Constitutive receptor activation” shall mean stabilization of a receptor in an active state by means other than binding of the receptor to its endogenous ligand or chemical equivalent thereof.

  “Contact” or “contacting” shall mean bringing at least two parts together, whether in an in vitro or in vivo system.

  The term “directly identify” or “direct identification” in relation to the phrase “candidate compound” refers to constitutively activated receptors, preferably constitutively activated orphan receptors, and most preferably constitutively activated G protein coupled cells. It is meant to screen candidate compounds against the surface orphan receptor and assess the compound potency of this compound. This phrase is in no way construed or understood to be encompassed by or includes the phrases “indirectly identified” or “indirectly identified”.

  “Endogenous” shall mean a substance that is naturally produced by a mammal. For example, without limitation, “endogenous” with respect to the term “receptor” shall mean that which is naturally produced by a mammal (eg, but not limited to a human) or a virus. Conversely, the term “non-endogenous” within this scope shall mean that which is not naturally produced by a mammal (eg, but not limited to a human) or virus. For example, but not limited to, a receptor that is not constitutively active in its endogenous form but becomes constitutively active when manipulated is referred to herein as a “non-endogenous, constitutively activated receptor”. Is most preferably called. Both terms can be used to describe both “in vivo” and “in vitro” systems. For example, but not limited to, in screening methods, endogenous or non-endogenous receptors may be associated with in vivo screening systems. Another example, but not limited to, screening of candidate compounds by means of in vivo systems is applicable when the mammalian genome is engineered to contain non-endogenous, constitutively activated receptors. is there.

  “G protein-coupled receptor fusion protein” and “GPCR fusion protein” are within the scope of the invention disclosed herein, each of at least one G protein, most preferably such G protein. Means an endogenous, constitutively activated GPCR or a non-endogenous, constitutively activated GPCR fused to an alpha (α) subunit, which is a subunit that binds GTP Here, however, the G protein is preferably of the same type as the G protein inherently coupled to the endogenous orphan GPCR. For example, without limitation, a GPCR fusion protein based on a particular GPCR includes a GPCR fused to Gsα where, in the endogenous state, the G protein “Gsα” is the predominant G protein coupled to the GPCR. Would be a non-endogenous protein. In some cases, the non-dominant G protein can be fused to a GPCR, as described below. The G protein can be fused directly to the c-terminus of the constitutively active GPCR or there may be a spacer between the two.

  “Host cell” shall mean a cell into which a “plasmid” and / or “vector” can be incorporated. In the case of a prokaryotic “host cell”, the “plasmid” is typically replicated as an autonomous molecule as a “host cell” replica (so in general, a “plasmid” is a eukaryotic “host cell”. Isolated for introduction into). In the case of a eukaryotic “host cell”, the “plasmid” is incorporated into the cellular DNA of the “host cell” so that the “plasmid” is replicated when the eukaryotic “host cell” is replicated. . Preferably, for the purposes of the invention disclosed herein, the “host cell” is eukaryotic, more preferably a mammal, and most preferably 293, 293T and COS-7. Selected from the group consisting of cells.

  “Indirect identification” or “indirectly identified” refers to a receptor for identification of an endogenous ligand specific for the endogenous receptor, determination of what interferes with and / or competes with the ligand-receptor interaction. It means a traditional approach to drug discovery process that involves screening candidate compounds and assessing the potency of compounds to affect at least one second messenger pathway associated with activated receptors.

  “Inhibit” or “inhibit” in the context of the term “reaction” shall mean that the reaction is reduced or prevented in the presence of the compound as opposed to in the absence of the compound.

  An “inverse agonist” is a receptor that binds to either the endogenous form of the receptor or a constitutively activated form of the receptor and is below the normal activity baseline level observed in the absence of an agonist or partial agonist. It is intended to mean a substance (eg, ligand, candidate compound) that inhibits the basal intracellular reaction initiated by the active form of or reduces GTP binding to the membrane. Preferably, the basal intracellular response is inhibited by at least 30%, more preferably at least 50%, and most preferably at least 75% in the presence of the inverse agonist compared to the baseline response in the absence of the inverse agonist. The

  “Known receptor” shall mean an endogenous receptor for which an endogenous ligand specific for that receptor has been identified.

  “Ligand” shall mean an endogenous and naturally occurring molecule specific for an endogenous and naturally occurring receptor.

  A “variant” or “mutation” with respect to a nucleic acid and / or amino acid sequence of an endogenous receptor is an endogenous such that a variant of the endogenous, non-constitutively activated receptor reveals constitutive activation of the receptor. It shall mean a specific change or multiple changes to the sex sequence. For a specific sequence equivalent, (a) the level of constitutive activation of the next mutated form of the human receptor is essentially the same as revealed by the first mutation of the receptor, and ( b) a percentage of sequence (amino acid and / or nucleic acid) homology between the next variant of the receptor and the first variant of the receptor is at least about 80%, more preferably at least about 90% and most preferably If at least about 95%, the next variant of the human receptor is considered equivalent to the first variant of the human receptor. Ideally, and most preferred cassettes disclosed herein to achieve constitutive activation include a single amino acid and / or codon change between the endogenous and non-endogenous forms of the GPCR. Due to the fact, the percentage of sequence homology should be at least 98%.

  A “non-orphan receptor” is an endogenous, naturally-occurring molecule that is specific for an endogenous, naturally-occurring ligand, where binding of the ligand to the receptor activates an intramolecular signaling pathway. Shall mean.

  “Orphan receptor” is intended to mean an endogenous receptor for which no specific endogenous ligand has been identified or known.

  “Pharmaceutical composition” shall mean a composition comprising at least one active ingredient, wherein the composition is for a particular efficacious outcome in a mammal (eg, but not limited to a human). Can be tested. One of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has the desired efficacious outcome based on the needs of the skilled worker.

  “Plasmid” shall mean a combination of “vector” and cDNA. In general, a “plasmid” is introduced into a “host cell” for the purpose of replicating and / or expressing cDNA as a protein.

“Second messenger” shall mean an intracellular response produced as a result of receptor activation. Second messengers can include, for example, inositol triphosphate (IP ₃ ), diacylglycerol (DAG), cyclic AMP (cAMP), and cyclic GMP (cGMP). Second messenger response can be measured for determination of receptor activation. Further, the second messenger response can be measured for direct identification of candidate compounds including, for example, inverse agonists, agonists, partial agonists and antagonists.

  “Stimulation” or “stimulate” in relation to the term “response” shall mean that the response increases in the presence of the compound as opposed to the absence thereof.

  An associated “vector” of cDNA is intended to mean a circular DNA capable of incorporating at least one cDNA and capable of being incorporated into a “host cell”.

The order of the following sections is determined for the sake of explanation efficiency and is not intended and should not be construed as limiting the following disclosure or claims.
A. INTRODUCTION Traditional studies of receptors have a priori assumption (based on history) that endogenous ligands must be identified first before discovery can proceed until discovery of antagonists and other molecules that can affect the receptor. I was always moving forward. Even when antagonists were first discovered, the search immediately proceeded to the search for endogenous ligands. This mode of thought continued in receptor research even after the discovery of constitutively activated receptors. What has not been recognized before is that the most useful for the discovery of receptor agonists, partial agonists, and inverse agonists is the active state of the receptor. For diseases resulting from overactive or underactive receptors, what are desired for therapeutic agents are compounds that reduce or enhance the activity of the receptor, respectively, It is not necessarily an agent that is an antagonist to an endogenous ligand. This is because compounds that reduce or enhance the activity of the active receptor state need not bind to the same site as the endogenous ligand. Thus, any search for therapeutic compounds, as taught by the methods of the present invention, should begin with screening compounds for ligand-independent activity states.
B. Identification of human GPCRs Human Genome Project research has led to the identification of a great deal of information about nucleic acid sequences located within the human genome. In the case of this study, genome sequence information is
Provided with no understanding or recognition as to whether specific genomic sequences contain or do not contain open reading frame information that translates human proteins. Several methods for identifying nucleic acid sequences within the human genome are within the recognition of those having ordinary skill in the art. For example, but not by way of limitation, the various human GPCRs disclosed herein have been discovered by examining the GeneBank ^™ database. Table B below lists some of the endogenous GPCRs we discovered, along with other GPCRs that are homologous to the disclosed GPCRs.

  Receptor homology is useful to gain an assessment of the role of receptors within the human body. In accordance with the progress described herein, we will disclose techniques for mutating these receptors to establish non-endogenous, constitutively activated forms of these receptors.

  The techniques disclosed herein also apply to other human, orphan GPCRs known in the art, which will become apparent as the description proceeds.

C. Receptor Screening Screening candidate compounds for the non-endogenous, constitutively activated forms of human GPCRs disclosed herein does not require the use of the receptor's endogenous ligand, and this cell surface receptor Allows direct identification of acting candidate compounds. Conventional and often commercially available techniques can be used to determine the region in the body where the endogenous form of the human GPCR disclosed herein is expressed and / or overexpressed. These techniques can also be used to determine associated disease / disorder states associated with receptor expression and / or overexpression, and such methods are disclosed herein.

  The creation of mutations that will reveal the constitutive activation of the human GPCR disclosed herein is based on the distance from the proline residue assumed to be located within TM6 of the GPCR. This algorithmic technique is disclosed in co-pending and commonly assigned patent specification PCT application number PCT / US99 / 23938, published as WO 00/22129, dated April 20, 2000, which is described in this book. It is incorporated herein by reference with other patent documents listed in the specification. This algorithmic technique is not asserted based on the conventional sequence “alignment” and is derived from the TM6 proline residues described above (or, of course, endogenous constitutive substitutions of such proline residues). Determined by the specified distance. Such activation may be obtained by mutating an amino acid located 16 amino acid residues from this residue (presumably located in the IC3 region of the receptor) to the most preferred lysine residue. Other amino acid residues may be useful for mutations at this position to achieve this goal.

