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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
• • 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
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

• the invention disclosed in this patent document relates to transmembrane receptors, and more particularly to human G protein-coupled receptors, and specifically to endogenous human GPCRs with particular emphasis on non- endogenous versions of the GPCRs that have been altered to establish or enhance constitutive activity of the receptor.
• the altered GPCRs are used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists having potential applicability as therapeutic agents.
• GPCR G protein-coupled receptor
• Receptors including GPCRs, for which the endogenous ligand has been identified are referred to as "known" receptors, while receptors for which the endogenous ligand has not been identified are referred to as "orphan" receptors.
• 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.
• GPCRs share a common structural motif. All these receptors have 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 "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).
• 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, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
• 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 produce 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 an endogenous 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 endogenous 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 an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed "constitutive receptor activation.”
• Figure 1 provides an illustration of second messenger B° 3 production from endogenous version RUP12 ("RUP12”) as compared with the control (“CMV").
• Figure 2 is a graphic representation of the results of a second messenger cell- based cyclic AMP assay providing comparative results for constitutive signaling of endogenous RUP13 ("RUP13”) and a control vector (“CMV").
• RUP13 endogenous RUP13
• CMV control vector
• FIG. 3 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP13 ("RUP13 wt”) and non-endogenous, constitutively activated RUP13 ("RUP13(A268K)”), utilizing 8XCRE-Luc reporter plasmid.
• Figure 4 is a graphic representation of the results of a [ 35 S]GTP ⁇ S assay
• RUP13:Gs Fusion Protein (“RUP13-Gs”) and a control vector (“CMV”).
• Figure 5 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP14 ("RUP14 wt”) and non-endogenous, constitutively activated RUP13 ("RUP14(L246K)”) 5 utilizing 8XCRE-Luc reporter plasmid.
• Figure 6 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP15 (“RUP15 wt”) and non-endogenous, constitutively activated RUP15 (“RUP15(A398K)”), utilizing 8XCRE-Luc reporter plasmid.
• Figure 7 is a graphic representation of the results of a second messenger cell- based cyclic AMP assay providing comparative results for constitutive signaling of endogenous RUP15 (“RUP15 wt”), non-endogenous, constitutively activated version of RUP15 (“RUP15(A398K)”) and a control vector ("CMV").
• Figure 8 is a graphic representation of the results of a [ S]GTP ⁇ S assay
• RUP15:Gs Fusion Protein (“RUP15-Gs”) and a control vector (“CMV”).
• FIG 9 provides an illustration of second messenger IP 3 production from endogenous version RUP17 (“RUP17”) as compared with the control (“CMV").
• FIG 10 provides an illustration of second messenger IP 3 production from endogenous version RUP21 ("RUP21”) as compared with the control (“CMV").
• Figure 11 is a diagrammatic representation of the signal measured comparing CMV, endogenous RUP23 (“RUP23 wt”) and non-endogenous, constitutively activated RUP23 (“RUP23(W275K)”), utilizing 8XCRE-Luc reporter plasmid.
• Figure 12 is a graphic representation of results from a primary screen of several candidate compounds against RUP13; results for "Compound A” are provided in well A2 and "Compound “B” are provided in well G9.
• 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.
• PARTIAL AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists.
• 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 or partial agonists.
• ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.
• 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, 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 subject 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 endogenous ligand 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 IDENTIFIED 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 inte ⁇ reted 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 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
• 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.
• the G protein preferably being of the same type as the G protein that naturally couples with endogenous orphan GPCR.
• endogenous protein comprising the GPCR fused to Gs ⁇ ; in some circumstances, as will
• 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 inco ⁇ orated therein.
• a Plasmid is typically replicated as a autonomous molecule as the Host Cell replicates (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 replicates, the Plasmid replicates.
• 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 INDIRECTLY 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 ligand-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., ligand, 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 partial agonists, or decrease GTP binding to membranes.
• the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist.
• KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified.
• LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, 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 about 80%, more preferably at least about 90% and most preferably at least 95%.
• NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway.
• ORPHAN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand 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).
• 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 piuposes 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.
• second messenger response can be measured for the direct identification of candidate compounds, including for example, inverse agonists, agonists, partial agonists and antagonists.
• 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.
• VECTOR in reference to cDNA shall mean a circular DNA capable of inco ⁇ orating at least one cDNA and capable of inco ⁇ oration into a Host Cell.
• any search for therapeutic compounds should start by screening compounds against the ligand-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, we will disclose techniques for mutating these receptors to establish non-endogenous, constitutively activated versions of these receptors .
• Receptor Screening candidate compounds against a non-endogenous, constitutively activated version of the human GPCRs disclosed herein allows for the direct identification of candidate compounds which act at this 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. 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.
• amino acid residue located 16 amino acid residues from this residue presumably located in the IC3 region of the receptor
• a lysine residue such activation may be obtained.
• Other amino acid residues may be useful in the mutation at this position to achieve this objective.
• 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.
• a search for diseases and disorders associated with the GPCR is relevant. For example, scanning both diseased and normal tissue samples for the presence of the GPCR now becomes more than an academic exercise or one which might be pursued along the path of identifying an endogenous ligand to the specific GPCR.
• Tissue scans can be conducted across a broad range of healthy and diseased tissues. Such tissue scans provide a preferred first step in associating a specific receptor with a disease and/or disorder.
• the DNA sequence of the human GPCR 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 preferably utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease.
• Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced.
• GTP [ 35 S]GTP ⁇ S, can be used to momtor enhanced binding to membranes which
• momtor G protein coupling to membranes in the absence and presence of ligand was reported by Traynor and Nahorski in 1995.
• the preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.
• G protein-coupled receptor assay i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists
• further screening to confirm that the compounds have interacted at the receptor site is preferred.
• 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.
• Gs, Gz and Gi. stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, inhibit this enzyme.
• Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of c AMP. 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 utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP).
• a candidate compound e.g., an inverse agonist to the receptor
• a variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELIS A- based format.
• Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes.
• Cyclic 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 called cAMP response elements and drives the expression of the gene.
• Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter
• the reporter protein such as ⁇ -galactosidase or luciferase can then be expressed.
• Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 , releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP ). Increased accumulation of IP is associated with activation of Gq- 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 Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of _P 3 ).
• Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-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.
• GPCR Fusion Protein The use of an endogenous, constitutively activate o ⁇ han GPCR or a non- endogenous, constitutively activated o ⁇ han GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provide an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto.
• the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist, agonist, partial 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 o ⁇ han 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. Because it is most preferred that screening take place by use of 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 o ⁇ han GPCR will continuously signal. In this regard, it is preferred that 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.
• an inverse agonist to the receptor
• 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 a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is import preferred for the screening of candidate compounds as disclosed herein.
• GPCR Fusion Protein The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator.
• the criteria of importance for such a GPCR Fusion Protein construct is 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 must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed.
• 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).
• G protein that couples to the non- endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e., a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.
• an endogenous Gi coupled receptor can be fused to a Gs protein - we believe that such a fusion construct, upon expression, "drives” or “forces” the endogenous GPCR to couple with, e.g., Gs rather than the "natural" Gi protein, such that a cyclase-based assay can be established.
• Gi, Gz and Go coupled receptors we prefer that 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 Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
• G Protein Fusion construct that utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein.
• a most preferred fusion construct can be accomplished
• G ⁇ q is deleted and the last five (5) amino acids at the C-terminal end of G ⁇ q is
• a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct".
• Gq 6 amino acid deletion
• Gi Protein resulting in a "Gq/Gi Fusion Construct”.
• this fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylgycerol, can be measured in lieu of cAMP production.
• a Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decrease 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 Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non- endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I, disclosed below), with the Gi linked GPCR.
• a signal enhancer e.g., a non- endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I, disclosed below
• constitutive activation of a Gs 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 Gi target (i.e., which decreases cAMP).
