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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a novel human DNA encoding HG07, a G protein-coupled receptor (GPCR) related to the leukotriene B4 receptor, the protein encoded by the DNA, and methods of identifying selective agonists and antagonists of the protein encoded by the DNA.
• GPCR G protein-coupled receptor
• GPCRs G-protein coupled receptors
• GDP guanosine diphosphate
• GTP guanosine triphosphate
• GPCRs possess common structural characteristics. They have seven hydrophobic domains, each about 20-30 amino acids long, linked by sequences of hydrophilic amino acids of varied length. These seven hydrophobic domains intercalate into the plasma membrane, giving rise to a protein with seven transmembrane domains, an extracellular amino terminus, and an intracellular carboxy terminus (Strader et al., 1994, Ann. Rev. Biochem. 63:101-132; Schertler et al., 1993, Nature 362:770-7721; Dohlman et al., 1991, Ann. Rev. Biochem. 60:653- 688).
• GPCRs are expressed in a wide variety of tissue types and respond to a wide range of ligands, e.g., protein hormones, biogenic amines, peptides, lipid derived messengers, etc. Given their wide range of expression and ligands, it is not surprising that GPCRs are involved in many pathological states. This has led to great interest in developing modulators of GPCR activity that can be used pharmacologically. For example, Table 1 of Stadel et al., 1997, Trends Pharmacol. Sci. 18:430-437, lists 37 different marketed drugs that act upon GPCRs. Accordingly, there is a great need to understand GPCR function and to develop agents that can be used to modulate GPCR activity.
• ligands e.g., protein hormones, biogenic amines, peptides, lipid derived messengers, etc. Given their wide range of expression and ligands, it is not surprising that GPCRs are involved in many pathological states. This has led to great interest in developing modulators of
• Leukotriene B4 (5(S),12(R)-dihydroxy-6,14-c «, 8,10,-trans- eicosatetraenoic acid) (LTB4) is involved in various immune responses, such as defenses against infection, and is involved in the process of inflammation as well.
• LTB4 induces chemotactic migration (Goetzl & Pickett, 1980, J. Immunol. 125:1789- 1791) and chemokinetic activity (Palmer et al., 1980, Prostaglandins 20:41 1-418) as well as aggregation of polymorphonuclear leukocytes (Ford-Hutchinson et al., 1980, Nature 286:264-265).
• LTB4 has been detected in the spinal fluid of multiple sclerosis patients (Neu et al., 1992, Acta Neurol. Scand. 86:586-587) and inhibition of the interaction of LTB4 with its receptor prevents the development of paralysis and the infiltration into the spinal chord by eosinophils in experimental allergic encephalomyelitis, a murine model of multiple sclerosis (Gladue et al., 1996, J. Exp. Med. 183:1893-1898).
• the transfected cells Upon exposure to LTB4, the transfected cells also displayed several functional responses characteristic of LTB4-responsive cells: an increase in intracellular calcium, D-my ⁇ -inositol 1 ,4,5-triphosphate (InsP3) accumulation, inhibition of adenylyl cyclase, and chemotaxis.
• IsP3 D-my ⁇ -inositol 1 ,4,5-triphosphate
• the present invention is directed to a novel human DNA that encodes a G-protein coupled receptor, HG07.
• the DNA encoding HG07 is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO. : 1.
• an HG07 protein encoded by the novel DNA sequence is substantially free from other proteins and has the amino acid sequence shown in SEQ.ID.NO. :2.
• Figure 1 shows the complete cDNA sequence of HG07 (SEQ.ID.NO.:l).
• Figure 2 shows the complete amino acid sequence of HG07 (SEQ.ID.NO.:2).
• Figure 3A-B shows the location of the HG07 open reading frame.
• the nucleotide sequence shown is a portion of (SEQ.ID.NO.: 1).
• the amino acid sequence shown is (SEQ.ID.NO.:2).
• Figure 4 shows the results of a Northern blot of HG07 mRNA in various human tissues.