D. Disease / Disorder Identification and / or Selection As described in detail below, most preferably non-endogenous, inverse agonists and agonists for constitutively activated GPCRs can be identified by the methodology of the present invention. Such inverse agonists and agonists are ideal candidates as lead compounds in drug discovery programs for treating diseases associated with this receptor. The ability to directly identify inverse agonists for GPCRs, thereby enabling the development of pharmaceutical compositions, allows for the search for diseases and disorders associated with GPCRs. For example, the scanning of both disease and normal tissue samples for the presence of GPCRs has gone beyond tracking along scholarly experiments or pathways to identify endogenous ligands to specific GPCRs. Tissue scans can be performed over a wide range of healthy and diseased tissues. Such tissue scans provide a preferred first step when associating specific receptors with diseases and / or disorders.

  Preferably, the DNA sequence of the human GPCR is used to generate a probe for (a) dot-blot analysis on tissue-mRNA and / or (b) RT-PCT identification of receptor expression in tissue samples. used. The presence of the receptor in the tissue source, or the presence of a receptor at a higher concentration in the diseased tissue compared to the diseased tissue or normal tissue, but is not limited to the treatment strategy and the disease associated with the disease. It can preferably be used to identify correlations. Receptors can be similarly localized to the area of the organ by this technique. Based on the known function of the specific tissue in which the receptor is localized, the putative functional role of the receptor can be inferred.

E. Screening candidate compounds Comprehensive GPCR screening assay technique When a G protein receptor becomes constitutively active, it binds to G proteins (eg, Gq, Gs, Gi, Gz, Go) and stimulates GTP binding to G proteins. Thus, the G protein acts as a GTPase and slowly hydrolyzes GTP to GDP, thereby deactivating the receptor under normal conditions. However, constitutively activated receptors continue to exchange GDP for GTP. [ ³⁵ S] GTPγS, a non-hydrolyzable analog of GTP, can be used to measure enhanced binding to membranes expressing constitutively activated receptors. It has been reported that [ ³⁵ S] GTPγS can be used to measure G protein coupling to the membrane in the absence or presence of a ligand. Among other examples well known and usable by those skilled in the art, an example of this measurement was reported in 1995 by Traynor and Nahorski. A preferred use of this assay system is the initial screening of candidate compounds, which are comprehensive for all G protein coupled receptors regardless of the specific G protein that interacts with the intracellular domain of the receptor. This is because it can be applied.
2. Specific GPCR Screening Assay Techniques Candidate compounds are identified using a “global” G protein-coupled receptor assay (ie, an assay to select compounds that are agonists, partial agonists, or inverse agonists) It is preferable to perform screening to confirm that interaction has occurred at the receptor site. For example, a compound identified in a “global” assay will not bind to the receptor, but will simply “uncouple” the G protein from the intracellular domain.

a. Gs, Gz and Gi
Gs stimulates the enzyme adenylate cyclase. Gi (and Gz and Go), on the contrary, inhibits this enzyme. Adenylate cyclase catalyzes the conversion of ATP to cAMP. Thus, constitutively activated GPCRs that couple to Gs proteins are associated with increased cellular levels of cAMP. Conversely, constitutively activated GPCRs coupled to Gi (or Gz, Go) proteins are associated with reduced cellular levels of cAMP. Generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt 8, From Neuron To Brain (3rd Ed.) Nichols, JG et al eds, Sinauer Associates, Inc. (1992) See). Thus, an assay that detects cAMP can be used to determine whether a candidate compound is, for example, an inverse agonist for a receptor (ie, such a compound would reduce the level of cAMP). Various methods known in the art can be used to measure cAMP. The most preferred method is by the use of anti-cAMP antibodies in an ELISA based format. Another type of assay that can be used is a whole cell second messenger reporter based assay. A promoter on a gene drives the expression of a protein encoding a particular gene. Cyclic AMP promotes the binding of cAMP-responsive DNA binding protein or transcription factor (CREB) to drive gene expression, which then binds to the promoter at a specific site called cAMP-responsive factor and directs gene expression. To drive. A reporter system can be constructed having a promoter containing multiple cAMP responsive elements in front of a reporter gene, eg, β-galactosidase or luciferase. Thus, constitutively activated Gs-coupled receptors cause cAMP accumulation and reporter protein expression which in turn activate the gene. Reporter proteins such as β-galactosidase or luciferase can then be detected using standard biochemical assays (Chenet al. 1995).

b. Go and Gq
Go and Gq are associated with the activation of the enzyme phospholipase C, which then hydrolyzes the phospholipid PIP ₂ and produces two intracellular messengers, diacycloglycerol (DAG) and inositol 1,4,5 Liberates triphosphoric acid (IP ₃ ). Increased accumulation of IP ₃ is associated with activation of Gq and Go related receptors. In general, Chapter 8 of “Indirect Mechanisms of Synaptic Transmission,” Chpt 8, From Neuron To Brain (3rdEd.) Nichols, JG et al eds, Sinauer Associates, Inc. (1992) reference. Assays that detect IP ₃ accumulation can be used to determine whether a candidate compound is, for example, an inverse agonist for a Gq or Go-related receptor (ie, such a compound reduces the level of IP ₃ ). . Gq-related receptors can also be tested using an AP1 reporter assay in which Gq-dependent phospholipase C causes activation of a gene containing the AP1 factor. Thus, activated Gq-related receptors reveal an increase in the expression of this gene, whereby the inverse agonist reveals a decrease in this expression, and the agonist reveals an increase in this expression. Commercially available assays are available for this assay.

3. GPCR fusion proteins Use of endogenous, constitutively active orphan GPCRs or non-endogenous, constitutively activated orphan GPCRs for use in screening candidate compounds for direct identification of inverse agonists, agonists and partial agonists The definition provides an interesting screening problem that the receptor is active even in the absence of endogenous ligand binding. Thus, such compounds are inverse agonists, agonists, partial agonists, for example to distinguish between non-endogenous receptors in the presence of a candidate compound and non-endogenous receptors in the absence of the compound For the purpose of such a distinction that allows an understanding as to whether or not it affects such a receptor, it is preferred to use a method that can enhance such a distinction. A preferred method is the use of “GPCR fusion proteins”.

  In general, once it has been determined that a non-endogenous orphan GPCR has been constitutively activated using the assay techniques described above (and others), the dominant G protein coupled to the endogenous GPCR is determined. it can. Coupling of the G protein to the GPCR provides a signal transduction pathway that can be assessed. Since it is most preferred that the screening be performed using a mammalian expression system, such a system is expected to have an endogenous G protein therein. Thus, by definition, in such systems, non-endogenous, constitutively activated orphan GPCRs continuously signal. In this regard, it is preferred that this signal is enhanced, for example in the presence of an inverse agonist at the receptor, and in particular within the scope of the screening, it may be possible to more easily distinguish between receptors contacted with the inverse agonist. More preferably, the signal is enhanced so that

  A “GPCR fusion protein” is intended to enhance the potency of a G protein coupled to a non-endogenous GPCR. “GPCR fusion proteins” are preferred for screening using non-endogenous, constitutively activated GPCRs because such methods increase the signal most preferably used in this screening technique. This is important in order to easily obtain a significant “signal to noise ratio”. Such significant ratios are advantageously introduced in the screening of candidate compounds disclosed herein.

  The construction of constructs useful for the expression of “GPCR fusion proteins” is within the recognition of those having ordinary skill in the art. Commercially available expression vectors and systems provide a variety of methods that can meet the specific needs of researchers. An important criterion for such a “GPCR fusion protein” construct is that both the endogenous GPCR sequence and the G protein sequence are in-frame (preferably the sequence of the endogenous GPCR is upstream of the G protein sequence). And the "stop" codon of the GPCR must be deleted or replaced so that the G protein can also be expressed during the expression of the GPCR. The GPCR may be linked directly to the G protein, or there may be between two spacer residues (preferably no more than about 12, this number will be understood by those of ordinary skill in the art. Can be easily confirmed). We prefer the use of spacers (for convenience) where some unused restriction sites effectively become spacers during expression. Most preferably, G proteins that couple to non-endogenous GPCRs are identified prior to creation of a “GPCR fusion protein” construct. Since only a few G proteins have been identified, constructs comprising the sequence of the G protein (ie, a universal G protein construct) can be used for insertion of the endogenous GPCR sequences within this specification. This provides efficiency within the scope of large-scale screening of a variety of different endogenous GPCRs with different sequences.

  As noted above, constitutively activated GPCRs coupled to Gi, Gz and Go are expected to inhibit cAMP formation, making assays based on these types of GPCRs of interest interesting (ie cAMP signals are Decreases upon activation, thus, for example, direct identification of inverse agonists (this will further reduce this signal). As disclosed herein, a “GPCR fusion protein” that is not based on an endogenous G protein of an endogenous GPCR in a study to establish usable cyclase-based assays for these types of receptors. We confirmed that it is possible to create. Thus, for example, an endogenous Gi-coupled receptor can be fused to a Gs protein, and when such a fusion construct is expressed, the endogenous GPCR is not a “native” Gi protein, for example with Gs We believe that we can “drive” or “force” to conjugate and establish cyclase based assays. Thus, for Gi, Gz and Go-coupled receptors, we found that when a “GPCR fusion protein” is used and the assay is based on detection of adenylate cyclase activity, the fusion construct is Gs (or the enzyme adenyl). It is preferably established using an equivalent G protein that stimulates the formation of acid cyclase.

  A “G protein fusion” construct using a “Gq protein” fused to a “Gs, Gi, Gz or Go protein” is equally effective. The most preferred fusion construct can be achieved using the “Gq protein”, wherein the first six amino acids of the G protein α-subunit (“Gαq”) are deleted and the last of the C-terminus of Gαq. Five amino acids are replaced with the corresponding amino acid of Gα of the relevant G protein. For example, a fusion construct can have Gq (deletion of 6 amino acids) fused to a “Gi protein”, resulting in a “Gq / Gi fusion construct”. We allow this fusion construct to couple an endogenous Gi-coupled receptor to a non-endogenous G protein, Gq, so that second messengers such as inositol triphosphate or diacylglycerol can be measured instead of cAMP production. I believe to force.