• 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., (Oslo et al., eds.).
• non-endogenous versions the human GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these versions of human GPCRs can also be utilized in research settings.
• in vitro and in vivo systems inco ⁇ orating 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.
• non-endogenous human GPCRs are useful as a research tool in that, because of their unique features, non-endogenous human GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefore is identified.
• Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.
• the disclosed human RUP8 was identified based upon the use of EST database (dbEST) information. While searching the dbEST, a cDNA clone with accession number AL121755 was identified to encode a novel GPCR.
• the following PCR primers were used for RT-PCR with human testis Marathon-Ready cDNA (Clontech) as templates: 5'-CTTGCAGACATCACCATGGCAGCC-3' (SEQ.ID .NO.:41; sense) and 5'-GTGATGCTCTGAGTACTGGACTGG-3' (SEQ.ID.NO.: 42; antisense). PCR was performed using Advantage cDNA polymerase (Clontech; manufacturing
• a 1.2kb PCR fragment was isolated and cloned into the pCRJJ-TOPO vector (Invifrogen) and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). See, SEQ.ID.NO.: 1.
• the putative amino acid sequence for RUP8 is set forth in SEQ.ID.NO. :2.
• b. hRUP9 (Seq. Id. Nos. 3 & 4)
• the disclosed human RUP9 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number ACO 11375 was identified as a human genomic sequence from chromosome 5.
• RUP9 The full length RUP9 was cloned by PCR using primers: 5'-GAAGCTGTGAAGAGTGATGC-3' (SEQ.ID.NO.:43; sense), 5'-GTCAGCAATATTGATAAGCAGCAG-3' (SEQ.ID.NO. :44; antisense) and human genomic DNA (Promega) as a template.
• Taq Plus Precision polymerase (Stratagene) was used for the amplification in a lOO ⁇ l reaction with 5% DMSO by the
• step 2 to step 4 repeated 35 times: 94°C for 1 minute; 94°C for
• a cDNA clone with accession number AC008754 was identified as a human genomic sequence from chromosome 19.
• the full length RUP10 was cloned by RT-PCR using primers: 5'-CCATGGGGAACGATTCTGTCAGCTACG-3' (SEQ.ID.NO.:45; sense) and 5'-GCTATGCCTGAAGCCAGTCTTGTG-3' (SEQ.ID.NO. :46; antisense) and human leukocyte Marathon-Ready cDNA (Clontech) as a template.
• Advantage cDNA polymerase (Clontech) was used for the amplification in a 50 ⁇ l reaction by the
• step 2 to step 4 repeated 35 times: 94°C for 30 seconds; 94°C
• Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem).
• the nucleic acid sequence of the novel human receptor RUP10 is set forth in SEQ.ID.NO.: 5 and the putative amino acid sequence thereof is set forth in SEQ.ID.NO. :6.
• the disclosed human RUP11 was identified based upon the use of GenBank database information. While searching the database, a cDNA clone with accession number AC013396 was identified as a human genomic sequence from chromosome 2.
• the full length RUPl 1 was cloned by PCR using primers: 5'-CCAGGATGTTGTGTCACCGTGGTGGC-3' (SEQ.ID.NO. :47; sense), 5'-CACAGCGCTGCAGCCCTGCAGCTGGC-3' (SEQ.ID.NO. :48; antisense) and human genomic DNA (Clontech) as a template.
• TaqPlus Precision DNA polymerase (Stratagene) was used for the amplification in a 50 ⁇ l reaction by the
• step 2 to step 4 repeated 35 times: 94°C for 3 minutes; 94°C for 20
• the nucleic acid sequence of the novel human receptor RUPl 1 is set forth in SEQ.ID.NO.: 7 and the putative amino acid sequence thereof is set forth in SEQ._D.NO.:8. e. hRUP12 (Seq. Id. Nos. 9 & 10)
• the disclosed human RUPl 2 was identified based upon the use of GenBank database.
