WO2004015427A2 - Methodes diagnostiques et therapeutiques s'adressant a des maladies associees au recepteur 19 couple a la proteine g (gpr19) - Google Patents

Methodes diagnostiques et therapeutiques s'adressant a des maladies associees au recepteur 19 couple a la proteine g (gpr19) Download PDF

Info

Publication number
WO2004015427A2
WO2004015427A2 PCT/EP2003/008142 EP0308142W WO2004015427A2 WO 2004015427 A2 WO2004015427 A2 WO 2004015427A2 EP 0308142 W EP0308142 W EP 0308142W WO 2004015427 A2 WO2004015427 A2 WO 2004015427A2
Authority
WO
WIPO (PCT)
Prior art keywords
gpr19
diseases
polypeptide
disorders
cancer
Prior art date
Application number
PCT/EP2003/008142
Other languages
English (en)
Other versions
WO2004015427A3 (fr
Inventor
Stefan Golz
Ulf Brüggemeier
Holger Summer
Original Assignee
Bayer Healthcare Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Healthcare Ag filed Critical Bayer Healthcare Ag
Priority to AU2003250165A priority Critical patent/AU2003250165A1/en
Publication of WO2004015427A2 publication Critical patent/WO2004015427A2/fr
Publication of WO2004015427A3 publication Critical patent/WO2004015427A3/fr