• Figure 5 shows an alignment of the amino acid sequence of HG07 with the amino acid sequence of a human leukotriene B4 receptor (SEQ.ID.NO. :3; GenBank accession number D89079, see Yokomizo et al., 1997, Nature 387:620- 624).
• substantially free from other proteins means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins.
• an HG07 protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1 %>, of non-HG07 proteins.
• Whether a given HG07 protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
• SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
• Substantially free from other nucleic acids means at least 90%, preferably 95%, more preferably 99%, and even more preferably
• an HG07 DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HG07 nucleic acids. Whether a given HG07 DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.
• “Functional equivalent” means a receptor which does not have exactly the same amino acid sequence as naturally occurring HG07, due to alternative splicing, substitutions, deletions, mutations, or additions, but retains substantially the same biological activity as HG07. Such functional equivalents will have significant amino acid sequence identity with naturally occurring HG07. Genes and DNA encoding such functional equivalents can be detected by reduced stringency hybridization with a DNA sequence encoding naturally occurring HG07.
• naturally occurring HG07 has the amino acid sequence shown as SEQ.ID.NO.:2 and is encoded by SEQ.ID.NO.:l.
• a nucleic acid encoding a functional equivalent has at least about 50% identity at the nucleotide level to SEQ.ID.NO. :1.
• a polypeptide has "substantially the same biological activity" as HG07 if that polypeptide has a Kd for a ligand that is no more than 5-fold greater than the K of HG07 having SEQ.ID.NO.:2 for the same ligand.
• a "conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
• isolated HG07 protein or "isolated HG07 DNA” is meant HG07 protein or DNA encoding HG07 that has been isolated from a natural source.
• isolated indicates that HG07 protein or DNA has been removed from its normal cellular environment.
• an isolated HG07 protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally.
• isolated does not imply that an isolated HG07 protein is the only protein present, but instead means that an isolated HG07 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the HG07 protein.
• an HG07 protein that is expressed in bacteria or even in eukaryotic cells which do not naturally (i.e., without human intervention) express it through recombinant means is an "isolated HG07 protein.”
• DNA encoding HG07 that is present in bacteria or even in eukaryotic cells which do not naturally (i.e., without human intervention) contain it through recombinant means is an "isolated DNA encoding HG07."
• One aspect of this invention is an isolated DNA comprising nucleotides encoding a polypeptide having the amino acid sequence SEQ.ID.NO. :2.
• This isolated DNA is substantially free from other nucleic acids and can be either single stranded or double stranded, i.e., paired with its complementary sequence.
• Another aspect of this invention is the identification and cloning of a cDNA which encodes HG07, a G protein-coupled receptor.
• This cDNA is substantially free from other nucleic acids and can be either single stranded or double stranded.
• the present invention provides a cDNA molecule substantially free from other nucleic acids having the nucleotide sequence shown in Figure 1 as SEQ.ID.NO.: 1.
• SEQ.ID.NO.: 1 contains an open reading frame (positions 158-1,324 of SEQ.ID.NO.: 1) encoding a protein of 389 amino acids (see Figure 3A-B).
• the present invention also provides a DNA molecule substantially free from other nucleic acids comprising the nucleotide sequence of positions 158-1,324 of SEQ.ID.NO.:l .
• the present invention also provides recombinant DNA molecules comprising the nucleotide sequence of positions 158-1,324 of SEQ.ID.NO.: 1.
• GPCR G-protein coupled receptor
• HG07 RNA is widely expressed in humans as a transcript of about 2.4 kb, especially in cells of the immune system (peripheral blood lymphocytes (PBLs) and spleen). This argues for a role for HG07 in such immune system functions as inflammation, responses to infection, chemotaxis of lymphocytes, etc..
• the novel DNA sequences of the present invention encoding HG07, in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to which HG07 is not naturally linked, to form "recombinant DNA molecules" containing HG07.
• the novel DNA sequences of the present invention can be inserted into vectors in order to direct recombinant expression of HG07.
• Such vectors may be comprised of DNA or RNA; for most purposes DNA vectors are preferred.
• Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode HG07.