• the disclosed human RUPl 3 was identified based upon the use of GenBank database. While searching the database, a cDNA clone with accession number ACO 11780 was identified to encode a new GPCR, having significant homology with GPCR fish GPRX-ORYLA.
• the full length RUP13 was cloned by PCR using primers: 5'-GCCTGTGACAGGAGGTACCCTGG-3' (SEQ.ID.NO. :51; sense) 5'-CATATCCCTCCGAGTGTCCAGCGGC-3' (SEQ.ID.NO.:52; antisense) and human genomic DNA (Clontech) as template.
• TaqPlus Precision DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4
• the disclosed human RUPl 4 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AL137118 was identified as a human genomic sequence from chromosome 13. The full length RUP14 was cloned by PCR using primers: 5'-GCATGGAGAGAAAATTTATGTCCTTGCAACC-3' (SEQ.ID.NO.:53; sense) 5'-CAAGAACAGGTCTCATCTAAGAGCTCC-3' (SEQ.ID.NO.:54; antisense) and human genomic DNA (Promega) as a template. Taq Plus Precision polymerase (Stratagene) and 5% DMSO were used for the amplification by the following cycle with step 2 and step 3 repeated 35 times: 94°C for 3 minute; 94°C for 20 seconds;
• a 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem) (see, SEQ.ID.NO.: 13 for nucleic acid sequence and SEQ.ID.NO.: 14 for deduced amino acid sequence).
• the sequence of RUPl 4 clones isolated from human genomic DNA matched with the sequence obtained from database. h. hRUP15 (Seq. Id. Nos. 15 & 16)
• the disclosed human RUPl 5 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC016468 was identified as a human genomic sequence.
• the full length RUPl 5 was cloned by PCR using primers:
• a 1.5 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Terminator kit (P.E.
• a 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the T7 sequenase kit (Amsham). See, SEQ.ID.NO.: 17 for nucleic acid sequence and SEQ.ID.NO.: 18 for deduced amino acid sequence.
• the sequence of RUPl 6 clones matched with four unordered segments of AL136106, indicating that the RUP 16 cDNA is composed of 4 exons.
• j. hRUP17 (Seq. Id. Nos. 19 & 20)
• the disclosed human RUP 17 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC023078 was identified as a human genomic sequence from chromosome 11. The full length RUP 17 was cloned by PCR using primers:
• a 970bp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO.: 19 for nucleic acid sequence and SEQ.ID.NO. :20 for deduced amino acid sequence. k. hRUP18 (Seq. Id. Nos. 21 & 22) The disclosed human RUP 18 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC008547 was identified as a human genomic sequence from chromosome 5. The full length RUP 18 was cloned by PCR using primers: 5'-GGAACTCGTATAGACCCAGCGTCGCTCC-3' (SEQ.ID.NO.:61; sense, 5' of the initiation codon),
• a cDNA clone with Accession Number AC026331 was identified as a human genomic sequence from chromosome 12.
• the full length RUP 19 was cloned by PCR using primers: 5'-CTGCACCCGGACACTTGCTCTG-3' (SEQ.ID.NO.:63; sense, 5' of initiation codon),
• step 2 to 4 repeated 35 times: 94°C for 1 min; 94°C for 15 sec;
• a l.lkp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :23 for nucleic acid sequence and SEQ.ID.NO. :24 for deduced amino acid sequence.
• m. hRUP20 (Seq. Id. Nos. 25 & 26)
• the disclosed human RUP20 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AL161458 was identified as a human genomic sequence from chromosome 1. The full length RUP20 was cloned by PCR using primers:
• a 1.0 kp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :25 for nucleic acid sequence and SEQ.ID.NO. :26 for deduced amino acid sequence.
• n. hRUP21 (Seq. Id. Nos. 27 & 28)
• the disclosed human RUP21 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC026756 was identified as a human genomic sequence from chromosome 13.
• RUP21 The full length RUP21 was cloned by PCR using primers: 5'- GGAGACAACCATGAATGAGCCAC -3' (SEQ._D.NO.:67; sense) 5'-TATTTCAAGGGTTGTTTGAGTAAC -3' (SEQ.ID.NO.:68; antisense) and human genomic DNA (Promega) as template.
• Taq Plus Precision polymerase (Stratagene) was used for the amplification in a lOOul reaction with 5% DMSO by the
• a 1,014 bp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :27 for nucleic acid sequence and
• the disclosed human RUP22 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC027026 was identified as a human genomic sequence from chromosome
• a 970bp PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye
• the disclosed human RUP23 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession
• RUP23 was cloned by PCR using primers: 5 '-CCTGGCGAGCCGCTAGCGCCATG-3 ' (SEQ.ID.NO.:71; sense, ATG as the initiation codon),
• a 1.0 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystem). See, SEQ.ID.NO. :31 for nucleic acid sequence and SEQ.ID.NO. :32 for deduced amino acid sequence. q. hRUP24 (Seq. Id. Nos. 33 & 34) The disclosed human RUP25 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC026331 was identified as a human genomic sequence from chromosome 12.
• RUP25 The full length RUP25 was cloned by PCR using primers: 5'-GCTGGAGCATTCACTAGGCGAG-3' (SEQ.ID.NO.:73; sense, 5 'of initiation codon), 5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.:74; antisense, 3' of stop codon) and human genomic DNA (Promega) as template.
• Advantage cDNA polymerase mix (Clontech) was used for the amplification with 5% DMSO by the following cycle with
• a 1.2kb PCR fragment was isolated from 1% agarose gel and cloned into the pCRH-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :33 for nucleic acid sequence and
• the disclosed human RUP25 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC026331 was identified as a human genomic sequence from chromosome 12. The full length RUP25 was cloned by PCR using primers: 5'-GCTGGAGCATTCACTAGGCGAG-3' (SEQ._D.NO.:75; sense, 5 'of initiation codon), 5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.: 76; antisense, 3' of stop codon) and human genomic DNA (Promega) as template. Advantage cDNA polymerase mix (Clontech) was used for the amplification with 5% DMSO by the following cycle with
• step 2 to 4 repeated 35 times: 94°C for 1 minute; 94°C for 15 seconds; 56°C for 20 seconds 72°C for 1 minute 30 seconds and 72°C for 5 minutes.
• a 1.2kb PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :35 for nucleic acid sequence and SEQ.ID.NO. :36 for deduced amino acid sequence. s. hRUP26 (Seq. Id. Nos. 37 & 38) The disclosed human RUP26 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC023040 was identified as a human genomic sequence from chromosome 2.
• RUP26 The full length RUP26 was cloned by RT-PCR using RUP26 specific primers: 5 '-AGCCATCCCTGCCAGGAAGCATGG-3 ' (SEQ.ID.NO. :77; sense, containing initiation codon) 5 '-CCAGACTGTGGACTCAAGAACTCTAGG-3 ' (SEQ.ID.NO.:78; antisense, containing stop codon) and human pancreas Marathon - Ready cDNA (Clontech) as template.
• Advantage cDNA polymerase mix (Clontech) was used for the amplification in a lOO ⁇ l reaction with 5% DMSO by the following cycle with step 2 to 4 repeated 35 times: 94°C for 5 minute;
• a l.lkb PCR fragment was isolated from 1% agarose gel and cloned into the pCRH-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :37 for nucleic acid sequence and SEQ.ID.NO. :38 for deduced amino acid sequence. t. hRUP27 (Seq. Id. Nos. 39 & 40)
• the disclosed human RUP27 was identified based upon the use of GeneBank database information. While searching the database, a cDNA clone with Accession Number AC027643 was identified as a human genomic sequence from chromosome 12. The full length RUP27 was cloned by PCR using RUP27 specific primers: 5 '-AGTCCACGAACAATGAATCCATTTCATG-3 ' (SEQ.ID.NO. :79; sense, containing initiation codon),
• a l.lkb PCR fragment was isolated from 1% agarose gel and cloned into the pCRII-TOPO vector (Invifrogen) and completely sequenced using the ABI Big Dye Termiantor Kit (P.E. Biosystem). See, SEQ.ID.NO. :35 for nucleic acid sequence and
• Preparation of non-endogenous human GPCRs may be accomplished on human GPCRs using Transformer Site-DirectedTM Mutagenesis Kit (Clontech) according to the manufacturer instructions.
• Two mutagenesis primers are utilized, most preferably a lysine mutagenesis oligonucleotide that creates the lysine mutation, and a selection marker oligonucleotide.
• the codon mutation to be inco ⁇ orated into the human GPCR is also noted, in standard form (Table D):
• Preparation of non-endogenous human GPCRs can also be accomplished by using QuikChangeTM Site-DirectedTM Mutagenesis Kit (Stratagene, according to manufacturer's instructions). Endogenous GPCR is preferably used as a template and two mutagenesis primers utilized, as well as, most preferably, a lysine mutagenesis oligonucleotide and a selection marker oligonucleotide (included in kit). For convenience, the codon mutation inco ⁇ orated into the novel human GPCR and the respective oligonucleotides are noted, in standard form (Table E):
• mammalian cells Although a variety of cells are available to the art for the expression of proteins, it is most 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 GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor- coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems - thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as that obtained from mammalian cells.
• COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan. a. Transient Transfection
• tube A was prepared by mixing 4 ⁇ g DNA (e.g., pCMV vector; pCMV vector with
• tube B was prepared by
• Stable Cell Lines Gs Fusion Protein Approximately 12x10° 293 cells are plated on a 15cm tissue culture plate. Grown in DME High Glucose Medium containing ten percent fetal bovine serum and one percent sodium pyruvate, L-glutamine, and anti-biotics. Twenty-four hours
• the cells are transfected using 12 ⁇ g
• High Glucose Medium without serum The medium is aspirated from the plates and the cells are washed once with medium without serum. The DNA, lipofectamine, and medium mixture is added to the plate along with lOmL of medium without serum.
• the medium is aspirated and 25ml of medium containing serum is added. Twenty-four hours following transfection, the medium is aspirated again, and fresh medium with serum is added. Forty-eight hours following transfection, the medium is aspirated and medium with
• serum is added containing geneticin (G418 drug) at a final concentration of 500 ⁇ g/mL.
• geneticin G418 drug
• the transfected cells now undergo selection for positively transfected cells containing the G418 resistant gene.
• the medium is replaced every four to five days as selection occurs.
• cells are grown to create stable pools, or split for stable clonal selection.
• a G protein-coupled receptor 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 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
• the assay utilizes the ability of G protein coupled receptors to stimulate
• the assay can,
• the assay is generic and has application to drug discovery at all G protein-coupled receptors.
• membrane protein e.g, 293 cells expressing the Gs Fusion
• 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 following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.
• Transfected cells were harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10ml of PBS is gently added to each dish of cells followed by careful aspiration. 1ml of Sigma cell dissociation buffer and 3ml of PBS are added to each plate. Cells were pipeted off the plate and the cell suspension was collected into a 50ml conical centrifuge tube. Cells were then centrifuged at room temperature at 1,100 ⁇ m for 5 min. The cell pellet was carefully re-suspended into an appropriate volume of PBS (about 3ml/plate). The cells were then counted using a hemocytometer and additional PBS was added to give the appropriate number of cells (with a final volume of about 50 ⁇ l/well).
• cAMP standards and Detection Buffer comprising 1 ⁇ Ci of tracer [ 125 I cAMP (50 ⁇ l] to 11 ml Detection Buffer) was prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer was prepared fresh for screening and
• Detection Mix containing tracer cAMP was then added to the wells. Plates were then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well were then extrapolated from a standard cAMP curve which was contained within each assay plate.
• TSHR is a Gs 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 Gi 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 Gi coupled receptor can be accomplished by co-transfecting, most preferably, non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP.
• TSHR-A623I non-endogenous, constitutively activated TSHR
• Gs coupled receptor an endogenous, constitutively active Gs coupled receptor
• tube A will be prepared by mixing 2 ⁇ g DNA of each receptor transfected into the
• 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
• transfection mixture lipofectamine (Gibco BRL) in 1.2ml serum free DMEM. Tubes A and B will then be admixed by inversions (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
• 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
• a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is designed for cell-based assays, however, 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 quantitated 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 fransfection. Media will be carefully aspirated off and discarded. 10ml of PBS will be gently added to each dish of cells followed by careful aspiration. 1ml of Sigma cell dissociation buffer and 3ml of PBS will be added to each plate. Cells will be pipeted off the plate and the cell suspension will be collected into a 50ml conical centrifuge tube. Cells will then be centrifuged at room temperature at 1,100 ⁇ m 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, 3ul of test compound (12uM 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-11 and H12. 50ul of Stimulation Buffer will be added to all wells. Selected compounds (e.g., TSH) will be added to appropriate 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. lOO ⁇ l of Detection Mix containing tracer cAMP will then be added to the wells. Plates were then 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
• 293 and 293T cells are plated-out on 96 well plates at a density of 2 x 10 4 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions.
• a DNA/lipid mixture is prepared for each 6- well transfection as follows: 260ng of plasmid DNA in lOO ⁇ l of DMEM were 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 pCMV alone, and lOng of a GPRS expression plasmid (GPRS in pcDNA3 (Invifrogen)).
• the 8XCRE-Luc reporter plasmid was
• vector SRIF- ⁇ -gal was obtained by cloning the rat somatostatin
• the 8xCRE-Luc reporter plasmid was generated by replacing the
• Gq-associated receptors A method to detect Gq stimulation depends on the known property of Gq- 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.
• Gq- associated receptors One method to detect Gq stimulation depends on the known property of Gq- 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 Gq 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
• cells comprising the receptors can be plated onto 24 well plates, usually lxlO 5 cells/well (although his umber can be optimized.
• cells can be transfected by firstly mixing 0.25 ⁇ g DNA in 50 ⁇ l serum free DMEM/well and 2 ⁇ l lipofectamine in 50 ⁇ l serumfree DMEM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are washed with 0.5 ml PBS and 400 ⁇ l of serum free media is mixed with the fransfection
• 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) is added/well with 0.25 ⁇ Ci of 3 H-myo-inositol/ well and the cells
• the column is washed with 10 mis 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 ammomum formate.
• the columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammomum formate and rinsed twice with dd H 2 O and stored at 4°C in water.
• the Gs ⁇ protein vector contains a variety of well-known
• a RUP13-Gs ⁇ Fusion Protein construct was made as follows: primers were
• the sense and anti-sense primers included the restriction sites for Xbal and EcoRV, respectively, such that spacers
• Reaction temperatures and cycle times for RUP 15 were 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
• a RUP15-Gs ⁇ Fusion Protein construct was made as follows: primers were
• Nucleotides in lower caps are included as spacers in the restriction sites between the G protein and RUPl 5.
• the sense and anti-sense primers included the restriction sites for EcoRV and Xbal, respectively, such that spacers (attributed to the restriction sites) exists between the G protein and RUPl 5. PCR was then utilized to secure the respective receptor sequences for fusion
• Reaction temperatures and cycle times for RUP 15 were 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
• the design of a Gq (del)/Gi fusion construct can be accomplished as follows: the N-terminal six (6) amino acids (amino acids 2 through 7, having the sequence of
• Plasmid 63313 which contains the mouse G ⁇ q- wild type version with a
• PCR product will be cloned into a pCRII-TOPO vector (Invifrogen) and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystem). Inserts from a TOPO clone containing the sequence of the fusion construct will be shuttled into the expression vector pcDNA3.1(+) at the Hindlll/BamHI site by a 2 step cloning process.
