Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

• the present invention relates to transmembrane receptors, in some embodiments to
• G protein-coupled receptors and, in some preferred embodiments, to endogenous GPCRs that are altered to establish or enhance constitutive activity of -the receptor.
• the constitutively activated GPCRs will be used for the direct identification of candidate compounds as receptor agonists or inverse agonists having applicability as therapeutic agents.
• GPCR G protein-coupled receptor
• GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription pharmaceuticals have been developed. For example, in 1999, of the top 100 brand name prescription drugs, the following drugs interact with GPCRs (diseases and/or disorders treated are indicated in parentheses):
• GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane- 1 (TM-1), transmebrane-2 (TM-2), etc.).
• the transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or AREN-0309 PATENT
• extracellular side of the cell membrane (these are referred to as "extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).
• the transmembrane helices are also joined by strands of amino acids between transmembrane- 1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular” side, of the cell membrane (these are referred to as "intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively).
• the "carboxy" (“C”) terminus of the receptor lies in the intracellular space within the cell, and the "amino" (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
• GPCRs are "promiscuous" with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, G q , G s , Gi, G z and G 0 are G proteins that have been identified.
• Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Although not wishing to be bound to theory, it is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein. Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an "inactive" state and an "active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G- protein) and produces a biological response.
• a receptor may be stabilized in an active state by a ligand or a compound such as a drug.
• Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a Ugand binding to the receptor. Stabilization by such ligand-independent means is termed "constitutive receptor activation.”
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.:2, non-endogenous, constitutively activated versions of the same, and host cells comprising the same- Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO.:l and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.:4, non-endogenous, constitutively activated versions of the same, and host cells comprising the same..
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO.:3, non-endogenous, constitutively activated versions of the same, and host cells comprising the same. Some embodiments of the present invention relate to G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.:6, non-endogenous, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO.:5 and host cells comprising the same. Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.:8, non-endogenous, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO.:7, non-endogenous, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.:10, non-endogenous, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO.:9 and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.:12, non-endogenous, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO. : 11 , and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.: 14, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO. : 13 and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.: 16, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO. : 15 and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO.: 18, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO.:17 and host cells comprising the same.
• Some embodiments of the present invention relate to a G protein-coupled receptor encoded by an amino acid sequence of SEQ.ID.NO. :20, constitutively activated versions of the same, and host cells comprising the same.
• Some embodiments of the present invention relate to a plasmid comprising a vector and the cDNA of SEQ.ID.NO. : 19 and host cells comprising the same.
• Figure 1 is a graphic representation of activation of RUP32, G q (del)/Gi, RUP32 co-transfected with G q (del)/Gj, and CMV (control; expression vector) in a second messenger assay measuring the accumulation of inositol phosphate (IP 3 ) utilizing 293 cells.
• FIG. 2 provides an illustration of second messenger IP 3 production from endogenous version RUP35 and RUP36 as compared with the control ("CMV").
• CMV control
• AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes, hi some embodiments, AGONISTS are those materials not previously known to activate the intracellular response when they bind to the receptor or to enhance GTP binding to membranes.
• ANTAGONIST shall mean materials (e.g., ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists.
• ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist.
• ANTAGONISTS are those materials not previously known to activate the intracellular response when they bind to the receptor or to enhance GTP binding to membranes.
• CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique.
• the phrase "candidate compound” does not include compounds which were publicly known to be compounds selected from the group consisting of inverse agonist, agonist or antagonist to a receptor, as previously determined by an indirect identification process ("indirectly identified compound”); more preferably, not including an indirectly identified compound which has previously been determined to have therapeutic efficacy in at least one mammal; and, most preferably, not including an indirectly identified compound which has previously been determined to have therapeutic utility in humans.
• COMPOSITION means a material comprising at least one component; a "pharmaceutical composition” is an example of a composition.
• COMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality; i.e. the ability to activate/inhibit a AREN-0309 PATENT signal transduction pathway, as opposed to receptor binding affinity. Exemplary means of detecting compound efficacy are disclosed in the Example section of this patent document.
• CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides) which generally comprise a nucleoside (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to a phosphate group and which, when translated, encodes an amino acid.
• A adenosine
• G guanosine
• C cytidine
• U uridine
• T thymidine
• CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subjected to constitutive receptor activation.
• a constitutively activated receptor can be endogenous or non-endogenous.
• CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptor in the active state by means other than binding of the receptor with its Ugand or a chemical equivalent thereof.
• CONTACT or CONTACTING shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system.
• DIRECTLY IDENTIFYING or DIRECTLY IDENTIFHED in relationship to the phrase "candidate compound”, shall mean the screening of a candidate compound against a constitutively activated receptor, preferably a constitutively activated orphan receptor, and most preferably against a constitutively activated G protein-coupled cell surface orphan receptor, and assessing the compound efficacy of such compound.
• This phrase is, under no circumstances, to be interpreted or understood to be encompassed by or to encompass the phrase "indirectly identifying" or "indirectly identified.”
• ENDOGENOUS shall mean a material that a mammal naturally produces.
• ENDOGENOUS in reference to, for example and not limitation, the term "receptor,” shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus.
• the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus.
• a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active is most preferably referred to herein as a "non-endogenous, constitutively activated receptor.”
• Both terms can be utilized to describe both "in vivo" and “in vitro” systems.
• the endogenous or non-endogenous receptor may be AREN-0309 PATENT in reference to an in vitro screening system.
• screening of a candidate compound by means of an in vivo system is viable.
• FUSION PROTEIN in the context of the invention disclosed herein, each mean a non- endogenous protein comprising an endogenous, constitutively activate GPCR or a non- endogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha ( ) subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR.
• G protein "G s " is the predominate G protein that couples with the GPCR
• a GPCR Fusion Protein based upon the specific GPCR would be a non-endogenous protein comprising the GPCR fused to G s ⁇ ; in some circumstances, as will be set forth below, a non-predominant G protein can be fused to the GPCR.
• the G protein can be fused directly to the C-terminus of the constitutively active GPCR or there may be spacers between the two.
• HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein.