• plasmids modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode HG07.
• DNA sequences that hybridize to SEQ.ID.NO. :1 under stringent conditions include DNA sequences that hybridize to SEQ.ID.NO. :1 under stringent conditions.
• a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10° " cpm of 32p_ ⁇ a beled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1 X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
• the degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon encodes a particular amino acid.
• This allows for the construction of synthetic DNA that encodes the HG07 protein where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequence of SEQ.ID.NO.:l, but still encodes the same HG07 protein as SEQ.ID.NO.:l.
• Such synthetic DNAs are intended to be within the scope of the present invention. If it is desired to express such synthetic DNAs in a particular host cell or organism, the codon usage of such synthetic DNAs can be adjusted to reflect the codon usage of that particular host, thus leading to higher levels of expression of HG07 protein in the host.
• Another aspect of the present invention includes host cells that have been engineered to contain and or express DNA sequences encoding HG07.
• Such recombinant host cells can be cultured under suitable conditions to produce HG07.
• An expression vector containing DNA encoding HG07 can be used for expression of HG07 in a recombinant host cell.
• Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey, and rodent origin, and insect cells including but not limited to, Drosophila and silkworm derived cell lines.
• L cells L-M(TK") ATCC CCL 1.3
• L cells L-M ATCC CCL 1.2
• 293 ATCC CRL 1573
• Raji ATCC CCL 86
• CV-1 ATCC CCL 70
• COS-1 ATCC CRL 1650
• COS-7 ATCC CRL 1651
• CHO-K1 ATCC CCL 61
• 3T3 ATCC CCL 92
• NIH/3T3 ATCC CRL 1658
• HeLa ATCC CCL 2
• C127I ATCC CRL 1616
• BS-C-1 ATCC CCL 26
• MRC-5 ATCC CCL 171
• HEK 293 cells and Chinese hamster ovary (CHO) cells are particularly suitable for expression of the HG07 protein because these cells express a large number of G-proteins.
• HEK 293 cells and Chinese hamster ovary (CHO) cells are particularly suitable for expression of the HG07 protein because these cells express a large number of G-proteins.
• at least one of these G-proteins will be able to functionally couple the signal generated by interaction of HG07 and its ligands, thus transmitting this signal to downstream effectors, eventually resulting in a measurable change in some assayable component, e.g., cAMP level, expression of a reporter gene, hydrolysis of inositol lipids, or intracellular Ca2+ levels.
• HG07 Other cells that are particularly suitable for expression of the HG07 protein are immortalized melanophore pigment cells from Xenopus laevis. Such melanophore pigment cells can be used for functional assays using recombinant expression of HG07 in a manner similar to the use of such melanophore pigment cells for the functional assay of other recombinant GPCRs (Graminski et al., 1993, J. Biol. Chem. 268:5957-5964; Lerner, 1994, Trends Neurosci. 17:142-146; Potenza & Lerner, 1992, Pigment Cell Res. 5:372-378).
• mammalian expression vectors can be used to express recombinant HG07 in mammalian and other cells.
• Commercially available mammalian expression vectors which are suitable include, but are not limited to, pCR2.1 (Invitrogen), pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1 , pCR3.1 (Invitrogen), EBO-pSN2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146).
• HG07 can be purified to a level that is substantially free from other proteins by conventional techniques, e.g., salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography, hydrophobic interaction chromatography, and preparative gel electrophoresis.
• the present invention includes HG07 protein substantially free from other proteins.
• the amino acid sequence of the full-length HG07 protein is shown in Figure 2 as SEQ.ID.NO. :2.
• the present invention includes HG07 protein substantially free from other proteins having the amino acid sequence of SEQ.ID.NO.:2.
• this invention includes modified HG07 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as HG07. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085).
• the present invention includes polypeptides where one amino acid substitution has been made in SEQ.ID.NO.:2 wherein the polypeptides still retain substantially the same biological activity as HG07.
• the present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.NO.:2 wherein the polypeptides still retain substantially the same biological activity as HG07.
• the present invention includes embodiments where the above-described substitutions are conservative substitutions.