4). Target Gi coupled GPCR using signal enhancer Gs coupled GPCR
Cotransfection (cAMP-based assay)
Gi-coupled receptors are known to inhibit adenylate cyclase, thus reducing the level of cAMP production, which can make evaluation of cAMP levels interesting. An effective means of measuring the reduction of cAMP production as an indicator of constitutive activation of receptors that are primarily coupled to Gi upon activation is a signal enhancer, such as Gs upon activation using a Gi-linked GPCR. This can be achieved by co-transfecting a primarily conjugated non-endogenous, constitutively activated receptor (eg, TSHR-A623I disclosed below). As is apparent, constitutive activation of Gs-coupled receptors can be determined based on increased production of cAMP. Constitutive activation of Gi-coupled receptors leads to a decrease in cAMP production. The cotransfection method is therefore intended to take advantage of these “opposite” effects. For example, co-transfection of a non-endogenous, constitutively activated Gs-coupled receptor (“signal enhancer”) with an endogenous Gi-coupled receptor (“target receptor”) provides a reference cAMP signal (ie, This “reduction” is associated with an intrinsic increase in cAMP levels established by constitutively activated Gs-coupled receptor signal enhancers, although Gi-coupled receptors reduce cAMP levels). In so doing, by co-transfection of the signal enhancer and a constitutively activated form of the target receptor, cAMP is further reduced (reference) by the increased functional activity of the Gi target (ie, this reduces cAMP). Against).

  Screening candidate compounds using a cAMP-based assay can then be accomplished under two conditions. The first condition leads to an “opposite” effect with respect to the Gi-coupled target receptor, ie an inverse agonist of the Gi-coupled target receptor increases the measured cAMP signal, whereas an agonist of the Gi-coupled target receptor produces this signal. descend. The second condition is that, as is clear, candidate compounds identified directly using this method should be evaluated independently to confirm that they do not target the signal enhancing receptor. (This can be done before or after screening for co-transfected receptors).

F. Medicinal Chemistry Generally, but not necessarily, direct identification of candidate compounds is preferably performed in conjunction with compounds generated via combinatorial chemistry techniques, which allows thousands of compounds to be analyzed for this analysis. Randomly prepared. In general, the result of such screening is a compound having a unique core structure. These compounds are then preferably subjected to additional chemical modifications around the preferred core structure to further enhance their pharmaceutical properties. Such techniques are known to those skilled in the art and are not dealt with in detail herein.

G. Pharmaceutical Compositions Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known to those skilled in the art. Suitable pharmaceutically acceptable carriers are available to those skilled in the art. See, for example, “Remington's Pharmaceutical Sciences, 16th. Edition, 1980, Mack Publishing Co., (Oslo et al., Eds.)”.

H. Other Benefits A preferred use of the non-endogenous forms of human GPCRs disclosed herein is for direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as drugs) These types of human GPCRs can also be used in research settings. For example, in vivo and in vitro systems incorporating GPCRs have been applied to further elucidate and understand the role that these receptors play in both normal and diseased human states, and to understand the signaling cascade Can be used to understand the role of genetic activation. The value of non-endogenous human GPCRs is due to their unique characteristics that non-endogenous human GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand is identified, Their usefulness as a research tool will increase. Other uses of the disclosed receptors will be apparent to those skilled in the art, among others, upon review of this specification.

  The following examples are presented for purposes of illustration of the invention and are not intended to be limiting. Although specific nucleic acid and amino acid sequences are disclosed herein, one of ordinary skill in the art can make the same or substantially similar results as reported below with minor modifications to these sequences. Has the ability to gain. Conventional methods of application or understanding of sequence cassettes from one sequence to another (eg, rat receptor to human receptor, or human receptor A to human receptor B) generally apply to sequence alignment techniques. Based on this, the sequences are aligned in the study to determine a common region. The mutation methods disclosed herein are not dependent on this method, but instead are based on algorithmic methods and positional distances from conserved proline residues located within the TM6 region of human GPCRs. Once this method is established, those skilled in the art have the ability to make minor changes to this to obtain essentially the same results (ie, constitutive activation) as disclosed herein. I believe that. Such modification methods are considered to be within the scope of the present disclosure.

Example 1
Endogenous human GPCR
1. Identification of human GPCRs The disclosed endogenous human GPCRs were identified based on a survey of GeneBank ^™ database information. While searching the database, the following cDNA clones were identified as apparent below (Table C):

2. Full length cloning a. hRUP8 (SEQ ID NOS: 1 and 2)
The disclosed human RUP8 was identified based on the use of EST database (dbEST) information. While investigating dbEST, a cDNA clone with accession number AL121755 was identified as encoding a novel GPCR. The following PCR primers were used as templates for RT-PCR using human testis marathon-ready cDNA (Clontech).
5′-CTTGCAGACATCATACCGCAGCC-3 ′ (SEQ ID NO: 41, sense) and 5′-GTGATGCTCTGAGTACTGGACTACT-3 ”(SEQ ID NO: 42, antisense)
PCR was performed using Advantage cDNA polymerase (Clontech, according to manufacturing guidelines) in a 50 ul reaction by the following cycle: 94 ° C. for 30 seconds, 94 ° C. for 10 seconds, 65 ° C. for 20 seconds, 1.5 minutes 72 ° C and 7 minutes at 72 ° C. Cycles 2-4 were repeated 35 times.

  The 1.2 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). See SEQ ID NO: 1, and the deduced amino acid sequence of RUP8 is shown in SEQ ID NO: 2.

b. hRUP9 (SEQ ID NOs: 3 and 4)
The disclosed human RUP9 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC011375 was identified as the human genomic sequence from chromosome 5. Full length RUP9 was used as primer 5′-GAAGCTGTGAAGAGTGATGC-3 ′ (SEQ ID NO: 43, sense),
5′-GTCAGCAAATTGATAAGCAGCAG-3 ′ (SEQ ID NO: 44, antisense)
And cloned by PCR using human genomic DNA (Promega) as template. Taq Plus Precision polymerase (Stratagene) was used in a 100 μl reaction with 5% DMSO for amplification by 35 repetitions of steps 2 to 4 in the following cycle: 1 min at 94 ° C. 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 2 minutes, and 72 ° C for 5 minutes.

  The 1.3 Kb PCR fragment was isolated and cloned from a 1% agarose gel into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 3, and the deduced amino acid sequence of RUP8 is set forth in SEQ ID NO: 4. The sequence of the RUP9 clone isolated from human genomic DNA matched the sequence obtained from the database.

c. hRUP10 (SEQ ID NOS: 5 and 6)
The disclosed human RUP10 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC008754 was identified as the human genomic sequence from chromosome 19. The full length RUP10 was used as primers 5'-CCATGGGGACACATTCTGTCAGCTACCG-3 '(SEQ ID NO: 45, sense) and 5'-GCTATGCCCTGAAGCCAGTCTTTGTG-3' (SEQ ID NO: 46, antisense).
And cloned by RT-PCR using human leukocyte marathon ready cDNA (Clontech) as template. Advantage cDNA polymerase (Clontech) was used for amplification by 35 iterations of steps 2 to 4 in the following cycle in a 50 μl reaction: 94 ° C. for 30 seconds, 94 ° C. for 10 seconds, 62 ° C. for 20 seconds, 1 72 minutes at 72 ° C. and 7 minutes at 72 ° C. A 1.0 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (PEBiosystem). The nucleic acid sequence of the novel human receptor RUP10 is set forth in SEQ ID NO: 5 and its deduced amino acid sequence is set forth in SEQ ID NO: 6.

d. hRUP11 (SEQ ID NOs: 7 and 8)
The disclosed human RUP11 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC013396 was identified as the human genomic sequence from chromosome 2. The full-length RUP11 was used as a primer 5′-CCAGGATGTTGTGTCCACCGTGGTGGC-3 ′ (SEQ ID NO: 47, sense),
5′-CACAGCCGCTGCAGCCCTGCAGCTGGC-3 ′ (SEQ ID NO: 48, antisense)
And cloned by PCR using human genomic DNA (Clontech) as template. Tack Plus Precision DNA Polymerase (Stratagene) was used in a 50 μl reaction for amplification by 35 cycles of Step 2 to Step 4 in the following cycle: 3 minutes at 94 ° C., 20 seconds at 94 ° C., 20 seconds at 67 ° C. 1.5 minutes at 72 ° C and 7 minutes at 72 ° C. The 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (PEBiosystem). The nucleic acid sequence of the novel human receptor RUP11 is set forth in SEQ ID NO: 7 and its deduced amino acid sequence is set forth in SEQ ID NO: 8.

e. hRUP12 (SEQ ID NOs: 9 and 10)
The disclosed human RUP12 has been identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AP000808 was identified as encoding a novel GPCR with significant homology to rat RTA and human mas1 oncogene GPCRs. Full length RUP12 was used as primer 5′-CTTCCTCTCGTAGGGATGAACCAGAC-3 ′ (SEQ ID NO: 49, sense)
5′-CTCGCACAGGTGGGGAAGCACCTGTG-3 ′ (SEQ ID NO: 50, antisense)
And cloned by PCR using human genomic DNA (Clontech) as template. Tack Plus Precision DNA Polymerase (Stratagene) was used for amplification by 35 repetitions of steps 2 to 4 in the following cycle: 94 ° C for 3 minutes, 94 ° C for 20 seconds, 65 ° C for 20 seconds, 72 ° C for 2 minutes, And 7 minutes at 72 ° C. A 1.0 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI Big Dye Terminator Kit (PEBiosystem) (SEQ ID NO: 9 and the deduced amino acid for the nucleic acid sequence) See SEQ ID NO: 10 for sequence).

f. hRUP13 (SEQ ID NOS: 11 and 12)
The disclosed human RUP13 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC011780 was identified as encoding a novel GPCR with significant homology to GPCR fish GRPX-ORYLA. Full length RUP13 was used as primer 5′-GCCTGTGACAGAGGTACCCCTGG-3 ′ (SEQ ID NO: 51, sense)
5′-CATATCCCTCCGAGTGTCCAGCGGC-3 ′ (SEQ ID NO: 52, antisense)
And cloned by PCR using human genomic DNA (Clontech) as template. Tack Plus Precision DNA Polymerase (Stratagene) was used for amplification by 35 repetitions of steps 2 to 4 in the following cycle: 94 ° C for 3 minutes, 94 ° C for 20 seconds, 65 ° C for 20 seconds, 72 ° C for 2 minutes, And 7 minutes at 72 ° C. A 1.35 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI Big Dye Terminator Kit (PEBiosystem) (SEQ ID NO: 11 and the deduced amino acid for the nucleic acid sequence). See SEQ ID NO: 12 for sequence).
g. hRUP14 (SEQ ID NOS: 13 and 14)
The disclosed human RUP14 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AL137188 was identified as the human genome sequence from chromosome 13. Full length RUP14 was used as primer 5′-GCATGGGAGAGAAAATTTATGTCCTTGCAACC-3 ′ (SEQ ID NO: 53, sense)
5′-CAAGAAACAGGTCTCATCTAAGAGCTCC-3 ′ (SEQ ID NO: 54, antisense)
And cloned by PCR using human genomic DNA (Promega) as template. Tack Plus Precision Polymerase (Stratagene) and 5% DMSO were used for amplification by 35 repetitions of Step 2 and Step 3 in the following cycle: 3 minutes at 94 ° C., 20 seconds at 94 ° C., 2 minutes at 58 ° C., 10 minutes 72 ° C.