• a Plasmid is typically replicated as a autonomous molecule as the Host Cell repUcates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell repUcates, the Plasmid repUcates.
• the Host Cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells.
• INDIRECTLY IDENTIFYING or INDD ECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate compounds against the receptor for determination of those which interfere and/or compete with the Ugand-receptor interaction, and assessing the efficacy of the compound for affecting at least one second messenger pathway associated with the activated receptor.
• INHIBIT or INHIBITING in relationship to the term "response” shall mean that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
• INVERSE AGONISTS shall mean materials (e.g., Ugand, candidate compound) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which is observed in the absence of agonists, or decrease GTP binding to membranes.
• the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, at least 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%, and most preferably at least 99% as compared with the baseline response in the absence of the inverse agonist.
• KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous Ugand specific for that receptor has been identified.
• LIGAND shall mean a molecule specific for a naturally occurring receptor.
• MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor.
• a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if (a) the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor; and (b) the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least 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%, and most preferably at least 99%.
• some preferred cassettes disclosed herein for achieving constitutive activation include a single amino acid and/or codon change between the endogenous and the non-endogenous forms of the GPCR, it is preferred that the percent sequence homology should be at least 98%.
• NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an identified ligand wherein the binding of a Ugand to a receptor activates an intracellular signaling pathway.
• ORPHAN RECEPTOR shall mean an endogenous receptor for which the Ugand specific for that receptor has not been identified or is not known.
• PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, and not limitation, a human).
• PLASMID shall mean the combination of a Vector and cDNA.
• a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein.
• SECOND MESSENGER shall mean an intracellular response produced as a result of receptor activation.
• a second messenger can include, for example, inositol triphosphate (IP 3 ), diacycglycerol (DAG), cyclic AMP (cAMP), and cyclic GMP (cGMP).
• Second messenger response can be measured for a determination of receptor activation, hi addition, second messenger response can be measured for the direct identification of candidate compounds, including for example, inverse agonists, agonists, and antagonists.
• SIGNAL TO NOISE RATIO shall mean the signal generated in response to activation, amplification, or stimulation wherein the signal is above the background noise or the basal level in response to non-activation, non-ampUfication, or non-stimulation.
• SPACER shall mean a translated number of amino acids that are located after the last codon or last amino acid of a gene, for example a GPCR of interest, but before the start codon or beginning regions of the G protein of interest, wherein the translated number amino acids are placed in-frame with the beginnings regions of the G protein of interest.
• the number of translated amino acids can be tailored according to the needs of the skilled artisan and is generally from about one amino acid, preferably two amino acids, more preferably three amino acids, more preferably four amino acids, more preferably five amino acids, more preferably six amino acids, more preferably seven amino acids, more preferably AREN-0309 PATENT eight amino acids, more preferably nine amino acids, more preferably ten amino acids, more preferably eleven amino acids, and even more preferably twelve amino acids.
• STIMULATE or STIMULATING in relationship to the term "response” shall mean that a response is increased in the presence of a compound as opposed to in the absence of the compound.
• SUBSTANTIALLY shall refer to a result which is within 40% of a control result, preferably within 35%, more preferably within 30%, more preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within 10%, more preferably within 5%, more preferably within 2%, and most preferably within 1% of a control result.
• a test receptor may exhibit substantially similar results to a control receptor if the transduced signal, measured using a method taught herein or similar method known to the art-skilled, if within 40% of the signal produced by a control signal.
• VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell.
• any search for therapeutic compounds should start by screening compounds against the Ugand-independent active state.
• Receptor homology is useful in terms of gaining an appreciation of a role of the receptors within the human body. As the patent document progresses, techniques for mutating these receptors to establish non-endogenous, constitutively activated versions of these receptors will be discussed.
• Screening candidate compounds against a non-endogenous, constitutively activated version of the GPCRs disclosed herein allows for the direct identification of candidate compounds which act at the cell surface receptor, without requiring use of the receptor's endogenous ligand.
• routine, and often commercially available techniques one can determine areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed.
• the expression location of a receptor in a specific tissue provides a scientist with the abiUty to assign a physiological functional role of the receptor. It is also possible using these techniques to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document.
• expression of a receptor in diseased organs can assist one in determining the magnitude of the cUnical relevance of the receptor.
• inverse agonists and agonists to the non- endogenous, constitutively activated GPCR can be identified by the methodologies of this invention.
• Such inverse agonists and agonists are ideal candidates as lead compounds in drug discovery programs for treating diseases related to this receptor. Because of the ability to directly identify inverse agonists to the GPCR, thereby allowing for the development of pharmaceutical compositions, a search for diseases and disorders associated with the GPCR is relevant.
• the expression location of a receptor in a specific tissue provides a scientist with the ability to assign a physiological function to the receptor.
• tissue scans can be conducted across a broad range of healthy and diseased tissues. Such tissue scans provide a potential first step in associating a specific receptor with a disease and/or disorder. Furthermore, expression of a receptor in diseased organs can assist one in determining the magnitude of clinical relevance of the receptor.
• the DNA sequence of the GPCR can be used to make a probe/primer.
• the DNA sequence is used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples.
• the presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue can be used to correlate location to function and indicate the receptor's physiological role/function and create a treatment regimen, including but not limited to, a disease associated with that function/role.
• Receptors can also be localized to regions of organs by this technique.
• the putative physiological function of the receptor can be deduced.
• proteins located/expressed in areas of the thalamus are associated with sensorimotor processing and arousal (see, Goodman & Gilman's, The Pharmacological Basis of Therapeutics, 9 th Edition, page 465 (1996)).
• Proteins expressed in the hippocampus or in Schwann cells are associated with learning and AREN-0309 PATENT memory, and myehnation of peripheral nerves, respectively (see, Kandel, E. et al.,
• G protein receptor When a G protein receptor becomes constitutively active, it binds to a G protein (e.g., G q , G s , Gêt G z , Go) and stimulates the binding of GTP to the G protein.
• the G protein then acts as a GTPase and hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated.
• constitutively activated receptors continue to exchange GDP to GTP.