Links

Classifications

    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • 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 present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human GPR19 and its regulation for the treatment of hematological diseases, cancer, cardio- vascular diseases and disorders of the peripheral and central nervous system in mammals.
  • GPR19 is a seven transmembrane G protein coupled receptor (GPCR) [O'Dowd BF et al, (1996), Montpetit A.and Colltt D., (1999), EP 1130094, WO 200022129]. Many medically significant biological processes are mediated by signal transduction pathways that involve G-proteins [Lef owitz, (1991)].
  • GPCRs G-protein coupled receptors
  • the family of G-protein coupled receptors (GPCRs) includes receptors for hormones, neurotransmitters, growth factors, and viruses.
  • GPCRs include receptors for such diverse agents as dopamine, calcitonine, adrenergic hormones, endotheline, cAMP, adenosine, acetylcholine, serotonine, histamine, thrombin, kinine, follicle stimulating hor- mone, opsins, endothelial differentiation gene-1, rhodopsins, odorants, cytomegalovirus, G-proteins themselves, effector proteins such as phospholipase C, adenyl cy- clase, and phosphodiesterase, and actuator proteins such as protein kinase A and protein kinase C.
  • GPCRs possess seven conserved membrane-spanning domains connecting at least eight divergent hydrophilic loops. GPCRs, also known as seven transmembrane, 7TM, receptors, have been " characterized as including these seven conserved hydro- phobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most GPCRs have single conserved cysteine residues in each of the first two extracellular loops, which form disulfi.de bonds that are believed to sta- bilize functional protein structure. The seven transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 is being implicated with signal transduction.
  • Phosphorylation and lipidation (palmirylation or farnesylation) of cysteme residues can influence signal transduction of some GPCRs.
  • Most GPCRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus.
  • GPCRs such as the beta-adrenergic receptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
  • the ligand binding sites of GPCRs are believed to comprise hy- drophilic sockets fonned by several GPCR transmembrane domains.
  • the hydrophilic sockets are surrounded by hydrophobic residues of the GPCRs.
  • the hydrophilic side of each GPCR transmembrane helix is postulated to face inward and form a polar ligand binding site.
  • TM3 is being implicated with several GPCRs as having a ligand binding site, such as, the TM3 aspartate residue.
  • TM5 serines, a TM6 aspara- gine, and TM6 or TM7 phenylalanines or tyrosines also are implicated in ligand binding.
  • GPCRs are coupled inside the cell by heterotrimeric G-proteins to various intracellular enzymes, ion channels, and transporters. Different G-protein alpha-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of GPCRs is an important mechanism for the regulation of some GPCRs.
  • the effect of hormone binding is the activation of the enzyme, adenylate cy- clase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding.
  • a G-protein connects the hormone receptor to adenylate cyclase.
  • G-protein exchanges GTP for bound GDP when activated by a hormone receptor.
  • the GTP-carrying form then binds to activated adenylate cyclase.
  • Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form.
  • the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
  • TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle
  • CT fluorescence above background
  • Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action.
  • the TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue.
  • Expression profil- ing has been used in identifying genes, which are up- or downregulated in a variety of diseases.
  • An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drug treatment or in patients versus healthy individuals.
  • the premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets.
  • physiological or environmental stimuli e.g., drugs
  • the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.
  • GPR19 is accessible in public databases by the accession number NM_006143 and is given in SEQ ID NO:l.
  • the amino acid sequence of GPR19 is depicted in SEQ ID NO:2.
  • GPR19 is described as an orphan receptor [O'Dowd BF et al, (1996)] .
  • the receptor
  • GPR19 is published in [O'Dowd BF et al., (1996)], [Montpetit A.and Colltt D., (1999)], EP 1130094, WO 200022129. It was shown that GPR 19 maps to the chromosome 12pl2.3 region frequently rearranged in cancer cells [Montpetit A. and Colltt D., (1999)]. The expression of GPR19 in caudate nucleus, putamen thalamus and frontal cortex, hypothlamus and pituitary was previously described [O'Dowd BF et al, (1996)]. GPR19 is also known as G protein-coupled receptor GPR-NGA.
  • the invention relates to novel disease associations of GPR19 polypeptides and poly- nucleotides.
  • the invention also relates to novel methods of screening for therapeutic agents for the treatment of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal.
  • the invention also relates to pharmaceutical compositions for the treatment of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising a GPR19 polypeptide, a GPR19 polynucleotide, or regulators of GPR19 or modulators, of GPR19 activity.
  • the invention further comprises methods of diagnosing hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal.
  • Fig. 1 shows the nucleotide sequence of a GPR19 receptor polynucleotide (SEQ ID NO:l).
  • Fig. 2 shows the amino acid sequence of a GPR19 receptor polypeptide (SEQ ID NO:2).
  • Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:3).
  • Fig. 4 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:4).
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ ID NO:5).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double- stranded nucleic acids or may be chemically synthesized. They are useful in detect- ing the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • Chimeric molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following GPR19 characteristics: cellular location, distribution, ligand-binding affinities, interchain affini- ties, degradation/turnover rate, signaling, etc.
  • GPR19 polypeptide refers to those forms, fragments, or domains of a GPR19 polypeptide which retain the biological and/or antigenic activity of a GPR19 polypeptide.
  • Naturally occurring GPR19 polypeptide refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • “Derivative” refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins. '
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide tlirough a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide.
  • Ohgopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic ac- tivity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists. "Standard expression” is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use- as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used.herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • domestic e.g., cats, dogs, etc.
  • agricultural e.g., cows, horses, sheep, etc.
  • test species e.g., mouse, rat, rabbit, etc.
  • GPR19 polynucleotide within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a. group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ ID NO: 1,
  • nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii);
  • nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code;
  • polypeptide encoded by said nucleic acid molecule has GPR19 activity.
  • GPR19 polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • polypeptides having the sequence of SEQ ID NO: 2 (i) polypeptides having the sequence of SEQ ID NO: 2, (ii) polypeptides comprising the sequence of SEQ ID NO : consult 2 j
  • polypeptides which show at least 99%, 98%, 95%, .90%, or 80% homology with a polypeptide of (i), (ii), or (iii);
  • polypeptide has GPR19 activity.
  • nucleotide sequences encoding a GPR19 have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of GPR19, and use in generation of antisense DNA or RNA, their chemi-
  • nucleotides encoding a GPR19 disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • nucleotide sequences which encode a GPR19, its derivatives or its vari- ants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring GPR19 polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding GPR19 polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a GPR19 polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to GPR19 polynucleotides include an assortment of cloning vectors such as plasmids, c ⁇ smids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for GPR19-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding GPR19.
  • Such probes may also be used for the detection of similar GPCR encoding sequences and should preferably show at least 40% nucleotide identity to
  • hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ ID NO: 1 or from genomic sequences including promoter; enhancers or introns of the native gene.
  • Hybridization probes may be . labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such GPR19 encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity. Hybridization conditions and probes can be adjusted in well- characterized ways to achieve selective hybridization of human-derived probes. Stringent conditions, within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7 % (w/v) SDS.
  • Nucleic acid molecules that will hybridize to GPR19 polynucleotides under stringent conditions can be identified functionally.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express GPR19; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of GPR19; and detecting poly- morphisms of GPR19.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes GPR19.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise dis- crete nucleotide sequences employed under optimized conditions for identification of
  • GPR19 in specific tissues or diagnostic use.
  • the same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • PCR primers i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with GPR19.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence; For example, appropriate nucleic acid " 10 primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.
  • PCR methods for amplifying nucleic acid will utilize at least two primers. One of 15 . these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction. The other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the Gi ⁇ t amplification cycle) and of priming nu- 20 cleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer. Conditions for conducting such amplifications, particularly under preferred stringent hybridization conditions; are well known.
  • nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are .well known in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • GPR19 polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • Chromosome-based techniques such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be narrowed further using loss of heterozygosity analysis (LOH), in which disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker.
  • LHO loss of heterozygosity analysis
  • the first experiments used re- striction- fragment length polymorphisms (RFLPs) [Johnson, (1989)], or hyper- variable minisatellite DNA [Barnes, 2000].
  • a gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization.
  • This approach suffers from several drawbacks.
  • the method requires that each sample has equal input amounts of the nucleic acid and that the amplification efficiency between sam- pies is identical until the time of analysis.
  • it is difficult using the conventional methods of PCR quantitation such as gel electrophoresis or plate capture hybridization to determine that all samples are in fact analyzed during the log phase of the reaction as required by the method.
  • Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product.
  • the basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently referred to in the literature simply as the "TaqMan method" [Pia- tak,(1993), Science; Heid, (1996); Gibson, (1996); Holland. (1991)].
  • the probe used. in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes.
  • the 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well.
  • the probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fiuorophors occurs and the quencher quenches emission from the reporter.
  • the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby re- leasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector.
  • a nucleic acid polymerase such as Taq polymerase
  • One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 or 4700 HT manu- factored by Applied Biosystems, Inc. in Foster City, Calif.
  • the ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube arrangement.
  • the instrument includes a laser for exciting the labels and is capable of measuring the fluorescence spectra intensity from each tube with continuous monitoring during PCR amplification. Each tube is re-examined every 8.5 seconds.
  • Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis.
  • the increase in emission intensity is plot- ted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification.
  • the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which- each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.
  • the fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.
  • a variety of options are available for measuring the amplification products as they. are formed.
  • One. method utilizes labels, such as dyes, which only bind to double stranded DNA.
  • amplification product which is double stranded
  • dyes it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product.
  • certain dyes fluoresce only when bound to amplification product. Examples of dyes which can be used in methods of this general type include, but are not limited to, Syber Green. TM. and Pico Green from Molecular Probes, Inc.
  • These detection methods involve some alteration to the structure or conformation of a probe hybridized to, the locus between the amplification primer pair.
  • the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process.
  • the alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.
  • some methods involve the degradation or digestion of the probe during the extension reaction. These methods are a consequence of the 5'-3' nuclease activ- ity associated with some nucleic acid polymerases. Polymerases having this activity cleave mononucleotides or small oligonucleotides from an oligonucleotide probe annealed to its complementary sequence located within the locus.