• the present invention includes embodiments where the above-described substitutions do not occur in the ligand-binding domain of HG07.
• HG07 When deciding which amino acid residues of HG07 may be substituted to produce polypeptides that are functional equivalents of HG07, one skilled in the art would be guided by a comparison of the amino acid sequence of HG07 with the amino acid sequences of related proteins, e.g., the human leukotriene B4 receptor disclosed in Yokomizo et al., 1997, Nature 387:620-624. See also Figure 5 of the present application. Such a comparison would allow one skilled in the art to minimize the number of amino acid substitutions made in regions that are highly conserved between HG07 and the related protein. Accordingly, the present invention includes embodiments where the substitutions are conservative and do not occur in positions where HG07 and the human leukotriene B4 receptor share the same amino acid (see Figure 5).
• polypeptides that are functional equivalents of HG07 and have changes from the HG07 amino acid sequence that are small deletions or insertions of amino acids could also be produced by following the same guidelines, i.e., minimizing the differences in amino acid sequence between HG07 and related proteins.
• Small deletions or insertions are generally in the range of about 1 to 5 amino acids. The effect of such small deletions or insertions on the biological activity of the modified HG07 polypeptide can easily be assayed by producing the polypeptide synthetically or by making the required changes in DNA encoding HG07 and then expressing the DNA recombinantly and assaying the protein produced by such recombinant expression.
• Assays that could be used include simple binding assays to determine if the modified HG07 polypeptide is capable of binding the same ligands, with approximately the same affinity, as the naturally occurring HG07 protein.
• functional assays such as assays for increases in intracellular calcium, D-my ⁇ -inositol 1,4,5-triphosphate (InsP ⁇ ) accumulation, inhibition of adenylyl cyclase, or chemotaxis.
• Such assays can be based upon similar assays for the leukotriene B4 receptor disclosed in Yokomizo et al, 1997, Nature 387:620-624.
• the present invention also includes C-terminal truncated forms of HG07, particularly those which encompass the extracellular portion of the receptor, but lack the intracellular signaling portion of the receptor.
• Such truncated receptors are useful in various binding assays described herein, for crystallization studies, and for structure-activity-relationship studies.
• the present invention also includes chimeric HG07 proteins.
• Chimeric HG07 proteins consist of a contiguous polypeptide sequence of HG07 fused in frame to a polypeptide sequence of a non-HG07 protein.
• the N-terminal domain and seven transmembrane spanning domains of HG07 fused at the C-terminus in frame to a G protein would be a chimeric HG07 protein.
• the present invention also includes HG07 proteins that are in the form of multimeric structures, e.g., dimers.
• HG07 proteins that are in the form of multimeric structures, e.g., dimers.
• Such multimers of other G-protein coupled receptors are known (Hebert et al, 1996, J. Biol. Chem. 271, 16384-16392; Ng et al, 1996, Biochem. Biophys. Res. Comm. 227, 200-204; Romano et al, 1996, J. Biol. Chem. 271, 28612-28616).
• the present invention also includes isolated forms of HG07 proteins.
• the present invention includes methods of identifying compounds that specifically bind to HG07 protein, as well as compounds identified by such methods.
• the specificity of binding of compounds having affinity for HG07 is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from such cells. Expression of the cloned receptor and screening for compounds that bind to HG07 or that inhibit the binding of a known ligand of HG07 to such cells, or membranes prepared from such cells, provides an effective method for the rapid selection of compounds with high affinity for HG07.
• Such ligands or compounds can be radiolabeled, but can also be nonisotopic compounds that can be used to displace bound radiolabeled ligands or that can be used as activators in functional assays.
• Compounds identified by the above method are likely to be agonists or antagonists of HG07 and may be peptides, proteins, or non-proteinaceous organic molecules.
• the present invention includes assays by which HG07 agonists and antagonists may be identified.
• Methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of HG07.
