JP2010166925A - Endogenous and non-endogenous version of human g protein-coupled receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new human G protein-coupled receptor and a mutated (non-endogenous) version for evidence of constitutive activity. <P>SOLUTION: There are provided a human G protein-coupled receptor for which the endogenous ligand is unknown, and a mutant (non-endogenous) version for evidence of constitutive activity, wherein the G protein-coupled receptor having a specific amino acid sequence and its constitutively activated version are provided, a cDNA encoding the human G protein-coupled receptor, and a plasmid containing a vector are provided, and host cells containing the plasmid are provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

(Quotations to related applications)
This application is filed with US patent application Ser. No. 09 / 170,496 (filed with the USPTO on October 13, 1998) and its corresponding PCT application number PCT / US99 / 23938 (WO 00/00 on April 20, 2000). Published as 22129). This application claims the benefit of priority from the following provisional applications (all of the following provisional applications were filed with the US Patent and Trademark Office on the indicated date via the US Express Mail: ): US Provisional Patent Application No. 60 / 253,404 (filed November 27, 2000); US Provisional Patent Application Number 60 / 255,366 (filed December 12, 2000); US Provisional Patent Application Number 60/270 286 (submitted on February 20, 2001); U.S. Provisional Patent Application No. 60 / 282,356 (filed Apr. 6, 2001) (this provisional application is U.S. Provisional Patent Application No. 60 / 270,266 (2001)). US Provisional Patent Application No. 60 / 282,032 (filed April 6, 2001); US Provisional Patent Application No. 60 / 282,358 (April 2001) 6th U.S. Provisional Patent Application No. 60 / 282,365 (submitted on April 6, 2001); U.S. Provisional Patent Application No. 60 / 290,917 (submitted on May 14, 2001); U.S. Provisional Patent Application No. 60 / 309, 208 (submitted on July 31, 2001); these disclosures are incorporated by reference in their entirety).

(Field of Invention)
The present invention relates to transmembrane receptors, in some embodiments, to G protein coupled receptors, and in some embodiments, to endogenous GPCRs that have been altered to establish or enhance the constitutive activity of the receptor. . In some embodiments, this constitutively activated GPCR is used for direct identification of candidate compounds as receptor agonists or inverse agonists with applicability as therapeutic agents.

(Background of the Invention)
Although a large number of receptor classes exist in humans, it is demonstrated by the G protein coupled receptor (GPCR) class that it is by far the most abundant and therapeutically relevant. It is estimated that there are about 30,000-40,000 genes in the human genome, of which about 2% are estimated to encode GPCRs. Receptors for which endogenous ligands have been identified (including GPCRs) are termed “known” receptors, whereas receptors for which no endogenous ligand has been identified are termed “orphan” receptors.

GPCRs represent an important area for drug development: from about 20 out of 100 known GPCRs, about 60% of all prescription drugs have been developed. For example, in 1999, of the top 100 trade name prescription drugs, the following drugs interact with GPCRs (diseases and / or disorders to be treated are indicated in parentheses):
Claritin (R) (allergy) Prozac (R) (depression) Vasotec (R) (hypertension) Paxil (R) (depression) Zoloft (R) (depression) Zyprexa (R) (psychiatric) Cozaar ( (Registered trademark) (hypertension) Imitrex (registered trademark) (migraine) Zantac (registered trademark) (reflux) Propulsid (registered trademark) Risperdal (registered trademark) (schizophrenia) Servent (registered trademark) (asthma) Pepcid (R) (reflux) Gaster (R) (ulcer) Atrovent (R) (bronchospasm) Effexor (R) (depression) Depakote (R) (）) Cardura (R) (prostatic hypertrophy) All gra (registered trademark) (allergy) Lupron (registered trademark) (prostate cancer) Zoladex (registered trademark) (prostate cancer) Diprivan (registered trademark) (sensory loss) BuSpar (registered trademark) (anxiety) Ventolin (registered trademark) ( Bronchospasm) Hytrin (R) (hypertension) Wellbutrin (R) (depression) Zyrtec (R) (rhinitis) Plavix (R) (myocardial infarction / seizure) Topol-XL (R) (hypertension)
Tenormin (registered trademark) (angina) Xalatan (registered trademark) (glaucoma)
Singulair (registered trademark) (asthma) Diovan (registered trademark) (hypertension) Harnal (registered trademark) (prostatic hypertrophy) (Non-patent Document 1).

  GPCRs share a common structural motif, which has seven sequences of 22-24 hydrophobic amino acids that form seven α helices, each of which spreads across the membrane. (Each spread is identified by a number, ie transmembrane 1 (TM-1), transmembrane 2 (TM-2), etc.). This transmembrane helix is located between the transmembrane 2 and transmembrane 3, between the transmembrane 4 and transmembrane 5 and between the transmembrane 6 and transmembrane 7 by the amino acid strands (ie, outside the cell membrane). , "Extracellular" side) (these are called "extracellular" region 1 (EC-1), extracellular region 2 (EC-2), and extracellular region 3 (EC-3), respectively) ). This transmembrane helix is also introduced into the cell membrane by amino acid strands between transmembrane 1 and transmembrane 2, between transmembrane 3 and transmembrane 4 and between transmembrane 5 and transmembrane 6 ( That is, they are connected on the “intracellular” side (these are “intracellular” region 1 (IC-1), intracellular region 2 (IC-2), and intracellular region 3 (IC-3), respectively). be called). The “carboxy” (“C”) terminus of this receptor is in the intracellular space in the cell, and the “amino” (“N”) terminus of this receptor is in the extracellular space outside the cell.

In general, when an endogenous ligand binds to this receptor (often referred to as “activation” of the receptor), it allows intracellular coupling between the intracellular region and the intracellular “G protein”. There are region conformational changes. GPCRs are reported to be “promiscuous” with respect to G proteins, ie, GPCRs can interact with more than one G protein. Kenakin, T .; , 43 Life Sciences 1095 (1988). Currently, G _q , G _s , G _i , G _Z and G _O are the identified G proteins, although other G proteins exist. A ligand-activated GPCR coupled to a G protein initiates a signal transduction cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cell activation or cell inhibition. Without wishing to be bound by theory, it is thought that the IC-3 loop and carboxy terminus of this receptor interact with the G protein.

Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations, an “inactive” state and an “active state”. An inactive receptor cannot connect to the intracellular signaling pathway and initiate signaling to produce a biological response. Changing the receptor conformation to the active state allows linkage to this transduction pathway (via the G protein) and produces a biological response.
Receptors can be stabilized in the active state by ligands or compounds (eg, drugs). Due to recent discoveries (including but not limited to modifications to the amino acid sequence of this receptor), there is a different means for ligands and drugs to stimulate and stabilize the receptor in its active state conformation. Provided. These means effectively stabilize the receptor in the active state by mimicking the effect of ligand binding to the receptor. Such stabilization by ligand independent means is referred to as “constitutive receptor activity”.
Prior art document information relating to the invention of this application includes the following.

Med Ad News 1999 Data

(Summary of the Invention)
Disclosed herein are endogenous and non-endogenous versions of human GPCRs and their uses.

  Some embodiments of the invention relate to a G protein coupled receptor encoded by the amino acid sequence of SEQ ID NO: 2, a non-endogenous constitutively activated version, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 1 and a host cell comprising the same.

  Some embodiments of the invention relate to a G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 4, a non-endogenous constitutively activated version, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 3, a non-endogenous constitutively activated version thereof, and a host cell comprising the same.

  Some embodiments of the invention relate to a G protein coupled receptor encoded by the amino acid sequence of SEQ ID NO: 6, a non-endogenous constitutively activated version, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 5, and a host cell comprising the same.

  Some embodiments of the invention relate to a G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 8, a non-endogenous constitutively activated version, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 7, a non-endogenous constitutively activated version thereof, and a host cell comprising the same.

  Some embodiments of the invention relate to a G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 10, a non-endogenous constitutively activated version, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 9, and a host cell comprising the same.

  Some embodiments of the invention relate to the G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 12, its non-endogenous constitutively activated version, and host cells containing it.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 11, and a host cell comprising the same.

  Some embodiments of the invention relate to the G protein coupled receptor encoded by the amino acid sequence of SEQ ID NO: 14, a constitutively activated version thereof, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 13, and a host cell comprising the same.

  Some embodiments of the invention relate to the G protein coupled receptor encoded by the amino acid sequence of SEQ ID NO: 16, a constitutively activated version thereof, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 15, and a host cell comprising the same.

  Some embodiments of the invention relate to the G protein coupled receptor encoded by the amino acid sequence of SEQ ID NO: 18, a constitutively activated version thereof, and a host cell comprising the same.

  Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 17, and a host cell comprising the same.

  Some embodiments of the invention relate to the G protein coupled receptor encoded by the amino acid sequence of SEQ ID NO: 20, a constitutively activated version thereof, and a host cell comprising the same.

Some embodiments of the invention relate to a vector and a plasmid comprising the cDNA of SEQ ID NO: 19, and a host cell comprising the same.
The present invention provides the following.
(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 version of the G protein coupled receptor of item 1.
(Item 3) A plasmid comprising a vector and the cDNA of SEQ ID NO: 1.
(Item 4) A host cell comprising the plasmid according to item 3.
(Item 5) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 4.
(Item 6) A non-endogenous constitutively activated version of the G protein coupled receptor of item 5.
(Item 7) A plasmid comprising a vector and the cDNA of SEQ ID NO: 3.
(Item 8) A host cell comprising the plasmid according to 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 version of the G protein-coupled receptor of item 9.
(Item 11) A plasmid comprising a vector and the cDNA of SEQ ID NO: 5.
(Item 12) A host cell comprising the plasmid according to 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 version of the G protein coupled receptor of item 13.
(Item 15) A plasmid comprising a vector and the cDNA of SEQ ID NO: 7.
(Item 16) A host cell comprising the plasmid according to 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 version of the G protein coupled receptor of item 17.
(Item 19) A plasmid comprising a vector and the cDNA of SEQ ID NO: 9.
(Item 20) A host cell comprising the plasmid according to 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 version of the G protein-coupled receptor of item 21.
(Item 23) A plasmid comprising a vector and the cDNA of SEQ ID NO: 11.
(Item 24) A host cell comprising the plasmid according to item 23.
(Item 25) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 14.
26. A non-endogenous constitutively activated version of the G protein coupled receptor of claim 25.
(Item 27) A plasmid comprising a vector and the cDNA of SEQ ID NO: 13.
(Item 28) A host cell comprising the plasmid according to item 27.
(Item 29) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 16.
30. A non-endogenous constitutively activated version of the G protein coupled receptor of item 29.
(Item 31) A plasmid comprising a vector and the cDNA of SEQ ID NO: 15.
(Item 32) A host cell comprising the plasmid according to item 31.
(Item 33) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 18.
34. A non-endogenous constitutively activated version of the G protein coupled receptor of claim 33.
(Item 35) A plasmid comprising a vector and the cDNA of SEQ ID NO: 17.
(Item 36) A host cell comprising the plasmid according to item 35.
(Item 37) A G protein-coupled receptor encoded by the amino acid sequence of SEQ ID NO: 20.
38. A non-endogenous constitutively activated version of the G protein coupled receptor of claim 37.
(Item 39) A plasmid comprising a vector and the cDNA of SEQ ID NO: 19.
(Item 40) A host cell comprising the plasmid according to item 39.