  • the 3' end of the upstream primer provides the initial binding site for the nucleic acid polymerase.
  • the nucleic acid polymerase displaces a portion of the 5' end of the ' probe and through its nuclease. activity cleaves mononucleotides or oligonucleotides from the probe.
  • the upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. In the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe.
  • the 5'-3' exonuclease activity of the nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.
  • the two end sections can hybridize with each other to form a hairpin loop.
  • the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye.
  • Hybridized probe in contrast, results in a linearized conformation in which the extent of quenching is decreased.
  • the labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.
  • the primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified.
  • the primers are cho- sen to have at least substantial complementarity with the different strands of the nucleic acid being amplified.
  • the primers are selected in such that they flank the probe, i.e. are located upstream and downstream of the probe.
  • the primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization.
  • the length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe.
  • the primer typically includes 15-30 nucleotides.
  • the length of the primer may be more or less depending on the. complexity of the primer binding site and the factors listed above.
  • the labels used for labeling the probes or primers of the current invention and which can provide the signal corresponding to the quantity of amplification product can take a variety of forms. As indicated above with regard to the 5' fluorogenic nuclease method, a fluorescent signal is one signal which can be measured. However, measurements may also be made, for example, by monitoring radioactivity, colorimetry, absorption, magnetic parameters, or enzymatic activity. Thus, labels which can be employed include, but are not limited to, fiuorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).
  • a number of labels useful for attachment to probes or primers are commercially available including fluorescein and various fluorescein derivatives such as FAM, HEX, TET and JOE (all which are available from Applied Biosystems, Foster City, Calif); lucifer yellow, and coumarin derivatives.
  • Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide.
  • the label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer. In some instances, a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe.
  • the probe includes multiple labels
  • a number of diseases are associated with changes in the copy number of a certain gene.
  • the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.
  • Fusion proteins are useful for generating antibodies against GPR19 polypeptides and for use in various assay systems; For example, fusion proteins can be used to identify proteins which interact with portions of GPR19 polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a GPR19 fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment can comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, or 275 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length GPR19.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), autofiuorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, NSN- G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag,' Lex a D ⁇ A binding domain (DBD) fusions, GAL4 D ⁇ A binding domain fusions, herpes simplex virus (HSN) BPI 6 protein fusions and
  • G-protein fusions for example G(alpha)16, Gs, Gi.
  • a fusion protein also can be engineered to contain a cleavage site located adjacent to the GPR19.
  • GPR19 polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated GPR19 polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise GPR19 nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • GPR19 cD ⁇ A molecules can be made with standard molecular biology techniques, using GPR19 mR ⁇ A as a template. GPR19 cD ⁇ A molecules can thereafter be replicated using molecular biology techniques known in the art.
  • An amplification tech- nique such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic D ⁇ A or cD ⁇ A as a template.
  • synthetic chemistry techniques can be used to synthesizes GPR19 polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode GPR19 having, for example, an amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof.
  • PCR-based methods can be used to extend nucleic acid sequences encoding human GPR19, for example to detect upstream sequences of GPR19 gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and liga- tions also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser acti- vated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially pref- erable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • GPR19 can be obtained, for example, by purification from human cells, by expression of GPR19 polynucleotides, or by direct chemical synthesis.
  • GPR19 can be purified from any human cell which expresses the receptor, including those which have been transfected with expression constructs which express GPR19.
  • a purified GPR19 is separated from other compounds which normally associate with
  • GPR19 in the cell such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion
  • GPR19 polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding GPR19 . and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo ge- netic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding GPR19. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMN; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMN;
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host util- ized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacte- rial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La oUa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La oUa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e..g, heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding GPR19, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected.
  • vectors which direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • BLUESCRIPT a sequence encoding GPR19 can be ligated into the vector in frame with se- quences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pIN vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads fol- lowed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • sequences encoding GPR19 can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV ca be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • GPR19 An insect system also can be used to express GPR19.
  • Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding GPR19 can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • GPR19 Successful insertion of GPR19 will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which GPR19 can be expressed.
  • a number of viral-based expression systems can be used to express GPR19 in mammalian host cells.
  • sequences encoding GPR19 can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing GPR19 in infected host cells [Engelhard, 1994)].
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding GPR19. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding GPR19, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the
  • ATG initiation codon should be provided.
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed GPR19 in the desired fashion.
  • modifications ' of the polypeptide include, but are not limited to, acetylation, car- boxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post- translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express GPR19 can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective me- dium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced GPR19 sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems can be used to recover transformed cell lines.
  • herpes include, but are not limited to, the herpes, simplex virus thymidine kinase [Logan, (1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in tk ⁇ or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the amino glycosides, neomy- cin and G-418 [Wigler, (1980)]
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981]. Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • marker gene expression suggests that a GPR19 polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding GPR19 is inserted within a marker gene sequence, transformed cells containing sequences which encode GPR19 can be identi- fied by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding GPR19 under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of GPR19 polynucleotide.
  • host cells which contain a GPR19 polynucleotide and which express GPR19 can be identified by a variety of procedures known to those of skill in the art.
  • DNA-DNA or DNA-RNA " hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding GPR19.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding GPR19 to detect transformants which contain a GPR19 polynucleotide.
  • a variety of protocols for detecting and measuring the expression of GPR19, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioim- munoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioim- munoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non- interfering epitopes on GPR19 can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to poly-, nucleotides encoding GPR19 include oligo labeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding GPR19 can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with GPR19 polynucleotides can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the poly- peptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing GPR19 polynucleotides can be de- signed to contain signal sequences which direct secretion of soluble GPR19 through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound GPR19.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS exten- sion/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and GPR19 also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing GPR19 and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by
  • Sequences encoding GPR19 can be synthesized, in whole or in part, using chemical methods well known in the art.
  • GPR19 itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of GPR19 can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic GPR19 can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of GPR19 can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a vari- ant polypeptide or a fusion protein.
  • GPR19 polynucleotides possessing non-naturally occurring codons.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter GPR19 polynucleotides for a variety, of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of GPR19.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of GPR19.
  • Fab fragments thereof
  • F(ab') 2 fragments thereof
  • Fv fragments thereof
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid.
  • An antibody which specifically binds to an epitope of GPR19 can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, im- munohistochemical assays, immunoprecipitati ⁇ ns, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, im- munohistochemical assays, immunoprecipitati ⁇ ns, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradi- ometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation, between an immunogen and an antibody which specifically binds to the GPR19 immunogen.
  • an antibody which specifically binds to GPR19 provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to GPR19 do not detect other proteins in immunochemical assays and can im- munoprecipitate GPR19 from solution.
  • GPR19 can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • GPR19 can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides,. oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • BCG Bacilli Calmette-Gueri ⁇
  • Monoclonal antibodies which specifically bind to GPR19 can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma tech- nique [Roberge, (1995)].
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing resi- dues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining, regions.
  • Antibodies wliich specifically bind to GPR19 can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to GPR19.
  • Antibodies with related specificity, but of distinct idio- typic composition can be generated by chain shuffling from random combinatorial , immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispecific,. and can be bivalent or tetrava- lent. Construction of tetravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding se- quence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to GPR19 also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin li- braries or panels of highly specific binding reagents.
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antibodies according to i s invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which GPR19 is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of GPR19 gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of an- other nucleotide with non-phosphodiester internucleotide linkages such alkyl- phosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, al- kylphosphohates, phosphoramidates, phosphate esters, carbamates, acetamidate, car- boxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of GPR19 gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the GPR19 gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature [Nicholls, (1993)].
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the -complementary sequence of a GPR19 polynucleotide.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a GPR19 polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent GPR19 nucleotides, can provide sufficient targeting specificity for GPR19 mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an an- tisense-sense pair. to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular GPR19 polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a GPR19 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'- substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)]. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribo- zyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a GPR19 polynucleo- tide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a GPR19 polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete
  • hybridization region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a GPR19 RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features wliich may render the target inoperable. Suitability of candidate GPR19 RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NO: 1 and its complement provide sources of suitable hybridization region sequences.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease GPR19 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA oc- curs only when both a ribozyme and a target gene are induced in the cells.