• a non-hydrolyzable analog of GTP [ 35 S]GTP ⁇ S, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [ 35 S]GTP ⁇ S can be used to monitor G protein coupling to membranes in the absence and presence of Ugand.
• a compound identified by the "generic" assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.
• G s stimulates the enzyme adenylyl cyclase.
• Gj (and G z and Go), on the other hand, inhibits adenylyl cyclase.
• Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the G s protein are associated with increased cellular levels of cAMP.
• GPCRs that couple Gi (or G z , Go) protein are associated with decreased cellular levels of cAMP. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 rd Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992).
• assays that detect cAMP can be utiUzed to determine if a candidate compound is, e.g., an inverse AREN-0309 PATENT agonist to the receptor (i.e., such a compound would decrease the levels of cAMP).
• CycUc AMP drives gene expression by promoting the binding of a cAMP- responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites (cAMP response elements) and drives the expression of the gene.
• CREB cAMP-responsive DNA binding protein or transcription factor
• Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., ⁇ -galactosidase or luciferase.
• a constitutively activated G s -linked receptor causes the accumulation of cAMP that then activates the gene and leads to the expression of the reporter protein.
• the reporter protein such as ⁇ -galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
• G 0 and G q are examples of the reporter protein that can then be detected using standard biochemical assays (Chen et al. 1995).
• G q and G 0 are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 , releasing two intracellular messengers: diacycloglycerol (DAG) and inositol 1,4,5-triphoisphate (IP 3 ).
• DAG diacycloglycerol
• IP 3 inositol 1,4,5-triphoisphate
• Increased accumulation of IP is associated with activation of G q - and Go-associated receptors. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 rd Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992).
• Assays that detect IP accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a G q - or Go-associated receptor (i.e., such a compound would decrease the levels of IP 3 ).
• G q - associated receptors can also be examined using an API reporter assay wherein G q - dependent phospholipase C causes activation of genes containing API elements; thus, activated G q -associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression.
• Commercially available assays for such detection are available.
• an endogenous, constitutively activated GPCR or a non-endogenous, constitutively activated GPCR for use in screening of candidate compounds for the direct identification of inverse agonists, agonists provide an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous Ugand bound thereto.
• an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist or agonist or have no affect on such a receptor, it is preferred that an approach be utilized that can enhance such differentiation.
• a preferred approach is the use of a GPCR Fusion Protein.
• a non-endogenous GPCR has been constitutively activated using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. In some embodiments it is preferred that screening take place using a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated GPCR will continuously signal.
• this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
• the GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR.
• the GPCR Fusion Protein is preferred for screening with either an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is utilized in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein.
• GPCR Fusion Protein AREN-0309 PATENT construct include but are not limited to, that the endogenous GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence), and that the "stop" codon of the GPCR be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed.
• inventions include constructs wherein the endogenous GPCR sequence and the G protein sequence are not in-frame and or the "stop" codon is not deleted or replaced.
• the GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Based upon convenience it is preferred to use a spacer.
• the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct.
• a construct comprising the sequence of the G protein (i.e., a universal G protein construct (see Examples)) be available for insertion of an endogenous GPCR sequence therein; this provides for further efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.
• G z and G 0 are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging (i.e., the cAMP signal decreases upon activation thus making the direct identification of, e.g., inverse agonists (which would further decrease this signal), challenging.
• the cAMP signal decreases upon activation thus making the direct identification of, e.g., inverse agonists (which would further decrease this signal), challenging.
• inverse agonists which would further decrease this signal
• an endogenous G; coupled receptor can be fused to a G s protein -such a fusion construct, upon expression, "drives” or “forces” the endogenous GPCR to couple with, e.g., G s rather than the "natural" Gj protein, such that a cyclase-based assay can be estabUshed.
• Gj, G z and G 0 coupled receptors in some embodiments it is preferred that when a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with G s (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
• G Protein Fusion construct that utilizes a G q Protein fused with a G s , Gj, G z or G 0 Protein.
• a preferred fusion construct can be accomplished with a G q Protein wherein the first six (6) amino acids of the G-protein ⁇ - subunit ("G ⁇ q") is deleted and the last five (5) amino acids at the C-terminal end of G ⁇ q is replaced with the corresponding amino acids of the G ⁇ of the G protein of interest.
• a fusion construct can have a G q (6 amino acid deletion) fused with a Gj Protein, resulting in a "G q /Gj Fusion Construct".
• This fusion construct will forces the endogenous Gj coupled receptor to couple to its non-endogenous G protein, G q , such that the second messenger, for example, inositol triphosphate or diacylgycerol, can be measured in lieu of cAMP production.
• G q non-endogenous G protein
• a Gj coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decreases the level of cAMP production, which can make assessment of cAMP levels challenging.
• An effective technique in measuring the decrease in production of cAMP as an indication of constitutive activation of a receptor that predominantly couples Gj upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non-endogenous, constitutively activated receptor that predominantly couples with G s upon activation (e.g., TSHR-A623I, disclosed below), with the Gj linked GPCR.
• a signal enhancer e.g., a non-endogenous, constitutively activated receptor that predominantly couples with G s upon activation (e.g., TSHR-A623I, disclosed below
• constitutive activation of a G s AREN-0309 PATENT coupled receptor can be determined based upon an increase in production of cAMP.
• cAMP By then co-transfecting the signal enhancer with a constitutively activated version of the target receptor, cAMP would be expected to further decrease (relative to base line) due to the increased functional activity of the Gj target (i.e., which decreases cAMP).
• Screening of candidate compounds using a cAMP based assay can then be accomplished, with two 'changes' relative to the use of the endogenous receptor/G-protein fusion: first, relative to the G; coupled target receptor, "opposite" effects will result, i.e., an inverse agonist of the Gj coupled target receptor will increase the measured cAMP signal, while an agonist of the G; coupled target receptor will decrease this signal; second, as would be apparent, candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or after screening against the co-transfected receptors).
• Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16 th Edition, 1980, Mack Publishing Co., (Osol et al., eds.).
• non-endogenous versions of the GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists or agonists (preferably for use as pharmaceutical agents), other uses of these versions of GPCRs exist.
• in vitro and in vivo systems incorporating GPCRs can be utilized to further elucidate and understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade.
