WO1998016557A1 - Dosages pour recepteurs lies a la proteine g - Google Patents

Dosages pour recepteurs lies a la proteine g Download PDF

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WO1998016557A1
WO1998016557A1 PCT/US1996/020510 US9620510W WO9816557A1 WO 1998016557 A1 WO1998016557 A1 WO 1998016557A1 US 9620510 W US9620510 W US 9620510W WO 9816557 A1 WO9816557 A1 WO 9816557A1
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receptor
subunit
cell
amino acid
seq
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Ikuo Nishimoto
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The General Hospital Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4722—G-proteins
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4719—G-proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the field of the invention is GTP-binding proteins and the receptors to which they link.
  • G proteins are the best characterized and the most versatile. They elicit biological functions which include hormone signalling, neurotransmission, chemotaxis, and perception of light, smell, and taste. G proteins couple to various cell surface receptors (G-linked receptors) and activate various intracellular effectors. Each G protein is made up of a G ⁇ subunit and a G ⁇ subunit. The specificity of G proteins' coupling to receptors and downstream signalling molecules is conferred by the various G ⁇ subunits.
  • the G ⁇ molecules are classified into two categories: one is a class of sensory-organ-specific G proteins (e.g., G ⁇ t , G ⁇ olf , and G ⁇ gust ) , and the other is a less tissue-specific class consisting of G ⁇ g , the G family (i.e., G ⁇ i;L , G ⁇ i2 , G ⁇ i3 , Ga ol , G ⁇ o2 , and G ⁇ 2 ) , the G ⁇ 12 family (i.e., G ⁇ 12 and G ⁇ 13 ) , and the G ⁇ q family (i.e, G ⁇ cron, G ⁇ ⁇ , G ⁇ 14 , and G ⁇ 16 ) . It is likely that more members of each class will be discovered.
  • G ⁇ g the G family
  • the G family i.e., G ⁇ i;L , G ⁇ i2 , G ⁇ i3 , Ga ol , G ⁇ o2 , and G ⁇ 2
  • G ⁇ s -based chimeric system for identifying the G ⁇ subunit of a G protein to which a given G-linked receptor couples.
  • a series of G ⁇ s / ⁇ ⁇ chimeras (G ⁇ ⁇ : any G ⁇ subunit except G ⁇ g ) can be made with a first amino acid sequence corresponding to a region of G ⁇ s (SEQ ID NO: 21) encompassing G g 's residues 236-356, followed by a second amino acid sequence 4-30 amino acids long and corresponding to a segment of G ⁇ x , which segment ends at (and includes) G ⁇ ⁇ 's C-terminal residue.
  • the first amino acid sequence should contain the effector portion of G ⁇ s , and preferably will contain residues 1-389 of SEQ ID NO: 21.
  • the second amino acid sequence should contain the receptor-coupling portion of G ⁇ ⁇ , and preferably is 4 or 5 amino acids in length (e.g., as represented by SEQ ID NOs:22-30).
  • two identical samples of cells are provided, wherein the cells co-express a given G-linked receptor and a given G ⁇ s / ⁇ ⁇ chimera.
  • the second sample of cells is contacted with a ligand of the G- linked receptor.
  • AC activity as anisfested by the rate of cAMP formation, is measured in both samples of cells.
  • a significant increase in cAMP formation in the second sample as compared to the first sample indicates that that particular G ⁇ ⁇ can couple to the receptor.
  • Cells co-expressing a given G-linked receptor and a given G ⁇ g / ⁇ ⁇ chimera can be established by introducing into the cells a recombinant nucleic acid construct permitting expression of the receptor and a second recombinant nucleic acid construct permitting expression of the chimera.
  • recombinant is meant that the nucleic acid (or polypeptide) molecule is the product of artificial genetic manipulation.
  • a G ⁇ s / ⁇ ⁇ chimera is a polypeptide which includes the AC-coupling portion (e.g., amino acid residues 236-356) of G ⁇ s (SEQ ID NO: 21) as well as the receptor-coupling portion of G ⁇ x .
  • the receptor-coupling portion can be 4-30 amino acids long and usually corresponds to the extreme C-terminal region of G ⁇ ⁇ .
  • the chimeric polypeptide can also include an additional peptide sequence such as one that serves as an epitope tag, so long as the additional sequence does not interfere with the functioning of the chimera.
  • G- linked receptor is meant any naturally occurring cell surface receptor, or any functional recombinant variant thereof, that couples to a G protein.
  • ligand is meant any molecule that binds and activates a receptor.
  • a ligand can be, for example, the natural, physiological activator of the receptor (e.g., a hormone), a biologically active analogue thereof, or an antibody which binds to and thereby activates the receptor.
  • the chimeras of the invention can also be used in a method of screening compounds for their ability to modulate the interaction between a given G-linked receptor and the G ⁇ (i.e., G ⁇ ⁇ ) subunit of a non-G s G protein known to couple to the receptor.
  • G ⁇ i.e., G ⁇ ⁇
  • two identical samples of cells are provided, wherein the cells co-express the G-linked receptor and a G ⁇ g / ⁇ ⁇ chimera. Both samples of cells are contacted with a ligand of the G-linked receptor. The second sample is additionally contacted with a test compound. cAMP formation is then measured in both samples.