• RT-PCR was applied to confirm the expression and to determine 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. 20 ⁇ l of the reaction will be loaded on a 1.5% agarose gel to analyze the RT-PCR products. Table J below lists the receptors, the cycle conditions and the primers utizilized.
• Membranes comprising the constitutively active orphan GPCR Fusion Protein of interest and for use in the direct identification of candidate compounds as inverse agomsts, agonists or partial 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 m for 17 minutes at 4°C). Thereafter, the supernatant will be obtained.
• Membrane Protein The Protein concentration of the membranes will be determined 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
• Duplicate tubes will be prepared, one including the membrane, and one as a control "blank". Each contained 800ul Binding Buffer. Thereafter, lO ⁇ l of Bradford Protein Standard (lmg/ml) will be added to each tube, and lO ⁇ l of membrane Protein will then be added to just one tube (not the blank). Thereafter, 200ul of Bradford Dye Reagent will be added to each tube, followed by vortex of each. After five (5) minutes, the tubes will be re-vortexed and the material therein will be transferred to cuvettes. The cuvettes will then be read using a CECIL 3041 spectrophotometer, at wavelength 595.
• GDP Buffer consisted of 37.5 ml Binding Buffer and 2mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 ⁇ M GDP (final concenfration of GDP in each well was 0.1 ⁇ M GDP); each well comprising a candidate compound, has a final volume of 200ul consisting of lOO ⁇ l GDP Buffer (final concenfration, 0.1 ⁇ M GDP), 50ul Membrane Protein in Binding Buffer, and 50 ⁇ l
• Candidate compounds will be preferably screened using a 96-well plate format
• the 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 should be rinsed in three reservoirs comprising water (IX), ethanol (IX) and water (2X) - excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes.
• 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. Thereafter, 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 then be stopped by spinning of the plates at 4000
• Another assay approach to directly identified candidate compound was accomplished by utilizing a cyclase-based assay. In addition to direct identification, this assay approach can be utilized as an independent approach to provide confirmation of
• a modified Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) was preferably utilized for direct identification of candidate compounds as inverse agonists and agonists to constitutively activated orphan GPCRs in accordance with the following protocol.
• Transfected cells were harvested approximately three days after transfection.
• Membranes were prepared by homogenization of suspended cells in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl 2 . Homogenization was performed on ice using a Brinkman PolyfronTM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 X g for 15 minutes at 4°C. The resulting pellet was then 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 was then stored at -80°C until utilized.
• the membrane pellet On the day of direct identification screening, the membrane pellet as slowly thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCL2, to yield a final protein concenfration of 0.60mg/ml (the resuspended membranes are 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) were prepared and maintained in accordance with the manufacturer's instructions.
• Assay Buffer was prepared fresh for screening and contained 20mM HEPES, pH 7.4, lOmM MgCl 2 , 20mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 ⁇ M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer was then stored on ice until utilized.
• Candidate compounds identified as per above if frozen, thawed at room temperature) were 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. This admixture was then incubated for 30 minutes at room temperature, with gentle shaking.
• FIG. 12 A representative screening assay plate (96 well format) result is presented in Figure 12. Each bar represents the results for a different compound in each well, plus RUP13-Gs ⁇ Fusion Protein construct, as prepared in Example 5(a) above.
• representative results presented in Figure 12 also provide standard deviations based upon the mean results of each plate ("m") and the mean plus two arbifrary preference for selection of inverse agonists as "leads" from the primary screen involves selection of candidate compounds that that reduce the per cent response by at least the mean plate response, minus two standard deviations.
• an arbitrary preference for selection of an agonists as "leads" from the primary screen involves selection of candidate compounds that increase the per cent response by at least the mean plate response, plus the two standard deviations.
• the candidate compounds in the following wells were directly identified as putative inverse agonist (Compound A) and agonist (Compound B) to RUP 13 in wells A2 and G9, respectively. See, Figure 12.
• the vector utilized be pCMV.
• This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Boulevard., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Pmpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #203351. //

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