• the endogenous receptors be "orphan receptors", i.e., the endogenous Ugand for the receptor has not been identified.
• the modified, non-endogenous GPCRs can be used to understand the role of endogenous receptors in the human body before the endogenous Ugand therefore is identified.
• Such receptors can also be used to further elucidate known receptors and the pathways through which they transduce a signal.
• Other uses of the disclosed receptors will become apparent to those in the art based upon, ter alia, a review of this patent document.
• AC073957 was identified as a human genomic sequence from chromosome 7.
• a 1.16kb 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 (P.E. Biosystems). See, SEQ.ID.NO. :1 for the nucleic acid sequence and SEQ.ID.NO. :2 for the putative amino acid sequence.
• b. hRUP29 (Seq. Id. Nos. 3 & 4)
• the disclosed human RUP29 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC0083865 was identified as a human genomic sequence from chromosome 7. The full length RUP29 was cloned by PCR using primers: 5'-GTATGCCTGGCCACAATACCTCCAGG-3' (SEQ.ID.NO. :23; sense, containing the initiation codon),
• a 930bp 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 (P.E. Biosystems).
• RACE Rapid amplification of cDNA ends
• RUP29 specific primer (1) having the sequence: 5-GGTACCACAATGACAATCACCAGCGTCC-3 '(SEQ.ID.NO. :25) and API primer (Clontech) were used for the first-round PCR reaction
• RUP29 specific primer (2) having the following sequence: AREN-0309 PATENT
• GGAACGTGAGGTACATGTGGATGTGCAGC-3 ' (SEQ.ID.NO. :26) and AP2 primer (Clontech) were used for the second-round PCR reaction.
• the products of the RACE reactions were isolated and cloned into the pCRII-TOPO vector (rnvitrogen) and sequenced. See, SEQ.ID.NO. :3 for the nucleic acid sequence and SEQ.ID.NO. :4 for the putative amino acid sequence.
• c. hRUP30 (Seq. Id. Nos. 5 & 6) The disclosed human RUP30 was identified based upon the use of GenBank database information.
• a cDNA clone with Accession Number AC055863 was identified as a human genomic sequence from chromosome 17.
• the full length RUP30 was cloned by 5 'RACE -PCR with a human pancreas Marathon-ReadyTM cDNA (Clontech) as template and the following oligonucleotide: 5'-GCAGTGTAGCGGTCAACCGTGAGCAGG-3'(SEQ.ID.NO.:27; sense, containing the initiation codon), and API primer (Clontech) were used for the first round of RT-PCR and oligonucleotide:
• 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 sequenced using the ABI Big Dye Terminator kit (P.E. Biosystems). See, SEQ.ID.NO.:5 for the nucleic acid sequence and SEQ.ID.NO. :6 for the putative amino acid sequence.
• AREN-0309 PATENT d. hRUP31 (Seq. Id. Nos. 7 & 8)
• the disclosed human RUP31 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession
• 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 (P.E. Biosystems). See, SEQ.ID.NO. :7 for the nucleic acid sequence and SEQ.ID.NO. :8 for the putative amino acid sequence. e. hRUP32 (Seq. Id. Nos. 9 & 10)
• the disclosed human RUP32 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AL513524 was identified as a human genomic sequence from chromosome 6. The full length RUP32 was cloned by PCR using primers: 5'-GCGTTATGAGCAGCAATTCATCCCTGCTGG-3' (SEQ.ID.NO.:33; sense, containing the initiation codon),
• 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 (P.E. Biosystems). See, SEQ.ID.NO. :9 for the nucleic acid sequence and SEQ.ID.NO.: 10 for the putative amino acid sequence.
• AREN-0309 PATENT f. hRUP33 (Seq. Id. Nos. 11 & 12)
• the disclosed human RUP33 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession
• 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 (P.E. Biosystems). See, SEQ.ID.NO.: 11 for the nucleic acid sequence and
• the disclosed human RUP34 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AL513524 was identified as a human genomic sequence from chromosome 6. The full length RUP34 was cloned by PCR using primers:
• 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
• the disclosed human RUP35 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession
• 5'-GTGGCAGACAGCGATATACCTGTCAATGG-3' SEQ.ID.NO.:40; antisense
• AP2 primer Clontech
• DNA fragments generated by the 5' RACE-PCR were cloned into the pCRTI-TOPO vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene).
• RUP35 was cloned by RT-PCR, using primers 5'-GCGCTCATGGAGCACACGCACGCCCAC-3' (SEQ.ID.NO. :41; sense, ATG as the initiation codon) and
• 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 (P.E. Biosystems). See, SEQ.ID.NO.:15 for the nucleic acid sequence and SEQ.ID.NO. : 16 for the putative amino acid sequence. i. hRUP36 (Seq. Id. Nos. 17 & 18) The disclosed human RUP36 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC090099 was identified as a human genomic sequence from chromosome 11. The full length RUP36 was cloned by PCR using primers:
• 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 (P.E. Biosystems). See, SEQ.ID.NO.: 17 for the nucleic acid sequence and SEQ.ID.NO. : 18 for the putative amino acid sequence.
• j. hRUP37 (Seq. Id. Nos. 19 & 20)
• the disclosed human RUP37 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with Accession Number AC090099 was identified as a human genomic sequence from chromosome 11. The full length RUP37 was cloned by PCR using primers:
• a 969 base pair was isolated from a 1%% agarose gel and cloned into the pCRII- TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:19 for the nucleic acid sequence and SEQ.ID.NO.:20 for the putative amino acid sequence.
• Example 2
• Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using, inter alia, Transformer Site-DirectedTM Mutagenesis Kit (Clontech) according to the manufacturer instructions.
• two mutagenesis primers are used, preferably a lysine mutagenesis oUgonucleotide that creates the lysine mutation, and a selection marker oligonucleotide.
• the codon mutation to be incorporated into the human GPCR is also noted, in standard form (Table D):
• mammalian cells Although a variety of cells are available to the art-skilled for the expression of proteins, it is preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a
• tube A was prepared by mixing 4 ⁇ g DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 0.5 ml serum free DMEM (Gibco BRL); tube B was prepared by mixing 24 ⁇ l lipofectamine (Gibco BRL) in 0.5ml serum free DMEM. Tubes A and B were admixed by inversion (several times), followed by incubation at room temperature for 30- 45min. The admixture is referred to as the "transfection mixture”.
• Plated 293 cells were washed with 1XPBS, followed by addition of 5 ml serum free DMEM. One ml of the transfection mixture were added to the cells, followed by incubation for 4hrs at 37°C/5% CO 2 . The transfection mixture was removed by aspiration, followed by the addition of 10ml of DMEM/10% Fetal Bovine Serum. Cells were incubated at 37°C/5% CO 2 . After 48hr incubation, cells were harvested and utiUzed for analysis. b.
• 293 cells Approximately 12x10 6 293 cells will be plated on a 15cm tissue culture plate, and grown in DME High Glucose Medium containing 10% fetal bovine serum and one percent sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours following plating of 293 cells (to approximately -80% confluency), the cells will be transfected using 12 ⁇ g of DNA. The 12 ⁇ g of DNA is combined with 60 ⁇ l of lipofectamine and 2mL of DME High Glucose Medium without serum. The medium will be aspirated from the plates and the cells washed once with medium without serum. The DNA, Upofectamine, and medium mixture will be added to the plate along with lOmL of medium without serum.
• the medium will be aspirated and 25ml of medium containing serum wiU be added. Twenty-four hours following transfection, the medium will be aspirated again, and fresh medium with serum will be added. Forty-eight hours following transfection, the medium will be aspirated and medium with serum will be added containing geneticin (G418 drug) at a final concentration of 500 ⁇ g/mL.
• G418 drug geneticin
• a G protein-coupled receptor When a G protein-coupled receptor is in its active state, either as a result of Ugand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein.
• the alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP.
• the non-hydrolyzable GTP analog, [ 35 S]GTP ⁇ S can be utilized to demonstrate enhanced binding of [ 35 S]GTP ⁇ S to membranes expressing constitutively activated receptors.
• [ S]GTP ⁇ S binding to measure constitutive activation include but are not Umited to the following: (a) it is generically applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade.
• the assay takes advantage of the ability of G protein coupled receptors to stimulate
• [ 35 S]GTP ⁇ S binding to membranes expressing the relevant receptors can, therefore, be used in the direct identification method to screen candidate compounds to constitutively activated G protein-coupled receptors.
• the assay is generic and has application to drug discovery at all G protein-coupled receptors.
• the [ 35 S]GTP ⁇ S assay is incubated in 20 mM HEPES and between 1 and about
• membrane protein e.g., 293 cells expressing the G s Fusion Protein; this amount can be adjusted for optimization
• 10 ⁇ M GDP this amount can be changed for optimization
• Adenylyl Cyclase A Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes.
• the Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP.
• the cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody.
• the foUowing serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.
• Transfected cells will be harvested approximately twenty four hours after transient transfection. Media will be carefully aspirated and discarded. Ten ml of PBS will gently be added to each dish of cells followed by careful aspiration. One ml of Sigma cell dissociation buffer and 3ml of PBS will be added to each plate. Cells will be pipetted off the plate and the cell suspension collected into a 50ml comcal centrifuge tube. Cells will be centrifuged at room temperature at 1,100 rpm for 5 min. The cell pellet will be carefully re- suspended into an appropriate volume of PBS (about 3ml/plate).
• cAMP standards and Detection Buffer comprising 1 ⁇ Ci of tracer [ 125 I cAMP (50 ⁇ l] to 11 ml Detection Buffer) will be prepared and maintained in accordance with the manufacturer's instructions.
• Assay Buffer will be prepared fresh for screening and contained 50 ⁇ l of Stimulation Buffer, 3 ⁇ l of test compound (12 ⁇ M final assay concentration) and 50 ⁇ l cells, Assay Buffer will be stored on ice until utilized.
• the assay will be initiated by addition of 50 ⁇ l of cAMP standards to appropriate wells followed by addition of 50 ⁇ l of PBSA to wells H-ll and H12. Fifty ⁇ l of Stimulation Buffer will be added to all wells. DMSO (or selected candidate compounds) will be added to appropriate AREN-0309 PATENT wells using a pin tool capable of dispensing 3 ⁇ l of compound solution, with a final assay concentration of 12 ⁇ M test compound and lOO ⁇ l total assay volume. The cells will then be added to the wells and incubated for 60 min at room temperature. One hundred ⁇ l of Detection Mix containing tracer cAMP will then be added to the wells.
• TSHR is a G s coupled GPCR that causes the accumulation of cAMP upon activation.
• TSHR will be constitutively activated by mutating amino acid residue 623 (i.e., changing an alanine residue to an isoleucine residue).
• a Gj coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging.
• An effective technique for measuring the decrease in production of cAMP as an indication of constitutive activation of a Gj coupled receptor can be accompUshed by co-transfecting, most preferably, non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active G s coupled receptor) as a "signal enhancer" with a Gj linked target GPCR to estabUsh a baseline level of cAMP.
• TSHR-A623I non-endogenous, constitutively activated TSHR
• Gj linked target GPCR to estabUsh a baseline level of cAMP.
• This approach will be utilized to effectively generate a signal when a cAMP assay is used; this approach is preferably used in the direct identification of candidate compounds against G; coupled receptors. It is noted that for a Gj coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal.
• tube A will be prepared by mixing 2ug DNA of each receptor transfected into the mammatian cells, for a total of 4ug DNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc.) in 1.2ml serum free DMEM (Irvine Scientific, Irvine, CA); tube B will be prepared by mixing 120 ⁇ l lipofectamine (Gibco BRL) in 1.2ml serum free AREN-0309 PATENT
• DMEM fetal calf serum
• Tubes A and B will then be admixed by inversion (several times), followed by incubation at room temperature for 30-45min. The admixture is referred to as the "transfection mixture".
• Plated 293 cells will be washed with 1XPBS, followed by addition of 10ml serum free DMEM.
• 2.4ml of the transfection mixture will then be added to the cells, followed by incubation for 4hrs at 37°C/5% CO 2 .