  • a significant decrease (or increase) of the cAMP level in the second sample as compared to the first sample indicates that the compound is capable of inhibiting (or enhancing) the interaction between the G-linked receptor and that particular G ⁇ ⁇ .
  • signal-transducing output is meant the end result of the signalling initiated by a liganded G-linked receptor. Such an end result can be, for example, cell growth inhibition, cell proliferation, or secretion of a protein.
  • G ⁇ chimeric polypeptide containing the sequence of a G ⁇ linking to a desirable effector, the receptor-coupling region (e.g., the 4-30 residues at the C-terminal end) of which sequence is replaced with that of a G ⁇ to which the G-linked receptor normally couples.
  • the receptor-coupling region e.g., the 4-30 residues at the C-terminal end
  • Such a chimeric polypeptide can be employed in a method of therapy for a condition associated with the function or lack of function of that receptor in a patient's cells.
  • the G ⁇ s / ⁇ Q polypeptide can be introduced into the target cell by introducing into the cell a recombinant nucleic acid construct that permits expression of the chimeric polypeptide.
  • a construct can, for instance, be derived from a herpes simplex viral vector, or any other vector able to transfect neural cells.
  • SST somatostatin
  • SSTR SST receptors
  • Somatostatin is a 14 amino acid cyclic peptide hormone which was originally isolated from the hypothalamus.
  • Biologically active analogues of SST include, but are not limited to, (1) naturally occurring analogues, such as SST-28 (FEBS Lett. 282: 363-367, 1991) and SST-25 (Gen CompEndocinol 81: 365-372, 1991); and (2) artificial compounds, such as octreotide (New Engl. J. Med. 334: 246-254, 1995), RC-160 (Buscail et al., PNAS 92: 1580-1584, 1995), RC-160-I and RC-160-II (Cancer Res. 54: 5895-5901, 1994), SMS 201-995 (Kubota et al., J.
  • Another method of inhibiting tumor growth is useful for tumor cells the growth of which is stimulated via an endogenous, hyperactive G-linked receptor.
  • endogenous is meant that the receptor is expressed in the cell absent any artificial genetic manipulation.
  • hyperactive is meant that the G-linked receptor is more active, or active for a longer period of time, than it is in a normal cell. Hyperactivity of a G-linked receptor can be caused by, for example, certain mutations in the receptor's peptide sequence, an unusually high level of the receptor's ligand, and/or a ligand that dissociates from the receptor at a rate lower than normal.
  • One can then introduce into the tumor cell a G ⁇ 12 or G 13 chimeric molecule, the C-terminal 5 residues of which are replaced with those of the G ⁇ that the hyperactive receptor normally couples to.
  • the hyperactivity of the receptor is transduced via the G ⁇ 12 or G ⁇ 13 chimeric molecule to downstream growth-inhibitory effectors, which counteract at least in part growth-stimulatory signals normally transduced by the receptor and its cognate G protein.
  • the chimera will transduce a signal that results in apoptosis of the tumor cell.
  • the chimeric molecule can be introduced into the target cell in vivo , in vitro , or ex vivo in a carrier such as saline and/or liposomes. It can also be expressed by a recombinant nucleic acid construct that has been introduced into the cell.
  • Fig. 1 is a schematic representation of the G ⁇ s chimeras constructed in the study.
  • G ⁇ s wt denotes wild-type G ⁇ s .
  • Sequences of the last 5 C-terminal residues of the chimeras are illustrated, and referred to as SEQ ID NOs : 22-31. These sequences are identical between G ⁇ i:L and G ⁇ i2 , between G ⁇ ol and G ⁇ o2 , and between G ⁇ q and G ⁇ ⁇ .
  • Fig. 2A is a bar graph showing the effects of SST on cholera toxin (CTX) -stimulated AC activity in cells expressing SSTR3.
  • CCTX cholera toxin
  • Cells were transfected with 0.125 ⁇ g of pCMV6-SSTR3 and 0.125 ⁇ g of pCMV6 vector.
  • cells were treated for 30 min with or without 1 ⁇ M SST, in the presence of (1) 1 mM IBMX, or (2) lmM IBMX plus 250 ng/ml CTX.
  • cAMP formation was subsequently measured. All values are "means ⁇ S.E.” of quadruplicated experiments.
  • Fig. 2B & Fig. 2C are bar graphs showing the effects of SST on cAMP formation in cells expressing a G ⁇ s chimera with (Fig. 2C) or without (Fig. 2B) SSTR3.
  • Cells were transfected with 0.125 ⁇ g of plasmid encoding a G ⁇ g chimera and 0.125 ⁇ g of either pCMV6-SSTR3 (Fig. 2C) or pCMV6 (Fig. 2B) .
  • Fig. 2C pCMV6-SSTR3
  • Fig. 2B pCMV6
  • AC activity levels are represented as percentage relative to the basal AC activity level in cells expressing G ⁇ g / ⁇ i;L alone. All values are "means ⁇ S.E.” of quadruplicated experiments. Similar results were found at least three times for each chimera.