  • Regulators as used herein refer to compounds that affect the activity of a GPR19 in vivo and/or in vivo. Regulators can be agonists and antagonists of a GPR19 polypeptide and can be compounds that exhert their effect on the GPR19 activity via the expression, via post-translational modifications or by other means.
  • Agonists of GPR19 are molecules which, when bound to GPR19, increase or prolong the activity of GPR19.
  • Agonists of GPR19 include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate GPR19.
  • Antagonists of GPR19 are molecules wliich, when bound to GPR19, decrease the amount or the duration of the activity of GPR19. Antagonists include proteins, nucleic acids, carbohydrates, anti- bodies, small molecules, or any other molecule which decrease the activity of
  • modulate refers to a change in the activity of GPR19 polypeptide. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunologi- cal properties of GPR19.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • the invention provides methods (also referred to herein as “screening assays") for identifying compounds which can be used for the treatment of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the methods 0 entail the identification of candidate or test compounds or agents (e.g., peptides, pep- tidomimetics, small molecules or other molecules) which bind to GPR19 and/or have a stimulatory or inhibitory effect on the biological activity of GPR19 or its expression and then determining which of these compounds have an effect on symtoms or diseases regarding the hematological and cardiovascular diseases, disorders of the 5 peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases in an in vivo assay.
  • candidate or test compounds or agents e.g., peptides, pep- tidomimetics, small molecules or other molecules
  • Candidate or test compounds or agents which bind to GPR19 and/or have a stimulatory or inhibitory effect on the activity or the expression of GPR19 are identified 0 either in assays that employ cells which express GPR19 on the cell surface (cell- based assays) or in assays with isolated GPR19 (cell-free assays).
  • the various assays can employ a variety of variants of GPR19 (e.g., full-length GPR19, a biologically active fragment of GPR19, or a fusion protein which includes all or a portion of GPR19).
  • GPR19 can be derived from any suitable mammalian species 5 (e.g., human GPR19, rat GPR19 or murine GPR19).
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known GPR19 ligand to GPR19.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of GPR19.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of GPR19 0 mRNA or GPR19 protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symtoms of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a membrane-bound (cell surface expressed) form of GPR19.
  • Such assays can employ full-length GPR19, a biologically active fragment of GPR1-9, or a fusion protein which includes all or a portion of GPR19.
  • the test compound can be ob- tained by any suitable means, e.g., from conventional compound libraries. Determining the ability of the test compound to bind to a membrane-bound form of GPR19 can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the GPR19 -expressing cell can be.
  • the test compound can be labelled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmis- sion or by scintillation counting.
  • the test compound can be enzymati- cally labelled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting GPR19 expressing cell with a known compound which binds to GPR19 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the GPR19 expressing cell, wherein determining the ability of the test compound to interact with the GPR19 expressing cell comprises determining the ability of the test compound to preferentially bind the GPR19 expressing cell as compared to the known compound.
  • the assay is a cell-based assay comprising contacting a cell
  • a membrane-bound form of GPR19 e.g., full-length GPR19, a biologi- cally active fragment of GPR19, or a fusion protein which includes all or a portion of GPR19
  • a test compound e.g., a membrane-bound form of GPR19
  • determining the ability of the test compound to modulate e.g., stimulate or inhibit
  • Determining the ability of the test compound to modulate the activity of the membrane-bound form of GPR19 can be accomplished by any method suitable for measuring the activity of GPR19, e.g., any method suitable for measuring the activity of a G-protein coupled receptor or other seven- transmembrane receptor (described in greater detail below).
  • the activity of a seven- transmembrane receptor can be measured in a number of ways, not all of which are suitable for any given receptor. Among the measures of activity are: alteration in intracellular Ca 2+ concentration, activation of phospholipase C, alteration in intracellular inositol triphosphate (_P 3 ) concentration, alteration in intracellular diacyl- glycerol (DAG) concentration, and alteration in intracellular adenosine cyclic 3', 5'- onophosphate (cAMP) concentration.
  • Determining the ability of the test compound to modulate the activity of GPR19 can be accomplished, for example, by determining the ability of GPR19 to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which GPR19 binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses GPR19, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • the target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a GPR19 ligand, through the cell membrane and into the cell.
  • the target GPR19 molecule can be, for example, a second intracellular protein wliich has catalytic activity or a protein which facilitates the association of downstream signaling molecules with GPR19.
  • Determining the ability of GPR19 to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding.
  • determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determimng the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, IP 3 , etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • the present invention also includes cell-free assays.
  • assays involve contacting a form of GPR19 (e.g., full-length GPR19, a biologically active fragment of GPR19, or a fusion protein comprising all or a portion of GPR19) with a test compound and determining the ability of the test compound to bind to GPR19. Binding of the test compound to GPR19 can be determined either directly or indirectly as described above.
  • the assay includes contacting GPR19 with a known compound which binds GPR19 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with GPR19, wherein determining the ability of the test compound to interact with GPR19 comprises determining the ability of the test compound to preferentially bind to GPR19 as compared to the known compound.
  • the cell-free assays of the present invention are amenable to use of either a membrane-bound form of GPR19 or a soluble fragment thereof.
  • a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • non-ionic detergents such as n-oc
  • GPR19 or a GPR19 target molecule
  • binding of a test compound to GPR19, or interaction of GPR19 with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reac- tants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or GPR19, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of GPR19 can be determined using standard techniques.
  • GPR19 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin- coated plates (Pierce Chemical).
  • biotinylation kit N-hydroxy-succinimide
  • streptavidin- coated plates Piereptavidin- coated plates
  • target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such- complexes include inimunodetection of complexes using antibodies reactive with GPR19 or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with GPR19 or target molecule.
  • the screening assay can also involve monitoring the expression of GPR19.
  • regulators of expression of GPR19 can be identified in a method in which a cell is contacted with a candidate compound and the expression of GPR19 protein or mRNA in the cell is determined. The level of expression of GPR19 protein or mRNA the presence of the candidate compound is compared to the level of expres- sion of GPR19 protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of GPR19 based on this comparison. For example, when expression of GPR19 protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GPR19 protein or mRNA expression.
  • the candidate compound when expression of GPR19 protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GPR19 protein or mRNA expression.
  • the level of GPR19 protein or mRNA expression in the cells can be determined by methods described below.
  • the test compound is preferably a small molecule which binds to and occupies the active site of GPR19 polypeptide, thereby making the ligand bind- ing site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • Potential ligands which bind to a polypeptide of the invention include, but are not limited to, the natural ligands of known GPR19 GPCRs and analogues or derivatives thereof.
  • either the test compound or the GPR19 polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to GPR19 polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a GPR19 polypeptide can be determined without labeling either, of the interactants.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a microphysiometer can be used to detect binding of a test compound with a GPR19 polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • Determining the ability of a test compound to bind to GPR19 also can be accom- plished using a technology such as real-time Bimolecular Interaction Analysis (BIA)
  • BIA is a technology for studying biospeci- fic interactions in real time, without labeling any of the interactants (e.g., BIA- coreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • a GPR19-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with GPR19 and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs.
  • polynucleotide encoding GPR19 can be fused to a polynucleotide encoding the DNA binding do- main of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey” or “sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait” and the “prey” proteins are able to interact in vivo to form an protein- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity.
  • reporter gene e.g., LacZ
  • a reporter gene e.g., LacZ
  • Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with GPR19.
  • either the GPR19 (or polynucleotide) or the test compound can be bound to a solid support.
  • suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach GPR19-like polypeptide (or poly- nucleotide) or test compound to a solid support, including use of covalent and non- covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to GPR19 (or a polynucleotide encoding for GPR19) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • GPR19 is a fusion protein comprising a domain that allows binding of GPR19 to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed GPR19; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • GPR19 or a polynucleotide encoding GPR19
  • test compounds can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated GPR19 or a polynucleotide encoding biotinylated GPR19
  • test compounds can be prepared from biotin-NHS
  • GST-immobilized complexes include immunodetection of complexes using anti- bodies which specifically bind to GPR19 polypeptide or test compound, enzyme- linked assays which rely on detecting an activity of GPR19 polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a GPR19 polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a GPR19 polypeptide or polynucleotide can be used in a cell-based assay system. A GPR19 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to GPR19 or a polynucleotide encoding GPR19 is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease GPR19 activity of a GPR19 polypeptide.
  • the GPR19 activity can be measured, for example, using methods described in the specific examples, below.
  • GPR19 activity can be measured after contacting either a purified GPR19, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound which decreases GPR19 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing GPR19 activity.
  • a test compound which in- creases GPR19 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing GPR19 activity.
  • such an assay may be employed for screening for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
  • the screen may be em- ployed for identifying a compound which activates the receptor by contacting such cells with compounds to be screened and determining whether each compound generates a signal, i.e., activates the receptor.
  • GPR19 for example, transfected CHO cells
  • compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g., signal transduction or pH changes, can be measured to determine whether the potential compound activates or inhibits the receptor.
  • a second messenger response e.g., signal transduction or pH changes
  • Another such screening technique involves introducing RNA encoding GPR19 into Xenopus oocytes to transiently express the receptor. The receptor oo- cytes can then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.
  • Another screening technique involves expressing GPR19 in cells in which the receptor is linked to a phospholipase C or D.
  • Such cells include endothelial cells, smooth muscle cells, embryonic kidney cells, etc.
  • the screening may be accom- pushed as described above by quantifying the degree of activation of the receptor from changes in the phospholipase activity.
  • test compounds which increase or decrease GPR19 gene expression are identified.
  • the term "correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids,, the same or related to a nucleic acid sequence encoding GPR19, by northern analysis or re- latime PCR is indicative of the presence of nucleic acids encoding GPR19 in a sam- pie, and thereby correlates with expression of the transcript from the polynucleotide encoding GPR19.
  • microarray refers to an array of dis- tinct polynucleotides or oligonucleotides arrayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • a GPR19 polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of GPR19 polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a regulator of expression based on this comparison.