• the transfection mixture will then be removed by aspiration, followed by the addition of 25ml of DMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37°C/5% CO 2 . After 24hr incubation, cells will be harvested and utilized for analysis.
• a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) although designed for cell-based assays, can be modified for use with crude plasma membranes depending on the need of the skilled artisan.
• the Flash Plate wells will contain a scintillant coating which also contains a specific antibody recognizing cAMP.
• the cAMP generated in the wells can be quantified by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.
• Transfected cells will be harvested approximately twenty four hours after transient transfection. Media will be carefully aspirated and discarded. Ten ml of PBS will be gently added to each dish of cells followed by careful aspiration. One ml of Sigma cell dissociation buffer and 3ml of PBS will be added to each plate. Cells will be pipetted off the plate and the cell suspension will be collected into a 50ml conical centrifuge tube. Cells will be centrifuged at room temperature at 1,100 rpm for 5 min. The cell pellet will be carefully re-suspended into an appropriate volume of PBS (about 3ml/plate).
• cAMP standards and Detection Buffer comprising 1 ⁇ Ci of tracer [ 125 I cAMP (50 ⁇ l] to 11 ml Detection Buffer) will be prepared and maintained in accordance with the manufacturer's instructions.
• Assay Buffer should be prepared fresh for screening and contained 50 ⁇ l of Stimulation Buffer, 3 ⁇ l of test compound (12 ⁇ M final assay concentration) and 50 ⁇ l cells, Assay Buffer can be stored on ice until utilized.
• the assay can be initiated by addition of 50 ⁇ l of cAMP standards to appropriate wells followed by addition of 50 ⁇ l of PBSA to wells H-l 1 and H12. Fifty ⁇ l of Stimulation Buffer will be added to all wells. Selected compounds (e.g., TSH) will be added to appropriate wells AREN-0309 PATENT using a pin tool capable of dispensing 3 ⁇ l of compound solution, with a final assay concentration of 12 ⁇ M test compound and lOO ⁇ l total assay volume. The ceUs will then be added to the wells and incubated for 60 min at room temperature. One hundred ⁇ l of Detection Mix containing tracer cAMP will then be added to the wells. Plates will then be incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well will then be extrapolated from a standard cAMP curve which is contained within each assay plate.
• Selected compounds e.g., TSH
• CRE-LUC Reporter Assay (G s -associated receptors) 293 and 293T cells will be plated-out on 96 well plates at a density of 2 x 10 4 cells per well and will be transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions.
• BBL Lipofectamine Reagent
• a DNA/lipid mixture will be prepared for each 6-well transfection as follows: 260ng of plasmid DNA in lOO ⁇ l of DMEM are gently mixed with 2 ⁇ l of lipid in lOO ⁇ l of DMEM (the 260ng of plasmid DNA consisted of 200ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMN alone, and lOng of a GPRS expression plasmid (GPRS in pcD ⁇ A3 (Invitrogen)).
• the 8XCRE-Luc reporter plasmid is prepared as follows: vector SRIF- ⁇ -gal will be obtained by cloning the rat somatostatin promoter (-71/+51) at BglN-Hindlll site in the p ⁇ gal-Basic Vector (Clontech). Eight (8) copies of cAMP response element will be obtained by PCR from an adenovirus template Ad ⁇ CF126CCRE8 (see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF- ⁇ -gal vector at the Kpn-BglV site, resulting in the 8xCRE- ⁇ -gal reporter vector.
• the 8xCRE-Luc reporter plasmid will be generated by replacing the beta-galactosidase gene in the 8xCRE- ⁇ -gal reporter vector with the luciferase gene obtained from the pGL3- basic vector (Promega) at the Hindlll-BamHI site. Following 30 min. incubation at room temperature, the D ⁇ A/lipid mixture will be diluted with 400 ⁇ l of DMEM and lOO ⁇ l of the diluted mixture will be added to each well. One hundred ⁇ l of DMEM with 10%) FCS will be added to each well after a 4hr incubation in a cell culture incubator. The following day the transfected cells will be changed with 200 ⁇ l well of DMEM with 10% FCS.
• a method to detect G q stimulation depends on the known property of G q - dependent phospholipase C to cause the activation of genes containing API elements in their promoter.
• a PathdetectTM AP-1 cis-Reporting System (Stratagene, Catalogue # 219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate were 410 ng pAPl-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.
• SRF-LUC Reporter Assay G q - associated receptors
• G q stimulation depends on the known property of G q - dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter.
• a PathdetectTM SRF-Luc-Reporting System (Stratagene) can be utilized to assay for G q coupled activity in, e.g., COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian TransfectionTM Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions.
• 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
• CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
• ceUs comprising the receptors are plated onto 24 well plates, usually lxl 0 5 cells/well (although his number can be optimized.
• cells are transfected by firstly mixing 0.25ug DNA in 50 ⁇ l serum free DMEM/well and 2 ⁇ l lipofectamine in 50 ⁇ l serum free DMEM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are then washed with 0.5 ml PBS and 400 ⁇ l of serum free media and then mixed with the transfection media and added to the cells.
• the cells are incubated for 3-4 hrs at 37°C/5%CO 2 and then the transfection media is removed and replaced with lml/well of regular growth media.
• the cells are labeled with 3 H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol- free/serum free media (GIBCO BRL) are added/well with 0.25 ⁇ Ci of 3 H-myo-inositol/ well and the cells incubated for 16-18 hrs overnight at 37°C/5%CO 2 .
• GEBCO BRL inositol- free/serum free media
• the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing inositol- free/serum free media 10 ⁇ M pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50 ⁇ l of lOx ketanserin (ket) to final concentration of lO ⁇ M.
• the cells are then incubated for 30 min at 37°C.
• the cells are then washed with 0.5 ml PBS and 200 ⁇ l of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added to each well.
• the solution is kept on ice for 5-10 min (or until cells are lysed) and then neutralized by 200 ⁇ l of fresh/ice cold neutralization solution (7.5 % HCL).
• the lysate is then transferred into 1.5 ml Eppendorf tubes and 1 ml of chloroform/methanol (1:2) is added/tube.
• the solution is vortexed for 15 sec and the upper phase is applied to a Biorad AG1-X8TM anion exchange resin (100-200 mesh). First, the resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded onto the column.
• the column is then washed with 10 ml of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60mM Na- formate.
• the inositol tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/ 1 M ammonium formate.
• the columns are regenerated by washing with 10 ml of 0.1 M formic aci ⁇ VBM ammonium formate and rinsed twice with dd H 2 O and stored at 4°C in water.
• RUP35 and RUP36 receptor are endogenously, constitutively active.
• RUP35 evidences about a six (6) fold increase in intracellular inositol phosphate accumulation when compared to pCMV and
• RUP36 evidences about a four (4) fold increase when compared to pCMV.
• the design of the constitutively activated GPCR-G protein fusion construct can be accomplished as follows: both the 5' and 3' ends of the rat G protein G s ⁇ (long form; Itoh, H. et al., 83 PNAS 3776 (1986)) is engineered to include a HindHI (5'-AAGCTT-3') sequence thereon. Following confirmation of the correct sequence (including the flanking Hindlll sequences), the entire sequence is shuttled into pcDNA3.1(-) (Invitrogen, cat. no. N795-20) by subcloning using the Hindlll restriction site of that vector. The correct orientation for the G s ⁇ sequence will be determined after subcloning into pcD ⁇ A3.1(-).
• the modified pcDNA3.1(-) containing the rat G s ⁇ gene at Hindi ⁇ sequence is then verified; this vector will then be available as a "universal" G s ⁇ protein vector.
• the pcDNA3.1(-) vector contains a variety of well-known restriction sites upstream of the Hindi ⁇ site, thus beneficially providing the ability to insert, upstream of the G s protein, the coding sequence of an endogenous, constitutively active GPCR.
• This same approach can be utilized to create other "universal" G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan can be utilized.
• the important criteria is that the sequence for the GPCR be upstream and in-frame with that of the G protein.
• Spacers in the restriction sites between the G protein and the GPCR are optional.
• the sense and anti-sense primers included the restriction sites for Xbal and EcoRN, respectively, such that spacers (attributed to the restriction sites) exist between the G protein and the GPCR.
• PCR will then be utilized to secure the respective receptor sequences for fusion within the G s ⁇ universal vector disclosed above, using the following protocol for each: lOOng cD ⁇ A for GPCR will be added to separate tubes containing 2 ⁇ l of each primer (sense and anti-sense), 3 ⁇ l of lOmM d ⁇ TPs, lO ⁇ l of lOXTaqPlusTM Precision buffer, l ⁇ l of TaqPlusTM Precision polymerase (Stratagene: #600211), and 80 ⁇ l of water.
• Reaction temperatures and cycle times for the GPCR will be as follows with cycle steps 2 through 4 were repeated 35 times: 94°C for 1 min; 94°C for 30 seconds; 62°C for 20 sec; 72°C 1 min 40sec; and 72°C 5 min.
• PCR products will be run on a 1% agarose gel and then purified.
• the purified products will be digested with Xbal and EcoRN and the desired inserts purified and ligated into the G s universal vector at the respective restriction sites.
• the positive clones will be isolated following transformation and determined by restriction enzyme digestion; expression using 293 cells will be accompUshed following the protocol set forth infra. Each positive clone for GPCR- G s Fusion Protein will be sequenced to verify correctness. b.
• G q (6 amino acid deletion)/Gi Fusion Construct
• the design of a G q (del)/G; fusion construct was accomplished as follows: the ⁇ - terminal six (6) amino acids (amino acids 2 through 7), having the sequence of TLESIM (SEQ.ID.NO. :47) G ⁇ q-subunit was deleted and the C-terminal five (5) amino acids, having the sequence EYNLV (SEQ.ID.NO.:48) was replaced with the corresponding amino acids of the G ⁇ i Protein, having the sequence DCGLF (SEQ.ID.NO.:49).
• This fusion construct was obtained by PCR using the following primers:
• Plasmid 63313 which contains the mouse G ⁇ q-wild type version with E hemagglutinin tag as template. Nucleotides in lower caps are included as spacers. TaqPlus ® Precision DNA polymerase (Stratagene) was utilized for the amplification by the following cycles, with steps 2 through 4 repeated 35 times: 95°C for
• the PCR AREN-0309 PATENT product will be cloned into a pCP I-TOPO vector (Invitrogen) and sequenced using the
• RT-PCR was applied to confirm the expression and to dete ⁇ nine the tissue distribution of several novel human GPCRs. Oligonucleotides utilized were GPCR- specific and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) were utilized for the amplification in a 40 ⁇ l reaction according to the manufacturer's instructions. Twenty ⁇ l of the reaction will be loaded on a 1.5% agarose gel to analyze the RT-PCR products. Table E, below, lists the receptors, the cycle conditions and the primers utilized, and also lists exemplary diseases/disorders linked to the receptors.
• Diseases and disorders related to receptors located in these tissues or regions include, but are not limited to, cardiac disorders and diseases (e.g. thrombosis, myocardial infarction; atherosclerosis; cardiomyopathies); kidney disease/disorders (e.g., renal failure; renal tubular acidosis; renal glycosuria; nephrogenic diabetes insipidus; cystinuria; polycystic kidney disease); eosinophilia; leukocytosis; leukopenia; ovarian cancer; sexual dysfunction; polycystic ovarian syndrome; pancreatitis and pancreatic cancer; irritable bowel syndrome; colon cancer; Crohn's disease; ulcerative colitis; diverticulitis; Chronic Obstructive Pulmonary Disease (COPD); Cystic Fibrosis; pneumonia; pulmonary hypertension; tuberculosis and lung cancer; Parkinson's disease; movement disorders and ataxias; learning and memory disorders; eating disorders (e.g., anorexia
• Membranes comprising the constitutively active orphan GPCR Fusion Protein of interest and for use in the direct identification of candidate compounds as inverse agonists or agonists are preferably prepared as follows: a. Materials "Membrane Scrape Buffer” is comprised of 20mM HEPES and lOmM EDTA, pH 7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; “Binding Buffer” is comprised of 20mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4 b. Procedure All materials will be kept on ice throughout the procedure.