  • Fig. 2D is a bar graph converted from Fig. 2B, showing the ratios of cAMP levels in the presence vs. absence of SST in transfected cells.
  • Fig. 3A & Fig. 3B are bar graphs showing the effects of SST on AC activity in cells expressing a Ga s chimera with (Fig. 3A) or without (Fig. 3B) SSTR3.
  • Cells were transfected with 0.125 ⁇ g of plasmid encoding a G ⁇ chimera and either 0.125 ⁇ g of pCMV6-SSTR3 (Fig. 3A) or pCMV6 (Fig. 3B) .
  • AC activity levels are represented as percentage relative to the basal AC activity level in cells expressing G ⁇ g / ⁇ q alone. All values are "means ⁇ S.E.” of quadruplicated experiments. Similar results were found at least three times for each chimera.
  • Fig. 3C is a bar graph showing the effects of SST on cAMP formation in cells expressing SSTR3 and a G ⁇ s chimera derived from the G ⁇ i or G family. All the indicated chimeras were tested in parallel. Experiments were performed as described in the legend for Figs. 2A and 2B. All values are "means ⁇ S.E.” of quadruplicated experiments. Similar results were found at least three times for each chimera.
  • Fig. 3D is a bar graph converted from Fig. 3C, showing the ratios of cAMP levels in the presence vs. absence of SST in transfected cells.
  • Fig. 4A is a bar graph showing the stimulation of inositol phosphate (IP) production in cells transfected with (1) 0.125 ⁇ g of pCMV6-SSTR3, and (2) 0.125 ⁇ g of plasmid encoding the intact G ⁇ 16 .
  • IP inositol phosphate
  • cells were treated for 5 min with or without 1 ⁇ M SST, and IP production was measured.
  • PTX pertussis toxin
  • cells were treated with 10 ng/ml PTX for 3 h and with SST as described above.
  • Fig. 4B is a bar graph showing the stimulation of IP production in cells transfected with (1) 0.125 ⁇ g of pCMV6-SSTR3, and (2) 0.125 ⁇ g of plasmid encoding intact G ⁇ 14 . Experiments were performed as described in Fig. 4A's legend.
  • Fig. 4C is a bar graph showing the stimulation of IP production in cells transfected with (1) 0.125 ⁇ g of pCMV6-SSTR3, and (2) 0.125 ⁇ g of plasmid encoding intact G ⁇ . Experiments were performed as described in Fig. 4A's legend.
  • Fig. 4D is a bar graph showing the stimulation of IP production in cells transfected with (1) 0.125 ⁇ g of plasmid encoding parathyroid hormone receptor (PTHR) , and (2) 0.125 ⁇ g of plasmid encoding intact G ⁇ . Experiments were performed as described in Fig. 4A's legend.
  • PTHR parathyroid hormone receptor
  • Fig. 5A is a bar graph showing the effects of SST on AC activity in cells expressing a SSTR and G ⁇ g / ⁇ 12 .
  • Cells were transfected with (1) 0.125 ⁇ g of plasmid encoding G ⁇ s / ⁇ 12 , and (2) 0.125 ⁇ g of pCMV6-SSTRl, pCMV6- SSTR2, pCMV6-SSTR3, pCMV6-SSTR5, pCDNAI-SSTR4 , or pCMV6.
  • cells were stimulated with 1 ⁇ M SST and cAMP formation was measured.
  • Fig. 5B is a bar graph converted from Fig. 5A, showing the ratios of cAMP levels in the presence vs. absence of SST.
  • Fig. 5C is a bar graph showing the effects of SST on AC activity in cells expressing a SSTR and G ⁇ g / ⁇ 13 .
  • Cells were transfected with (1) 0.125 ⁇ g of plasmid encoding G ⁇ g / ⁇ 13 , and (2) 0.125 ⁇ g of pCMV6-SSTRl, pCMV6- SSTR2, pCMV6-SSTR3, pCMV6-SSTR5, pCDNAI-SSTR4 , or pCMV6.
  • cells were stimulated with 1 ⁇ M SST and cAMP formation was measured.
  • Fig. 5D is a bar graph converted from Fig. 5C, showing the ratios of cAMP levels in the presence vs. absence of SST.
  • G ⁇ subtype coupling can be assigned for any given G-linked receptor.
  • the following examples are meant to illustrate, but not limit, the methods of the present invention.
  • Other suitable modifications and adaptations of the conditions which are obvious to those skilled in the art are within the scope and spirit of the invention.
  • genetically engineered variants of G-linked receptors can be substituted for the naturally occurring receptors.
  • Standard transfection techniques other than the lipofection technique illustrated below, e.g., calcium phosphate precipitation, biolistic transfer, DEAE- Dextran, and viral-vector methods, can also be employed in the invention.
  • COS cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and antibiotics, as described previously (Ikezu et al., J. Biol. Chem. 270: 29224-29228, 1995) Transient transfection was performed by lipofection as previously described (Ikezu et al. , J. Biol. Chem. 270: 29224-29228, 1995) .
  • 2xl0 4 cells were seeded onto a 24-well plate and cultured in complete growth medium for 24 h.
  • the cells were subsequently transfected with 0.25 ⁇ g of plasmid and 1 ⁇ l LipofectAMINETM (GIBCO-BRL) for another 24 h in serum-free DMEM, and cultured in complete growth medium for an additional 24 h. Measurement of AC activity
  • Intact-cell AC activity was assessed by measuring cAMP formation as described previously (Ikezu et al., J. Biol. Chem. 270: 29224-29228, 1995).
  • cells were labeled with 6 ⁇ Ci/ l of [ 3 H]adenine (Du Pont-NEN) for 24 h, and then treated with ligands of the G-linked receptor of interest (e.g. , somatostatin-14 for a somatostatin receptor) and 1 mM IBMX (3-isobutyl-l-methylxanthine) for 30 min.
  • the resultant radioactive cAMP was separated on two-step ion-exchange columns. Specific accumulation of cAMP was expressed as [cAMP/(ADP + ATP)] x 10 3 , which represents intact-cell AC activity.
  • Statistical analysis was performed with Student's t test. Measurement of PI Turnover
  • PI phosphatidyl inositol turnover was assessed by measuring IP (inositol phosphates) production.
  • IP inositol phosphates
  • 4xl0 4 cells were seeded onto a 24-well plate, cultured in complete growth medium for 24 h, and transfected for 24 h as described above. The culture medium was replaced with the labeling medium [inositol-free RPMI supplemented with dialyzed fetal calf serum and 10 ⁇ Ci/ml of [ 3 H]myo-inositol (Du Pont-NEN)].
  • the cells were washed four times with inositol-free RPMI and treated with 1 ⁇ l somatostatin (SST) in inositol-free RPMI at 37 °C for 5 min. After discarding the medium, the cells in 0.2 ml fresh medium were lysed on the plate by 0.8 ml of ice-cold 12.5% (final concentration: 10%) TCA, and the lysate was put on ice for 20 min before centrifugation. Supernatant of the lysate was mixed well with 1 ml of saturated diethyl ether to extract acid.
  • SST somatostatin
  • constructs (designated pCMV6-SSTRl, pCMV6-SSTR2, pCMV6-SSTR3, and pCMV6-SSTR5) , all of which were derived from a pCMV6 vector (the SSTRl and 2 constructs: pCMV6b; the SSTR3 and 5 constructs: pCMV6c) , contain the SSTR coding sequences under the transcriptional control of the cytomegalovirus promoter.
  • the SSTR4 expression construct (pcDNAI-SSTR4) was made by inserting the SSTR4 cDNA (Bito et al. , J. Biol. Chem. 269: 12722-12730, 1994) in pBluescript (Strategene) into pcDNAI (Invitrogen) .
  • the G ⁇ s chimeras were constructed as follows. First, PCR was performed to add Aflll and Xbal sites at the 3' end of the wild type G ⁇ g cDNA using the following two primers: ATCTGGAATAACAGATGGCTGC (SEQ ID N0:1) and
  • the PCR product was digested with Bglll and Xbal, and subcloned into pcDNAI-G ⁇ s (i.e., the original plasmid containing the wild type G ⁇ s cDNA) which had been predigested with the same enzymes .
  • the resultant construct designated G ⁇ g -AX, was sequenced to confirm the presence of Aflll and Xbal sites. Subsequently, the construct was digested with Aflll and Xbal, and ligated with two synthetic oligonucleotides to add sequence encoding the carboxyl-terminal five residues of a non-G ⁇ g subunit.
  • the oligonucleotides were:
  • CTAGATTAACACAAACCGATGTATC SEQ ID NO: 10 (for G ⁇ s / ⁇ z ) ;
  • G ⁇ g Stimulation of G ⁇ g , but not any other G ⁇ , results in an increase in adenylyl cyclase (AC) activity.
  • AC adenylyl cyclase
  • G ⁇ g / ⁇ x G ⁇ ⁇ : any type of G ⁇ except G ⁇ g ) chimeras wherein the last five C-terminal residues of the G ⁇ s polypeptide are replaced with those of G ⁇ x . If a receptor couples to G ⁇ x , it will, upon binding to its ligand, recognize and activate the G ⁇ g / ⁇ ⁇ chimera, thereby resulting in G ⁇ s -mediated AC stimulation in the cell.
  • G ⁇ g / ⁇ x chimeras consisting of G ⁇ g 1-389 (which lacks the original five C-terminal residues of G ⁇ g ) and the five C-terminal residues of each known G ⁇ were constructed.
  • the five C-terminal residues are identical between G ⁇ i:L and G ⁇ i2 , between G ⁇ Ql and G ⁇ o2 , and between G ⁇ q and G ⁇ ⁇ ;L .
  • G ⁇ s / ⁇ i;L G ⁇ s / ⁇ i3 , G ⁇ g / ⁇ 0 , G ⁇ g / ⁇ z , G ⁇ B / ⁇ q , G ⁇ s / ⁇ 12 , G ⁇ g / ⁇ 13 , G ⁇ g / ⁇ 14 , and G ⁇ s / ⁇ 16 , respectively (Fig. 1).
  • the residues 1-389 (SEQ ID N0:21) of G ⁇ g are the following:
  • the experimental strategy was to transiently express a G ⁇ g / ⁇ ⁇ cDNA along with a SSTR cDNA, and then to compare AC activities in the presence and absence of SST. If treatment with SST promotes cAMP formation only in cells expressing the SSTR and a given G ⁇ g / ⁇ ⁇ , one can assume the linkage of the SSTR to that G ⁇ g / ⁇ ⁇ and therefore to that G ⁇ ⁇ .
  • the G ⁇ g chimeras were each expressed as a 52-kDa protein at similar levels in COS cells, consistent with expected molecular weight.
  • G ⁇ s / ⁇ ⁇ chimeras where G ⁇ ⁇ is derived from the G ⁇ i family (G ⁇ G ⁇ 0 , and G ⁇ z ) were tested for their ability to transduce AC-stimulatory signal initiated by SST-bound SSTR3.
  • SSTR3 has been shown to function as a G ⁇ coupled receptor and to suppress AC activity in various cell types (Yasuda et al., J. Biol. Chem. 267: 20422-20428, 1992; Ya ada et al., Mol. Endocrinol. 6: 2136-2142, 1992; Kaupmann et al., FEBS Lett.
  • G ⁇ proteins known to inhibit AC are members of the G ⁇ j ⁇ family, which include the Ga ' s (i.e., G ⁇ ilf G ⁇ i2 , G ⁇ i3 ) , the G ⁇ 0 's (i.e., G ⁇ ol and G ⁇ o2 ) , and Ga z (Wong et al., Nature 351: 63-65, 1991), the present data suggest that SSTR3 may inhibit cAMP formation exclusively through the G ⁇ i 's.
  • Fig. 2A Since SST significantly reduced cAMP formation when SSTR3 was transfected without G ⁇ g / ⁇ 14 or G ⁇ g / ⁇ 16 (Fig. 2A) , it is conceivable that the net stimulation of AC by SSTR3 through these two chimeras may have been considerably larger than what was observed. In contrast, in cells co- expressing SSTR3 and G ⁇ ./ ⁇ , no stimulation of AC was observed under the same conditions.
  • Figs. 3C and 3D show results of the experiments wherein the linkage of SSTR3 to chimeras derived from the G ⁇ and G ⁇ families were examined in parallel. Again, the results demonstrated that SSTR3 may link to Ga 14 and G ⁇ 16 , in addition to the G ⁇ i's, but not to any other members of the G ⁇ i and G ⁇ families .
  • inability of a G-linked receptor to couple to a given G ⁇ s / ⁇ ⁇ indicates the inability of the receptor to recognize the C terminus of that particular G ⁇ x , and therefore rules out the coupling between the receptor and the intact G ⁇ ⁇ .
  • the present study suggests for the first time that SSTR3 may not couple to G ⁇ Q , G ⁇ z , G ⁇ q , G ⁇ 11; G ⁇ 12 , or Ga 13 (see below for G ⁇ 12 and G ⁇ 13 ) .
  • ability of a G- linked receptor to couple to a given G ⁇ s / ⁇ ⁇ indicates that the receptor is capable of coupling to that particular G ⁇ ⁇ .
  • the present chimeric system is extremely useful in identifying potential receptor-G ⁇ linkage, especially for G ⁇ 's which have less established signal-transducing effectors and which therefore are less amenable to assaying.
  • the present system can be employed to identify G ⁇ 12 - or G ⁇ 13 -coupled receptors.
  • G ⁇ 12 and G ⁇ 13 have been implicated in pivotal cellular functions (Voyno-Yasenetskaya et al., Oncogene 9: 2559-2565, 1994 and Voyno-Yasenetskaya et al., J. Biol. Chem. 269: 4721-4724, 1994), receptors to which they couple remain elusive.
  • G ⁇ 12 and G ⁇ 13 couple to any of the 5 known subtypes of SSTR's
  • SST-induced cAMP formation was measured in cells co-expressing a given SSTR and either G ⁇ g /G ⁇ 12 or G ⁇ s / GQ: 13 - F gs.
  • 5A-5D shows that G ⁇ s /G ⁇ 12 was activated by SSTR2 , 4, and 5 in the order of SSTR5 >> SSTR2 ⁇ SSTR4 , while G ⁇ g /G ⁇ 13 was activated almost exclusively by SSTR5.
  • the stimulation of G ⁇ g /G ⁇ 12 and G ⁇ s /G ⁇ 13 by liganded SSTR5 yielded a more than 5 fold increase in the cAMP level (Figs. 5B and 5D) .
  • Table I shows that, in the presence of G ⁇ s / ⁇ 12 , the stimulation of cAMP formation by SSTR5 and SSTR2 is SST-dosage-dependent and biphasic. At low SST concentrations, cAMP formation was slightly but reproducibly inhibited, whereas at higher concentrations, cAMP formation was strongly stimulated. It is conceivable that the former effect may be mediated by
  • SST-28 had a more potent effect on the function of SSTR5 than the naturally occurring SST (i.e., SST-14) , regarding both of their inhibitory and stimulatory effects.
  • the present system can also be used to investigate proteins with a G-linked-receptor-like structure (e.g., with multiple transmembrane domains) but having unknown functions.
  • the system can also be used to investigate proteins which have only a single-transmembrane domain but which are suspected of being a G-linked receptor.
  • insulin-like growth factor II receptor Murayama et al., J. Biol. Chem. 265: 17456- 17462, 1990
  • amyloid precursor protein APP
  • sperm ⁇ - 1, 4-galactosyltransferase Gong et al., Science, 269: 1718-1721, 1995.
  • epidermal growth factor receptor Sun et al., Proc. Natl. Acad. Sci.
  • One aspect of the present invention is a method of identifying a compound that can modulate the interaction between a G-linked receptor and the G ⁇ subunit of a non- G s G protein known to couple to the receptor.
  • two samples of cells are provided, both of which express (a) the receptor of interest, and (b) a chimeric polypeptide containing amino acid residues 1-389 of G ⁇ g (SEQ ID NO: 21) followed by the C-terminal 5 amino acid residues of the non-G ⁇ s G ⁇ subunit known to couple to the receptor.
  • a ligand of the G-linked receptor is administered to both cell samples.
  • the second cell Prior to, subsequent to, or at the same time as the ligand administration, the second cell is contacted with a candidate compound.
  • the activity of adenylyl cyclase in each cell sample is determined and compared as described above.
  • a statistically significant (i.e., p ⁇ 0.05 in Student's t test) change of the AC activity in the second cell sample as compared to the first cell sample indicates that the compound may be capable of modulating the interaction between the G-linked receptor and the coupling G ⁇ subunit.
  • a statistically significant decrease of the AC activity in the compound-contacted cells will suggest that the compound may block the interaction.
  • the efficacy of the compound can be confirmed by a second assay using the full length G ⁇ subunit instead of the chimera.
  • Cell lines that can be used in connection with this method include those of mammalian origin, such as COS cells and HEK 293 cells (American Type Culture Collection) . Maintenance and transfection of such cells can be performed using well known methods. Proteins (a) and (b) (see above) can be introduced into the target cells via transfection of nucleic acid constructs encoding them. Techniques for making nucleic acid constructs are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) ; examples of such techniques have been illustrated above.
  • G-linked receptors of interest include, but are not limited to, those described in U.S. Patent No.5, 559, 209 , herein incorporated by reference (e.g., insulin-like growth factor II receptor, muscarinic acetylcholine receptor, ⁇ 2 -adrenergic receptor, adenosine receptor, thrombin receptor, transforming growth factor ⁇ receptor, T cell receptor, PTH/PTHrP receptor, calcitonin receptor, endothelin receptor, angiotensin receptor, platelet activating factor receptor, thromboxane A 2 receptor, any of the somatostatin receptors, D 2 -dopamine receptor, ⁇ -butyric acid receptor) , and amyloid protein precursor (APP) .
  • insulin-like growth factor II receptor e.g., insulin-like growth factor II receptor, muscarinic acetylcholine receptor, ⁇ 2 -adrenergic receptor, adenosine receptor, thro
  • APP has at least 10 isoforms, one of which (APP 695 ) is preferentially expressed in neuronal tissue (Sandbrink et al., J. Biol. Chem. 269: 1510, 1994).
  • the construction of a baculovirus construct containing the APP 695 cDNA has been described (Nishimoto et al., Nature 362: 75-79, 1993) .
  • Similar cloning techniques can be employed to create APP 695 mammalian expression constructs based on mammalian expression vectors such as pCDNAI and pCMV6.
  • Constitutively active variants of the G-linked receptors can also be used in the present screening method, eliminating the need for their ligands.
  • three constitutively active APP 695 mutants designated Ile-APP, Phe-APP, and Gly-APP, have been identified in familial Alzheimer's Disease patients (Ya atsuji et al., Science 272: 1349-1352, 1996; and references therein) .
  • These three mutants have mis-sense mutations in which Val 642 in the transmembrane domain of APP 695 is replaced by lie, Phe, or Gly, respectively.
  • the chimeras of the invention can alternatively be used in a method of altering the signal-transducing output of a G-linked receptor.
  • Abnormalities of G-linked receptor functions have been implicated in many significant diseases such as familial Alzheimer's disease (Nishimoto et al. , Nature 362: 75-79, 1993; Yamatsuji et al., Science 272: 1349-1352, 1996; Okamoto et al. , The EMBO J. 15: 3769-3777, 1996; Ikezu et al., The EMBO J.
  • Amyloid protein precursor (APP) , a G-linked cell surface receptor, has been shown to be mutated and constitutively active in at least some forms of familial Alzheimer's Disease (Okamoto et al., The EMBO J. 15: 3769-3777, 1996; and references therein). APP is known to couple to G Q , the activation of which inhibits adenylyl cyclase (Okamoto et al., The EMBO J. 15: 3769- 3777, 1996 and references therein) .
  • the present invention provides a method for augmenting adenylyl cyclase activity in brain neurons of a mammal, and preferably, of a familial Alzheimer's patient.
  • a G ⁇ s subunit in which the C- terminal 5 aa residues are replaced with those of G ⁇ Q is introduced into the brain neurons of the mammal.
  • This chimeric G ⁇ molecule will compete with the endogenous G ⁇ 0 for the binding of APP, and upon binding to APP, will transduce stimulatory signals to adenylyl cyclase, thereby counteracting the inhibitory signals transduced by native G 0 .
  • This chimeric molecule can be introduced into the target cell by overexpressing within the target cell a nucleic acid construct comprising a promoter sequence operably linked to a sequence encoding the protein.
  • the nucleic acid construct is typically derived from a non-replicating linear or circular DNA or RNA vector, or from an autonomously replicating plasmid or viral vector; or the construct is integrated into the host genome.
  • These nucleic acid constructs can be made with methods well known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989) . Any vector that can transfect a brain neuron may be used in the method of the invention.
  • a preferred vector is a herpes simplex viral (HSV) vector or an appropriately modified version of this vector.
  • HSV herpes simplex viral
  • a therapeutic composition containing this vector may be used alone or in a mixture, or in chemical combination, with one or more materials, including other proteins or recombinant vectors that increase the biological stability of the recombinant vectors, or with materials that increase the therapeutic composition's ability to penetrate the target tissue selectively.
  • the therapeutic compositions of the invention is typically administered in a pharmaceutically acceptable carrier (e.g., physiological saline), which is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • a pharmaceutically acceptable carrier e.g., physiological saline
  • Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington 's Pharmaceutical Sciences , a standard reference text in this field, and in the USP/NF.
  • the therapeutic compositions of the invention can be administered in dosages determined to be appropriate by one skilled in the art. It is expected that the dosages will vary, depending upon the pharmacokinetic and pharmacodynamic characteristics of the particular agent, and its mode and route of administration, as well as the age, weight, and health of the recipient; the nature and extent of the disease; the frequency and duration of the treatment; the type of, if any, concurrent therapy; and the desired effect.
  • the therapeutic compositions may be administered to a patient by any appropriate mode, e.g., via applying drops or spray onto the nasal mucosa, or via injection into the nasal mucosa, as determined by one skilled in the art. Alternatively, it may be desired to administer the treatment surgically to the target tissue. The treatments of the invention may be repeated as needed, as determined by one skilled in the art.
  • the invention includes a method of inhibiting tumor growth by expressing an exogenously introduced SSTR5 protein, e.g., a recombinant protein comprising (a) SSTR5, or (b) a biologically active fragment thereof, in a tumor cell.
  • SSTR5 protein e.g., a recombinant protein comprising (a) SSTR5, or (b) a biologically active fragment thereof
  • Recombinant G ⁇ 12 or G ⁇ 13 polypeptides can also be introduced into the target cell.
  • the recombinant SSTR5 present on the cell surface will be stimulated and will thereby inhibit growth of the tumor cell via endogenous or recombinant G ⁇ 12 and G ⁇ 13 .
  • This aspect of the invention is useful in cancer treatments using SST-related drugs (i.e., SST or SST analogues) .
  • SST-related drugs i.e., SST or SST analogues
  • Such treatments frequently lead to loss of SSTR's naturally expressed on cancer cells, thereby desensitizing the cells to the SST-related drugs.
  • Introduction of recombinant SSTR5 into the cancer cells solves this problem, at least temporarily; further transfetions may be necessary to maintain the effect, if the recombinant SSTR5 is lost as well. All cancers, including highly malignant ones such as pancreatic cancer and small cell lung cancer, can be treated by the present method.
  • the recombinant SSTR5 protein can be introduced into the cancer cells by overexpressing within the cells a nucleic acid construct comprising a mammalian promoter sequence operably linked to a sequence encoding the protein.
  • the construct primarily targets fast-proliferating cells, and can, for example, be derived from retroviral, adenoviral, adeno-associated- viral, or herpes simplex viral vectors, or any appropriately modified versions of these vectors.
  • Retroviral vectors are particularly appropriate, as they selectively integrate into the genome of replicating cells, such as tumor cells.
  • Gly Gly Gin Arg Asp Gin Arg Arg Lys Trp lie Gin Cys Phe Asn Asp 225 230 235 240
  • Val Thr Ala lie lie Phe Val Val Ala Ser Ser Ser Tyr Asn Met Val
  • Lys Ser Lys lie Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr 305 310 315 320

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Abstract

L'invention porte sur des polypeptides chimères dérivés des sous-unités Gα de diverses protéines G, et sur des procédés d'utilisation des polypeptides chimères dans des thérapies et dans le criblage d'agents thérapeutiques potentiels.
PCT/US1996/020510 1996-10-11 1996-12-16 Dosages pour recepteurs lies a la proteine g WO1998016557A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1999014344A1 (fr) * 1997-09-13 1999-03-25 Glaxo Group Limited Chimeres de proteines g
WO2000006722A1 (fr) * 1998-07-28 2000-02-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Clonage par interaction de genes a base de cellules eukaryiotes
WO2001036481A1 (fr) * 1999-11-17 2001-05-25 Yung Hou Wong Nouvelles protéine g-alpha à plus grande promiscuité avec les gprc
WO2001072802A1 (fr) * 2000-03-28 2001-10-04 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine de liaison 14 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2001079438A2 (fr) * 2000-03-29 2001-10-25 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine de liaison 9 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2002024867A2 (fr) * 2000-09-22 2002-03-28 University Of Aarhus Nouvelles compositions et nouvelles methodes pour le diagnostic et le traitement des lymphomes et des leucemies
WO2003027276A2 (fr) * 2001-09-24 2003-04-03 University Of Aarhus Nouvelles compositions et procedes relatifs aux lymphomes et aux leucemies
EP1328663A2 (fr) * 2000-09-28 2003-07-23 New England Medical Center Hospitals, Inc. Dosages d'identifications de recepteurs presentant des alterations de signaux de transduction
DE10233516A1 (de) * 2002-07-23 2004-02-12 Aventis Pharma Deutschland Gmbh Verfahren zur Identifizierung von Substanzen
WO2005047318A1 (fr) * 2003-11-11 2005-05-26 Astrazeneca Ab Variant d'epissage de gnal et ses applications
US7067277B1 (en) 1999-12-23 2006-06-27 H. Lundbeck A/S Chimeric G proteins and uses thereof
US7674584B2 (en) 2004-09-30 2010-03-09 Ge Healthcare Uk Limited Method for measuring binding of a test compound to a G-protein coupled receptor
WO2017129998A1 (fr) * 2016-01-29 2017-08-03 Heptares Therapeutics Limited Protéines g

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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014344A1 (fr) * 1997-09-13 1999-03-25 Glaxo Group Limited Chimeres de proteines g
EP1533379A1 (fr) * 1997-09-13 2005-05-25 Glaxo Group Limited Chiméras de Protéine G
US6509447B1 (en) 1997-09-13 2003-01-21 Glaxo Welcome Inc. G protein chimeras and methods of screening compounds
WO2000006722A1 (fr) * 1998-07-28 2000-02-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Clonage par interaction de genes a base de cellules eukaryiotes
US6462178B1 (en) 1999-11-17 2002-10-08 Yong Hou Wong G protein
WO2001036481A1 (fr) * 1999-11-17 2001-05-25 Yung Hou Wong Nouvelles protéine g-alpha à plus grande promiscuité avec les gprc
US7067277B1 (en) 1999-12-23 2006-06-27 H. Lundbeck A/S Chimeric G proteins and uses thereof
WO2001072802A1 (fr) * 2000-03-28 2001-10-04 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine de liaison 14 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2001079438A3 (fr) * 2000-03-29 2002-02-28 Biowindow Gene Dev Inc Nouveau polypeptide, proteine humaine de liaison 9 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2001079438A2 (fr) * 2000-03-29 2001-10-25 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine de liaison 9 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2002024867A3 (fr) * 2000-09-22 2003-08-14 Univ Aarhus Nouvelles compositions et nouvelles methodes pour le diagnostic et le traitement des lymphomes et des leucemies
WO2002024867A2 (fr) * 2000-09-22 2002-03-28 University Of Aarhus Nouvelles compositions et nouvelles methodes pour le diagnostic et le traitement des lymphomes et des leucemies
EP1328663A2 (fr) * 2000-09-28 2003-07-23 New England Medical Center Hospitals, Inc. Dosages d'identifications de recepteurs presentant des alterations de signaux de transduction
EP1328663A4 (fr) * 2000-09-28 2005-06-01 New England Medical Center Inc Dosages d'identifications de recepteurs presentant des alterations de signaux de transduction
WO2003027276A3 (fr) * 2001-09-24 2004-02-12 Univ Aarhus Nouvelles compositions et procedes relatifs aux lymphomes et aux leucemies
WO2003027276A2 (fr) * 2001-09-24 2003-04-03 University Of Aarhus Nouvelles compositions et procedes relatifs aux lymphomes et aux leucemies
DE10233516A1 (de) * 2002-07-23 2004-02-12 Aventis Pharma Deutschland Gmbh Verfahren zur Identifizierung von Substanzen
WO2005047318A1 (fr) * 2003-11-11 2005-05-26 Astrazeneca Ab Variant d'epissage de gnal et ses applications
US7674584B2 (en) 2004-09-30 2010-03-09 Ge Healthcare Uk Limited Method for measuring binding of a test compound to a G-protein coupled receptor
WO2017129998A1 (fr) * 2016-01-29 2017-08-03 Heptares Therapeutics Limited Protéines g
GB2558968A (en) * 2016-01-29 2018-07-25 Heptares Therapeutics Ltd G Proteins
CN108699123A (zh) * 2016-01-29 2018-10-23 赫普泰雅治疗有限公司 G蛋白
JP2019507590A (ja) * 2016-01-29 2019-03-22 ヘプタレス セラピューティックス リミテッド Ｇタンパク質
US10738287B2 (en) 2016-01-29 2020-08-11 Heptares Therapeutics Limited G proteins
AU2017212788B2 (en) * 2016-01-29 2021-07-29 Nxera Pharma Uk Limited G proteins
GB2558968B (en) * 2016-01-29 2021-09-29 Heptares Therapeutics Ltd G Proteins
US11339383B2 (en) 2016-01-29 2022-05-24 Heptares Therapeutics Limited G proteins
JP2022084574A (ja) * 2016-01-29 2022-06-07 ヘプタレス セラピューティックス リミテッド Ｇタンパク質
CN108699123B (zh) * 2016-01-29 2023-12-08 赫普泰雅治疗有限公司 G蛋白

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