• the present invention includes a method for determining whether a substance is a potential agonist or antagonist of HG07 that comprises:
• step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
• the present invention also includes a method for determining whether a substance is capable of binding to HG07, i.e., whether the substance is a potential agonist or an antagonist of HG07, where the method comprises:
• test cells by transfecting cells with an expression vector that directs the expression of HG07 in the cells;
• test cells to the substance;
• step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
• the cells are eukaryotic cells.
• the cells are mammalian cells.
• the cells are L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CN-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), ⁇ IH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
• transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HG07, and electroporation.
• binding of the substance or agonist to HG07 can be measured by employing a labeled substance or agonist.
• the substance or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
• HG07 has an amino acid sequence of SEQ.ID.NO. :2.
• the above-described methods can be modified in that, rather than exposing the test cells to the substance, membranes can be prepared from the test cells and those membranes can be exposed to the substance. Such a modification utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al., 1992, Biochem. Biophys. Res. Comm. 184:260-268.
• the present invention provides a method for determining whether a substance is capable of binding to HG07 comprising:
• step (c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
• the present invention provides a method for determining whether a substance is capable of binding to HG07 comprising: (a) providing test cells by transfecting cells with an expression vector that directs the expression of HG07 in the cells;
• RNA encoding HG07 can be prepared, e.g., by in vitro transcription using a plasmid containing HG07 under the control of a bacteriophage T7 promoter, and the RNA can be microinjected into Xenopus oocytes in order to cause the expression of HG07 in the oocytes. Substances are then tested for binding to the HG07 expressed in the oocytes. Alternatively, rather than detecting binding, the effect of the substances on the electrophysiological properties of the oocytes can be determined.
• the present invention includes assays by which HG07 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HG07.
• One skilled in the art would be familiar with a variety of methods of measuring the functional responses of G-protein coupled receptors (see, e.g., Lerner, 1994, Trends Neurosci. 17:142-146 [changes in pigment distribution in melanophore cells]; Yokomizo et al., 1997, Nature 387:620-624 [changes in cAMP or calcium concentration; chemotaxis]; Howard et al., 1996,
• the present invention provides a method of identifying agonists and antagonists of HG07 comprising: (a) providing test cells by transfecting cells with an expression vector that directs the expression of HG07 in the cells;
• the substance is an agonist or antagonist of HG07; where the control cells are cells that have not been transfected with HG07 but have been exposed to the substance or are test cells that have not been exposed to the substance; where HG07 has the amino acid sequence SEQ.ID.NO. :2.
• the functional response is selected from the group consisting of: changes in pigment distribution in melanophore cells; changes in cAMP or calcium concentration; chemotaxis; changes in membrane currents in Xenopus oocytes; and changes in inositol phosphate levels.
• HG07 belongs to the class of proteins known as G-protein coupled receptors (GPCRs). GPCRs transmit signals across cell membranes upon the binding of ligand. The ligand-bound GPCR interacts with a heterotrimeric G-protein, causing the G ⁇ subunit of the G-protein to disassociate from the G ⁇ and G ⁇ subunits. The G ⁇ subunit can then go on to activate a variety of second messenger systems.
• GPCRs G-protein coupled receptors
• Offermanns described a system in which cells are transfected with expression vectors that result in the expression of one of a large number of GPCRs as well as the expression of one of the promiscuous G-proteins G ⁇ l5 or G ⁇ l6.
• the GPCR was activated and was able, via G ⁇ l5 or G ⁇ l6, to activate the ⁇ isoform of phospholipase C, leading to an increase in inositol phosphate levels in the cells.
• the present invention provides a method of identifying antagonists of HG07 comprising: (a) providing cells that expresses a chimeric HG07 protein fused at its C-terminus to a promiscuous G-protein;
• step (c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG07; (d) measuring the level of inositol phosphates in the cells; where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HG07.
• Another possibility for utilizing promiscuous G-proteins in connection with HG07 includes a method of identifying agonists of HG07 comprising:
• Intracellular calcium mobilization is typically assayed in whole cells under a microscope using fluorescent dyes or in cell suspensions via luminescence using the aequorin assay.
• the cells are eukaryotic cells.
• the cells are mammalian cells.