1, the second messenger _{assay, RUP32, G q (del)} / G i, G q (del) / G i and cotransfected RUP32, and CMV; graphically display activation (control expression vector) Yes, this assay uses 293 cells to measure the accumulation of inositol phosphates (IP ₃ ). FIG. 2 provides an illustration of the generation of second messenger IP ₃ from endogenous versions of RUP 35 and RUP 36 compared to control (“CMV”).

(Detailed explanation)
The scientific literature developed for receptors has adopted a number of terms to refer to ligands that have various effects on the receptors. For clarity and consistency, the following definitions are used throughout this patent application. To the extent that these definitions contradict other definitions for these terms, the following definitions govern:
“Agonist” is intended to mean a substance (eg, a ligand, candidate compound) that activates an intracellular response or enhances the binding of GTP to the membrane when bound to a receptor. In some embodiments, an “agonist” is a substance that has not previously been known to activate an intracellular response when bound to a receptor or to enhance binding of GTP to a membrane. is there.
As used herein, “amino acid abbreviations” are shown in Table A:
Table A

  An “antagonist” is a competitive binding 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 inhibiting the intracellular response by the agonist. It shall mean a substance (eg, a ligand, a candidate compound, etc.). Antagonists do not decrease the baseline intracellular response in the absence of agonist. In some embodiments, an antagonist is a substance that was not previously known to activate an intracellular response when bound to a receptor or to enhance the binding of GTP to the membrane.

  "Candidate compound" shall mean a molecule (eg, but not limited to a compound) that is capable of undergoing screening techniques. Preferably, the phrase “candidate compound” is a publicly known compound selected from the group consisting of an inverse agonist for a receptor, an agonist for a receptor, or an antagonist for a receptor as previously determined by an indirect identification process (“Indirectly identified compounds”); more preferably, does not include indirectly identified compounds previously determined to have therapeutic efficacy in at least one mammal; most preferably Does not include indirectly identified compounds previously determined to have therapeutic efficacy in humans.

  “Composition” shall mean a substance comprising at least one component; “Pharmaceutical composition” is an example of a composition.

  “Compound potency” shall mean a measure of the ability of a compound to inhibit or stimulate receptor function (ie, the ability to activate / inhibit a signaling pathway as opposed to receptor binding affinity). . Exemplary means of detecting compound potency are disclosed in the Examples section herein.

  "Codon" generally includes nucleosides (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) linked to a phosphate group, and when translated It shall mean the classification of three nucleotides (or nucleotide equivalents) that encode amino acids.

  "Constitutively activated receptor" shall mean a receptor that is subjected to constitutive receptor activation. The constitutively activated receptor may be endogenous or non-endogenous.

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

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

  “Directly identified” or “directly identified” in relation to the phrase “candidate compound” means a constitutively activated receptor (preferably a constitutively activated orphan receptor, most preferably a constitutively It is meant to screen candidate compounds against activated G protein-coupled cell surface orphan receptors and assess the compound potency of such compounds. This phrase should in no way be construed, nor should it be construed, encompassed, or understood by the phrase “indirectly identified” or “indirectly identified”.

  “Endogenous” shall mean a substance naturally produced by a mammal. For example, without limitation, “endogenous” with respect to the term “receptor” shall mean produced naturally by a mammal (eg, but not limited to a human) or a virus. In contrast, the term “non-endogenous” in this context shall mean not naturally produced by a mammal (eg, but not limited to a human) or a virus. For example, but not limited to, a receptor that has been engineered to be constitutively active but not constitutively active in its endogenous form is most preferably referred to herein as “non-endogenous. , Called constitutively activated receptor. Both terms can be used to describe both “in vivo” and “in vitro” systems. For example, but not limited to, in a screening approach, endogenous or non-endogenous receptors can be with respect to an in vitro screening system. By way of further example, but not by way of limitation, screening of candidate compounds by an in vivo system is feasible if the mammalian genome is engineered to contain a non-endogenous constitutively activated receptor.

“G protein-coupled receptor fusion protein” and “GPCR fusion protein” in the context of the invention disclosed herein each refers to at least one G protein (most preferably the alpha ( α) refers to a non-endogenous protein comprising an endogenous constitutively activated GPCR or a non-endogenous constitutively activated GPCR fused to a subunit, which is a subunit that binds GTP) However, this G protein is preferably of the same type as the G protein naturally conjugated to the endogenous orphan GPCR. For example, but not limited to, in the endogenous state, if the G protein “G _s α” is the dominant G protein coupled to a GPCR, a particular GPCR-based GPCR fusion protein is fused to G _s α. Non-endogenous proteins, including GPCRs; in some situations, non-dominant G proteins can be fused to GPCRs, as shown below. The G protein can be fused directly to the C-terminus of the constitutively activated GPCR, or there can be a spacer between the two.

  “Host cell” shall mean a cell capable of having a plasmid and / or vector incorporated therein. In the case of prokaryotic host cells, the plasmid is typically replicated as an autologous molecule when the host cell replicates (generally this plasmid is subsequently isolated for introduction into a eukaryotic host cell. In the case of a eukaryotic host cell, the plasmid is integrated into the cellular DNA of the host cell so that the plasmid replicates when the eukaryotic host cell replicates. In some embodiments, the host cell is a eukaryotic host cell, more preferably a mammalian host cell, and most preferably from the group consisting of 293 cells, 293T cells, and COS-7 cells. Selected.

  “Indirectly identifying” or “indirectly identified” means a traditional approach to the drug discovery process, which involves identifying an endogenous ligand specific for an endogenous receptor, its ligand -Screening candidate compounds against the receptor to determine compounds that interfere with and / or compete with receptor interactions, and assess the potency of the compound for affecting at least one second messenger associated with the activated receptor. Process.

  “Inhibit” or “inhibition” in the context of the term “response” means that the response is reduced or prevented in the presence of a compound as opposed to in the absence of that compound. Shall.

  An “inverse agonist” binds to either the endogenous form of a receptor or a constitutively activated form of the receptor and initiates a baseline intracellular response initiated by the activated form of that receptor in the absence of an agonist. Is intended to mean a substance (eg, ligand, candidate compound) that inhibits below normal baseline level activity observed in or reduces GTP binding to the membrane. Preferably, the baseline intracellular response is at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, at least compared to the baseline response in the absence of the opposite agonist. 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, most preferably at least 99%, and vice versa in the presence of agonist Is done.

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

  “Ligand” shall mean a molecule specific for a naturally occurring receptor.

  “Mutant” or “mutation” refers to a nucleic acid sequence and / or amino acid sequence of an endogenous receptor such that a mutant form of the endogenous non-constitutively activated receptor exhibits constitutive activation of the receptor. It is intended to mean a specific change to such an endogenous sequence. (A) when the level of constitutive activation of the mutated form of the human receptor is substantially the same as that indicated by the initial mutation of that receptor, with respect to the equivalent to a particular sequence; and (b The percent homology of the sequence (amino acid sequence and / or nucleic acid sequence) between the mutated form of the receptor and the first mutation of the receptor is at least 80%, at least 85%, at least 90%, at least If it is 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, most preferably at least 99%, the mutated form of the human receptor is the first of the human receptor Is considered equivalent to the mutation. In some embodiments, some preferred cassettes disclosed herein for achieving constitutive activation are single amino acid changes between the endogenous and non-endogenous forms of the GPCR. Due to the fact that and / or contains a single codon change, it is preferred that this percent sequence homology should be at least 98%.

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

  “Orphan receptor” is intended to mean an endogenous receptor for which a specific ligand for the receptor has not been identified or is unknown.

  A “pharmaceutical composition” is a composition comprising at least one active ingredient, whereby the composition is of a specified effective outcome in a mammal (eg, a human, but not limited to). It shall mean a composition subject to investigation. Those skilled in the art will understand and appreciate the appropriate techniques for determining whether an active ingredient has the desired efficacious outcome based on the needs of the artisan.

  “Plasmid” shall mean a combination of a vector and cDNA. Generally, a plasmid is introduced into a host cell for the purpose of cDNA replication and / or expression 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 to determine receptor activation. In addition, second messenger response can be measured for direct identification of candidate compounds, including, for example, inverse agonists, agonists and antagonists.

  “Signal-to-noise ratio” is the signal generated in response to activation, amplification or stimulation, where the signal is above the background level in response to background noise or inactivation, unamplification or non-stimulation. Also means high signal.

  A “spacer” is a number of translated amino acids that are located after the last codon or amino acid of a gene (eg, GPCR of interest), but before the start codon or region of the G protein of interest. Thus, here, a number of translated amino acids shall mean a number of translated amino acids arranged in-frame with the start region of the target G protein. The number of translated amino acids can be adjusted according to the needs of those skilled in the art and is generally about 1 amino acid, preferably 2 amino acids, more preferably 3 amino acids, more preferably 4 amino acids, more preferably 5 amino acids, more preferably Is from 6 amino acids, more preferably 7 amino acids, more preferably 8 amino acids, more preferably 9 amino acids, more preferably 10 amino acids, more preferably 11 amino acids, and even more preferably 12 amino acids.

  In the context of the term “response”, “stimulate” or “stimulating” shall mean that the response increases in the presence of the compound as opposed to in the absence of the compound. .

  “Substantially” means within 40% of the control result, preferably within 35%, more preferably within 30%, more preferably within 25%, more preferably within 20%, more preferably within 15% of the control result. It is intended to refer to results that are within%, more preferably within 10%, more preferably within 5%, more preferably within 2%, and most preferably within 1%. For example, with respect to receptor functionality, the signal transmitted is 40% of the signal produced by the control signal as measured using the methods taught herein or similar methods known to those skilled in the art. If within%, the test receptor may show substantially similar results as the control receptor.

  With respect to cDNA, a vector shall mean circular DNA capable of incorporating at least one cDNA and capable of incorporating into a host cell.

  The order of the following sections is presented for display efficiency, and neither the following disclosure nor the claims are intended and should not be construed as limiting.

(A. Introduction)
Traditional studies of receptors typically show that endogenous ligands must first be identified before discovery can proceed to find antagonists and other molecules that can affect this receptor (historical Based on prior assumptions). Even when antagonists were first known, this search was immediately extended to search for endogenous ligands. This form of thought persisted in receptor studies even after constitutively activated receptors were found. What has not been recognized so far is that it is the receptor in the active state that is most useful for finding agonists and inverse agonists of this receptor. For diseases resulting from overactive receptors or under-active receptors, what is desired in therapeutic drugs is not necessarily a drug that is an antagonist to an endogenous ligand, respectively, the active state of the receptor Is a compound that acts to decrease or increase the activity of the receptor. 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, as taught by the methods of the invention, any search for therapeutic compounds should be initiated by screening the compounds to eliminate ligand-independent activity states.

(B. Identification of human GPCR)
The efforts of the Human Genome Project have identified excessive information about nucleic acid sequences located within the human genome; the fact in this effort is that any particular genomic sequence contains or contains open reading frame information that translates human proteins. The gene sequence information has become available without understanding or recognizing whether it is obtained or not. Several methods for identifying nucleic acid sequences within the human genome are within the purview of those skilled in the art. For example, and without limitation, the various human GPCRs disclosed herein have been found by reviewing the GenBank ^™ database. Table B below lists some endogenous GPCRs we have found, along with other GPCRs that are homologous to the disclosed GPCRs.

  Receptor homology is useful for obtaining the correct recognition of the role of this receptor within the human body. As the patent literature progresses, techniques for mutating these receptors to establish non-endogenous constitutively activated versions of these receptors are considered.

  The techniques disclosed herein are also applicable to other human GPCRs known in the art, as will be apparent to those skilled in the art.

(C. Receptor screening)
Screening for candidate compounds, eliminating the non-endogenous constitutively activated version of the GPCR disclosed herein, is a candidate that acts on a cell surface receptor without requiring the use of the receptor's endogenous ligand. Allows direct identification of compounds. Conventional and often commercially utilized techniques can be used to determine areas in the body where the endogenous version of the human GPCR disclosed herein is expressed and / or overexpressed. The location of the receptor expression in a particular tissue provides the scientist with the ability to assign the physiological functional role of this receptor. Using these techniques it is also possible to determine the relevant disease / disorder status associated with the expression and / or overexpression of this receptor; such an approach is disclosed in this patent document . In addition, the expression of the receptor in the diseased organ can help determine the clinically relevant magnitude of this receptor.

  The constitutive activation of the GPCR disclosed herein is based on the distance from the proline residue predicted to be located within TM6 of the GPCR; this algorithmic technique is co-pending and assigned to the same person Patent Document PCT Application No. PCT / US99 / 23938 (published on April 20, 2000 as WO 00/22129) (incorporated herein by reference with other patent documents listed herein) Is disclosed). Algorithmic techniques are not predicted based on the traditional sequence “alignment”, but rather from the TM6 proline residues described above (or, of course, endogenous constitutive substitutions for such proline residues). Predicted for a specific distance. By mutating the amino acid residue present at position 16 amino acid residues from the residue (possibly present in the IC3 region of the receptor) to a lysine residue most preferably, constitutive activation of the receptor is obtained. Can be. Other amino acid residues may be useful in mutations at this position to achieve this goal and are discussed in detail below.

(D. Identification and / or selection of disease / disorder)
As shown in greater detail below, inverse agonists and agonists for non-endogenous 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 search for diseases and disorders associated with GPCRs is appropriate because of the ability to directly identify inverse agonists for GPCRs, thereby enabling the development of pharmaceutical compositions. The location of receptor expression in a particular tissue provides scientists with the ability to assign physiological functions to the receptor. For example, scanning both diseased tissue samples and normal tissue samples for the presence of GPCRs is now more than can be accomplished with academic challenges or pathways that identify endogenous ligands for a particular GPCR. . Tissue scanning can be performed over a wide range of healthy and diseased tissues. Such tissue scanning provides a potential first step in associating specific receptors with diseases and / or disorders. Furthermore, the expression of the receptor in the diseased organ can help to identify in determining the magnitude of clinical relevance of the receptor.

  GPCR DNA sequences can be used to generate probes / primers. In some preferred embodiments, this DNA sequence is used to generate probes for: (a) dot-blot analysis on tissue mRNA, and / or (b) RT of expression of this receptor in tissue samples. -PCR identification. The presence of the receptor in the tissue source or diseased tissue, or the presence of this receptor in the diseased tissue at an elevated concentration compared to normal tissue can be used to correlate position to function and receptor physiology Treatment regimens can be generated and treatment regimens can be generated including, but not limited to, diseases associated with that function / role. Receptors can also be localized to regions of the organ by this technique. Based on the known or putative role / function of the particular tissue in which the receptor is localized, the putative physiological function of this receptor can be inferred. For example, and without limitation, proteins that are located and / or expressed in the area of the thalamus are associated with sensorimotor processing and arousal (Goodman and Gilman, The Pharmaceutical Basis of Therapeutics, 9th edition, page 465 (1996). See Proteins expressed in hippocampal or Schwann cells are associated with learning and memory and myelination of peripheral nerves, respectively (Kandel, E. et al., Essentials of Neural Science and Behavior, 657, 680 and 28, respectively). Page (1995)).

(E. Screening of candidate compounds)
(1. Comprehensive GPCR screening assay technology)
When the G protein receptor becomes constitutively active, it binds to the G protein (eg, G _q , G _s , G _i , G _z , Go) and stimulates GTP binding to the G protein. The G protein then acts as GTPase and hydrolyzes GTP to GDP, thereby inactivating the receptor under normal conditions. However, constitutively activated receptors continue to exchange GDP for GTP. The non-hydrolyzable analog of GTP [ ³⁵ S] GTPγS can be used to monitor enhanced binding to membranes expressing constitutively activated receptors. It has been reported that [ ³⁵ S] GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring was reported in 1995 by Raynor and Nahorski, among other examples well known and available to those skilled in the art. The use of this assay system is typically for initial screening of candidate compounds. This system is universally applicable to all G protein coupled receptors, regardless of the specific G protein that interacts with the intracellular domain of the receptor.

(2. Specific GPCR screening assay technology)
Once a candidate compound has been identified using a “global” G protein coupled receptor assay (ie, an assay to select compounds that are agonists or inverse agonists), confirm that the compound interacted at the receptor site Further screening is preferred. For example, a compound identified by a “global” assay may not bind to the receptor, but instead may simply “uncouple” the G protein from the intracellular domain.

(A. G _s , G _z and G _i )
G _s stimulates the enzyme adenylyl cyclase. On the other hand, _{G i} (and _{G z} and Go) inhibit adenylyl cyclase. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, conjugating _{G s} protein, the GPCR that is constitutively activated, associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple G _i (or G _z , Go) proteins are associated with reduced cellular levels of cAMP. See generally, “Indirect Mechanicals of Synchronic Transmission”, Chapter 8, From Neuron To Brain (3rd edition) Nichols, J. et al. G. Et al., Sinauer Associates, Inc. (1992). Thus, an assay that detects cAMP can be utilized to determine whether a candidate compound is, for example, an inverse agonist for its receptor (ie, such a compound reduces the level of cAMP). Various approaches known in the art for measuring cAMP can be utilized; the most preferred approach relies on the use of anti-cAMP antibodies in an ELISA-based format. Another type of assay that can be utilized is a whole cell second messenger reporter-based assay. A promoter on a gene drives the expression of the protein encoded by that particular gene. Cyclic AMP drives gene expression by promoting the binding of cAMP-responsive DNA binding protein or transcription factor (CREB), which then binds to the promoter at a specific site (cAMP responsive element), and Drives gene expression. A reporter system having a promoter, wherein the promoter comprises a plurality of cAMP response elements in front of a reporter gene (eg, β-galactosidase or luciferase) can be constructed. Therefore, constitutively activated G _s coupled receptor causes cAMP accumulation, this is then the gene activated, directs the expression of the reporter protein. The reporter protein (eg, β-galactosidase or luciferase) can then be detected using standard biochemical assays (Chen et al. 1995).

(B. G _o and G _q )
G _q and _{G o} are associated with activation of the enzyme phospholipase C, which in turn, the phospholipid PIP ₂ by hydrolysis, to release the following two intracellular messengers: diacylglycerol (diacycloglycerol) (DAG) And inositol 1,4,5-triphosphate (IP ₃ ). Increased accumulation of IP ₃ is associated with activation of _{G q} associated receptors and _{G o} associated receptors. See generally, “Indirect Mechanicals of Synchronic Transmission”, Chapter 8, From Neuron To Brain (3rd edition) Nichols, J. et al. G. Et al., Sinauer Associates, Inc. (1992). Using the assays that detect IP ₃ accumulation candidate compound, for example, an agonist of inverse relative to G _q associated receptors or Go-associated receptors (i.e., such a compound may reduce the level of IP ₃₎ or You can decide whether or not. G _q related receptors also obtained and tested using the AP1 reporter assay, where, G _q-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated G _q related receptors The increase in the expression of such genes is demonstrated, whereby the opposite agonist against it demonstrates a decrease in such expression, and the agonist demonstrates the increase in such expression. Commercially available assays for such detection are available.

(3. GPCR fusion protein)
In the use of endogenous constitutively activated GPCRs or non-endogenous constitutively activated GPCRs for use in screening candidate compounds for direct identification of inverse agonists, clearly Agonists provide an interesting screening challenge in that the receptor is active even in the absence of endogenous ligands that bind to it. Thus, such a compound can be an inverse agonist or agonist, eg, by discrimination such as discrimination between a non-endogenous receptor in the presence of a candidate compound and a non-endogenous receptor in the absence of that compound. Or non-endogenous receptor in the presence of the candidate compound and non-existence in the absence of the compound, with the aim of enabling an understanding of whether or not it has any effect on such a receptor. In order to distinguish from endogenous receptors, it is preferable to utilize an approach that can enhance such discrimination. A preferred approach is the use of GPCR fusion proteins.

  In general, using the assay techniques described above (as well as other techniques), once it has been determined that the non-endogenous GPCR is constitutively activated, the dominant G protein coupled to the endogenous GPCR is determined. Is possible. Coupling of the G protein to the GPCR provides a signaling pathway that can be evaluated. In some embodiments, it is preferred to screen using a mammalian expression system, such a system is expected to have an endogenous G protein therein. Clearly, therefore, in such a system, non-endogenous constitutively activated GPCRs continuously signal. In some embodiments, this signal is enhanced so that, for example, in the absence of the reverse agonist for the receptor, between this receptor when contacted with the reverse agonist (particularly in a screening context). It is preferred that there is a higher probability that it can be more easily identified.

  GPCR fusion proteins are intended to enhance the efficacy of G protein coupling with non-endogenous GPCRs. GPCR fusion proteins are preferred for screening using either endogenous constitutively active GPCRs or non-endogenous constitutively activated GPCRs. This is because such an approach increases the signal utilized in such screening techniques. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is preferred for screening candidate compounds as disclosed herein.

  The construction of constructs useful for expression of GPCR fusion proteins is within the skill of the art. Commercially available expression vectors and systems provide various approaches that can be tailored to the investigator's specific needs. An important criterion for the construction of such GPCR fusion protein constructs is that both the endogenous GPCR sequence and the G protein sequence are in-frame (preferably the sequence for the endogenous GPCR is upstream of the G protein sequence. And the GPCR “stop” codon has been deleted or replaced so that the G protein can also be expressed during expression of the GPCR. Other embodiments include constructs in which the endogenous GPCR and G protein sequences are not in frame and / or the “stop” codon has not been deleted or replaced. The GPCR can be linked directly to the G protein or there can be a spacer residue between the two (preferably less than about 12 but this number can be readily ascertained by one skilled in the art). It is preferable to use a spacer based on convenience. Preferably, the G protein that couples to the non-endogenous GPCR has been identified prior to making the GPCR fusion protein construct. Since only a few G proteins have been identified, a construct containing the sequence of the G protein (ie, a universal G protein construct (see Examples)) is due to the insertion of the endogenous GPCR sequence therein. This provides additional efficiency in the context of large-scale screening of a variety of different endogenous GPCRs with different sequences.

As described _above, conjugated to G i, _{G z} and _{G o,} the GPCR that is constitutively activated, is expected to inhibit the formation of cAMP, This difficulty assays based upon these types of GPCR (Ie, the cAMP signal decreases when activated, thus making it difficult to directly identify, for example, the reverse agonist, which further reduces this signal). As disclosed herein, in an effort to establish viable cyclase-based assays for these types of receptors, we make GPCR fusion proteins that are not based on GPCR endogenous G proteins. Confirmed that it is possible. Thus, for example, an endogenous G _i coupled receptor can be fused to a G _s protein—such a fusion construct, upon expression, converts an endogenous GPCR into a “native” G _i protein, eg, a G “Drive” or “force” to be coupled to _s , so that a cyclase-based assay can be established. Thus, G _i coupled receptors, G _z-coupled receptor and G _o-coupled receptors, in some embodiments, GPCR fusion protein is used, and if the assay is based upon detection of adenylyl cyclase activity, fusion constructs, It is preferably established using G _s (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).

Equally effective are G protein fusion constructs that utilize G _q protein fused to G _s protein, G _i protein, G _z protein or _Go protein. In some embodiments, preferred fusion construct can be accomplished using a G _q proteins, where the first 6 amino acids of G protein α- subunit ( "Gαq") is deleted, The last 5 amino acids at the C-terminal of Gaq are substituted with the corresponding amino acids of Gα of the target G protein. For example, a fusion construct may have Gq (6 amino acid deletion) fused to a G _i protein, resulting in a “G _q / G _i fusion construct”. This fusion construct, endogenous G _i coupled receptor is forced to conjugate to its non-endogenous G protein G _q, as a result, second messenger (e.g., inositol triphosphate or diacylglycerol) is, cAMP generation Can be measured instead of

(4. Co-transfection of the target _{G i} coupled GPCR and signal enhancer _{G s} coupled GPCR (cAMP Based Assays))
G _i coupled receptors, are known to inhibit adenylyl cyclase, therefore, reduces the level of cAMP production, which can make it difficult to evaluate the cAMP level. When activated as an indication of constitutive activation of the receptor that preferentially coupled with G _i, 1 single effective technique in measuring the decrease in production of cAMP, the signal enhancer (e.g., when activated G _s and preferentially conjugated to non-endogenous, constitutively activated receptor) (e.g., be achieved by TSHR-A623I) a G _i coupled GPCR and cotransfected disclosed below. As is apparent, constitutive activation of G _s coupled receptors may be determined based on the increase in production of cAMP. Constitutive activation of G _i coupled receptors leads to decrease in cAMP production. Thus, co-transfection approaches are intended to take advantage of these “reverse” effects. For example, co-transfection of a non-endogenous constitutively activated G _s coupled receptor (the "signal enhancer") with the endogenous G _i coupled receptor (the "target receptor") may provide the cAMP signal baseline to (i.e., the G _i coupled receptors reduces cAMP levels, this "decrease" is established by G _s coupled signal enhancer that is constitutively activated, in compared to the substantial increase in cAMP levels is there). Then the signal enhancer, by targeting a receptor cotransfected with constitutive activation version, cAMP is G _i target increased functional activity (i.e., which decreases the cAMP) due to , Expected to decrease further (compared to baseline).

Then, screening of candidate compounds using cAMP-based assays of two compared to the use of endogenous receptor / G-protein fusions using the "change" may be achieved: First, the G _i coupled target receptor in comparison, results in a "reverse" effect (i.e., inverse agonists of G _i coupled target receptor will increase the cAMP signal measured, whereas agonists of Gi-coupled target receptor will decrease this signal Secondly, as is apparent, candidate compounds that are directly identified using this approach should be evaluated independently to ensure that they do not target signal-enhancing receptors ( This can be done before or after screening for co-transfected receptors).

(F. Pharmaceutical chemistry)
In general, but not always, direct identification of candidate compounds is performed in relation to compounds generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis Is done. In general, the results of such screening are compounds with unique core structures; these compounds can then be subjected to further chemical modifications around the preferred core structure to further enhance their medical properties. . Such techniques are known to those skilled in the art and are not dealt with in detail in this patent document.

(G. Pharmaceutical composition)
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. (See Osol et al.).

(H. Other usefulness)
A preferred use of the non-endogenous version of the GPCR disclosed herein may be for direct identification of candidate compounds as inverse agonists or agonists (preferably for use as pharmaceutical agents) There are other uses for these versions of GPCRs. For example, utilizing in vitro and in vivo systems incorporating GPCRs, we can further elucidate and understand the role that these receptors play in the human (both normal and diseased) state and understand the signaling cascade When applying this to understand, the role of constitutive activation can be understood. In some embodiments, it is preferred that the endogenous receptor is an “orphan receptor,” ie, no endogenous ligand for the receptor has been identified. Thus, in some embodiments, a modified non-endogenous GPCR can therefore be used to understand the role of endogenous receptors in the human body before the endogenous ligand is identified. Such receptors can also be used to further elucidate known receptors and the pathways through which they transmit signals. Other uses of the disclosed receptors will be apparent to those skilled in the art based upon, among other things, a review of this patent document.

(Example)
The following examples are presented for the purpose of elucidating the present invention and not for the purpose of limitation. Although specific nucleic acid and amino acid sequences are disclosed herein, one of ordinary skill in the art will be able to perform minor or minor changes to these sequences while achieving the same or substantially similar results as reported below. It is considered capable of making modifications. Traditional approaches for the application or understanding of sequence cassettes from one sequence to sequence cassettes from another sequence (eg, from rat receptor to human receptor or from human receptor A to human receptor B) are generally In particular, it is estimated against sequence alignment techniques, whereby sequences are aligned in an effort to determine common regions. The mutational approach disclosed herein does not rely on this approach, but instead is based on an algorithmic approach and positional distance from a conserved proline residue located within the TM6 region of a human GPCR. Once this approach has been ensured, those skilled in the art are capable of making minor modifications thereto to achieve substantially the same results (ie, constitutive activation) disclosed herein. It is believed that there is. Such a modified approach is considered to be within the scope of this disclosure.

Example 1: Endogenous human GPCRS
(1. Identification of human GPCR)
Disclosed endogenous human GPCRs were identified based on a summary of GenBank ^™ database information. While searching this database, the following cDNA clones were identified as demonstrated below (Table C).

(2. Full length cloning)
(A. HRUP28 (SEQ ID NOS: 1 and 2))
The disclosed human RUP28 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AC073957 was identified as the human genome sequence from chromosome 7.

Full length RUP28 was cloned by PCR using the following primers and human adult liver Marathon-Ready ^™ cDNA (Clontech) as a template:
5′-CAGAGCTCTGGTGGCCCACCCTGTCC-3 ′ (SEQ ID NO: 21; sense, 5 ′ side of start codon), 5′-CTGCGTCCCACCAGAGTCACGTCTCC-3 ′ (SEQ ID NO: 22; antisense, 3 ′ side of stop codon). Advantage ^™ cDNA polymerase (Clontech) was used for amplification in a 50 μl reaction by repeating steps 2 to 4 35 times: 95 ° C. for 5 minutes; 94 ° C. for 30 seconds; 58 ° C. for 30 seconds 1 minute 30 seconds at 72 ° C; and 7 minutes at 72 ° C.

  A 1.16 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 1 for the nucleic acid sequence and SEQ ID NO: 2 for the deduced amino acid sequence.

(B. HRUP29 (SEQ ID NOs: 3 and 4))
The disclosed human RUP29 was identified based on the use of GenBank database information. While searching this database, a cDNA clone with accession number AC0083865 was identified as a human genome sequence from chromosome 7.

  Full length RUP29 was cloned by PCR using the following primers and human genomic DNA as a template:

TaqPlus® Precision DNA polymerase (Stratagene) was used for amplification in a 50 μl reaction by repeating steps 2 to 4 35 times: 94 ° C. for 5 minutes; 94 ° C. for 30 seconds; 54 30 ° C. for 30 seconds; 72 ° C. for 1 minute 30 seconds; and 72 ° C. for 7 minutes.

  A 930 bp PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems).

Rapid amplification of cDNA ends (RACE) was performed using human leukocyte and ovarian Marathon-Ready ^™ cDNA (Clontech) to determine the exact 5 ′ end of the RUP29 cDNA. RUP29-specific primer (1) (with sequence: 5-GGTACCACAATGACAATCACCAGCGTCC-3 ′ (SEQ ID NO: 25)) and AP1 primer (Clontech) were used for the first PCR reaction, and RUP29-specific primer (2) (Having the following sequence: 5′-GGAACGTGAGGTACATTGTGGATGTGCAGC-3 ′ (SEQ ID NO: 26)) and AP2 primer (Clontech) were used for the second PCR reaction. The product of the RACE reaction was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced. See SEQ ID NO: 3 for the nucleic acid sequence and SEQ ID NO: 4 for the deduced amino acid sequence.

(C. HRUP30 (SEQ ID NOS: 5 and 6))
The disclosed human RUP30 was identified based on the use of GenBank database information. During the search of this database, the cDNA clone with accession number AC055863 was identified as the human genome sequence from chromosome 17.

Full length RUP30 as a template for human pancreatic Marathon-Ready ^™
Cloned by 5'RACE-PCR using cDNA (Clontech) and the following oligonucleotide: 5'-GCAGTGTAGCGGTCAACCGTGAGCAGGG-3 '(SEQ ID NO: 27; including sense, start codon), and AP1 primer (Clontech) for the first time The RT: PCR was used, and the oligonucleotides: 5'-TGAGCAGGATGGCGATCCAGACTGAGGCGTGG-3 '(SEQ ID NO: 28, including antisense, stop codon) and AP2 primer (Clontech) were used for the second PCR. DNA fragments generated by 5′RACE-PCR were cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using SP6 / T7 primers (Stratagene).

Based on the sequence of the 5 ′ RACE product, full-length RUP30 was cloned by RT-PCR using the following primers as well as human pancreatic Marathon-Ready ^™ cDNA (Clontech) as a template:

Taq DNA polymerase (Stratagene) was used for amplification in a 50 μl reaction by repeating steps 2 to 4 35 times: 94 ° C. for 40 seconds; 94 ° C. for 20 seconds; 64 ° C. for 20 seconds; 2 minutes at 72 ° C; and 5 minutes at 72 ° C.

  A 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and several clones were used using the ABI Big Dye Terminator Kit (PE Biosystems). Sequenced. See SEQ ID NO: 5 for the nucleic acid sequence and SEQ ID NO: 6 for the deduced amino acid sequence.

(D. HRUP31 (SEQ ID NOs: 7 and 8))
The disclosed human RUP31 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AL356214 was identified as the human genome sequence from chromosome 10.

Full length RUP31 was cloned by RT-PCR using the following primers and human mammary gland Marathon-Ready ^™ cDNA (Clontech) as template:

Advantage ^™ cDNA polymerase (Clontech) was used for amplification in a 50 μl reaction by the following cycle, repeating steps 2 to 4 35 times: 94 ° C. for 40 seconds; 94 ° C. for 20 seconds; 66 ° C. for 20 seconds 1 minute 30 seconds at 72 ° C; and 5 minutes at 72 ° C.

  A 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 7 for the nucleic acid sequence and SEQ ID NO: 8 for the deduced amino acid sequence.

(E. HRUP32 (SEQ ID NOs: 9 and 10))
The disclosed human RUP32 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AL513524 was identified as the human genome sequence from chromosome 6.

  Full length RUP32 was cloned by PCR using the following primers and human genomic DNA (Clontech) as a template:

TaqPlus® Precision DNA polymerase (Stratagene) was used for amplification by the following cycle of repeating steps 2 to 4 35 times: 94 ° C. for 3 minutes; 94 ° C. for 20 seconds; 58 ° C. for 20 seconds 1 minute 30 seconds at 72 ° C; and 7 minutes at 72 ° C.

  A 1.06 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 9 for the nucleic acid sequence and SEQ ID NO: 10 for the deduced amino acid sequence.

(F. HRUP33 (SEQ ID NOS: 11 and 12))
The disclosed human RUP33 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AL513524 was identified as the human genome sequence from chromosome 6.

  Full length RUP33 was cloned by PCR using the following primers and human genomic DNA (Clontech) as a template:

TaqPlus® Precision DNA polymerase (Stratagene) was used for amplification by the following cycle of repeating steps 2 to 4 35 times: 94 ° C. for 3 minutes; 94 ° C. for 20 seconds; 56 ° C. for 20 seconds 1 minute 30 seconds at 72 ° C; and 7 minutes at 72 ° C.

  A 1.1 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 11 for the nucleic acid sequence and SEQ ID NO: 12 for the deduced amino acid sequence.

(G. HRUP34 (SEQ ID NOS: 13 and 14))
The disclosed human RUP34 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AL513524 was identified as the human genome sequence from chromosome 6.

  Full length RUP34 was cloned by PCR using the following primers and human genomic DNA (Clontech) as a template:

TaqPlus® Precision DNA polymerase (Stratagene) was used for amplification by the following cycle of repeating steps 2 to 4 35 times: 94 ° C. for 3 minutes; 94 ° C. for 20 seconds; 60 ° C. for 20 seconds 1 minute 30 seconds at 72 ° C; and 7 minutes at 72 ° C.

  A 1.27 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 13 for the nucleic acid sequence and SEQ ID NO: 14 for the deduced amino acid sequence.

(H. HRUP35 (SEQ ID NOS: 15 and 16))
The disclosed human RUP35 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AC021089 was identified as the human genome sequence from chromosome 16.

The 5 ′ sequence of RUP35 was determined by 5 ′ RACE-PCR using human fetal brain Marathon-Ready ^™ cDNA (Clontech) as a template. Oligonucleotide 5′-GGTATGAGACCGTGTGTACTTGAGC-3 ′ (SEQ ID NO: 39; sense) and AP1 primer (Clontech) were used for the first RT-PCR, and oligonucleotide 5′-GTGGCAGACGCGATATACCTGTCAATGG-3 ′ (SEQ ID NO: 40; Sense) and AP2 primer (Clontech) were used for the second round of PCR. The DNA fragment generated by 5′RACE-PCR was cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using SP6 / T7 primer (Stratagene).

Based on the sequence of the 5 ′ RACE product, full length RUP35 was cloned by RT-PCR using the following primers and human brain Marathon-Ready ^™ cDNA (Clontech) as template:

Advantage ^™ cDNA polymerase (Clontech) was used for amplification in a 100 μl reaction by repeating steps 2 to 4 45 times: 95 ° C. for 2 minutes; 95 ° C. for 20 seconds; 60 ° C. for 20 seconds 1 minute 30 seconds at 72 ° C; and 5 minutes at 72 ° C.

  A 1.0 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 15 for the nucleic acid sequence and SEQ ID NO: 16 for the deduced amino acid sequence.

(I. HRUP36 (SEQ ID NOS: 17 and 18))
The disclosed human RUP36 was identified based on the use of GenBank database information. While searching this database, the cDNA clone with accession number AC090099 was identified as the human genome sequence from chromosome 11.

Full length RUP36 was cloned by PCR using the following primers and human genomic DNA (Clontech) as a template:
5'-CATCTGGTTTGGTTCCCAGGGGCACCAG-3 '(SEQ ID NO: 43; sense, 5' to the start codon),
5′-GACAGTGTGCCTCTCAAAGTCCCGTCTGACTG-3 ′ (SEQ ID NO: 44; antisense, 3 ′ to stop codon). TaqPlus® Precision DNA polymerase (Stratagene) was used for amplification in a 50 μl reaction by repeating steps 2 to 4 30 times: 95 ° C. for 5 minutes; 95 ° C. for 30 seconds; 70 30 seconds at 72 ° C; 1 minute 30 seconds at 72 ° C; and 7 minutes at 72 ° C.

  A 1.0 kb PCR fragment was isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 17 for the nucleic acid sequence and SEQ ID NO: 18 for the deduced amino acid sequence.

(J. HRUP37 (SEQ ID NO: 19 and 20))
The disclosed human RUP37 was identified based on the use of GenBank database information. While searching the database, a cDNA clone with accession number AC090099 was identified as the human genome sequence from chromosome 11.

  Full length RUP37 was cloned by PCR using the following primers and human genomic DNA (Clontech) as a template: 5'-CGTTTCCAGGGCATCATAGACTGGGG-3 '(SEQ ID NO: 45; sense); 5'-GCAGCATTGCTCTCAAAGTCCTGCTCTG-3' (SEQ ID NO: 46; Antisense). TaqPlus® Precision DNA polymerase (Stratagene) was used for amplification by the following cycle of repeating steps 2 to 4 35 times: 95 ° C. for 5 minutes; 95 ° C. for 30 seconds; 62 ° C. for 30 seconds 1 minute 30 seconds at 72 ° C; and 7 minutes at 72 ° C.

  969 base pairs were isolated from a 1% agarose gel and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (PE Biosystems). See SEQ ID NO: 19 for the nucleic acid sequence and SEQ ID NO: 20 for the deduced amino acid sequence.

Example 2: Preparation of non-endogenous constitutively activated GPCR
Those skilled in the art will be able to select techniques for mutation of nucleic acid sequences. Presented below are the approaches utilized to generate several non-endogenous versions of the human GPCR disclosed above. The mutations disclosed below are based on an algorithmic approach, whereby the conserved proline (or endogenous conservative substitution for it) residue (located in the TM6 region of the GPCR near the TM6 / IC3 interface) The 16th amino acid (located in the IC3 region of the GPCR) is preferably mutated to an amino acid residue of alanine, histidine, arginine or lysine, most preferably an amino acid residue of lysine.

(1. Transformer Site-Directed ^TM mutagenesis)
Preparation of non-endogenous human GPCRs can be accomplished in human GPCRs using, among other things, the Transformer Site-Directed ^™ Mutagenesis Kit (Clontech) according to the manufacturer's instructions. In some embodiments, two mutagenesis primers are used, preferably a lysine mutagenesis oligonucleotide that creates a lysine mutation and a selectable marker oligonucleotide. For convenience, codon mutations to be incorporated into human GPCRs are also noted in standard form (Table D):

(Example 3: Receptor expression)
Various cells are available to those skilled in the art for protein expression, but mammalian cells are preferably utilized. The main reason for this is estimated based on practical matters. Thus, for example, the use of yeast cells for the expression of GPCRs is possible, but may not include receptor couplings, genetic mechanisms and secretory pathways that have evolved for mammalian systems (in fact not included in yeast) ) Introducing non-mammalian cells into this protocol, and therefore the results obtained in non-mammalian cells are less favorable than the results obtained with mammalian cells, although with potential applications. Of the mammalian cells, COS-7 cells, 293 cells, and 293T cells are particularly preferred, but the particular mammalian cells utilized can be estimated based on the particular needs of those skilled in the art.

(A. Transient transfection)
On the first day, 293 cell wells in 6 × 10 ⁶ cells / 10 cm dish were plated. On day 2, two reaction tubes were prepared (the following ratios for each tube are per plate): Tube A was added to 4 μg DNA (eg, in 0.5 ml serum free DMEM (Gibco BRL)). prepared by mixing pCMV vector; such as pCMV vector with receptor cDNA); 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) followed by incubation at room temperature for 30-45 minutes. This mixture is referred to as a “transfection mixture”. Plated 293 cells were washed with 1 × PBS, followed by addition of 5 ml serum-free DMEM. 1 ml of the transfection mixture was added to the cells, followed by incubation at 37 ° C / 5% CO ₂ for 4 hours. The transfection mixture was removed by aspiration followed by the addition of 10 ml DMEM / 10% Fetal Bovine Serum. Cells were incubated at 37 ℃ / 5% CO _2. After 48 hours incubation, cells were harvested and utilized for analysis.

(B. Stable cell line)
Approximately 12 × 10 ⁶ 293 cells are plated on 15 cm tissue culture plates and grown in DME High Glucose Medium containing 10% fetal calf serum and 1 percent sodium pyruvate, L-glutamine and antibiotics . 24 hours after plating of 293 cells (to about 80% confluency), the cells are transfected with 12 μg of DNA. 12 μg of DNA is combined with 60 μl Lipofectamine and 2 mL serum free DME High Glucose Medium. Medium is aspirated from the plate and cells are washed once with serum free medium. DNA, lipofectamine and media mixture are added to this plate along with 10 mL of serum free media. After incubation at 37 ° C. for 4-5 hours, the medium is aspirated and 25 ml of serum-containing medium is added. Twenty-four hours after transfection, the medium is again aspirated and fresh serum-containing medium is added. Forty-eight hours after transfection, the medium is aspirated and serum-containing medium containing geneticin (G418 drug) at a final concentration of 500 μg / mL is added. Transfected cells are then selected for positively transfected cells containing the G418 resistance gene. The medium is changed every 4-5 days with selection. During selection, cells are grown to create a stable pool or split for stable clonal selection.

Example 4: Assay for determination of constitutive activity of non-endogenous GPCRs
Various approaches are available for assessing the constitutive activity of non-endogenous human GPCRs. The following are exemplary; those skilled in the art will have the ability to determine techniques that are preferentially beneficial to those skilled in the art.

(1. Membrane binding assay: [ ³⁵ S] GTPγS assay)
When a G protein coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to the G protein and releases GDP and subsequent to the G protein. Stimulates GTP binding. The α subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes GTP to GDP, at which point the receptor is normally inactivated. The constitutively activated receptor continues to exchange GDP for GTP. [ 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 include, but are not limited to: (a) it is globally applicable to all G protein coupled receptors. Yes; (b) it is proximal to the membrane surface and reduces the possibility of picking up molecules that affect the intracellular cascade.

This assay takes advantage of the ability of G protein coupled receptors to stimulate [ ³⁵ S] GTPγS binding to membranes expressing the relevant receptors. Therefore, this assay can be used in a direct identification method to screen candidate compounds for constitutively activated G protein coupled receptors. This assay is comprehensive and applies to drug discovery at all G protein coupled receptors.

[ ³⁵ S] GTPγS assay was performed using 20 mM HEPES and MgCl ₂ (pH 7.4) between 1 mM and about 20 mM (this amount can be adjusted for optimization of results, but 20 mM is preferred), about ^[35 S] ^GTPγS between 0.3nM and about 1.2 nM (this amount, which can be adjusted for optimization of results, 1.2 is preferable) binding buffer containing, and 12.5μg ~75μg of membrane proteins (e.g., 293 cells expressing the G _s fusion protein; this amount can be adjusted for optimization) in, and 10 [mu] M GDP (this amount can be changed for optimization) Incubate for 1 hour. Wheat germ agglutinin beads (25 μl; Amersham) are then added and the mixture is incubated for an additional 30 minutes at room temperature. The tubes are then centrifuged at 1500 × g for 5 minutes at room temperature and then counted in a scintillation counter.

(2. Adenylyl cyclase)
A Flash Plate ^™ Adenylyl Cycle kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with the crude plasma membrane. The Flash Plate well can include a scintillant coating that also includes a specific antibody that recognizes cAMP. CAMP produced in the wells can be quantified by direct competition for binding of the radioactive cAMP tracer to the cAMP antibody. The following serves as a simple protocol for measuring changes in cAMP levels in whole cells expressing this receptor.

  Transfected cells are harvested approximately 24 hours after transient transfection. Carefully aspirate and discard the medium. 10 ml of PBS is gently added to each dish of cells followed by careful aspiration. Add 1 ml Sigma cell dissociation buffer and 3 ml PBS to each plate. Cells are pipetted from the plate and the cell suspension is collected in a 50 ml conical centrifuge tube. The cells are centrifuged for 5 minutes at 1,100 rpm at room temperature. Carefully resuspend the cell pellet in an appropriate volume of PBS (approximately 3 ml / plate). Cells are then counted using a hemocytometer and additional PBS is added to obtain the appropriate number of cells (to a final volume of about 50 μl / well).

Prepare and maintain cAMP standard and Detection Buffer (1 μCi tracer for 11 ml Detection Buffer [includes ¹²⁵ I cAMP (50 μl)) according to manufacturer's instructions. Prepare and contain 50 μl Stimulation Buffer, 3 μl test compound (12 μM final assay concentration) and 50 μl cells Store Assay Buffer on ice until use. Start by adding 50 μl of cAMP standard followed by 50 μl PBSA to wells H-11 and H12 Add 50 μl Stimulation Buffer to all wells DMSO (or selected candidate compounds) is added to the appropriate wells using a pin tool that can dispense 3 μl of compound solution at a final assay concentration of 12 μM test compound and a total assay volume of 100 μl. Add and incubate at room temperature for 60 minutes, then add 100 μl Detection Mix containing tracer cAMP to the wells, incubate the plate for an additional 2 hours, and then count in a Wallac MicroBeta ^™ scintillation counter. The cAMP / well values are then extrapolated from the standard cAMP curve included in each assay plate.

(Cell-Based cAMP for 3.G _i-conjugated target GPCR)
TSHR, when activated causes the accumulation of cAMP, a _{G s} coupled GPCR. TSHR is constitutively activated by mutating amino acid residue 623 (ie, changing an alanine residue to an isoleucine residue). G _i coupled receptors are believed to inhibit adenylyl cyclase, therefore, it reduces the level of cAMP production, and may thereby assessment of cAMP levels attractive. TSHR G _i coupled receptor effective technique as an indicator of constitutive activation for measuring the decrease in production of cAMP as "signal enhancer", which is constitutively activated (non-endogenous most preferably) (TSHR-A623I) (or an endogenous, constitutively active _{G s} coupled receptors), and co-transfected with _{G i} coupled target GPCR, it may be accomplished by establishing a baseline level of cAMP. When non-endogenous version of the G _i coupled receptor is produced, then the Target GPCR of the non-endogenous version, co-transfected with this signal enhancer, and the may be used in the screening is the substance. Using this approach, when using a cAMP assay, it produces a signal effectively; this approach is preferably used in the direct identification of candidate compounds against G _i coupled receptors. Regard G _i coupled GPCR, when using this approach, an agonist of the inverse of the target GPCR will increase the cAMP signal and an agonist will be noted that to reduce the cAMP signal.

On day 1, 2 × 10 ⁴ 293 cells / well are plated. On day 2, prepare two reaction tubes (the following ratios for each tube are per plate): Tube A contains 2 μg of DNA for each receptor transfected into mammalian cells (4 μg total) DNA) (eg, pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc.) prepared by mixing in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, CA) Tube B is prepared by mixing 120 μl Lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tube A and tube B are then mixed by inversion (several times) followed by incubation at room temperature for 30-45 minutes. This mixture is referred to as a “transfection mixture”. Plated 293 cells are washed with 1 × PBS, followed by addition of 10 ml serum free DMEM. Then 2.4 ml of the transfection mixture is added to the cells, followed by incubation at 37 ° C./5% CO ₂ for 4 hours. The transfection mixture is then removed by aspiration followed by the addition of 25 ml of DMEM / 10% Fetal Bovine Serum. Cells are then incubated at 37 ℃ / 5% CO _2. After 24 hours of incubation, the cells are collected and utilized for analysis.

The Flash Plate ^™ Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A), although designed for cell-based assays, is for use on crude plasma membranes, depending on the needs of those skilled in the art. Can be modified. The Flash Plate well contains a scintillant coating that also contains a specific antibody that recognizes cAMP. CAMP produced in the wells can be quantified by direct competition for binding of the radioactive cAMP tracer to the cAMP antibody. The following serves as a simple protocol for measuring changes in cAMP levels in whole cells expressing this receptor.

  Transfected cells are harvested approximately 24 hours after transient transfection. Carefully aspirate and discard the medium. 10 ml of PBS is gently added to each dish of cells followed by careful aspiration. Add 1 ml Sigma cell dissociation buffer and 3 ml PBS to each plate. Cells are pipetted from the plate and the cell suspension is collected in a 50 ml conical centrifuge tube. The cells are centrifuged for 5 minutes at 1,100 rpm at room temperature. Carefully resuspend the cell pellet in an appropriate volume of PBS (approximately 3 ml / plate). Cells are then counted using a hemocytometer and additional PBS is added to obtain the appropriate number of cells (to a final volume of about 50 μl / well).

Prepare and maintain cAMP standard and Detection Buffer (1 μCi tracer for 11 ml Detection Buffer [includes ¹²⁵ I cAMP (50 μl)) according to manufacturer's instructions. Should be prepared and should contain 50 μl of Stimulation Buffer, 3 μl of test compound (12 μM final assay concentration) and 50 μl of cells, Assay Buffer can be stored on ice until utilized. The assay can be initiated by the addition of 50 μl cAMP standard to the appropriate wells followed by the addition of 50 μl PBSA to wells H-11 and H12 50 μl Stimulation Buffer. Add to all wells: Selected compound (eg TSH) is added to appropriate wells using a pin tool that can dispense 3 μl of compound solution at a final assay concentration of 12 μM test compound and 100 μl total assay volume Cells are then added to the wells and incubated for 60 minutes at room temperature, then 100 μl Detection Mix containing the tracer cAMP is added to the wells, then the plates are incubated for an additional 2 hours, followed by Wallac Count in a MicroBeta scintillation counter.The cAMP / well values are then extrapolated from the standard cAMP curve included in each assay plate.

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

(B. AP1 reporter assay (G _q- coupled receptor))
Method of detecting a G _q stimulus that causes the activation of genes containing AP1 elements in the promoter, depends on the known property of G _q dependent phospholipase C. Pathdetect ^™ AP-1 cis-Reporting System (Stratagene, Catalog # 219073) except that the components of the calcium phosphate precipitate are 410 ng pAP1-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP. Available according to the protocol set above.

(C.SRF-Luc Reporter Assay _{(G q-coupled} receptor))
One method of detecting G _q stimulation, serum response factor that cause the activation of genes containing the promoter, depends on the known property of G _q dependent phospholipase C. Pathdetect ^TM SRF-Luc-Reporting utilizing System (Stratagene), may be assayed (e.g., in COS7 cells) for _{G q} coupled activity. Cells are transfected with the plasmid components of this system and the indicated expression plasmids encoding endogenous or non-endogenous GPCRs using the Mammalian Transfection ^™ Kit (Stratagene, Catalog # 200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity was measured in the media of the transfected cells and Control variability in transfection efficiency) is combined in the calcium phosphate precipitate according to the manufacturer's instructions. Half of the precipitate is evenly distributed among the three wells in a 96 well plate and the cells are maintained in serum free medium for 24 hours. Where indicated, cells are incubated with 1 μM Angiotensin for the last 5 hours. Cells are then lysed and assayed for luciferase activity using Luclite ^™ Kit (Packard, Cat. # 6016911) and “Trilux 1450 Microbeta” liquid scintillation and luminescence counter (Wallac) according to the manufacturer's instructions. This data can be analyzed using GraphPad Prism ^™ 2.0a (GraphPad Software Inc.).

(D. Intracellular IP ₃ Accumulation Assay _{(G q-coupled} receptor))
On day 1, cells containing the receptor (endogenous and / or non-endogenous) are plated into 24-well plates, usually at 1 × 10 ⁵ cells / well (but this number can be optimized). On day 2, the cells are transfected by first mixing 0.25 μg DNA in 50 μl serum-free DMEM / well and 2 μl lipofectamine in 50 μl serum-free DMEM / well. The solution is gently mixed and incubated at room temperature for 15-30 minutes. The cells are then washed with 0.5 ml PBS and 400 μl of serum free medium, then mixed with transfection medium and added to the cells. Cells are incubated at 37 ° C./5% CO ₂ for 3-4 hours, then the transfection medium is removed and replaced with 1 ml / well normal growth medium. On day 3, cells are labeled with ³ H-myo-inositol. Briefly, media is removed and cells are washed with 0.5 ml PBS. Then 0.5 ml of inositol-free / serum-free medium (GIBCO BRL) per well was added to the wells with 0.25 μCi of ³ H-myo-inositol per well and the cells were allowed for 16-18 hours. Incubate overnight at 37 ° C./5% CO ₂ . On day 4, the cells were washed with 0.5 ml PBS and 0.45 ml assay medium with 0.4 μm assay medium and 10 μM without inositol / serum free medium, 10 μM pargyline, 10 mM lithium chloride. Add 50 μl of 10 × ketanserin (ket) to a final concentration of The cells are then incubated for 30 minutes at 37 ° C. Cells are then washed with 0.5 ml PBS and 200 μl of fresh / ice cold stop solution (1 M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added to each well. This 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 to a 1.5 ml Eppendorf tube and 1 ml of chloroform / methanol (1: 2) is added per tube. This solution is vortexed for 15 seconds and the top layer is applied to Biorad AG1-X8 ^™ anion exchange resin (100 mesh to 200 mesh). First, the resin is washed with 1: 1.25 W / V water and 0.9 ml of the upper layer is loaded onto the column. The column is then washed with 10 ml 5 mM myo-inositol and 10 ml 5 mM Na-borate / 60 mM Na-formate. Inositol tris phosphate is eluted in scintillation vials containing 10 ml of scintillation cocktail containing 2 ml of 0.1 M formic acid / 1 M ammonium formate. The column was regenerated by washing with 10 ml of 0.1M formic acid / 3M ammonium formate and dd
Rinse twice with H ₂ O and store in water at 4 ° C.

Please refer to FIG. In FIG. 1, 293 cells are treated with G _q protein “G _q (del)” containing 6 amino acid deletions; G _q protein “G _q (del) / G _i ” fused to G _i protein; endogenous RUP32; And RUP32 containing G _q (del) (“RUP32 + G _q (del) / G _i ”). This data _is based on measuring the IP ₃ accumulation of RUP32 cotransfection of _{G q (del) / G i} , indicating that the RUP32 is not intrinsically coupled to _{G q} protein. However, when RUP32 was co-transfected with a G _q (del) / G _i fusion protein, RUP 32 was forced to couple to the G _q protein. RUP27 + G _q (del) / G _i demonstrates an approximately 9-fold increase in IP3 accumulation when compared to endogenous RUP32. This data _demonstrates that that may be used to screen for G _q (del) / G i obtained Fusion Construct is co-transfected with GPCR and agonists or inverse agonists.

  Please refer to FIG. In FIG. 2, 293 cells were transfected with RUP35 and RUP36 receptors and compared against the control pCMV. This data indicates that both RUP35 and RUP36 receptors are endogenous and constitutively active. RUP35 demonstrates an approximately 6-fold increase in intracellular inositol phosphate accumulation when compared to pCMV and RUP36 demonstrates an approximately 4-fold increase when compared to pCMV.

(Example 5: Preparation of fusion protein)
(A.GPCR: _{G s} fusion construct)
The design of constitutively activated GPCR-G fusion construct of the protein can be achieved as follows: rat G protein _{G s} alpha; 5 of (long form Itoh, H et al., 83 PNAS 3776 (1986). ) Both the 'end and the 3' end are manipulated to include a HindIII (5'-AAGCTT-3 ') sequence therein. After confirmation of the correct sequence (including flanking HindIII sequences), the entire sequence is shuttled into pcDNA3.1 (-) (Invitrogen, cat. No. V795-20) by subcloning into the HindIII restriction site of the vector. To do. The exact orientation for the G _s α sequence is determined after subcloning into pcDNA3.1 (−). The modified pcDNA3.1 (-) containing the rat G _s α gene in the HindIII sequence is then identified; this vector is then available as a “universal” G _s α protein vector. The pcDNA3.1 (-) vector contains a variety of well-known restriction sites upstream of the HindIII site, thus upstream of _{G s} protein, beneficially the ability to insert the endogenous, constitutively active GPCR coding sequence provide. Using this same approach, other “universal” G protein vectors can be made, and of course, other commercially available or patented vectors known to those skilled in the art can be utilized. In some embodiments, an important criterion is that the sequence for the GPCR is upstream and in frame of the sequence of the G protein.

  The spacer at the restriction site between the G protein and the GPCR is optional. The sense and antisense primers contained restriction sites for XbaI and EcoRV, respectively, so that a spacer (due to the restriction site) was present between the G protein and the GPCR.

PCR is then used to ensure that each receptor sequence for fusion is within the G _s α universal vector disclosed above, using the following protocol for each: cDNA for GPCR (100 ng) is added to a separate tube containing: 2 μl of each primer (sense and antisense), 3 μl of 10 mM
dNTP, 10 μl of 10 × TaqPlus ^™ Precision buffer, 1 μl TaqPlus ^™ Precision polymerase (Stratagene: # 600211) and 80 μl water. The reaction temperature and number of cycles for GPCRs are as follows, repeating cycle steps 2 to 4 35 times: 94 ° C. for 1 minute; 94 ° C. for 30 seconds; 62 ° C. for 20 seconds; 72 ° C. for 1 40 minutes; and 5 minutes at 72 ° C. The PCR product is run on a 1% agarose gel and then purified. The purified product was digested with XbaI and EcoRV, and purified the desired insert, and ligated into the G _s universal vector at the respective restriction site. Positive clones are isolated after transformation and determined by restriction enzyme digestion; expression using 293 cells is achieved according to the protocol shown below. It was sequenced each positive clones for GPCR-G _s fusion proteins to confirm correct.

(B. G _q (6 amino acid deletion) / G _i fusion construct)
The design of the G _q (del) / G _i fusion construct was accomplished as follows: deletion of the Gαq subunit, which is the N-terminal 6 amino acids (amino acids 2-7) having the sequence TLESSIM (SEQ ID NO: 47) And the C-terminal 5 amino acids having the sequence EYNLV (SEQ ID NO: 48) were replaced with the corresponding amino acids of the Gαi protein having the sequence DCGLF (SEQ ID NO: 49). This fusion construct was obtained by PCR using the following primers and plasmid 63313 as template (including mouse Gαq-wild type version with hemagglutinin tag): 5′-gatAAGCTTCCATGGCGTGCTGCCTGAGCGAGG-3 ′ (SEQ ID NO: 50) And 5'-gatsGGATCCCTTAGAACAGGGCGCAGGTCCTTCAGGTTCAGCTGCAGGATGGTG-3 '(SEQ ID NO: 51). Lower cap nucleotides are included as spacers.

  TaqPlus® Precision DNA polymerase (Stratagene) was utilized for amplification by the following cycle of repeating steps 2 to 4 35 times: 95 ° C. for 2 minutes; 95 ° C. for 20 seconds; 56 ° C. for 20 seconds 2 minutes at 72 ° C; and 7 minutes at 72 ° C. PCR products are cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator kit (PE Biosystems). The insert from the TOPO clone containing the sequence of the fusion construct is shuttled to the expression vector pcDNA3.1 (+) to the HindIII / BamHI site by a two-step cloning process.

(Example 6: Tissue distribution of disclosed human GPCR: RT-PCR)
RT-PCR was applied to confirm expression and the tissue distribution of several new human GPCRs was determined. The oligonucleotide utilized was GPCR specific and a human multi-tissue cDNA panel (MTC, Clontech) was used as a template. Taq DNA polymerase (Stratagene) was utilized for amplification in a 40 μl reaction according to the manufacturer's instructions. 20 μl of the reaction is loaded onto a 1.5% agarose gel and the RT-PCR product is analyzed. Table E below lists the receptors, cycle conditions and primers utilized and also lists exemplary diseases / disorders associated with these receptors.

  Diseases and disorders associated with receptors located in these tissues or regions include, but are not limited to: cardiac disorders and diseases (eg, thrombosis, myocardial infarction; atherosclerosis; cardiomyopathy ); Kidney disease / disorder (eg, renal failure; tubular acidosis; renal diabetes; nephrogenic diabetes insipidus; cystineuria; polycystic kidney disease); eosinophilia; leukocytosis; leukopenia Ovarian cancer; sexual dysfunction; polycystic ovary syndrome; pancreatitis and pancreatic cancer; irritable bowel syndrome; colon cancer; Crohn's disease; ulcerative colitis; diverticulitis; chronic obstructive pulmonary disease (COPD); Fibrosis; pneumonia; pulmonary hypertension; tuberculosis and lung cancer; Parkinson's disease; movement disorders and ataxia; learning and memory disorders; eating disorders (eg, anorexia; bulimia etc.); obesity; cancer; Myasthenia gravis; cardiovascular disorder; prostate cancer; prostatitis; kidney disease / disorder (eg, renal failure; tubular acidosis; renal diabetes; nephrogenic diabetes insipidus; cystineuria; polycystic kidney disease Sensorimotor processing disorders and arousal disorders; Obsessive compulsive disorder; Testicular cancer; Persistent erection; Prostatitis; Hernia; Endocrine disorders; Sexual dysfunction; Allergy; Depression; Psychiatric disorder; Migraine; Perfusion; Schizophrenia; Bronchospasm; sputum; prostate hypertrophy; anxiety; rhinitis; angina; and glaucoma. Thus, the methods of the present invention may also be useful in the diagnosis and / or treatment of these and other diseases and disorders.

Example 7: Protocol: direct identification of inverse agonists and agonists
(A. [ ³⁵ S] GTPγS assay)
Endogenous constitutively active GPCRs are used, for example, for direct identification of candidate compounds as inverse agonists because they are not fully understood, but the intra-assay variability is exacerbated Can be. In some embodiments, GPCR fusion proteins as disclosed above are also utilized with non-endogenous constitutively activated GPCRs. When such proteins are used, intra-assay variability appears to be substantially stabilized, thereby providing an effective signal-to-noise ratio. This has the beneficial result of allowing more robust identification of candidate compounds. Thus, in some embodiments, it is preferred to use GPCR fusion proteins for direct identification, and when utilized, it is preferred to utilize the following assay protocol.

(1. Preparation of membrane)
Membranes comprising a constitutively active orphan GPCR fusion protein of interest and for use in direct identification of candidate compounds as inverse agonists or agonists are preferably prepared as follows:
(A. Material)
“Membrane Scrape Buffer” is composed of 20 mM HEPES and 10 mM EDTA (pH 7.4); “Membrane Wash Buffer” is composed of 20 mM HEPES and 0.1 mM EDTA (pH 7.4); “Binding Buffer” Is 20 mM HEPES, 100 mM
Consists of NaCl and 10 mM MgCl ₂ (pH 7.4).

(B. Procedure)
All materials are kept on ice throughout the procedure. First, the medium is aspirated from the confluent monolayer of cells, followed by rinsing with 10 ml of cold PBS followed by aspiration. Thereafter, 5 ml of Membrane Scrape Buffer is added to scrape the cells; this is followed by transferring the cell extract to 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 Membrane Wash Buffer followed by centrifugation at 20,000 rpm for 17 minutes at 4 ° C. The supernatant is then aspirated and the pellet is resuspended in Binding Buffer. The resuspended pellet is then homogenized using a Brinkman Polytron ^™ homogenizer (15-20 second burst until the material is suspended). In the present specification, this is referred to as “membrane protein”.

(2. Bradford protein assay)
After homogenization, the protein concentration of the membrane is determined using, for example, the Bradford Protein Assay (protein is diluted to approximately 1.5 mg / ml, aliquoted and frozen (-80) for later use. When frozen, the protocol for use is as follows: On the day of the assay, the frozen membrane protein is thawed at room temperature, followed by vortexing and then Polytron approximately 12 × 1, Homogenize using 000 rpm for about 5-10 seconds; note that for multiple preparations, the homogenizer was thoroughly cleaned during the homogenization of the different preparations).

(A. Material)
Binding Buffer (as above); Bradford Dye Reagent; Bradford Protein Standard is utilized according to manufacturer's instructions (Biorad, cat. No. 5000006).

(B. Procedure)
Duplicate tubes are prepared, one containing the membrane and the other a control “blank”. Each contains 800 μl of Binding Buffer. Thereafter, 10 μl of Bradford Protein Standard (1 mg / ml) is added to each tube, and then 10 μl of membrane protein is added to only one tube (not blank). Thereafter, 200 μl of Bradford Dye Reagent is added to each tube followed by vortexing. After 5 minutes, vortex the tube again and transfer the material therein to the cuvette. The cuvette is then
Read at wavelength 595 using a 3041 spectrophotometer.

(3. Direct identification assay)
(A. Material)
GDP Buffer is 37.5ml Binding Buffer and 2mg
Consisting of GDP (Sigma, cat. No. G7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 μM GDP (final concentration of GDP in each well is 0.1 μM GDP). Each well containing candidate compounds was GDP Buffer (final concentration 0.1 μM GDP) (100 μl), Binding
Having a final volume of 200 μl, consisting of membrane protein in Buffer (50 μl) and [ ³⁵ S] GTPγS (0.6 nM) (50 μl) in Binding Buffer (2.5 μl [ ³⁵ S] GTPγS) per 10 ml Binding Buffer .

(B. Procedure)
Candidate compounds are preferably screened using a 96 well plate format, which can be frozen at -80 ° C. Membrane protein (or as a control, a membrane containing the expression vector excluding the GPCR fusion protein) is homogenized briefly until suspended. The protein concentration is then determined using, for example, the Bradford Protein Assay 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 is added to each well of the Wallac Scintistrip ^™ (Wallac). A 5 μl pin tool is then used to transfer 5 μl of the candidate compound into such wells (ie, 5 μl in a total assay volume of 200 μl is a 40: 1 ratio so that the final of the candidate compound The screening concentration is 10 μM). Further, to avoid contamination, after each transfer step, rinse the pin tool in three reservoirs containing water (1 ×), ethanol (1 ×) and water (2 ×) —excess liquid Shake off from the tool after each rinse, and dry the tool with paper and Kimwipe. Thereafter, 50 μl of membrane protein is added to each well (no GPCR fusion protein, control wells containing membrane were also utilized) and preincubated at room temperature for 5-10 minutes. Then [ ³⁵ S] GTPγS (0.6 nM) (50 μl) in Binding Buffer is added to each well, followed by incubation on a shaker for 60 minutes at room temperature (further, in this example, the plate is foiled Covered). The assay is stopped by spinning the plate at 4000 RPM for 15 minutes at 22 ° C. The plate is then aspirated with an 8-channel manifold and sealed with a plate cover. The plate is then read on a Wallac 1450 (according to the manufacturer's instructions) using the “Prot. # 37” setting.

(B. Cyclic AMP assay)
Another assay approach for directly identifying candidate compounds is achieved utilizing cyclase-based assays. In addition to direct identification, this assay approach can be utilized as an independent approach that provides confirmation of results from the [ ³⁵ S] GTPγS approach as described above.

A modified Flash Plate ^™ Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is preferably used for direct identification of inverse agonists and candidate compounds as agonists for GPCRs according to the following protocol.

Transfected cells are collected approximately 3 days after transfection. Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES (pH 7.4) and 10 mM MgCl _2. Homogenization is performed for about 10 seconds on ice using a Brinkman Polytron ^™ . The resulting homogenate is centrifuged at 49,000 xg for 15 minutes at 4 ° C. The resulting pellet is then resuspended in a buffer containing 20 mM HEPES (pH 7.4) and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 × g for 15 minutes at 4 ° C. To do. The resulting pellet is 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 obtain a final protein concentration of 0.60 mg / ml. (Place resuspended membrane on ice until use).

cAMP standards and Detection Buffer (2 μCi of tracer [ ¹²⁵ I] cAMP (100 μl) in 11 ml Detection Buffer) are prepared and maintained according to the manufacturer's instructions. Assay Buffer was prepared fresh for screening, and this was 20 mM HEPES (pH 7.4), 10 mM MgCl ₂ , 20 mM phosphocreatine (Sigma), 0.1 unit / ml creatine phosphokinase (Sigma), 50 μM. Contains GTP (Sigma) and 0.2 mM ATP (Sigma); Store Assay Buffer on ice until use.

  Candidate compounds identified above (when frozen, thaw at room temperature) are preferably transferred to 96-well plate wells (3 μl / well; 12 μM final assay concentration) with 40 μl membrane protein (30 μg / well) and 50 μl Assay. Add with Buffer. This mixture is incubated at room temperature for 30 minutes with gentle agitation.

After incubation, 100 μl Detection Buffer is added to each well, followed by incubation for 2-24 hours. The plates are then counted in a Wallac MicroBeta ^™ plate reader using “Prot. # 31” (according to manufacturer's instructions).

(C. Melanin-bearing cell screening assay)
A method for identifying candidate agonists or inverse agonists for GPCRs can be used to test cells of a pigment cell line that can disperse or aggregate the pigment in response to a particular stimulus and express an exogenous clone encoding GCPR. Can be implemented. If activation of the GPCR induces dye dispersion, the stimulator (eg, light) sets the initial state of the aggregation arrangement, where the dye is aggregating within the test cells. However, if activation of the GPCR induces dye aggregation, the cells are stimulated with a stimulating factor to set the initial state of dye placement in which the dye is dispersed. The test cell is then contacted with the compound and it is determined whether the dye placement within the cell has changed from the initial state of the dye placement. The dispersion of pigment cells appears dark in Petri dishes, while pigment cell aggregation appears bright, due to the coupling of candidate compounds to the GPCR.

  Materials and methods follow the disclosures of US Pat. No. 5,462,856 and US Pat. No. 6,051,386, each of which is incorporated by reference.

  Although various expression vectors are available to those of skill in the art, for purposes of utilization for both endogenous and non-endogenous human GPCRs, in some embodiments, the vector utilized is pCMV. It is preferable. This vector was introduced to the American Type Culture Collection (ATCC) (10801 University Blvd., Manassas, VA 20110-2209 USA) on October 13, 1998, in the Budapest Treaty Convention on the International Approval of Deposits of Microorganisms in Patent Procedure Deposited under. This DNA was tested by ATCC and determined to be alive. The ATCC has assigned the following accession number to pCMV: ATCC # 203351.

  References cited throughout this patent document, including copending patent applications and related patent applications, are fully incorporated by reference herein unless otherwise indicated. Modifications and extensions of the disclosed invention that fall within the purview of those skilled in the art are encompassed within the above disclosure and the following claims.

Claims (1)

Invention described in the specification.

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