  A 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI Big Dye Terminator Kit (PE Biosystem) (SEQ ID NO: 13 and inferred for the nucleic acid sequence) See SEQ ID NO: 14 for amino acid sequence). The sequence of the RUP14 clone isolated from human genomic DNA matched the sequence obtained from the database.

h. hRUP15 (SEQ ID NOS: 15 and 16)
The disclosed human RUP15 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC016468 was identified as the human genomic sequence. Full length RUP15 was used as primer 5′-GCTGTGGCCATGACGTCCCCTGCAC-3 ′ (SEQ ID NO: 55, sense)
5′-GGACAGTTCAAGGTTTGCCTTAGAAC-3 ′ (SEQ ID NO: 56, antisense)
And cloned by PCR using human genomic DNA (Promega) as template. Tack Plus Precision Polymerase (Stratagene) was used for amplification by 35 iterations of steps 2-4 in the following cycle: 94 ° C for 3 minutes, 94 ° C for 20 seconds, 65 ° C for 20 seconds, 72 ° C for 2 minutes and 7 minutes. 72 ° C.

  The 1.5 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 15 for the nucleic acid sequence and SEQ ID NO: 16 for the deduced amino acid sequence. The sequence of RUP15 clone isolated from human genomic DNA matched the sequence obtained from the database.

i. hRUP16 (SEQ ID NOs: 17 and 18)
The disclosed human RUP16 has been identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AL136106 was identified as the human genome sequence from chromosome 13. Full length RUP16 was used as primer 5′-CTTTCGATACTGCTCCCTATGCTC-3 ′ (SEQ ID NO: 57, sense, 5 ′ of start codon)
5'-GTAGTCCCACTGAAAGTCCCAGTGATCC-3 '(SEQ ID NO: 58, antisense, 3' of stop codon)
And cloned by PCR using human skeletal muscle marathon ready cDNA (Clontech) as template. Advantage cDNA polymerase (Clontech) was used for amplification by 35 iterations of steps 2-4 in the following cycle in a 50 ul reaction: 94 ° C. for 30 seconds, 94 ° C. for 5 seconds, 69 ° C. for 15 seconds, 1 minute 72 ° C. and 72 ° C. for 5 minutes.

  The 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the T7 sequenase kit (Amersham). See SEQ ID NO: 17 for the nucleic acid sequence and SEQ ID NO: 18 for the deduced amino acid sequence. The sequence of the RUP16 clone is consistent with 4 irregular fragments of AL136106, indicating that the RUP16 cDNA consists of 4 exons.

j. hRUP17 (SEQ ID NOS: 19 and 20)
The disclosed human RUP17 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC023078 was identified as the human genomic sequence from chromosome 11. Full length RUP17 is primer 5′-TTTCTGAGC ATG GATCCAACCATTCC-3 ′ (SEQ ID NO: 59, including sense and start codon)
5′-CGTTCTGACAGGGCAGAGGCTCTTC-3 ′ (SEQ ID NO: 60, antisense, 3 ′ of stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. Advantage cDNA polymerase mix (Clontech) was used for amplification by 30 iterations of steps 2-4 in the following cycle in a 100 ul reaction containing 5% DMSO: 1 min 94 ° C, 15 sec 94 ° C, 20 67 ° C for 1 second, 72 ° C for 1 minute 30 seconds and 72 ° C for 5 minutes.

  The 970 bp PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 19 for the nucleic acid sequence and SEQ ID NO: 20 for the deduced amino acid sequence.

k. hRUP18 (SEQ ID NOs: 21 and 22)
The disclosed human RUP18 has been identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC008547 was identified as the human genomic sequence from chromosome 5. Full length RUP18 was primer 5'-GGAACTCGTATAGACCCACGGTCGCTCC-3 '(SEQ ID NO: 61, sense, 5' of start codon),
5′-GGAGGTTGCGCCCTTAGCGACAGATGAACC-3 ′ (SEQ ID NO: 62, antisense, 3 ′ of stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. Tack Plus Precision DNA Polymerase (Stratagene) was used for amplification by 35 iterations of steps 2-4 in the following cycle in a 100 ul reaction containing 5% DMSO: 5 minutes at 95 ° C., 30 seconds at 95 ° C., 30 65 ° C for 2 seconds, 72 ° C for 2 minutes and 72 ° C for 5 minutes.

  The 1.3 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 21 for the nucleic acid sequence and SEQ ID NO: 22 for the deduced amino acid sequence.

l. hRUP19 (SEQ ID NOS: 23 and 24)
The disclosed human RUP19 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC026633 was identified as the human genomic sequence from chromosome 12. Full length RUP19 is primer 5′-CTGCACCCGGACACTTGCTCTG-3 ′ (SEQ ID NO: 63, sense, 5 ′ of start codon)
5′-GTCTGCCTGT TCA GTGCCACTCAAC-3 ′ (SEQ ID NO: 64, including antisense and stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. Tack Plus Precision DNA Polymerase (Stratagene) was used for amplification by 35 repetitions of steps 2-4 with 5% DMSO in the following cycle: 1 minute 94 ° C, 15 seconds 94 ° C, 20 seconds 70 ° C, 1 72 ° C for 30 minutes and 72 ° C for 5 minutes.

  The 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 23 for the nucleic acid sequence and SEQ ID NO: 24 for the deduced amino acid sequence.

m. hRUP20 (SEQ ID NOs: 25 and 26)
The disclosed human RUP20 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AL161458 was identified as the human genome sequence from chromosome 1. Full length RUP20 primer 5′-TATCTGCAATTCTATTCTAGCCTCTG-3 ′ (SEQ ID NO: 65, sense, start codon 5 ′),
5'-TGTCCCCTATAATAAAGTCACATGAATGC-3 '(SEQ ID NO: 66, antisense, 3' of stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. Advantage cDNA polymerase mix (Clontech) was used for amplification by 35 cycles of steps 2-4 in the following cycle using 5% DMSO: 1 min 95 ° C, 15 sec 95 ° C, 20 sec 60 ° C, 1 72 ° C for 30 minutes and 72 ° C for 5 minutes.

  The 1.0 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 25 for the nucleic acid sequence and SEQ ID NO: 26 for the deduced amino acid sequence.

n. hRUP21 (SEQ ID NOs: 27 and 28)
The disclosed human RUP21 has been identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC026675 was identified as the human genomic sequence from chromosome 13. Full length RUP21 was used as primer 5′-GGAGACAACCATGAATGAGCCCAC-3 ′ (SEQ ID NO: 67, sense)
5'-TATTTCAAGGGGTTGTTGAGTAAC-3 '(SEQ ID NO: 68, antisense)
And cloned by PCR using human genomic DNA (Promega) as template. Tack Plus Precision Polymerase (Stratagene) was used for amplification by 30 iterations of steps 2-4 in the following cycle in a 100 ul reaction containing 5% DMSO: 1 minute 94 ° C., 15 seconds 94 ° C., 20 seconds 55 ° C, 1 minute 30 seconds 72 ° C and 5 minutes 72 ° C.

  The 1,014 bp PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 27 for the nucleic acid sequence and SEQ ID NO: 28 for the deduced amino acid sequence.

o. hRUP22 (SEQ ID NOS: 29 and 30)
The disclosed human RUP22 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC027026 was identified as the human genomic sequence from chromosome 11. Full length RUP22 is primer 5'-GGCACCAGTGGAGGTTTTCTGAGC ATG- 3 '(SEQ ID NO: 69, including sense and start codon)
5′-CTGATGGAAGTAGAGGCTGTCCATCTC-3 ′ (SEQ ID NO: 70, antisense, 3 ′ of stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. Tack Plus Precision DNA Polymerase (Stratagene) was used for amplification by 30 iterations of steps 2-4 in the following cycle in a 100 ul reaction containing 5% DMSO: 94 ° C., 1 minute 94 ° C., 15 seconds 55 ° C, 20 seconds 72 ° C, 1.5 minutes 72 ° C, 5 minutes.

  The 970 bp PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 29 for the nucleic acid sequence and SEQ ID NO: 30 for the deduced amino acid sequence.

p. hRUP23 (SEQ ID NOS: 31 and 32)
The disclosed human RUP23 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC007104 was identified as the human genomic sequence from chromosome 4. Full length RUP23 was used as primer 5'-CCAGGCGAGCCCTAGTAGCCCC ATG- 3 '(SEQ ID NO: 71, sense, ATC as start codon)
5′-ATGAGCCCCTGCCAGGCCCC TCA GT-3 ′ (SEQ ID NO: 72, antisense, TCA as stop codon)
And cloned by PCR using human placenta marathon ready DNA (Clontech) as template. Advantage cDNA polymerase (Clontech) was used for amplification by 35 repeats of steps 2-4 in the following cycle in a 50 ul reaction: 95 ° C. for 30 seconds, 95 ° C. for 15 seconds, 66 ° C. for 20 seconds, 1 minute 72 ° C for 20 seconds and 72 ° C for 5 minutes.

  A 1.0 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 31 for the nucleic acid sequence and SEQ ID NO: 32 for the deduced amino acid sequence.

q. hRUP24 (SEQ ID NOs: 33 and 34)
The disclosed human RUP25 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC026633 was identified as the human genomic sequence from chromosome 12. Full length RUP25 is primer 5'-GCTGGAGCATTCACTAGGCAGAG-3 '(SEQ ID NO: 73, sense, start codon 5')
5′-AGATCCTGGTTCTTGGTGACAATG-3 ′ (SEQ ID NO: 74, antisense, 3 ′ of stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. The Advantage cDNA Polymerase Mix (Clontech) was used for amplification by 35 repeats of steps 2-4 with 5% DMSO: 1 min 94 ° C, 15 sec 94 ° C, 20 sec 56 ° C, 72 ° C for 1 minute 30 seconds and 72 ° C for 5 minutes.

  The 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 33 for the nucleic acid sequence and SEQ ID NO: 34 for the deduced amino acid sequence.

r. hRUP25 (SEQ ID NOs: 35 and 36)
The disclosed human RUP25 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC026633 was identified as the human genomic sequence from chromosome 12. Full length RUP25 is primer 5′-GCTGGAGCATTCACTAGGCGAG-3 ′ (SEQ ID NO: 75, sense, 5 ′ of start codon)
5′-AGATCCTGGTTCTTGGTGACAATG-3 ′ (SEQ ID NO: 76, antisense, 3 ′ of stop codon)
And cloned by PCR using human genomic DNA (Promega) as template. The Advantage cDNA Polymerase Mix (Clontech) was used for amplification by 35 repeats of steps 2-4 with 5% DMSO: 1 min 94 ° C, 15 sec 94 ° C, 20 sec 56 ° C, 72 ° C for 1 minute 30 seconds and 72 ° C for 5 minutes.

  The 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 35 for the nucleic acid sequence and SEQ ID NO: 36 for the deduced amino acid sequence.

s. hRUP26 (SEQ ID NOs: 37 and 38)
The disclosed human RUP26 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC023040 was identified as the human genomic sequence from chromosome 2. Full length RUP26 is RUP26 specific primer 5'-AGCCATCCCTGCCCAGGAAGC ATG G-3 '(SEQ ID NO: 77, including sense and start codon)
5′-CCAGACTGTGGAACTCAAGAACT CTA GG-3 ′ (SEQ ID NO: 78, including antisense and stop codon)
And cloned by RT-PCR using human placental marathon ready cDNA (Clontech) as template. Advantage cDNA polymerase mix (Clontech) was used for amplification by 35 iterations of steps 2-4 in the following cycle in a 100 ul reaction containing 5% DMSO: 94 ° C. for 5 minutes, 95 ° C. for 30 seconds, 30 ° C. 65 ° C for 2 seconds, 72 ° C for 2 minutes and 72 ° C for 5 minutes.

  The 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 37 for the nucleic acid sequence and SEQ ID NO: 38 for the deduced amino acid sequence.

t. hRUP27 (SEQ ID NOs: 39 and 40)
The disclosed human RUP27 was identified based on the use of gene bank database information. During the investigation, a cDNA clone with accession number AC027643 was identified as the human genomic sequence from chromosome 12. Full length RUP27 is converted to RUP17 specific primer 5′-AGTCCCACGAACA ATG AATCCATTTCATG-3 ′ (SEQ ID NO: 79, including sense and start codon)
5′-ATCATGTTCTAGACTCATGGTGATCC-3 ′ (SEQ ID NO: 80, antisense, 3 ′ of stop codon)
And cloned by PCR using human adult brain marathon ready cDNA (Clontech) as template. Advantage cDNA polymerase mix (Clontech) was used for amplification by 35 iterations of steps 2-4 in the following cycle in a 50 ul reaction containing 5% DMSO: 1 min 94 ° C., 10 sec 94 ° C., 20 58 ° C. for 1 second, 72 ° C. for 30 minutes and 72 ° C. for 5 minutes.

  The 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and fully sequenced using the ABI big dye terminator kit (P.E. Biosystem). See SEQ ID NO: 35 for the nucleic acid sequence and SEQ ID NO: 36 for the deduced amino acid sequence. The sequence of the RUP27 cDNA clone isolated from human brain was determined to match 5 irregular fragments of AC027643, indicating that the RUP27 cDNA consists of 5 exons.

Example 2
Preparation of non-endogenous, constitutively activated GPCRs Those skilled in the art are believed to have the ability to select techniques for mutation of nucleic acid sequences. The following are the methods used to create the non-endogenous forms of several human GPCRs disclosed above. The mutations disclosed below are the 16th amino acid (GPCR of GPCR) from the conserved proline (or its endogenous conservative substituent) residue (located in the TM6 region of the GPCR near the TM6 / IC3 interface). Is located within the IC3 region), preferably based on an algorithmic method that is mutated to alanine, histidine, arginine or lysine amino acid residues, most preferably to lysine amino acid residues.

1. Transformer Site Directed ^TM Mutagenesis Non-endogenous human GPCRs may be prepared for human GPCRs using the Transformer Site-Directed ^TM Mutagenesis Kit (Clontech) according to the manufacturer's guidelines. . Two mutagenesis primers are used, most preferably a lysine mutagenesis oligonucleotide that creates a lysine mutation, and a selectable marker oligonucleotide. For convenience, the codon mutations to be incorporated into the human GPCR are also recorded in standard form (Table D).

2. QuikChange ^™ Site Directed ^™ Mutagenesis Non-endogenous human GPCRs can also be prepared by the QuikChange ^™ Site-Directed ^™ Mutagenesis Kit (Stratagene, following manufacturer's guidelines). Endogenous GPCR is used as a preferred template and two mutagenesis primers are used, and most preferably a lysine mutagenesis oligonucleotide and a selectable marker oligonucleotide (included in the kit) are used. For convenience, the codon mutations incorporated into the new human GPCR and the respective oligonucleotides are recorded in standard form (Table E).

  Non-endogenous human GPCRs were then sequenced and the derived and confirmed nucleic acid and amino acid sequences displayed in the “Sequence Listing” attached to this patent specification, which is summarized in Table F below.

Example 3
Receptor expression Although various cells are available in the art for protein expression, the use of mammalian cells is most preferred. The main reason for this is dictated by practicality, ie the use of yeast cells, for example for the expression of GPCRs, to the extent possible with receptor couplings, genetic mechanisms and secretion pathways developed for mammalian systems. Non-mammalian cells that are not included (and certainly not in the case of yeast) are introduced into the protocol, so that the results obtained with non-mammalian cells are as high as those obtained from mammalian cells, if available. Is not preferred. Although the particular mammalian cells used can be determined by the specific requirements of the skilled worker, among the mammalian cells, COS-7, 293 and 293T cells are particularly preferred.

a. Transient transfection On the first day, ⁶ × 10 ⁶ / (293 cell well 10 cm dish) were plated out. On the second day, two reaction tubes were prepared (the ratio below for each tube is per plate). Tube A was prepared by mixing 4 μg DNA (eg, pCMV vector, pCMV vector containing receptor cDNA, etc.) in 0.5 ml of DMEM (GibcoBRL) without serum. Tube B was prepared by mixing 24 μl Lipofectamine (Gibco BRL) in 0.5 ml serum free DMEM. Tubes A and B were mixed by inversion (several times) and then incubated at room temperature for 30-45 minutes. This mixture is referred to as the “transfection mixture”. Plated 293 cells were washed with 1 × PBS and then 0.5 ml serum free DMEM was added. One ml of the transfection mixture was added to the cells and then incubated for 4 hours at 37 ° C / 5% CO ₂ . The transfection mixture was aspirated and then 10 ml of DMEM / 10% fetal calf serum was added. Cells were incubated at 37 ℃ / 5% CO _2. After 48 hours incubation, cells were harvested and used for analysis.

b. Stable cell line: Gs fusion protein Approximately 12 × 10 ⁶ 293 cells were plated on 15 cm tissue culture plates. Cultured in DME HighGlucose Medium containing 10% fetal bovine serum and 1% sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours after plating, 293 cells were approximately 80% confluent and the cells were transfected with 12 μg of DNA. 12 μg of DNA was mixed with 60 ul of lipofectamine and 2 mL of DME high glucose medium without serum. The medium was aspirated from the plate and the cells were washed once with serum free medium. DNA, lipofectamine, and media mixture were added to the plate along with 10 mL of serum free media. After 4-5 hours incubation at 37 ° C., the medium was aspirated and 25 ml of medium containing serum was added. The medium was aspirated again 24 hours after transfection and fresh medium containing serum was added. Forty-eight hours after transfection, the medium was aspirated and medium containing serum and geneticin (G418 drug) was added to a final concentration of 500 μg / mL. Transfected cells are now selected for positively transfected cells containing the G418 resistance gene. The media was changed every 4-5 days each time selection occurred. During selection, the cells are grown to create a stable pool or split for stable clonal selection.

Example 4 Assays for Determination of Constitutive Activity of Non-Endogenous GPCRs Various methods are utilized for the assessment of constitutive activity of non-endogenous human GPCRs. The following is an example. The ordinary skill in the art is believed to have the ability to determine such techniques that preferentially serve the needs of the skilled person.
1. Membrane binding assay: [ ³⁵ S] GTPγS assay When a G protein-coupled receptor is in its active state, as a result of either ligand binding or constitutive activation, the receptor binds to G protein and Stimulates free and subsequent binding of GTP to G protein. The α subunit of the G protein coupled receptor complex acts as a GTPase and slowly hydrolyzes GTP to GDP, at which time the receptor is normally deactivated. The constitutively activated receptor continues to exchange GTP for GDP. [ ³⁵ S] GTPγS, a non-hydrolyzable GTP analog, can be used to demonstrate enhanced binding of [ ³⁵ S] GTPγS to membranes expressing constitutively activated receptors. The advantages of using [ ³⁵ S] GTPγS binding to measure constitutive activation are: (a) it applies globally to all G protein coupled receptors, (b) it is close to the membrane surface It is difficult to pick up molecules that affect the intracellular cascade.

This assay takes advantage of the ability of G protein-coupled receptors to stimulate [ ³⁵ S] GTPγS to bind to membranes expressing the relevant receptors. This assay can therefore be used as a direct identification method for screening candidate compounds to known orphan and constitutively activated G protein coupled receptors. The assay is comprehensive and applies to drug discovery at all G protein coupled receptors.