  • the test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its ab- sence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • the test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of GPR19 mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of GPR19 polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labelled amino acids into GPR19.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses GPR19 polynucleotide can be used in a cell-based assay system.
  • the GPR19 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection.
  • the biological library ap- proach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds [Lam, (1997)]. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage.
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate GPR19 ex- pression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites, such as the interaction domain of the ligand with GPR19.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand.
  • the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure de- termined.
  • the methods of computer based numerical modeling can. be used to, complete the structure or improve its accuracy. Any recognized modeling method may be used, including pa- rameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These com- pounds found from this search are potential GPR19 modulating compounds.
  • these methods can be used to identify improved modulating compounds from an aheady known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition.
  • the altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • GPR19 is expressed in various human tissues.
  • CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system. .
  • CNS disorders include, but are not limited to brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.
  • Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, fron- totemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld- akob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis, within the meaning of the definition are also considered to be CNS disorders.
  • cognitive-related disorders such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities are also considered to be
  • Pain within the meaning of this definition, is also considered to be a CNS disorder. Pain can be associated with CNS disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back -surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord
  • Non-central neuropathic pain includes that associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopatliy, post-surgical pain, HIV/ AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropa- thy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia.
  • RSD reflex sympathetic dystrophy
  • Headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache tension-type like headache, cluster headache, and chronic paroxysmal hemicrania are also CNS disorders.
  • Visceral pain such as pancreatits, intestinal cystitis, dysmenorrhea, irritable Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction and pain syndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders.
  • a disorder of the nervous system are acute pain, for example postoperative pain, and pain after trauma.
  • GPR19 is highly expressed in various brain tissues such as cerebellum, cerebellum (right), cerebellum (left), postcentral gyrus, tonsilla cerebelli, Alzheimer cerebral cortex, occipital lobe, Alzheimer brain frontal lobe, temporal lobe, frontal lobe, parietal lobe, precentral gyrus, fetal brain, vermis cerebelli, cerebral cortex, hippocam- pus, cerebral peduncles and retina.
  • the expression in the above mentioned tissues suggests an association of GPR19 with nervous system diseases.
  • GPR19 can be used to treat or to diagnose diseases of the nervous system.
  • Heart failure is defmed as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable ' angina, unstable angina and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others.
  • the genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon and venous disorders.
  • Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.
  • the atherosclerotic remodeling process in- volves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.
  • the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic, debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.
  • the formation of the atherosclerotic lesion can be considered to occur in five over- lapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear.
  • Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic dis- eases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.
  • GPR19 is highly expressed in different cardiovascular related tissues such as aorta and sclerotic coronary artery. Expression in the above mentioned tissues suggests an association between GPR19 and cardiovascular diseases. GPR19 can be regulated to treat or to diagnose cardiovascular diseases.
  • Hematological disorders comprise diseases of the blood and all its constituents as well as diseases of organs and tissues involved in the generation or degradation of all the constituents of the blood. They include but are not limited to 1) Anemias, 2) Myeloproliferative Disorders, 3) Hemorrhagic Disorders, 4) Leukopenia, 5) Eosi- nophilic Disorders, 6) Leukemias, 7) Lymphomas, 8) Plasma Cell Dyscrasias, 9)
  • Disorders of the Spleen in the course of hematological disorders include, but are not limited to anemias due to defective or deficient hem synthesis, deficient erythropoiesis.
  • Disorders according to 2) include, but are not limited to polycythemia vera, tumor-associated erythrocytosis, myelofibrosis, thrombocythe- mia.
  • Disorders according to 3) include, but are not limited to vasculitis, thrombocy- topenia, heparin-induced thrombocytopenia, thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, hereditary and acquired disorders of platelet function, hereditary coagulation disorders.
  • Disorders according to 4) include, but are not limited to neutropenia, lymphocytopenia.
  • Disorders according to 5) include, but are not limited to hypereosinophilia, idiopathic hypereosinophilic syndrome.
  • Disorders according to 6) include, but are not limited to acute myeloic leukemia, acute lymphoblastic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome.
  • Disorders according to 7) include, but are not limited to Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, mycosis fungoi- des cutaneous T-cell lymphoma.
  • Disorders according to 8) include, but are not limited to multiple myeloma, macroglobulinemia, heavy chain diseases.
  • thrombocytopenic purpura iron deficiency anemia, megalo- blastic anemia (vitamin B12 deficiency), aplastic anemia, thalassemia, , malignant lymphoma bone marrow invasion, malignant lymphoma skin invasion, hemolytic uremic syndrome, giant platelet disease are considered to be hematological diseases too.
  • GPR19 is highly expressed in tissues of the hematological system such as bone marrow CD71+ cells and thrombocytes. The expression in the above mentioned -tissues suggests an association between GPR19 and hematological diseases. GPR19 can be regulated in order to treat or to diagnose hematological disorders.
  • Cancer disorders within the scope of this definition comprise any disease of an organ or tissue in mammals characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole.
  • Cancer diseases within the scope of the definition comprise benign neoplasms, dys- plasias, hyperplasias as well as neoplasms showing metastatic growth or any other transformations like e.g. leukoplakias which often precede a breakout of cancer.
  • Cells and tissues are cancerous when they grow more rapidly than normal cells, displacing or spreading into the surrounding healthy tissue or any other tissues of the body described as metastatic growth, assume abnormal shapes and sizes, show changes in their nucleocytoplasmatic ratio, nuclear polychrpmasia, and finally may cease.
  • Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cancer occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results.
  • the ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease. .
  • Cancers are generally said to be in one of three stages of growth: early, or localized, when a tumor is still confined to the tissue of origin, or primary site; direct extension, where cancer cells from the tumour have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, in which cancer cells have migrated to distant parts of the body from the primary site, via the blood or lymph systems, and have established secondary sites of infection.
  • Cancer is said to be malignant because of its tendency to cause death if not treated. Benign tumors usually do not cause death, although they may if they interfere with a normal body function by virtue of their location, size, or paraneoplastic side effects. Hence benign tumors fall under the definition of cancer within the scope of this definition as well.
  • cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue.
  • Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers of the breast and prostate, for example, are considered dependent on specific hormones for their existence.
  • cancer under the scope of the definition is not limited to simple benign neoplasia but comprises any other benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Cancers of the blood-forming tissues, 5) tumors of nerve tissues including the brain, 6) cancer of skin cells.
  • Cancer according to 1) occurs in epithelial tissues, which cover the outer body (the skin) and line mucous membranes and the inner cavitary structures of organs e.g. such as the breast, lung, the respiratory and gastrointestinal tracts, the endocrine glands, and the genitourinary system.
  • Ductal or glandular elements may persist in epithelial tumors, as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • Cancers of the pavement-cell epithelium of the skin and of certain mucous membranes, such as e.g. cancers of the tongue, lip, larynx, urinary bladder, uterine cervix, or penis, may be termed epidermoid or squamous-cell carcinomas of the respective tissues and are in the scope of the definition of cancer as well.
  • Cancer according to 2) develops in connective tissues, includ- ing fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma.
  • Cancer according to 3) is cancer that develops in both epithelial and connective tissue.
  • Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion.
  • Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal.
  • cancers and tumor diseases of I) the bone marrow and bone marrow derived cells (leukemias), II) the endocrine and exocrine glands like e.g. thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas IH) the breast, like e.g.
  • malignant osteogenic sarcoma benign osteoma, cartilage tumors; like malignant chondrosarcoma or benign chondroma; bone marrow tumors like malignant myeloma or benign eosinophilic granuloma, as well as metastatic tumors from bone tissues at other locations of the body;
  • X) the mouth, throat, larynx, and the esophagus XI) the urinary bladder and the internal and external organs and structures of the urogenital system of male and female like ovaries, uterus, cervix of the uterus, testes, and prostate gland, XII) the prostate, XIII) the pancreas, like ductal carcinoma of the pancreas;
  • XIV) the lymphatic tissue like lymphomas and other tumors of lymphoid origin, XV) the skin, XVI) cancers and tumor diseases of all anatomical structures belonging to the respiration and respiratory systems including tho-
  • GPR19 receptor is highly expressed in different cancer tissues such as breast, lung and ovary cancer. The expression in the above mentioned tissues suggests an association between GPR19 and cancer. GPR19 can be regulated and measured in order to diagnose and treat cancer.
  • the present invention provides for both prophylactic and therapeutic methods for hematological diseases, cancer, cardiovascular diseases and disorders of the periph- eral and central nervous system.
  • the regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of GPR19.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally- occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or any small molecule.
  • the agent stimulates one or more of the biological activities of GPR19. Examples of such stimulatory agents include the active GPR19 and nucleic acid molecules encoding a portion of GPR19.
  • the agent inhibits one or more of the biological activities of GPR19. Ex- amples of such inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of GPR19 or a protein in the GPR19 signaling pathway.
  • the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of GPR19 or of any protein in the GPR19 signaling pathway.
  • the method involves administering a regulator of GPR19 as therapy to compensate for reduced or undesirably low expression or activity of GPR19 or a protein in the GPR19 signaling pathway.
  • Stimulation of activity or expression of GPR19 is desirable in situations in which activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity or expression of GPR19 is desirable in situations in which activity or expression of GPR19 is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • compositions suitable for admimstration can be incorporated into pharmaceutical compositions suitable for admimstration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
  • Supplementary active compounds can also be incorporated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of GPR19 expression or activity (and or a regulator of the activity or expression of a protein in the GPR19 signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically accept- able carrier.
  • pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use.
  • the instructions would specify use of the pharmaceutical composition for treatment of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the instructions would specify use of the pharmaceutical composition for treatment of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disor- ders and inflammation diseases.
  • An antagonist of GPR19 may be produced using methods which are generally known in the art.
  • purified GPR19 may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind GPR19.
  • Antibodies to GPR19 may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially prefereed for therapeutic use.
  • the polynucleotides encoding GPR19 may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding GPR19 may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding GPR19.