• the cells are L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CN-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), ⁇ IH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
• the cells are transfected with expression vectors that direct the expression of HG07 and the promiscuous G-protein in the cells.
• step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
• the promiscuous G-protein is selected from the group consisting of G l5 or G ⁇ l6.
• Expression vectors containing G ⁇ l5 or G ⁇ l6 are known in the art. See, e.g., Offermanns; Buhl et al, 1993, FEBS Lett. 323:132-134; Amatruda et al, 1993, J. Biol. Chem. 268:10139-10144.
• the above-described assay can be easily modified to form a method to identify antagonists of HG07.
• Such a method is also part of the present invention and comprises:
• the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells.
• the cells are L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CN-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), ⁇ IH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
• conditions under which steps (b) and (c) of the method are practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
• the cells are transfected with expression vectors that direct the expression of HG07 and the promiscuous G-protein in the cells.
• the promiscuous G-protein is selected from the group consisting of G ⁇ l5 or G ⁇ l6.
• HG07 has an amino acid sequence of SEQ.ID.NO. :2.
• Another functional asssay that can be used to identify agonists and antagonists of HG07 relies on the ability of leukotriene B4 to induce the chemotaxis or chemokinesis of certain cell types through action at the leukotriene receptor (see, e.g., Yokomizo et al., 1997, Nature 387:620-624; Ng et al., 1991, J. Immunol. 147:3096-3103). Given the high degreee of similarity in amino acid sequence between HG07 and the leukotriene B4 receptor (about 46%>, see Figure 5), is is expected that agonists of HG07 will be able to induce the chemotaxis or chemokinesis of cells.
• the present invention provides a method of identifying agonists of HG07 comprising:
• test cells by transfecting cells with an expression vector that directs the expression of HG07 in the cells;
• test cells exposing the test cells to a substance;
• the substance is an agonist of binding to HG07; where the control cells are cells that have not been transfected with HG07 but have been exposed to the substance or are test cells that have not been exposed to the substance; where HG07 has the amino acid sequence SEQ.ID.NO. :2.
• the cells are CHO cells.
• HG07 Given the resemblance between HG07 and the leukotriene
• LTB4 (LTB4) receptor it is expected that agonists and antagonists of HG07 will have pharmacological activity and be useful in a manner similar to that in which agonists and antagonists of the LTB4 receptor are useful.
• Agonists and antagonists of the LTB4 receptor are useful in the treatment of rheumatoid arthritis, gout, psoriasis, inflammatory bowel disease (U.S. Patent No. 5,684,162), chronic lung diseases, endotoxic shock, septic shock, and adult respiratory distress syndrome (U.S. Patent No. 5,552,441).
• HG07 appears to be most similar to the LTB4 receptor, and thus is expected to be pharmacologically useful in a manner similar to that of the LTB4 receptor, it is also expected that HG07 may have affinity for other related leukotrienes, e.g., leukotrienes A4, C4, D4, and E4. Antagonists of the leukotriene D4 receptor, such as zafirlukast, montelukast, and pranlukast, have been shown to have utility in the treatment of asthma (Tan & Spector, July/August 1997, Science and
• HG07 may also have utility in the treatment of asthma.
• the present invention includes pharmaceutical compositions comprising agonists and antagonists of HG07.
• the agonists and antagonists are generally combined with pharmaceutically acceptable carriers to form pharmaceutical compositions. Examples of such carriers and methods of formulation of pharmaceutical compositions containing agonists and antagonists and carriers can be found in Remington's Pharmaceutical Sciences.
• To form a pharmaceutically acceptable composition suitable for effective administration such compositions will contain a therapeutically effective amount of the agonists and antagonists.
• Therapeutic or prophylactic compositions are administered to an individual in amounts sufficient to treat or prevent conditions where HG07 activity is abnormal.
• the effective amount can vary according to a variety of factors such as the individual's condition, weight, gender, and age. Other factors include the mode of administration. The appropriate amount can be determined by a skilled physician.
• compositions can be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents can be desirable.
• compositions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
• the compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
• they can also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
• compositions can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.
• compositions can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
• the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
• the dosage regimen utilizing the compositions is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular composition thereof employed.
• a physician of ordinary skill can readily determine and prescribe the effective amount of the composition required to prevent, counter or arrest the progress of the condition.
• Optimal precision in achieving concentrations of composition within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the composition's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a composition.
• the present invention also includes methods of expressing HG07 in recombinant systems and then utilizing the recombinantly expressed HG07 for counter-screening.
• screening compounds in order to identify potential pharmaceuticals that specifically interact with a target receptor, it is necessary to ensure that the compounds identified are as specific as possible for the target receptor. To do this, it is necessary to screen the compounds against as wide an array as possible of receptors that are similar to the target receptor.
• the compounds that are potential pharmaceuticals that interact with receptor A it is necessary not only to ensure that the compounds interact with receptor A (the "plus target") and produce the desired pharmacological effect through receptor A, it is also necessary to determine that the compounds do not interact with receptors B, C, D, etc. (the "minus targets").
• HG07 proteins and DNA encoding HG07 proteins have utility in counter-screens. That is, they can be used as "minus targets" in counter-screens in connection with screening projects designed to identify compounds that specifically interact with other G-protein coupled receptors.
• the DNA of the present invention can be used in chromosomal mapping studies in order to identify the chromosomal location of the HG07 gene or of genes encoding proteins related to HG07.
• Such mapping studies can be carried out using well-known genetic and/or chromosomal mapping techniques such as, e.g., linkage analysis with respect to known chromosomal markers or in situ hybridization. See, e.g., Verma et al., 1988, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, NY.
• this information can be compared with the locations of known disease-causing genes contained in genetic map data (such as the data found in the genome issue of Science (1994, 265:1981-2144). In this way, one can correlate the chromosomal location of the HG07 gene or of genes encoding proteins related to HG07 with the locations of known disease-causing genes and thus help to limit the region of DNA containing such disease-causing genes. This will simplify the process of cloning such disease-causing genes.
• RFLPs restriction fragment length polymorphisms
• Nucleotide sequences that are complementary to the HG07 sequences disclosed herein can be synthesized for use in antisense therapy.
• Such antisense molecules can be DNA, stable derivatives of DNA such as phosphorothioates or methyl phosphonates, RNA, stable derivatives of RNA such as 2'-0-alkyl RNA, or other forms of HG07 antisense molecules.
• HG07 antisense molecules can be introduced into cells by a variety of methods, e.g., microinjection, liposome encapsulation, or by expression from vectors harboring the antisense sequence.
• HG07 antisense therapy is expected to be particularly useful in the treatment of conditions where it is beneficial to reduce HG07 activity.
• the present invention also includes antibodies to the HG07 protein.
• Such antibodies may be polyclonal antibodies or monoclonal antibodies and are useful in treating disorders of the immune system that involve the inappropriate expression or activity of the HG07 protein.
• the antibodies of the present invention are raised against the entire HG07 protein or against suitable antigenic fragments of the protein that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art. Methods of identifying suitable antigenic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.
• HG07 protein or an antigenic fragment is injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice.
• an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice.
• the animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen.
• the injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
• HG07 protein or an antigenic fragment is injected into an appropriate non- human host animal as above for the production of polyclonal antibodies.
• the animal is generally a mouse.
• the animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497.
• Gene therapy may be used to introduce HG07 polypeptides into the cells of target organs.
• Nucleotides encoding HG07 polypeptides can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, and polio virus based vectors. Alternatively, nucleotides encoding HG07 polypeptides can be transferred into cells for gene therapy by non- viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with HG07 polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate HG07 activity.
• a cDNA fragment encoding full-length HG20 can be isolated from an appropriate human cDNA library by using the polymerase chain reaction (PCR) employing suitable primer pairs. Such primer pairs can be selected based upon the cDNA sequence for HG20 shown in Figure 1 as SEQ.ID.NO.: 1. Suitable primer pairs would be, e.g. :
• the above primers contain BamHI sites in their 5' ends to facilitate cloning of the amplified cDNA into suitable vectors, e.g., pcDNA3.1.
• suitable vectors e.g., pcDNA3.1.
• the above primers are meant to be illustrative. One skilled in the art would recognize that a variety of other suitable primers can be designed.
• PCR reactions can be carried out with a variety of thermostable enzymes including but not limited to AmpliTaq, AmpliTaq Gold, or Vent polymerase.
• reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM MgCl2, 200 ⁇ M for each dNTP, 50 mM KC1, 0.2 ⁇ M for each primer, 10 ng of DNA template, 0.05 units/ ⁇ l of AmpliTaq.
• the reactions are heated at 95°C for 3 minutes and then cycled 35 times using the cycling parameters of 95°C, 20 seconds, 62°C, 20 seconds, 72°C, 3 minutes.
• suitable PCR protocols can be found in PCR Primer. A Laboratory Manual, edited by C.W. Dieffenbach and G.S. Dveksler, 1995, Cold Spring Harbor Laboratory Press; or PCR Protocols: A Guide to Methods and Applications, Michael et al, eds., 1990, Academic Press .
• a suitable cDNA library from which a clone encoding HG07 can be isolated would be a human cDNA library made from RNA from prostate, ovary, pancreas, peripheral blood lymphocytes, or spleen.
• Such libraries can be prepared by methods well-known in the art.
• several commercially available libraries would be suitable, e.g., cDNA libraries from Stratagene, Inc., La Jolla, CA, USA, such as human ovary, catalog #937217, human pancreas, catalog #937208, or human fetal spleen, catalog #937205).
• the primary clones of such libraries can be subdivided into pools with each pool containing approximately 20,000 clones and each pool can be amplified separately.
• a cDNA fragment encoding an open reading frame of 389 amino acids (SEQ.ID.NO. :2) can be obtained.
• This cDNA fragment can be cloned into a suitable cloning vector or expression vector.
• the fragment can be cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, Ca).
• HG07 protein can then be produced by transferring an expression vector encoding HG07 into a suitable host cell and growing the host cell under appropriate conditions. HG07 protein can then be isolated by methods well known in the art.
• a cDNA clone encoding HG07 can be isolated from a cDNA library using as a probe oligonucleotides specific for HG07 and methods well known in the art for screening cDNA libraries with oligonucleotide probes. Such methods are described in, e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II.
• Oligonucleotides that are specific for HG07 and that can be used to screen cDNA libraries can be readily designed based upon the cDNA sequence of HG07 shown in Figure 1 as SEQ.ID.NO.: 1 and can be synthesized by methods well-known in the art.
• Genomic clones containing the HG07 gene can be obtained from commercially available human PAC or BAC libraries, e.g., from Research Genetics, Huntsville, AL. Alternatively, one may prepare genomic libraries, especially in PI artificial chromosome vectors, from which genomic clones containing the HG07 can be isolated, using probes based upon the HG07 nucleotide sequences disclosed herein. Methods of preparing such libraries are known in the art (Ioannou et ⁇ /., 1994, Nature Genet. 6:84-89).
• the data shown in Figure 4 were obtained by the use of human multi- tissue Northern blots purchased from Clontech (Palo Alto, CA). The blots were hybridized with 32p_ ⁇ a belled probes prepared from a 805 bp BamHI-Apal fragment of HG07. Hybridizations were carried out using the ExpressHyb buffer supplied by Clontech under conditions suggested by Clontech. The blots were then washed in 0.1X SSC/0.1% SDS at 60°C and exposed by autoradiography.

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• 1999
  • 1999-11-08 JP JP2000582410A patent/JP2003532371A/ja not_active Withdrawn
  • 1999-11-08 EP EP99964968A patent/EP1129103A1/en not_active Withdrawn
  • 1999-11-08 CA CA002351874A patent/CA2351874A1/en not_active Abandoned
  • 1999-11-08 WO PCT/US1999/026303 patent/WO2000029423A1/en not_active Application Discontinuation

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