[ ³⁵ S] GTPγS assay uses 20 mM HEPES and 1 to about 20 mM MgCl ₂ (this amount can be adjusted for optimization of results although 20 mM is preferred), pH 7.4, about 0.3 to about 1.2 nM Binding buffer containing [ ³⁵ S] GTPγS (this amount is preferably 1.2 but can be adjusted for optimization of results) and 12.5-75 μg membrane protein (eg 293 cells expressing Gs fusion protein) This amount can be adjusted for optimization) and incubated in 10 μM GDP (this amount can be adjusted for optimization) for 1 hour. Wheat germ agglutinin beads (25 μl, Amersham) were then added and the mixture was incubated for an additional 30 minutes at room temperature. Tubes were then centrifuged at 1500 xg for 5 minutes at room temperature and counted in a scintillation counter.

2. Adenylate Cyclase The Flash Plate ^™ adenylate cyclase kit (New England Nuclear; catalog number SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The flash plate well can include a scintillant coating that also contains specific antibodies that recognize cAMP. CAMP produced in the well can be quantified by direct competition for binding of the radioactive cAMP tracer to the cAMP antibody. The following description serves as a simple protocol for measuring changes in cAMP levels in whole cells expressing the receptor.

  Transfected cells were harvested approximately 24 hours after transient transfection. The medium was carefully aspirated and removed. 10 ml of PBS was gently added to each dish of cells and then carefully aspirated. 1 ml of Sigma cell separation buffer and 3 ml of PBS were added to each plate. Cells were pipetted from the dish and the cell suspension was collected in a 50 ml conical centrifuge tube. Cells were then centrifuged at 1,100 rpm for 5 minutes at room temperature. The cell pellet was carefully resuspended in an appropriate volume of PBS (approximately 3 ml / plate). Cells were then counted using a hemocytometer and additional PBS was added to give an approximate number of cells (final volume about 50 μl / well).

cAMP standards and “detection buffer” (11 ml of “detection buffer” prepared tracer [comprising 1 μCi of ¹²⁵ IcAMP (50 μl)) and maintained according to the manufacturer's instructions. This was freshly prepared for and contained 50 μl of “stimulation buffer”, 3 ul of test compound (12 uM final assay concentration) and 50 μl of cells, and “assay buffer” was kept on ice until use. Starting with the addition of 50 μl of cAMP standard to the appropriate wells, 50 ul of PBSA was then added to wells H-11 and H-12, 50 μl of “stimulation buffer” was added to all wells, DMSO (or selected candidate compound). Add 12 μM test compound to appropriate wells using a pin instrument capable of supplying 3 μl of compound solution The final assay concentration and total assay volume was 100 μl, then the cells were added to the wells and incubated for 60 minutes at room temperature, then 100 μl of the “detection mixture” containing the tracer cAMP was added to the wells, and the plates were then incubated for another 2 hours The counts were then counted in a Wallac MicroBeta scintillation counter and the cAMP / well values were then extrapolated from the standard cAMP curve included in each assay plate.

3. Cell-based cAMP for Gi-coupled target GPCR
TSHR is a Gs-coupled GPCR that causes cAMP accumulation upon activation. TSHR is constitutively activated by mutating amino acid residue 623 (ie, changing an alanine residue to an isoleucine residue). Gi-coupled receptors are expected to inhibit adenylate cyclase and, therefore, reduce the level of cAMP production, which allows assessment of cAMP level problems. An effective technique for measuring the reduction of cAMP production as an indicator of constitutive activation of Gi-coupled receptors is as a “signal enhancer” using a Gi-linked target GPCR to establish a reference level of cAMP. Most preferably, it can be achieved by co-transfection with a non-endogenous, constitutively activated TSHR (TSHR-A6231) (or endogenous, constitutively active Gs-coupled receptor). Once the non-endogenous form of the Gi-coupled receptor has been created, this non-endogenous form of the target GPCR is then co-transfected with a signal enhancer and it is this substance that can be used for screening. We used this method to generate a signal efficiently when using the cAMP assay. This method is preferably used for direct identification of candidate compounds for Gi-coupled receptors. When this method is used, the inverse agonist of the target GPCR will increase the cAMP signal and the agonist will decrease the cAMP signal for Gi-coupled receptors.

On the first day, plate out 2 × 10 ⁴ 293 and 293 cells / well. On the second day, prepare two reaction tubes (the ratio below for each tube is per plate). Tube A contains 2 μg DNA of each receptor transfected into mammalian cells in a total of 4 μg DNA (eg, pCMV vector, mutant THSR (in Irvine Scientific, Irvine, Calif.) Without 1.21 serum). Prepared by mixing with pCMV vector containing THSR-A6231I), THSR-A6231I and GPCR). Tube B is prepared by mixing 120 μl Lipofectamine (GibcoBRL) in 1.2 ml serum free DMEM. Tubes A and B were then inverted (several times) to mix well, then incubated at room temperature for 30-45 minutes. The mixture is referred to as the “transfection mixture”. Plated 293 cells are washed with 1 × PBS, then 10 ml of serum free DMEM is added. Then 2.4 ml of the transfection mixture is added to the cells and then incubated for 4 hours at 37 ° C./5% CO ₂ . The transfection mixture is then aspirated and then 25 ml of DMEM / 10% fetal calf serum is added. The cells are then incubated at 37 ° C./5% CO ₂ . After 24 hours incubation, cells are harvested and used for analysis.

However, the Flash Plate ^™ adenylate cyclase kit (New England Nuclear; catalog number SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes according to the requirements of the skilled person. The flash plate well can include a scintillant coating that also includes specific antibodies that recognize cAMP. CAMP produced in the well can be quantified by direct competition for binding of the radioactive cAMP tracer to the cAMP antibody. The following description serves as a simple protocol for measuring changes in cAMP levels in whole cells expressing the receptor.

  Transfected cells are harvested approximately 24 hours after transient transfection. Carefully aspirate and remove the media for disposal. Gently add 10 ml of PBS to each dish of cells and then aspirate carefully. Add 1 ml of Sigma cell separation buffer and 3 ml of PBS to each plate. Cells are pipetted from the dish and the cell suspension is collected in a 50 ml conical centrifuge tube. The cells are then centrifuged at 1,100 rpm for 5 minutes at room temperature. Carefully resuspend the cell pellet in an appropriate volume of PBS (approximately 3 ml / plate). The cells are then counted using a hemocytometer and additional PBS is added to give an approximate number of cells (final volume approximately 50 μl / well).

Prepare cAMP standard and “detection buffer” (trace buffer [comprising 1 μCi of ¹²⁵ IcAMP (50 μl) in 11 ml of “detection buffer”) and maintain according to manufacturer's instructions. Must be freshly prepared and contain 50 μl of “stimulation buffer”, 3 ul of test compound (12 uM final assay concentration) and 50 μl of cells, and “assay buffer” can be stored on ice until use. The assay can begin with the addition of 50 μl of cAMP standard to the appropriate wells, then 50 μl of PBSA is added to wells H-11 and H-12, 50 μl of stimulation buffer is added to the wells, and the selected compound (eg, TSH) is added to the compound. Using a pin device that can supply 3 μl of solution, add to appropriate wells and test 12 μM The final assay concentration of the product and the total assay volume of 100 μl, then the cells are added to the wells and incubated for 60 minutes at room temperature, then 100 μl of the “detection mixture” containing the tracer cAMP is added to the wells, then the plate is incubated for another 2 hours Then, count in a wolac microbeta scintillation counter and then extrapolate the cAMP / well value from the standard cAMP curve included in each assay plate.

4). Reporter-based assays a. Gre-Luc reporter assay (Gs-related receptor)
293 and 293t cells were plated out on 96-well plates at a density of 2 × 10 ⁴ cells per well and transfected with Lipofectamine reagent (BRL) the following day according to the manufacturer's instructions. DNA / lipid mixtures were prepared for each 6-well transfection as follows: 260 ng plasmid DNA in 100 μl DMEM was gently mixed with 2 μl lipid in 100 μl DMEM (200 ng of 8 × CRE-Luc reporter plasmid, endogenous receptor or non-endogenous 260 ng of plasmid DNA consisting of 50 ng of pCMV or pCMV alone comprising sex receptors and 10 ng of a GPRS expression plasmid (GPRS (Invitrogen) in pcDNA3). The 8XCRE-Luc reporter plasmid was prepared as follows: The vector SRIF-β-gal was the rat somatostatin promoter (−71 / + 51) at the BglV-HindIII site in the pβgal-Basic Vector (Clontech). Was obtained by cloning. Eight copies of the cAMP responder were obtained by PCR from the adenovirus template AdpCF126CCRE8 (see 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF-β-gal vector at the Kpn-BglV site. A gal reporter vector was obtained. The 8xCRE-Luc reporter plasmid was generated by replacing the beta-galactosidase gene in the 8xCRE-β-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the HindIII-BamHI site. After incubation at room temperature for 30 minutes, the DNA / lipid mixture was diluted with 400 μl of DMEM and 100 μl of the diluted mixture was added to each well. 100 μl of DMEM containing 10% FCS was added to each well after 4 hours incubation in a cell culture incubator. The next day, the transfected cells were replaced with 200 μl / well DMEM containing 10% FCS. After 8 days, the wells were changed to 100 μl / well of DMEM without phenol red after one wash with PBS. Luciferase activity was measured the following day using the LucLite ^™ reporter gene assay kit (Packard) according to the manufacturer's instructions and read using a 1450 MicroBeta ^™ scintillation and fluorescence counter (Wallac).

b. AP1 reporter assay (Gq-related receptor)
The method for detecting Gq stimulation relies on the known nature of Gq-dependent phospholipase C, which causes activation of genes containing the AP1 element in these promoters. The Pathdetect ^™ AP-1 cis-reporting system (Stratagene, catalog number # 219073) can be used according to the protocol described above for the CREB reporter assay, but the components of the calcium phosphate precipitate are 410 ng pAP1-Luc, 80 ng pCMV. -Receptor expression plasmid and 20ng CMV-SEAP.
c. Srf-Luc reporter assay (Gq-related receptor)
One method for detecting Gq stimulation relies on the known nature of Gq-dependent phospholipase C, which causes activation of genes that contain serum response factors in their promoters. The Pathdetect ^™ SRF-Luc-reporting system (Stratagene) can be used, for example, to assay for Gq-coupled activity in COS7 cells. Cells are transfected with the plasmid components of the system and expression encoding an endogenous or non-endogenous GPCR using the Mammalian Transfection ^TM kit (Stratagene, catalog number # 200285) according to the manufacturer's instructions The plasmid is shown. In summary, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid. Alkaline phosphatase activity was transfected to control variations in transfection effects between samples. Is measured into the calcium phosphate sediment according to the manufacturer's instructions. Half of the pellet is evenly distributed over 3 wells in a 96-well plate and kept on cells in serum free medium for 24 hours. In the last 5 hours, the cells are incubated with 1 μM angiotensin when indicated. The cells were then lysed and used with the manufacturer's Lucilite ^™ kit (Packard, catalog number # 6016911) and “Trilux 1450 microcrete” liquid scintillation and fluorescence counter (Wallac). Assay luciferase activity according to instructions. Data can be analyzed using GraphPadPrism ^™ 2.0a (GraphPad Software Inc.).

d. Intracellular IP ₃ accumulation assay (Gw-related receptor)
On the first day, cells comprising the receptor (endogenous and / or non-endogenous) can be plated on 24-well plates, usually 1 × 10 ⁵ cells / well (although this number can be optimized) . On the second day, the cells can be initially mixed and mixed with 0.25 μg DNA in 50 μl / well of DMEM without serum and 2 μl lipophetamine in 50 μl / well of DMEM without serum. The solution is gently agitated and incubated for 15-30 minutes at room temperature. Cells are washed with 0.5 ml PBS, 400 μl of serum free medium is mixed with transfection medium and added to the cells. Cells were then incubated for 3-4 hours at 37 ° C./5% CO ₂ and then the transfection medium was removed and replaced with 1 ml / well of normal growth medium. On the third day, the cells are labeled with ³ H-myo-inositol. In summary, the media is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free / serum-free medium (GIBCOBRL) was added per well with 0.25 μCi of ³ H-myo-inositol per well and the cells were incubated at 37 ° C./5 for 16-18 hours. Incubate with% CO ₂ . On the fourth day, the cells were washed with 0.5 ml PBS and 0.45 ml assay medium or 0.4 ml assay medium containing 10 μM pargyline 10 mM lithium chloride with no inositol / serum free medium and 10 × ketanserin (ketanserin, ket) Add 50 μl to a final concentration of 10 μM. The cells are then incubated for 30 minutes at 37 ° C. The cells are then washed with 0.5 ml PBS and 200 μl of fresh / ice cold stop solution (1M KOH; 18 mM Na borate; 3.8 mM EDTA) is added per well. The solution is kept on ice for 5-10 minutes or until cells are lysed and then neutralized with 200 μl of fresh / ice cold neutralization solution (7.5% HCL). The lysate is then transferred into a 1.5 ml eppendorf tube and 1 ml of chloroform / methanol (1: 2) is added per tube. The solution is stirred for 15 seconds and the upper phase is added to Biorad AG1-X8 ^™ anion exchange resin (100-200 mesh). The resin is first washed with 1: 1.25 W / V water and 0.9 ml of the upper phase is added onto the column. The column is washed with 10 ml of 5 mM myo-inositol and 10 ml of 5 mM Na borate / 60 mM Na formate. Elute in a scintillation vial containing 10 ml of scintillation cocktail containing 2 ml of 0.1 M formic acid / 1 M ammonium formate. The column is washed with 10 ml of 0.1M formic acid / 3M ammonium formate and rinsed twice with ddH ₂ O and stored in 4 ° C. water.

  Exemplary results are shown in Table G below.

  Exemplary results of the GTPγS assay for detecting the constitutive activation disclosed in Example 4 (1) above were achieved using “Gs: fusion protein construct” on human RUP13 and RUP15. Table H below displays the signal generated from this assay and the difference in signal as indicated.

Example 5
Fusion protein preparation a. GPCR: Gs fusion constructs The design of the constitutively activated GPCR-G protein fusion construct was performed as follows: 5 'of rat G protein Gsα (long form; Itoh et al., 83 PNAS 3776 (1986)) and Both 3 ′ ends were engineered to contain a HindIII (5′-AAGCTT-3 ′) sequence on it. After confirmation of the correct sequence (including the flanking HindIII sequence), the entire sequence was subcloned using the HindIII restriction site of the vector and into pcDNA3.1 (-) (Invitrogen catalog number V795-20). Shuttled. The correct orientation of the Gsα sequence was determined after subcloning into pcDNA3.1 (−). The pcDNA3.1 (−) containing the rat Gsα gene modified with the HindIII sequence was then confirmed. This vector is now available as a “universal” Gsα protein vector. The pcDNA3.1 (−) vector contains various well-known restriction sites upstream of the HindIII site, thus advantageously providing the ability to insert an endogenous, constitutively active GPCR coding sequence upstream of the Gs protein. This same method can be used to create other “universal” G-protein vectors, and of course other commercially available or proprietary vectors known to the skilled person can also be used, In that case, an important criterion is that the sequence of the GPCR is upstream and in-frame with that of the G protein.

  RUP13 is conjugated via Gs. For the exemplary “GPCR fusion protein” below, fusion to Gsα was achieved.

The RUP13- “Gsα fusion protein” construct was made as follows.
Primers were designed as follows.
5′-gats [TCTAGAAT] GGAGTCCCTCACCCATCCCCCCAG-3 ′ (SEQ ID NO: 97, sense)
5'-gats [GATATC] CGGTACTCCAGCCGGGGTGGAGGCGGC-3 '(SEQ ID NO: 98, antisense)
Lower case nucleotides are included as spacers within the restriction site (shown in parentheses) between the G protein and RUP13. The sense and antisense primers contained restriction sites for XbaI and EcoRV, respectively, so that a spacer (assigned to the restriction site) was present between the G protein and RUP15.

Then, PCR was used to secure each receptor sequence for fusion in the Gsα general-purpose vector disclosed above, and the following protocols were used respectively. 100 ng cDNA for RUP15, 2 μl of primer (sense and anti-sense), 3 μl of 10 mM dNTP, 10 μl of 10X Tack Plus ^™ Precision Buffer, 1 μl of Tack Plus ^™ Precision Polymerase (Stratagene: # 600211), and water, respectively Add to separate tubes containing 80 μL. The reaction temperature and cycle time for RUP15 were as follows, including 35 iterations of cycle steps 2-4. 94 ° C for 1 minute, 94 ° C for 30 seconds, 62 ° C for 20 seconds, 72 ° C for 1 minute 40 seconds, and 72 ° C for 5 minutes. The PCR product was run on a 1% agarose gel and then purified (data not shown). The purified product was digested with XbaI and EcoRV and the desired insert was purified and ligated into the Gs universal vector at the respective restriction sites. Positive clones were isolated after transformation and determined by restriction enzyme digestion. Expression using 293 cells was achieved using the protocol described herein. Each positive clone of RUP15- “Gs fusion protein” was sequenced to confirm accuracy. (See SEQ ID NO: 99 for the nucleic acid sequence and SEQ ID NO: 100 for the amino acid sequence).

    RUP15 is conjugated via Gs. For the exemplary “GPCR fusion protein” below, fusion to Gsα was achieved.

The RUP15- “Gsα fusion protein” construct was made as follows. Primers were designed as follows.
5′-TCTAGAAATGACGTCCCACCTGACCACCAACAGC-3 ′ (SEQ ID NO: 101, sense)
5′-gatatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3 ′ (SEQ ID NO: 102, antisense)
Lower case nucleotides are included as spacers within the restriction site between the G protein and RUP15. The sense and antisense primers contained restriction sites for EcoRV and XbaI, respectively, so that a spacer (attributed to the restriction site) was present between the G protein and RUP15.

Then, PCR was used to secure each receptor sequence for fusion in the Gsα general-purpose vector disclosed above, and the following protocols were used respectively. Separate 100 ng cDNA for RUP15 containing 2 μl of primers (sense and anti-sense), 3 μl of 10 mM dNTPs, 10 μl of 10X Tuck Plus ^™ Precision Buffer, 1 μL of Tack Plus ^™ Precision Polymerase (Stratagene: # 600211), and 80 μl of water, respectively. Added to the tube. The reaction temperature and cycle time for RUP15 were as follows, including 35 iterations of cycle steps 2-4. 94 ° C for 1 minute, 94 ° C for 30 seconds, 62 ° C for 20 seconds, 72 ° C for 1 minute 40 seconds, and 72 ° C for 5 minutes. The PCR product was run on a 1% agarose gel and then purified (data not shown). The purified product was digested. ) The purified product was digested with EcoRV and XbaI and the desired insert was purified and ligated into the Gs universal vector at the respective restriction sites. Positive clones were isolated after transformation and determined by restriction enzyme digestion. Expression using 293 cells was achieved using the protocol described herein. Each positive clone of RUP15- “Gs fusion protein” was sequenced to confirm accuracy. (See SEQ ID NO: 103 for the nucleic acid sequence and SEQ ID NO: 104 for the amino acid sequence).

b. Gq (6 amino acid deletion) / Gi fusion construct The design of the Gq (deletion) / Gi fusion construct could be accomplished as follows. 6 amino acids at the N-terminus (2 to 7 amino acids having the TERIMS sequence (SEQ ID NO: 129) Gaq-subunit and the C-terminal 5 amino acids having the sequence EYNLB (SEQ ID NO: 130) Is replaced with the corresponding amino acid of the “GαI protein” having the sequence DCGLF (SEQ ID NO: 131) This fusion construct is composed of the primers (SEQ ID NO: 133)
And plasmid 63313 containing mouse Gαq-wild type and obtained by PCR using a hemagglutinin tag as a template. Lowercase nucleotides are included as spacers.

  Tack Plus Precision DNA Polymerase (Stratagene) is used for replication with the following cycle comprising 35 repeats of steps 2-4. 95 ° C for 2 minutes, 95 ° C for 20 seconds, 56 ° C for 20 seconds, 72 ° C for 2 minutes, and 72 ° C for 7 minutes. The PCR product was cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI big dye terminator kit (P.E. Biosystems). The insert from the TOPO clone containing the sequence of the fusion construct was shuttled into the expression vector pcDNA3.1 (+) by a two-step cloning method at the HindIII / BamHI site.

Example 6
Tissue distribution of disclosed human GPCRs: RT-PCR
RT-PCR was applied to confirm the expression of several new human GPCRs and to determine tissue distribution. The oligonucleotides used were GPCR specific and a human multi-tissue cDNA panel (MTC, Clontech) as a template. Tack DNA polymerase (Stratagene) was used for amplification in a 40 μl reaction according to the manufacturer's instructions. 20 μl of the reaction was added on a 1.5% agarose gel and the RT-PCR product was analyzed. Table J below lists the receptors, cycle conditions and primers used.

Example 7
Protocol: Direct Identification of Inverse Agonists and Agonists [ ³⁵ S] GTPγS assay Although we have used endogenous, constitutively active GPCRs for reasons not fully understood for direct identification of candidate compounds, eg inverse agonists, the variation within the assay can be significant. is there. Preferably, as disclosed above, a “GPCR fusion protein” is also used with a non-endogenous, constitutively activated GPCR. We have observed that intra-assay variability is inherently stabilized when using such proteins, which appears to yield an effective signal-to-noise ratio. This has the advantageous result of allowing more robust identification of candidate compounds. Thus, when a “GPCR fusion protein” is used and utilized for direct identification, it is desirable to utilize the following assay protocol.

1. Membrane Preparation Membranes comprising the relevant constitutively active orphan “GPCR fusion protein” and for use in the direct identification of candidate compounds as inverse agonists, agonists or partial agonists are preferably prepared as follows: .

a. Materials "Membrane scrape buffer" comprises 20 mM HEPES and 10 mM EDTA, pH 7.4. “Membrane wash buffer” comprises 20 mM HEPES and 0.1 mM EDTA, pH 7.4. “Binding buffer” comprises 20 mM HEPES, 100 mM NaCl, and 10 mM MgCl ₂ , pH 7.4.

b. Operation Keep all materials on ice throughout the operation. First, the medium is aspirated from the confluent monolayer of cells, then rinsed with 10 ml cold PBS and then aspirated. Thereafter, 5 ml of “membrane scrape buffer” is added to the scraped cells, followed by transferring the cell extract into a 50 ml centrifuge tube (centrifuged at 20,000 rpm for 17 minutes at 4 ° C.). The supernatant is then aspirated and the pellet is resuspended in 30 ml of membrane “wash buffer” and then centrifuged at 20,000 rpm for 17 minutes at 4 ° C. The supernatant is then aspirated and the pellet is resuspended in “binding buffer”. This is then homogenized using a Brinkman polytron ^™ homogenizer (burst for 15-20 seconds until all material is suspended). This is referred to herein as a “membrane protein”.

2. Bradford protein assay Following homogenization, the protein concentration of the membrane is determined using the Bradford protein assay (protein is diluted to approximately 1.5 mg / ml and placed in aliquots for subsequent use. Can be frozen (−80 ° C.) When frozen, the protocol for use is as follows: On the day of the assay, the frozen “membrane protein” is thawed at room temperature, then vortexed and at about 12 × 1.000 rpm. Homogenize with polytron for about 5-10 seconds, note that for multiple preparations, clean the homogenizer thoroughly between different preparations)

a. Materials "Binding buffer" (as above), "Bradford staining reagent", "Bradford protein standard" are used according to the manufacturer's instructions (Biorad catalog number 500-0006).

b. Procedure Prepare two tubes, one containing the membrane and the other as the control “blank”. Each contains 800 ul of “binding buffer”. Thereafter, 10 μl of “Bradford protein standard” (1 mg / ml) is added to each tube, then 10 μl of membrane “protein” is added except for one tube (except the blank). Thereafter, 200 μl of “Bradford Staining Reagent” is added to each tube, followed by vortexing each. After 5 minutes, the tube is stirred again and the material therein is transferred to the cuvette. The cuvette is then read at a wavelength of 595 using a CECIL 3041 spectrophotometer.

3. Direct identification assay a. Material “GDP buffer” consists of 37.5 ml “binding buffer” and 2 mg GDP (Sigma catalog number G-7127), then a series of dilutions in “binding buffer” yields 0.2 μM GDP ( The final concentration of GDP in each well is 0.1 μM GDP). Each well containing a candidate compound is 100 μl “DPG buffer” (final concentration, 0.1 μM GDP), 50 μl “membrane protein” in “binding buffer”, and [ ³⁵ S in “binding buffer”. ] 200 ul final volume consisting of 50 μl GTPγS (0.6 nM) (2.5 μl [ ³⁵ S] GTPγS per 10 ml “binding buffer”).

b. Procedure Candidate compounds are preferably screened using a 96 well plate format (these can be frozen at -80 ° C). “Membrane protein” (or membrane with expression vector without “GPCR fusion protein” as a control) is briefly homogenized until suspended. The protein concentration is then determined using the “Bradford Protein Assay” set described above. The “membrane protein” (and control) is then diluted to 0.25 mg / ml in “binding buffer” (final assay concentration, 12.5 μg well). Thereafter, 100 μl of “GDP buffer” was added to each well of a Wallac Scintistrip ^™ (Wallac). A 5 ul pin instrument is then used to transfer 5 ul of the candidate compound into this well (ie 5 ul in a total assay volume of 200 μl is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 μM). Again, to prevent contamination, after each transfer step, wash the pin apparatus in three containers comprising water (1X), ethanol (1X) and water (2X). Shake off excess liquid from the appliance after each rinse and dry with paper and kimwipe. Thereafter, 50 μl of “membrane protein” is added to each well (control wells comprising membrane without “GPCR fusion protein” are also used) and preincubated at room temperature for 5-10 minutes. Thereafter, 50 μl of [ ³⁵ S] GTPγS (0.6 nM) in “binding buffer” is added to each well and then incubated for 60 minutes at room temperature in a shaker (also in this example the plate is covered with foil) ). The assay is then stopped by spinning the plate at 400 rpm for 15 minutes at 22 ° C. The plate is then aspirated using an 8-channel manifold and sealed using a plate cover. The plate is then read on the Wallac 1450 using the setting “Prot. $ 37” (according to the manufacturer's specifications)

B. Cyclic AMP Assay Another assay method for directly identifying candidate compounds is accomplished using a cyclase-based assay. In addition to direct identification, this assay can also be used as an independent method to provide confirmation of results from the [ ³⁵ S] GTPγS method as described above.

Modified Flashplate ^™ adenylate cyclase kit (New England Nuclear, Cat. No. SMP004A) is preferably used for direct identification of candidate compounds as inverse agonists and agonists for constitutively activated orphan GPCRs according to the following protocol It was done.

Transfected cells were harvested approximately 3 days after transfection. 20 mM HEPES, membranes were prepared by homogenization of cells suspended in buffer containing pH7.4 and 10 mM MgCl _2. Homogenization was performed on ice using Brinkman Polytron ^™ for about 10 seconds. The resulting homogenate is centrifuged at 49,000 Xg for 15 minutes at 4 ° C. The resulting pellet was then resuspended in a buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, and then centrifuged at 49,000 Xg for 15 minutes at 4 ° C. The resulting pellet was then stored at −80 ° C. until use. On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature and resuspended in a buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl ₂ to give a final protein concentration of 0.60 mg / ml. (Resuspend the membrane on ice until use).

cAMP standards and “detection buffer” (11 μl of “detection buffer” prepared tracer [comprising ¹² μci of ¹² IcAMP (100 μl)] and maintained according to the manufacturer's instructions. This was prepared for 20 mM HEPES, pH 7.4, 10 mM MgCl ₂ , 20 mM phosphocreatine (Sigma), 0.1 unit / ml creatine phosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP ( The “assay buffer” was then stored on ice until use.

  Candidate compounds identified as described above (if frozen, thaw at room temperature) are combined with 40 μl “membrane protein” (30 μg / well) and 50 μl “assay buffer”, preferably 96 plate wells (3 μl / well, 12 μM final assay concentration). The mixture was then incubated for 30 minutes at room temperature with gentle shaking.

After incubation, 100 μl of “detection buffer” was added to each well and then incubated for 2-24 hours. The plates were then counted on a Wallac Microbeta ^™ plate reader using “Prot # 31” (according to manufacturer's instructions).

  Representative screening assay plate (96 well format) results are shown in FIG. Each bar shows the results for the various compounds in each well and the RUP13-GαA fusion protein prepared in Example 5 (a) above. The representative results shown in FIG. 12 also give the standard deviation and average based on the average result (“m”) of each plate, and the average plus for selection of the inverse agonist as “lead” from the primary screen Two options include selection of candidate compounds reduced by at least twice the standard deviation from the percent response due to the average plate reaction. Conversely, an option for the selection of an agonist as a “lead” from the primary screen includes the selection of candidate compounds that are increased by at least twice the standard deviation to the response percentage by the average plate response. Based on these selection procedures, candidate compounds in the following wells were identified directly as putative inverse agonists (Compound A) and agonists (Compound B) for RUP13 in wells A2 and G9, respectively. See FIG. For clarity, the following is described. These compounds were directly identified without any knowledge of the endogenous ligand for this GPCR. Focusing on assay technology based on receptor function and not compound binding affinity, we can reduce the functional activity of this receptor (compound A) as well as increase the functional activity of the receptor (compound B) The compound can be confirmed. Based on the location of these receptors in lung tissue (see, eg, hRUP13 and hRUP21 in Example 6), drugs can be developed for possible therapeutic treatment of lung cancer.

  References cited within this specification, including co-pending and related patents, are hereby incorporated by reference in their entirety unless otherwise specified. Modifications and extensions of the disclosed invention which are within the judgment of the skilled worker are encompassed within the above disclosure and claims.

  While various expression vectors are available to those skilled in the art, it is most preferred that the vector used be pCMV for purposes of use for both endogenous and non-endogenous human GPCRs. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998, based on the provisions of the Budapest Treaty concerning the international recognition of the deposit of microorganisms in patent proceedings (10801 University Blvd., Manassas , VA 20110-2209 USA). The DNA was tested by ATCCD and determined to be available. The ATCC has assigned the following deposit number to pCMV: ATCC # 203351.

Claims (1)

Invention described in the specification.

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