• the media will be aspirated from a confluent monolayer of cells, followed by rinse with 10ml cold PBS, followed by aspiration. Thereafter, 5ml of Membrane Scrape Buffer will be added to scrape cells; this will be followed by transfer of cellular extract into 50ml centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4°C). Thereafter, the supernatant will be aspirated and the pellet will be resuspended in 30ml Membrane Wash Buffer followed by centrifugation at 20,000 rpm for 17 minutes at 4°C. The supernatant will then be aspirated and the pellet resuspended in Binding Buffer. The resuspended pellet will then be homogenized using a Brinkman PolyfronTM homogenizer (15-20 second bursts until the material is in suspension). This is referred to herein as "Membrane Protein". 2.
• protein concentration of the membranes will be determined, for example, using the Bradford Protein Assay (protein can be diluted to about 1.5mg/ml, aliquoted and frozen (-80°C) for later use; when frozen, protocol for use will be as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polyfron at about 12 x 1,000 rpm for about 5-10 seconds; it was noted that for multiple preparations, the homogenizer is thoroughly cleaned between homogenization of different preparations).
• Bradford Protein Assay protein can be diluted to about 1.5mg/ml, aliquoted and frozen (-80°C) for later use; when frozen, protocol for use will be as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polyfron at about 12 x 1,000 rpm for about 5-10 seconds; it was noted that for multiple preparations, the homogenizer is thoroughly
• Duplicate tubes will be prepared, one including the membrane, and one as a control "blank". Each contains 800 ⁇ l Binding Buffer. Thereafter, lO ⁇ l of Bradford
• each well comprising a candidate compound has a final volume of 200 ⁇ l consisting of lOO ⁇ l GDP Buffer (final concentration, 0.1 ⁇ M GDP), 50 ⁇ l Membrane Protein in Binding Buffer, and 50 ⁇ l [ 35 S]GTP ⁇ S (0.6 nM) in Binding Buffer (2.5 ⁇ l [ 35 S]GTP ⁇ S per 10ml Binding Buffer).
• Candidate compounds will be preferably screened using a 96-well plate format (these can be frozen at -80°C).
• Membrane Protein or membranes with expression vector excluding the GPCR Fusion Protein, as control
• Protein concentration will then be determined using, for example, the Bradford Protein Assay set forth above.
• Membrane Protein (and controls) will then be diluted to 0.25mg/ml in Binding Buffer (final assay concentration, 12.5 ⁇ g/well). Thereafter, 100 ⁇ l GDP Buffer is added to each well of a Wallac ScintistripTM (Wallac).
• a 5 ⁇ l pin-tool will then be used to transfer 5 ⁇ l of a candidate compound into such well (i.e., 5 ⁇ l in total assay volume of 200 ⁇ l is a 1 :40 ratio such that the final screening concentration of the candidate compound is lO ⁇ M).
• the pin tool is rinsed in three reservoirs comprising water (IX), ethanol (IX) and water (2X) - excess AREN-0309 PATENT liquid is shaken from the tool after each rinse and the tool is dried with paper and Kim wipes. Thereafter, 50 ⁇ l of Membrane Protein will be added to each well (a control well comprising membranes without the GPCR Fusion Protein was also utilized), and pre- incubated for 5-10 minutes at room temperature.
• Binding Buffer 50 ⁇ l of [ 35 S]GTP ⁇ S (0.6 nM) in Binding Buffer will be added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates were covered with foil). The assay will be stopped by spinning the plates at 4000 RPM for 15 minutes at 22°C. The plates will then be aspirated with an 8 channel manifold and sealed with plate covers. The plates will then be read on a Wallac 1450 using setting "Prot. #37" (as per manufacturer's instructions).
• Another assay approach to directly identify candidate compound will be accomplished utilizing a cyclase-based assay.
• this assay approach can be utilized as an independent approach to provide confirmation of the results from the [ 35 S]GTP ⁇ S approach as set forth above.
• a modified Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) will be preferably utiUzed for direct identification of candidate compounds as inverse agonists and agonists to GPCRs in accordance with the following protocol. Transfected cells will be harvested approximately three days after transfection.
• Membranes will be prepared by homogenization of suspended cells in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl 2 . Homogenization will be performed on ice using a Brinkman PolyfronTM for approximately 10 seconds. The resulting homogenate will be centrifuged at 49,000 X g for 15 minutes at 4°C. The resulting pellet will then be resuspended in buffer containing 20mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 X g for 15 minutes at 4°C. The resulting pellet will then be stored at -80°C until utilized.
• the membrane pellet On the day of direct identification screening, the membrane pellet will slowly be thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl 2 , to yield a final protein concentration of 0.60mg/ml (the resuspended membranes will be placed on ice until use).
• cAMP standards and Detection Buffer comprising 2 ⁇ Ci of tracer [ 125 I cAMP (100 ⁇ l] to 11 ml Detection Buffer) will be prepared and maintained in accordance with the AREN-0309 PATENT manufacturer's instructions.
• Assay Buffer will be prepared fresh for screening and contain 20mM HEPES, pH 7.4, lOmM MgCl 2 , 20mM phosphocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 ⁇ M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer will be stored on ice until utiUzed.
• Candidate compounds identified as per above if frozen, thawed at room temperature • will be added, preferably, to 96-well plate wells (3 ⁇ l/well; 12 ⁇ M final assay concentration), together with 40 ⁇ l Membrane Protein (30 ⁇ g/well) and 50 ⁇ l of Assay Buffer.
• a method for identifying candidate agonists or inverse agonists for a GPCR can be preformed by introducing tests cells of a pigment cell line capable of dispersing or aggregating their pigment in response to a specific stimulus and expressing an exogenous clone coding for the GCPR.
• a stimulant e.g., Ught
• the tests cells are then contacted with chemical compounds, and it is determined whether the pigment disposition in the cells changed from the initial state of pigment disposition. Dispersion of pigments cells due to the candidate compound coupUng to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light.
• the vector utilized be pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 AREN-0309 PATENT).

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