  • complementary molecules or fragments may be used to modulate GPR19 activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding GPR19.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing GPR19 in conjunction with a pharmaceutically ac- ceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of GPR19, antibodies to GPR19, and mimetics, agonists, antagonists, or inhibitors of GPR19.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical car- rier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of admimstration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), trans- dermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of ' tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporane- ous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremo- phor EMTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy sy- ringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • iSotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent wliich delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional de- sired ingredient from a previously sterile- filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propel- lant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can-also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. . .
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable,, biocompatible polymers can be used, such as ethylene vinyl acetate, poly- anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound cal- culated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the instructions for administration will specify use of the composition for hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the instructions for administration will specify use of the composition for hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the instructions for administration will specify use of the composition for hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • antibodies which specifically bind GPR19 may be used for the diagnosis of disorders characterized by the expression of GPR19, or in assays to monitor patients being treated with GPR19 or agonists, antagonists, and inhibitors of
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for GPR19 include methods which utilize the antibody and a label to detect GPR19 in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • GPR19 A variety of protocols for measuring GPR19, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of
  • GPR19 expression Normal or standard values for GPR19 expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to GPR19 under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, preferably by photometric means. Quantities of GPR19 expressed in subject samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding GPR19 may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of GPR19 may be conelated with disease.
  • the diagnos- ⁇ tic assay may be used to distinguish between absence, presence, and excess expression of GPR19, and to monitor regulation of GPR19 levels during therapeutic intervention.
  • Polynucleotide sequences encoding GPR19 may be used for the diagnosis of hema- tological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system associated with expression of GPR19.
  • the polynucleotide sequences encoding GPR19 may be used in Southern, Northern, or dot-blot analysis, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patient biopsies to detect altered GPR19 expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding GPR19 may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding GPR19 may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation pe- . riod, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nu- cleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding GPR19 in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding GPR19, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • Another technique for drug screening which, may be used provides for high through- put screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large num- bers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with GPR19, or fragments thereof, and washed.
  • Bound GPR19 is then detected by methods well known in the art.
  • Purified GPR19 can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • G-protein coupled receptors are ubiquitous in the mammalian host and are responsi- ble for many biological functions, including many pathologies. Accordingly, it is desirable to find compounds and drugs which stimulate a G-protein coupled receptor on the one hand and which can inhibit the function of a G-protein coupled receptor on the other hand.
  • compounds which activate the G-protein coupled receptor may be employed for therapeutic purposes, such as the treatment of asthma, Parkinson's disease, acute heart failure, urinary retention, and osteoporosis.
  • compounds which activate the receptors of the present invention are useful in treating various cardiovascular ailments such as caused by the lack of pulmonary blood flow or hypertension.
  • these compounds may also be used in treating various physiological disorders relating to abnormal control of fluid and electro- lyte homeostasis and in diseases associated with abnormal angiotensin-induced al- dosterone secretion.
  • compounds which inhibit activation of the G-protein coupled receptor may be employed for a variety of therapeutic, purposes, for example, for the treatment of hypotension and/or hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological dis- orders including schizophrenia, manic excitement, depression, delirium, dementia or severe mental retardation, dyskinesias, such as Huntington's disease or Tourett's syndrome, among others.
  • Compounds which inhibit G-protein coupled receptors have also been useful in reversing endogenous anorexia and in the control of bulimia.
  • a therapeutically effective dose refers to that amount of ac- tive ingredient which increases or decreases GPR19 activity relative to GPR19 activity wliich occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and . it can be expressed as the ratio, LD 50 /ED 50 .
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation- mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of GPR19 gene or the activity of GPR19 by at least about 10, preferably about 50, more preferably about 75, 90, or
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, in- eluding, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid mole- cules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ ID NO: 1, (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1, (iv)nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, wherein the polypeptide encoded by said nucleic acid molecule has GPR19 activity.
  • Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ ID NO: 2,
  • polypeptides comprising the sequence of SEQ ID NO: 2, (iii) polypeptides encoded by nucleic acid molecules of the invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii), wherein said purified polypeptide has GPR19 activity.
  • An object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising the steps of (i) contacting a test com- pound with a GPR19 polypeptide, (ii) detect binding of said test compound to said
  • GPR19 polypeptide E.g., compounds that bind to the GPR19 polypeptide are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising the steps of (i) determining the activity of a GPR19 polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound.
  • compounds that lead to a difference in the activity of the GPR19 polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising the steps of (i) determining the activity of a GPR19 polypeptide at a certain concentration of a test compound, (ii) determining the activity of a GPR19 polypeptide at the presence of a compound known to be a regulator of a GPR19 polypeptide.
  • compounds that show similar effects on the activity of the GPR19, polypeptide in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.
  • test compound displaces a ligand which is first bound to the polypeptide.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising the steps of (i) contacting a test compound with a GPR19 polynucleotide, (ii) detect binding of said test compound to said GPR19 polynucleotide.
  • a test compound with a GPR19 polynucleotide
  • detect binding of said test compound to said GPR19 polynucleotide are potential therapeutic agents for the treatment of such diseases.
  • Another object of the invention is the method of the above, wherein the nucleic acid molecule is RNA.
  • Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.
  • Another object of the invention is a method of the above, wherein the contacting step is in a cell-free system.
  • Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.
  • Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.
  • Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular dis- eases and disorders of the peripheral and central nervous system in a mammal comprising the steps of (i) determining the amount of a GPR19 polynucleotide in a sample taken from said mammal, (ii) determining the amount of GPR19 polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in the amount of GPR19 polynucleotide in said test mammal as compared to a diseased mammal.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous sys- tern in a mammal comprising a therapeutic agent which binds to a GPR19 polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, can- cer, cardiovascular diseases and disorders of the peripheral and central nervous sys- tem in a mammal, comprising a therapeutic agent which regulates the activity of a GPR19 polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising a therapeutic agent which regulates the activity of a GPR19 polypeptide, wherein said therapeutic agent is (i) a small molecule, (ii) an RNA molecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous sys- tern in a mammal comprising a GPR19 polynucleotide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous sys- tern in a mammal comprising a GPR19 polypeptide.
  • Another object of the invention is the use of regulators of a GPR19 for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular dis- eases and disorders of the peripheral and central nervous system in a mammal.
  • Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, cancer,, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal comprising the steps of (i) identifying a regulator of GPR19, (ii) determining whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical carrier.
  • Another object of the invention is the use of a regulator of GPR19 for the regulation of GPR19 activity in a mammal having a disease comprised in a group of diseases consisting of hematological diseases, cancer, cardiovascular diseases and disorders of the peripheral and central nervous system.
  • Example 1 Search for homologous sequences in public sequence data bases
  • the degree of homology can readily be calculated by known methods. Preferred methods to determine homology are, designed to give the largest match between the sequences tested. Methods to determine homology are codified in publicly available computer programs such as BestFit, BLASTP, BLASTN, and FASTA. The BLAST programs are publicly available from NCBI and other sources in the internet.
  • RNA from each cell or tissue source was first reverse transcribed. 85 g of total RNA was reverse transcribed using 1 mole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Gronin- gen, Netherlands) in a final volume of 680 1.
  • the first strand synthesis buffer and Omniscript reverse transcriptase (2 u/ 1) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 1 with water, yielding a final concentration of 12.5 ng/ 1 of starting RNA. •
  • GPR19 For relative quantitation of the distribution of GPR19 mRNA in cells and tissues the Applied Biosystems 7900 HT Sequence Detection system or Biorad iCycler was used according to the manufacturer's specifications and protocols. PCR reactions were set up to quantitate GPR19 and the housekeeping genes HPRT (hypoxanthine phosphoribosyltransferase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), -actin, and others. Forward and reverse primers and probes for GPR19 were designed using the Perkin Elmer ABI Primer ExpressTM software and were synthesized by TibMolBiol (Berlin, Germany). The GPR19 forward primer sequence was:
  • Probel SEQ ID NO: 4
  • FAM carboxyfluorescein suc- cinimidyl ester
  • TAMRA carboxytetramethylrhodamine
  • the following reagents were prepared in a total of 25 1 : lx TaqMan buffer A, 5.5 mM MgCl 2 , 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/ 1 AmpliTaq GoldTM, 0.01 U/ 1 AmpErase and Probel (SEQ ID NO: 4), GPR19 forward and reverse primers each at 200 nM, 200 nM GPR19 FAM/TAMRA-labelled probe, and 5 1 of template cDNA.
  • Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min.
  • the CT (threshold cycle) value is calculated as described in the "Quantitative deter- mination of nucleic acids" section.
  • the CF- value (factor for threshold cycle correction) is calculated as follows : 1. . PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.
  • CT H K G - values were calculated as described in the "Quantitative determination of nucleic acids" section.
  • CT HKG - n -mean value (CTH KG I -value + CTHKG2-value +... + CT HKG - n -value) / n
  • CTp anne i mean value (CT mean value of all HKG in all tested cDNAs)
  • CT C DNA-n CT value of the tested gene for the cDNA n
  • CF CDNA - ⁇ correction factor for cDNA n
  • CT cor-cDNA-n corrected CT value for a gene on cDNA n
  • highest CT cor- cDNA-n ⁇ 40 is defined as CT CO ⁇ -C DNA [high]
  • GPR19 The expression of GPR19 was investigated in the following tissues: cerebellum, cerebellum (right), cerebellum (left), postcentral gyrus, tonsiUa cerebelli , Alzheimer cerebral cortex, occipital lobe, Alzheimer brain frontal lobe, temporal lobe, frontal lobe, parietal lobe, precentral gyrus, fetal brain, vermis cerebelli, cerebral cortex, hippocampus, cerebral peduncles, retina, aorta, corpus callosum, lung tumor, spinal cord, brain, Alzheimer brain, bone marrow CD71+ cells, pons, cerebral meninges, penis, thrombocytes, ovary tumor, rectum, coronary artery sclerotic, thalamus, neu- roblastoma SH5Y cells, heart ventricle (left), breast, artery, testis, dorsal root ganglia, liver liver cirrhosis, vein, ileum chronic inflammation
  • Table 1 Relative expression ofGPR19 in various human tissues.
  • cerebral meninges 309 penis 286 thrombocytes 282 ovary tumor 260 rectum 232 coronary artery sclerotic 231 thalamus 228 neuroblastoma SH5Y cells 212 heart ventricle (left) 207 breast 199 artery 184 testis 176 dorsal root ganglia 124 liver liver cirrhosis ⁇ 123 vein 117 ileum chronic inflammation 95 aorta sclerotic 92 breast tumor 83 thymus 82 lymphnode 80 neuroblastoma IMR32 cells 70 neuroblastoma SK-N-MC cells 70 stomach tumor 64 cord blood CD71+ cells 63
  • HEP G2 cells 62 heart atrium (left) 58 fetal kidney 58 ileum 56
  • HUNEC cells 36 small intestine 34 uterus 31 stomach 30 fetal liver 28 leukocytes (peripheral blood) 26 kidney tumor 25 fetal heart 25 adrenal gland 24 uterus tumor 24 esophagus tumor 22 bone marrow 22 pericardium 20 liver 19 coronary artery smooth muscle primary cells 19 fetal lung 18 pancreas 18 skin 17 mammary gland 17 ileum tumor 15 lung 14
  • Jurkat T-cells 14 salivary gland 13 interventricular septum 13 trachea 12
  • HeLa cells (cervix tumor) 11 colon 11 heart 11 pancreas liver cirrhosis 10 bone marrow-CD 15+ cells 9 placenta 9 heart atrium (right) 8 adipose 7 prostate BPH 6 thyroid tumor 6 spleen liver cirrhosis 6 bone marrow CD34+ cells 5 fetal aorta 4 cervix A thyroid 3 kidney 3 fetal lung fibroblast IMR-90 cells 3 bladder 2 coronary artery 1
  • modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to transacting regulatory genes.
  • GPR19 is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli.
  • the vector is engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of ⁇ -galactosidasel
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • IPTG Isopropyl- -D-thio- galactopyranoside
  • the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease III or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the GPR19 cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts. Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR.
  • the resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., in- sect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metal- lothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced GPR19 are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • GPR19 can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofec- tamine (Gibco BRL catolog no. 18324-020) mediated gene transfer.
  • GPR19 is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension affinity purification system (Immu- nex Corp., Seattle, Washington).
  • the inclusion of a cleavable linker sequence such as Factor Xa or enterokinase (Invitrogen, Groningen, The Netherlands) between the purification domain and the GPR19 sequence is useful to facilitate expression of GPR19.
  • Functional chimeric GPCRs are constructed by combining. the extracellular receptive sequences of a new isoform with the transmembrane and intracellular segments of a known isoform for test purposes. This concept was demonstrated by Kobilka et al. (1988), Science 240:1310-1316) who created a series of chimeric ⁇ 2- ⁇ 2 adrenergic receptors (AR) by inserting progressively greater amounts of ⁇ 2-AR transmembrane sequence into ⁇ 2-AR.
  • the binding activity of known agonists changed as the molecule shifted from having more ⁇ 2 than ⁇ 2 conformation, and intermediate constructs demonstrated mixed specificity.
  • the specificity for binding antagonists however, correlated with the source of the domain VII.
  • T7G domain VH for ligand recognition was also found in chimeras utilizing two yeast ⁇ -factor receptors and is significant because the yeast receptors are classified as miscellaneous recep- tors. Thus, functional role of specific domains appears to be preserved throughout the GPCR family regardless of category.
  • Chimeric or modified GPCRs containing substitutions in the extracellular and transmembrane regions have shown that these portions of the receptor determine ligand binding specificity.
  • two Serine residues conserved in domain N of all adrenergic and D catecholainine GPCRs are necessary for potent agonist activity. These serines are believed to form hydrogen bonds with the catechol moiety of the agonists within the GPCR binding site.
  • an Asp residue present in domain III of all GPCRs which bind biogenic amines is believed to form an ion pair with the ligand amine group in the GPCR binding site.
  • GPCRs are expressed in heterologous expression systems and their biological activity assessed.
  • One heterologous system introduces genes for a mammalian GPCR and a mammalian G-protein into yeast cells.
  • the GPCR is shown to have appropriate ligand specificity and affinity and trigger appropriate biological activation (growth arrest and morphological changes) of the yeast cells.
  • An alternate procedure for testing chimeric receptors is based on the procedure utilizing the purinergic receptor (P u). Function is easily tested in cultured K562 human leukemia cells because these cells lack P 2 u receptors.
  • K562 cells are transfected with expression vectors containing either normal or chimeric P 2 u and loaded with fura-a, fluorescent probe for Ca . Activation of properly assembled and functional P 2 u receptors with extracellular UTP or ATP mobilizes intracellular Ca "1-1" which reacts with fura-a and is measured spectrofluorometrically.
  • chimeric genes are created by combining sequences for extracellular receptive segments of any new GPCR polypeptide with the nucleotides for the transmembrane and intracellular segments of the . known P 2 u molecule. Bathing the transfected K562 cells in micro wells containing appropriate ligands triggers binding and fluorescent activity defining effectors of the GPCR molecule. Once ligand and function are established, the P 2 u system is useful for defining, antagonists or inhibitors which block binding and prevent such fluorescent reactions.
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 ⁇ g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate GPR19 domain is analyzed to determine regions of high antigenicity.
  • Ohgopeptides comprising appropriate hydrophilic regions are synthe- sized and used in suitable immunization protocols to raise antibodies. The optimal
  • - amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an
  • KLH keyhole limpet hemocyanin
  • MBS M-maleimidobenzoyl-N-hydroxysuccinimide ester
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti- rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled GPR19 to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton-Dickinson, Palo Alto, CA
  • affinity purified, specific rabbit anti-mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% bovine serum albumin, (BSA), washed and incubated with supematants from hybridomas. After washing the wells are incubated with labeled GPR19 at 1 mg/ml.
  • BSA bovine serum albumin
  • Supematants with specific antibodies bind more labeled GPR19 than is detectable in the background. Then clones producing specific antibodies are expanded and subjected to two cycles of cloning at limiting dilution. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at least
  • 10 M " preferably 10 to 10 M " or stronger, are typically made by standard procedures.
  • GPR19 antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amoimt or distribution of GPR19 or downstream products of an active signaling cascade.
  • Diagnostic tests for GPR19 include methods utilizing antibody and a label to detect
  • polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like.
  • a variety of protocols for measuring soluble or membrane-bound GPR19, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmuno- assay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmuno- assay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal- based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on GPR19 is preferred, but a competitive binding assay may be employed.
  • Example 9 Purification of Native GPR19 Using Specific Antibodies
  • Native or recombinant GPR19 is purified by immunoaffinity chromatography using antibodies specific for GPR19.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH, antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway N.J.). Likewise, monoclonal antibodies are pre- pared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated Sepharose
  • Such immunoaffinity columns are utilized in the purification of GPR19 by preparing a fraction from cells containing GPR19 in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble GPR19 containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • a soluble GPR19-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of GPR19 (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and GPR19 is collected.
  • Example 10 Drug Screemng
  • This invention is particularly useful for screening therapeutic compounds by using GPR19 or binding fragments thereof in any of a variety of drug screening techniques.
  • GPR19 is a G protein coupled receptor any of the methods commonly used in the art may potentially be used to identify GPR19 ligands.
  • the activity of a G protein coupled receptor such as GPR19 can be measured using any of a variety of appropriate functional assays in which activation of the receptor results in an observable change in the level of some second messenger system, such as adenylate cy- clase, guanylylcyclase, calcium mobilization, or inositol phospholipid hydrolysis.
  • the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, are used for standard binding assays.
  • Measured for example, is the formation of complexes between GPR19 and the agent being tested.
  • the present invention provides methods of screening for drug canditates, drugs, or any other agents which affect signal transduction.
  • These methods comprise contacting such an agent with GPR19 polypeptide or a fragment thereof and assaying (i) for the presence of a complex between the agent and GPR19 polypeptide or fragment, or (ii) for the presence of a complex between GPR19 polypeptide or fragment and the cell.
  • the GPR19 polypeptide or fragment is typically labeled.
  • free GPR19 polypeptide or fragment is separated from that present in bound form, and the - amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to GPR19 or to interfere with the GPR19-agent complex.
  • Another technique for drug screening provides high throughput screening for com- pounds having suitable binding affinity to GPR19 polypeptides. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with GPR19 polypeptide and washed. Bound GPR19 polypeptide is then detected by methods well known in the art. Purified GPR19 are also coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies are used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in wliich neutralizing antibodies capable of binding GPR19 specifically compete with a test compound for binding to GPR19 polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic determinants with GPR19.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer model- ing or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to de- termine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • the inventive purified GPR19 is a research tool for identification, characterization and purification of interacting G or other signal transduction pathway proteins. Radioactive labels are incorporated into a selected GPR19 domain by various methods known in the art and used in vitro to capture interacting molecules. A preferred method involves labeling the primary amino groups in GPR19 with 125 I Bolton- Hunter reagent. This reagent has been used to label various molecules without concomitant loss of biological activity.
  • Labeled GPR19 is useful as a reagent for the purification of molecules with which it interacts.
  • membrane-bound GPR19 is covalently coupled to a chromatography column.
  • Cell-free extract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to GPR19.
  • GPR19-complex is recovered from the column, and the GPR19-binding ligand disassociated and subjected to N-terminal protein se- quencing.
  • the amino acid sequence information is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
  • antibodies are raised against GPR19, specifically monoclonal antibodies.
  • the monoclonal antibodies are screened to identify those which inhibit the binding of labeled GPR19. These monoclonal antibodies are then used therapeutically.
  • Example 13 Use and Administration of Antibodies, Inhibitors, or Antagonists
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more prefera- bly 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, its half-life and antigenic- ity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are preferred as LSTs, but organic or synthetic mole- cules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the par- ticular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.
  • Normal dosage amounts vary from 0.1 to 10 5 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art employ different formulations for different LSTs.
  • Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells.
  • Animal model systems wliich elucidate the physiological and behavioral roles of the GPR19 are produced by creating nonhuman transgenic animals in which the activity of the GPR19 is either increased or decreased, or the amino acid sequence of the expressed GPR19 is altered, by a variety of techniques.
  • these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a GPR19, by microinjection, electroporation, retro viral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these GPR19 sequences.
  • the technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and hence is useful for producing an animal that cannot express native GPR19s but does express, for example, an inserted mutant GPR19, which has replaced the native GPR19 in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added GPR19, resulting in overex- pression ofthe GPR19.
  • transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesium- chloride M2 medium. DNA or cDNA encoding GPR19 is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • micro- injection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected is put in a depression slide.
  • the needle is inserted into the pro- nucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transferred into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term.
  • microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un GPR19 humain associé aux maladies hématologiques, cancer, maladies cardio-vasculaires et troubles du système périphérique et central. Elle concerne également des méthodes servant à identifier les composés utiles pour traiter ou prévenir des maladies hématologiques, le cancer, des maladies cardio-vasculaires et des troubles du système nerveux périphérique et central. Elle concerne également des composés se fixant à GPR19 et/ou activant ou inhibant l'activité de ce dernier, ainsi que des compositions pharmaceutiques contenant ces composés.
PCT/EP2003/008142 2002-08-06 2003-07-24 Methodes diagnostiques et therapeutiques s'adressant a des maladies associees au recepteur 19 couple a la proteine g (gpr19) WO2004015427A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003250165A AU2003250165A1 (en) 2002-08-06 2003-07-24 Diagnostics and therapeutics for diseases associated with g-protein coupled receptor 19 (gpr19)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP02017627 2002-08-06
EP02017627.7 2002-08-06

Publications (2)

Publication Number Publication Date
WO2004015427A2 true WO2004015427A2 (fr) 2004-02-19
WO2004015427A3 WO2004015427A3 (fr) 2005-06-09

Family

ID=31502693

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/008142 WO2004015427A2 (fr) 2002-08-06 2003-07-24 Methodes diagnostiques et therapeutiques s'adressant a des maladies associees au recepteur 19 couple a la proteine g (gpr19)

Country Status (2)

Country Link
AU (1) AU2003250165A1 (fr)
WO (1) WO2004015427A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112778183A (zh) * 2019-11-05 2021-05-11 江苏豪森药业集团有限公司 含氮环类衍生物调节剂、其制备方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022129A1 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs couples a la proteine g humaine non endogenes et actives de façon constitutive
WO2001044295A2 (fr) * 1999-12-17 2001-06-21 Astrazeneca Ab Nouveau compose
WO2002061087A2 (fr) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques
WO2003000928A2 (fr) * 2001-06-25 2003-01-03 Buadbo Aps Innovation en matiere de therapie anti-cancereuse

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022129A1 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs couples a la proteine g humaine non endogenes et actives de façon constitutive
WO2001044295A2 (fr) * 1999-12-17 2001-06-21 Astrazeneca Ab Nouveau compose
WO2002061087A2 (fr) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques
WO2003000928A2 (fr) * 2001-06-25 2003-01-03 Buadbo Aps Innovation en matiere de therapie anti-cancereuse

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
O'DOWD B F ET AL: "A NOVEL GENE CODES FOR PUTATIVE G PROTEIN-COUPLED RECEPTOR WITH AN ABUNDANT EXPRESSION IN BRAIN" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 394, no. 3, 7 October 1996 (1996-10-07), pages 325-329, XP000984803 ISSN: 0014-5793 cited in the application -& DATABASE SWISS-PROT [Online] ID: GP19_HUMAN, 1 November 1997 (1997-11-01) Database accession no. Q15760 XP002262914 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112778183A (zh) * 2019-11-05 2021-05-11 江苏豪森药业集团有限公司 含氮环类衍生物调节剂、其制备方法和应用
CN112778183B (zh) * 2019-11-05 2024-05-10 江苏豪森药业集团有限公司 含氮环类衍生物调节剂、其制备方法和应用

Also Published As

Publication number Publication date
WO2004015427A3 (fr) 2005-06-09
AU2003250165A1 (en) 2004-02-25

Similar Documents

Publication Publication Date Title
WO2004015426A1 (fr) Agents diagnostiques et therapeutiques contre des maladies associees au recepteur 5 de la chemokine cxc humain (cxcr5)
US20050164298A1 (en) Diagnostics and therapeutics for diseases associated with growth hormone secretagogue receptor(ghs)
WO2003074069A2 (fr) Diagnostic et therapeutique destines a des maladies associees au recepteur peptidique n-formyl 1 (fpr1)
WO2003093816A2 (fr) Diagnostics et therapeutiques pour lutter contre des maladies associees au recepteur de l'oxytocine (oxtr)
WO2004042402A2 (fr) Moyens diagnostiques et therapeutiques destines a des maladies associees au mrgx1 humain
EP1369698A1 (fr) Méthodes de diagnostic et de traitment de maladies associées au récepteur de la somatostatine 5 (SSTR5)
US7491550B2 (en) Diagnostics and therapeutics for diseases associated with Neuromedin U2 receptor (NMU2)
WO2004044580A1 (fr) Diagnostics et thérapeutiques destinés à des maladies associées au récepteur humain 2 couplé aux protéines g (edg2) d'acide lysophosphatidique, de différentiation endothéliale
US20060057577A1 (en) Diagnostics and therapeutics for diseases associated with homo sapiens formyl peptide receptor-like 2
EP1369697A1 (fr) Méthodes de diagnostic et de traitement de maladies associées au récepteur de la somatostatine 4 (SSTR4)
WO2004013637A1 (fr) Moyens de diagnostic et moyens therapeutiques utiles pour les maladies associees au recepteur 37 couple a la proteine g (gpr37)
EP1365247A1 (fr) Diagnostic et traitement des maladies associées au récepteur 3 de l'arginine vasopressin
WO2003096020A2 (fr) Diagnostic et traitement de maladies liees au recepteur 8 des chimiokines (ccr8)
EP1369696A1 (fr) Méthodes de diagnostic et de traitement de maladies associées au récepteur de la somatostatine 3 (SSTR3)
WO2004019046A1 (fr) Moyens diagnostiques et therapeutiques pour maladies associees au recepteur de chimiokine cxc humain 6(cxcr6)
EP1365244A1 (fr) Diagnostic et traitement des maladies associées au récepteur 2 de l'arginine vasopressin
WO2003093828A1 (fr) Diagnostic et traitement de maladies associees au recepteur 1 de la neurotensine (nt1)
WO2005095990A1 (fr) Methodes diagnostiques et therapeutiques pour des maladies associees au recepteur 52 couple a la proteine g (gpr52)
WO2004015427A2 (fr) Methodes diagnostiques et therapeutiques s'adressant a des maladies associees au recepteur 19 couple a la proteine g (gpr19)
EP1367397A1 (fr) Méthodes de diagnostic et de traitement de maladies associées au récepteur de la somatostatine 1 (SSTR1)
EP1367396A1 (fr) Produits diagnostiques et thérapeutiques pour les maladies associées au récepteur 10 de chemokine (CCR10)
WO2003081234A2 (fr) Methodes diagnostiques et therapeutiques relatives a des maladies associees au recepteur 8 couple a la proteine g humaine (gpr8)
WO2004013638A1 (fr) Moyens de diagnostic et moyens therapeutiques pour les maladies associees au recepteur 45 couple a la proteine g humaine (gpr45)
WO2004106936A2 (fr) Agents diagnostiques et therapeutiques destines a des maladies associees au purinorecepteur p2y9 couple aux proteines g
WO2003080097A2 (fr) Methodes diagnostiques et therapeutiques relatives a des maladies associees au recepteur 7 couple a la proteine g humaine (gpr7)

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP