WO2001044453A1 - La 25678, nouvelle adenylate cyclase humaine - Google Patents

La 25678, nouvelle adenylate cyclase humaine Download PDF

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WO2001044453A1
WO2001044453A1 PCT/US2000/033797 US0033797W WO0144453A1 WO 2001044453 A1 WO2001044453 A1 WO 2001044453A1 US 0033797 W US0033797 W US 0033797W WO 0144453 A1 WO0144453 A1 WO 0144453A1
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polypeptide
compound
nucleic acid
adenylate cyclase
cell
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PCT/US2000/033797
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English (en)
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Rosana Kapeller-Libermann
Miyoung Chun
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Millennium Pharmaceuticals, Inc.
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Priority to AU20957/01A priority Critical patent/AU2095701A/en
Publication of WO2001044453A1 publication Critical patent/WO2001044453A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y406/00—Phosphorus-oxygen lyases (4.6)
    • C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
    • C12Y406/01001—Aodenylate cyclase (4.6.1.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a newly identified human adenylate cyclase.
  • the invention also relates to polynucleotides encoding the adenylate cyclase.
  • the invention further relates to methods using the adenylate cyclase polypeptides and polynucleotides as a target for diagnosis and treatment in adenylate cyclase-mediated or -related disorders.
  • the invention further relates to drug-screening methods using the adenylate cyclase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment.
  • the invention further encompasses agonists and antagonists based on the adenylate cyclase polypeptides and polynucleotides.
  • the invention further relates to agonists and antagonists identified by drug screening methods with the adenylate cyclase polypeptides and polynucleotides as a target.
  • Adenylate cyclase is a membrane-bound enzyme that acts as an effector protein in a receptor-effector system referred to as the c AMP signal transduction pathway. As such, it plays a key intermediate role in the conversion of extracellular signals, perceived by various receptors following binding of a particular ligand, into intracellular signals that, in turn, generate specific cellular responses.
  • GPCRs G-protein- coupled receptors
  • heterotrimeric G proteins that either stimulate or inhibit the catalytic subunit of adenylate cyclase in response to interaction of ligands with appropriate GPCRs; and the catalytic entity, adenylate cyclase.
  • GPCRs G-protein- coupled receptors
  • Each G protein contains a guanine nucleoti de-binding alpha subunit and a complex of tightly associated ⁇ - and ⁇ -subunits.
  • G protein When a G protein is activated following binding of a ligand to a GPCR, GDP is released from the ⁇ -subunit in exchange for GTP. Binding of the GTP results in conformational changes that yield dissociation of the GTP- bound ⁇ -subunit from the ⁇ - ⁇ -subunit complex. The resulting macromolecular complexes regulate catalytic activity of adenylate cyclase.
  • the receptor is a stimulatory receptor (R s )
  • G S a stimulatory G-protein
  • the receptor is an inhibitory receptor (Rj)
  • an inhibitory G-protein one of several known G J S
  • the G-protein ⁇ - ⁇ -subunit complex may interact with and influence adenylate cyclase activity independent of or in parallel with the GTP-bound ⁇ - subunit, depending upon the adenylate cyclase isoform involved.
  • adenylate cyclase When activated, the catalytic subunit of adenylate cyclase converts intracellular ATP into cAMP. This second messenger then activates protein kinases, particularly protein kinase A. Activation of this protein kinase causes the phosphorylation of downstream target proteins involved in a number of metabolic pathways, thus initiating a signal transduction cascade.
  • the extent to which adenylate cyclase converts ATP to cAMP is highly dependent on the state of phosphorylation of the various components of the hormone- sensitive adenylate cyclase system.
  • stimulatory and inhibitory receptors are desensitized and down-regulated following phosphorylation by various kinases, particularly cAMP-dependent protein kinases, protein kinase C, and other receptor- specific kinases that preferentially use agonist-bound forms of receptors as substrates.
  • various kinases particularly cAMP-dependent protein kinases, protein kinase C, and other receptor- specific kinases that preferentially use agonist-bound forms of receptors as substrates.
  • Adenylate cyclase activation may also occur through increased intracellular calcium concentration, especially in nervous system and cardiovascular tissues. After depolarization, the influx of calcium elicits the activation of calmodulin, an intracellular calcium-binding protein. In the cardiovascular system, this effect gives rise to the contraction of the blood vessels or cardiac myocytes.
  • the activated calmodulin has been shown to bind and activate some isoforms of adenylate cyclase.
  • Several novel isoforms of mammalian adenylate cyclase have been identified through molecular cloning.
  • Type I adenylate cyclase (CYA1) is primarily localized in brain tissues (see Krupinski et al (1989) Science 244:1558-1564; Gilman (1987) Ann. Rev. Biochem. 56:615-649, citing Salter et al. (1981) J. Biol. Chem. 256:9830-9833; Andreasen et al. (1983) Biochemistry 22:2151-2162; and Smigel et al (1986) J. Biol Chem. 261 :1976-1982 for bovine CYA1; and Villacres et al. (1993) Genomics 16:473-478 for human CYA1).
  • the type II adenylate cyclase (CYA2) is localized in brain and lung tissues (see Feinstein et al. (1991) Proc. Natl. Acad. Sci. USA
  • Type III adenylate cyclase (CYA3) is primarily localized in olfactory neuroepithelium and is thought to mediate olfactory receptor responses (Bakalyar and Reed (1990) Science 250: 1403-1406; Glatt and Snyder (1993) N ⁇ twre 361 :536-538; and Xia ( 1992) Neurosci. Lett. 144 : 169- 173).
  • Type IV adenylate cyclase most resembles type II, but is expressed in a variety of peripheral tissues and in the central nervous system (Gao and Gilman (1991) Proc. Natl. Acad. Sci. USA 88:10178-10182, for rat CYA4).
  • Type V adenylate cyclase (CYA5) (Ishikawa et al. (1992) J. Biol. Chem. 267:13553-13557; Premont et ⁇ /. (1992) Proc. Natl. Acad. Sci. USA 89:9809-9813; and Glatt and Snyder (1993) Nature 361 :536-538; Krupinski et al. (1992) J.
  • Biol. Chem. 267:24858-24862) and type VI adenylate cyclase (CYA6) (Premont et al. (1992) Proc. Natl. Acad. Sci. USA 89:9808-9813; Yoshimura and Cooper (1992) Proc. Natl. Acad. Sci. USA 89:6716-6720; Katsushika et al. (1992) Proc. Natl. Acad. Sci. USA 89:8774-8778; and Krupinski et al. (1992) J. Biol. Chem. 267:24858-24862) both exhibit a widely distributed expression pattern, with type V having high expression in heart and striatum, and type VI having high expression in heart and brain.
  • CYA6 type VI adenylate cyclase
  • Type VII adenylate cyclase (CYA7) is widely distributed, though may be absent from brain tissues (Krupinski et al (1992) J. Biol. Chem. 267:24858- 24862).
  • Type VIII adenylate cyclase (CYA8) is abundant in brain tissues (Krupinski et al. (1992) J Biol. Chem. 267:24858-24862; and Parma et al. (1991 ) Biochem.
  • Type IX adenylate cyclase (CYA9) is widely expressed, at high levels in skeletal muscle and brain (Premont et al. (1996) J. Biol. Chem. 271 :13900-13907). The different isoforms of adenylate cyclase exhibit unique patterns of regulatory responses (see Sunahara et al. ( ⁇ 996) Annu. Tev. Pharmacol. Toxicol 56:461-480).
  • G s a particular G protein
  • G s a particular G protein
  • the adenylate cyclases designated type I, III, and VIII are also stimulated by Ca /calmodulin in vitro, while type II, IV, V, VI, VII, and IX are not.
  • Type I is inhibited by G protein ⁇ - ⁇ -subunit complex, independently of G s activation, while Type II is highly stimulated by G protein ⁇ - ⁇ - subunit complex when simultaneously activated by Gs alpha subunit.
  • Type III in contrast, is not affected by G protein ⁇ - ⁇ -subunit complex.
  • Type V and type VI are both are inhibited by low levels of Ca 2+ , but appear to be unaffected by G protein ⁇ - ⁇ - subunit complex.
  • Type IX is unique in that it is stimulated by Mg 2+ , but is not affected by G protein ⁇ - ⁇ -subunit complex.
  • genes for these adenylate cyclases all encode proteins having molecular weights of approximately 120,000 and which range from 1064 to 1353 amino acid residues. These proteins are predicted to have a short cytoplasmic amino terminus followed by a first motif consisting of six transmembrane spans and a cytoplasmic (domain Ci), and then a second motif, also consisting of six transmembrane spans and a second cytoplasmic domain (domain C 2 ). The two cytoplasmic domains are approximately 40 kDa each and contain a region of homology (designated C ⁇ a and C 2a ) with each other and with the catalytic domains of membrane-bound guanylate cyclases.
  • these domains are considered to be nucleotide binding domains, and together have been shown to be sufficient to confer enzymatic activity (Tang and Gilman (1995) Science 268:1769-1772). Alterations in the cAMP signal transduction pathway have been associated with diseases such as asthma, cancer, inflammation, hypertension, atherosclerosis, and heart failure. Antihypertensive drug therapy involves modulation of adenylate cyclase levels (Marcil et al. (1996) Hypertension 25:83-90). In addition, studies of heart in human and animal models indicate that adenylate cyclase has a function in cardiomyopathy (Michael et al.
  • cAMP protein kinase A
  • adenylate cyclases are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize novel adenylate cyclases and tissues and disorders in which adenylate cyclases are differentially expressed.
  • the present invention advances the state of the art by providing a novel human adenylate cyclase and tissues and disorders in which expression of a human adenylate cyclase is relevant. Accordingly, the invention provides methods directed to expression of the adenylate cyclase.
  • a specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of the adenylate cyclase in specific tissues and disorders.
  • a further specific object of the invention is to provide compounds that modulate expression of the adenylate cyclase for treatment and diagnosis of adenylate cyclase- mediated or related disorders.
  • the invention is thus based on the identification and expression of a human adenylate cyclase, especially in specific tissues and disorders.
  • the invention provides methods of screening for compounds that modulate expression or activity of the adenylate cyclase polypeptides or nucleic acid (RNA or DNA) in the specific tissues or disorders.
  • the invention also provides a process for modulating adenylate cyclase polypeptide or nucleic acid expression or activity, especially using the screened compounds.
  • Modulation may be used to treat conditions related to aberrant activity or expression of the adenylate cyclase polypeptides or nucleic acids.
  • the invention also provides assays for determining the activity of or the presence or absence of the adenylate cyclase polypeptides or nucleic acid molecules in specific biological samples, including for disease diagnosis.
  • the invention also provides assays for determining the presence of a mutation in the polypeptides or nucleic acid molecules, including for disease diagnosis.
  • the invention provides isolated adenylate cyclase polypeptides, including a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence encoded by the cDNA deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209 as Patent Deposit No. PTA-1871 ("the deposited cDNA") on May 12, 2000.
  • ATCC American Type Culture Collection
  • the invention also provides an isolated adenylate cyclase nucleic acid molecule having the sequence shown in SEQ ID NO:2 or encoded by the deposited cDNA.
  • the invention also provides variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO:l or encoded by the deposited cDNA.
  • the invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO:2 or in the deposited cDNA.
  • the invention also provides fragments of the polypeptide shown in SEQ ID NO:l and nucleotide sequence shown in SEQ ID NO:2, as well as substantially homologous fragments of the polypeptide or nucleic acid.
  • the invention further provides nucleic acid constructs comprising the nucleic acid molecules described herein.
  • the nucleic acid molecules of the invention are operatively linked to a regulatory sequence.
  • the invention also provides vectors and host cells that express the adenylate cyclase and provides methods for expressing the adenylate cyclase nucleic acid molecules and polypeptides in specific cell types and disorders, and particularly recombinant vectors and host cells.
  • the invention also provides methods of making the vectors and host cells and provides methods for using them to produce adenylate cyclase nucleic acid molecules and polypeptides and to assay expression and cellular effects of expression of the adenylate cyclase nucleic acid molecules and polypeptides in specific cell types and disorders.
  • the invention also provides antibodies or antigen-binding fragments thereof that selectively bind the adenylate cyclase polypeptides and fragments.
  • the invention provides a computer readable means containing the nucleotide and/or amino acid sequences of the nucleic acids and polypeptides of the invention.
  • Figure 1 shows the adenylate cyclase nucleotide sequence (SEQ ID NO: 2) and the deduced amino acid sequence (SEQ ID NO: 1 ).
  • Figure 2 shows an analysis of the adenylate cyclase amino acid sequence: ⁇ turn and coil regions; hydrophilicity; amphipathic regions; flexible regions; antigenic index; and surface probability plot.
  • Figure 3 shows a hydrophobicity plot of the adenylate cyclase.
  • Figure 4 shows an analysis of the adenylate cyclase open reading frame for amino acids corresponding to specific functional sites of SEQ ID NO:l. Glycosylation sites are shown in the figure with the actual modified residue being the first amino acid. Protein kinase C phosphorylation sites are shown in the figure with the actual modified residue being the first amino acid. Casein kinase II phosphorylation sites are shown in the figure with the actual modified residue being the first amino acid. Tyrosine kinase phosphorylation sites are shown in the figure with the actual modified residue being the last amino acid. N-myristoylation sites are shown in the figure, with the actual modified residue being the first amino acid. In addition, amino acids corresponding to the guanylate cyclase signature are found at amino acids 394-417 and 1009-1032.
  • Figure 5 shows expression of the 25678 adenylate cyclase in various normal human tissues.
  • Figure 6 shows expression of the 25678 adenylate cyclase in various cardiovascular tissues.
  • Int. Mamm. internal mammary artery
  • CHF congestive heart failure
  • ISCH ischemic heart
  • myop myopathic heart.
  • the present invention is based, at least in part, on the identification of novel molecules, referred to herein as adenylate cyclase nucleic acid and polypeptide molecules, which play a key role in regulation of the cyclic AMP (cAMP) signal transduction pathway by virtue of their conversion of intracellular ATP into cAMP.
  • adenylate cyclase molecules modulate the activity of one or more proteins involved in cellular metabolism associated with cell maintenance, growth, or differentiation, e.g., cardiac, epithelial, or neuronal cell maintenance, growth, or differentiation.
  • the adenylate cyclase molecules of the present invention are capable of modulating the phosphorylation state of one or more proteins involved in cellular metabolism associated with cell maintenance, growth, or differentiation, e.g., cardiac, epithelial, or neuronal cell maintenance, growth or differentiation, via their indirect effect on cAMP-dependent protein kinases, particularly protein kinase A, as described in, for example, Devlin (1997) Textbook of Biochemistry with Clinical Correlations (Wiley-Liss, Inc., New York, NY).
  • the receptors which trigger activity of the adenylate cyclases of the present invention are targets of drugs as described in Goodman and Gilman (1996), The Pharmacological Basis of Therapeutics (9* ed.) Hartman & Limbard Editors, the contents of which are incorporated herein by reference.
  • a “signaling pathway” refers to the modulation (e.g., stimulation or inhibition) of a cellular function activity upon the binding of a ligand to a receptor.
  • Examples of such functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP 2 ), inositol 1 ,4,5-triphosphate (IP 3 ) and adenylate cyclase; polarization of the plasma membrane; production or secretion of molecules; alteration in the structure of a cellular component; cell proliferation, e.g., synthesis of DNA; cell migration; cell differentiation; and cell survival.
  • PIP 2 phosphatidylinositol 4,5-bisphosphate
  • IP 3 inositol 1 ,4,5-triphosphate
  • adenylate cyclase polarization of the plasma membrane
  • production or secretion of molecules alteration in the structure of
  • binding of a ligand to the receptor may stimulate an activity such as release of compounds, gating of a channel, cellular adhesion, migration, differentiation, etc., through phosphatidylinositol or cyclic AMP metabolism and turnover while in other cells, binding will produce a different result.
  • the cAMP turnover pathway is a signaling pathway.
  • cyclic AMP turnover and metabolism refers to the molecules involved in the turnover and metabolism of c AMP as well as to the activities of these molecules.
  • Cyclic AMP is a second messenger produced in response to ligand-induced stimulation of certain receptors.
  • binding of a ligand can lead to the activation of the enzyme adenyl cyclase, which catalyzes the synthesis of c AMP.
  • the newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase.
  • This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability of the potassium channel to open during an action potential.
  • the inability of the potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization.
  • the cGMP turnover pathway is also a signaling pathway.
  • cyclic GMP turnover and metabolism refers to the molecules involved in the turnover and metabolism of cGMP as well as to the activities of these molecules.
  • Cyclic GMP is a second messenger produced in response to ligand-induced stimulation of certain receptors.
  • binding of a ligand can lead to the activation of the enzyme guanyl cyclase, which catalyzes the synthesis of cGMP.
  • Synthesized cGMP can in turn activate a cGMP-dependent protein kinase.
  • the invention is directed to methods, uses and reagents applicable to methods and uses that are applied to cells, tissues and disorders of these cells and tissues wherein adenylate cyclase expression is relevant.
  • the adenylate cyclase is expressed in a variety of tissues as shown in Figures 5 and 6. Accordingly, the methods and uses of the invention as disclosed in greater detail below apply to these tissues, disorders involving these tissues, and particularly to the disorders with which gene expression is associated, as shown in these figures and as disclosed herein. Accordingly, the methods, uses and reagents disclosed in greater detail below especially apply to prostate, skeletal muscle, brain, and testis.
  • the protein sequences of the present invention can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J Mol. Biol. 275:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17) :3889-3402. When utilizing BLAST and Gapped
  • BLAST programs the default parameters of the respective programs (e.g. XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • the adenylate cyclase polypeptides are useful for producing antibodies specific for the adenylate cyclase, regions, or fragments. Regions having a high antigenicity index score are shown in Figure 2.
  • the invention provides methods using the adenylate cyclase, variants, or fragments, including but not limited to use in the cells, tissues, and disorders as disclosed herein.
  • the invention provides biological assays related to adenylate cyclases. Such assays involve any of the known functions or activities or properties useful for diagnosis and treatment of cyclic adenylate cyclase-related conditions. These include, but are not limited to, binding and/or activation by G-protein subunits, alpha, beta or gamma, hydrolysis of ATP or GTP and consequent modulation of cAMP and/or cGMP intracellular concentration, ability to be bound by specific antibody, GTP or ATP binding, and protein kinase A phosphorylation, as well as the various other properties and functions disclosed herein and disclosed in the references cited herein.
  • the invention provides drug screening assays, in cell-based or cell-free systems.
  • Cell-based systems can be native, i.e., cells that normally express the adenylate cyclase, as a biopsy, or expanded in cell culture.
  • cell-based assays involve recombinant host cells expressing the adenylate cyclase.
  • cells that are useful in this regard include, but are not limited to, those disclosed herein as expressing or differentially expressing the adenylate cyclase, such as those shown in Figures 5 and 6.
  • cells or tissues derived from prostate, skeletal muscle, brain, colon, ovary, aorta, testis, placenta, fetal heart, aorta with intimal proliferations, internal mammary artery, kidney, and saphenous vein Such cells can naturally express the gene or can be recombinant, containing one or more copies of exogenously-introduced adenylate cyclase sequences or genetically modified to modulate expression of the endogenous adenylate cyclase sequence.
  • This aspect of the invention particularly relates to cells derived from subjects with disorders involving the tissues in which the adenylate cyclase is expressed or derived from tissues subject to disorders including, but not limited to, those disclosed herein.
  • disorders may naturally occur, as in populations of human subjects, or may occur in model systems such as in vitro systems or in vivo, such as in non-human transgenic organisms, particularly in non-human transgenic animals.
  • assays can involve the identification of agents that interact with the adenylate cyclase protein.
  • This interaction can be detected by functional assays, such as the ability to be affected by an effector molecule, such as binding and/or activation by G- protein subunits or hydrolysis of ATP and/or GTP to modulate intracellular cAMP/cGMP concentrations.
  • Such interaction can also be measured by ultimate biological effects, such as phosphorylation of protein kinases, for example protein kinase A, and other downstream effectors in the signal transduction pathway, having biological effects on immunity/inflammation or cell proliferation, i.e., any of the effects of modulating the intracellular levels of the second messengers cAMP and cGMP.
  • Determining the ability of the test compound to interact with the adenylate cyclase can also comprise determining the ability of the test compound to preferentially bind to the polypeptide as compared to the ability of a known binding molecule (e.g., G- protein, calmodulin, GTP or ATP) to bind to the polypeptide.
  • a known binding molecule e.g., G- protein, calmodulin, GTP or ATP
  • the invention provides methods to identify proteins that interact with the adenylate cyclase in the tissues and disorders disclosed.
  • the proteins of the invention can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046- 12054; Bartel et al. (1993) Biotechniques 74:920-924; Iwabuchi et al. (1993) Oncogene 5:1693-1696; and Brent WO 94/10300), to identify other proteins (captured proteins) which bind to or interact with the proteins of the invention and modulate their activity.
  • the invention provides methods to identify compounds that modulate adenylate cyclase activity.
  • Such compounds can increase or decrease affinity or rate of binding to GTP or ATP, compete with GTP or ATP for binding to the adenylate cyclase, or displace GTP or ATP bound to the adenylate cyclase.
  • Such compounds can also increase or decrease affinity or rate of binding to calmodulin, compete with calmodulin for binding to the adenyl cyclase, or displace calmodulin bound to the adenyl cyclase.
  • Such compounds can also, for example, increase or decrease the affinity or rate of binding of one or more G-protein subunits, compete with the subunits for binding, or displace the subunits bound to the adenyl cyclase.
  • Both adenylate cyclase and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the adenylate cyclase. These compounds can be further screened against a functional adenylate cyclase to determine the effect of the compound on the adenylate cyclase activity.
  • Compounds can be identified that activate (agonist) or inactivate (antagonist) the adenylate cyclase to a desired degree.
  • Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject.
  • the subject can be a human subject, for example, a subject in a clinical trial or undergoing treatment or diagnosis, or a non-human transgenic subject, such as a transgenic animal model for disease.
  • the invention provides methods to screen a compound for the ability to stimulate or inhibit interaction between the adenylate cyclase protein and a target molecule that normally interacts with the adenylate cyclase protein.
  • the target can be an ATP or GTP, or another component of the signal pathway with which the adenylate cyclase protein normally interacts, including but not limited to, calmodulin, or a G-protein subunit (one or more of alpha, beta, or gamma).
  • the assay includes the steps of combining the adenylate cyclase protein with a candidate compound under conditions that allow the adenylate cyclase protein or fragment to interact with the target molecule, and to detect the formation of a complex between the adenylate cyclase protein and the target, or to detect the biochemical consequence of the interaction with the adenylate cyclase and the target, such as any of the associated effects of signal transduction such as protein kinase A phosphorylation, cAMP or cGMP turnover, and biological endpoints of the pathway.
  • signal transduction such as protein kinase A phosphorylation, cAMP or cGMP turnover, and biological endpoints of the pathway.
  • adenylate cyclase Determining the ability of the adenylate cyclase to bind to a target molecule can also be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA).
  • BiA Bimolecular Interaction Analysis
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). 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
  • test compounds of the present invention can 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; and synthetic library methods using affinity chromatography selection.
  • biological libraries are limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 72:145).
  • Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al.
  • peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D-
  • One candidate compound is a soluble full-length adenylate cyclase or fragment that competes for GTP or ATP binding.
  • Other candidate compounds include mutant adenylate cyclases or appropriate fragments containing mutations that affect adenylate cyclase function and thus compete for GTP or ATP. Accordingly, a fragment that competes for ATP or GTP, for example with a higher affinity, or a fragment that binds ATP or GTP but does not cyclize it, is encompassed by the invention.
  • Other fragments that are encompassed include, but are not limited to, those that will bind but not be activated by G-protein subunits, or bind but not be activated by calmodulin.
  • the invention provides other end points to identify compounds that modulate (stimulate or inhibit) adenylate cyclase activity.
  • the assays typically involve an assay of events in the signal transduction pathway that indicate adenylate cyclase activity.
  • the expression of genes that are up- or down-regulated in response to the adenylate cyclase dependent signal cascade can be assayed.
  • the regulatory region of such genes can be operably linked to a marker that is easily detectable, such as luciferase. Any of the biological or biochemical functions mediated by the adenylate cyclase can be used as an endpoint assay.
  • GTP cyclization and a decrease or increase in intracellular cAMP or cGMP concentrations or in protein kinase A activation.
  • Assays for adenylate cyclase function include, but are not limited to, those that are well known in the art and available to the person of ordinary skill in the art, for example, G-protein subunit binding and activation of adenyl cyclase such as that disclosed in Taussig et al. (1995), or Sunahara et al., herein above, effect on cAMP- or cGMP-dependent kinases, as described for example in Devlin, herein above, changes in intracellular cAMP and/or cGMP concentration, as described in Sunahara et al. , herein above, and stimulation by calmodulin in vitro, as disclosed in Sunahara et al. , herein above.
  • nucleotide triphosphate binding domains e.g., for ATP and GTP
  • nucleotide triphosphate binding domains can be assayed according to Tang et al. (1995), herein above. All of these references are incorporated herein by reference for these assays.
  • Binding and/or activating compounds can also be screened by using chimeric adenylate cyclase proteins in which one or more domains, sites, and the like, as disclosed herein, or parts thereof, can be replaced by their heterologous counterparts derived from other adenylate cyclase isoforms of the same family or from adenylate cyclase isoforms of any other adenylate cyclase family.
  • a catalytic region can be used that interacts with a different cyclic nucleotide specificity and/or affinity than the native adenylate cyclase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation.
  • a heterologous effector protein binding/activation sequence can replace the native sequence.
  • a different G-protein subunit can be bound or interact with the modified adenyl cyclase.
  • the adenyl cyclase is subject to different modulation by different stimulatory or inhibitory G-protein subunits based on inhibitory or stimulatory receptor interaction with the G-protein.
  • the site of modification by an effector protein for example phosphorylation by a protein kinase can be replaced with the site from a different effector protein. This could also provide the use of a different signal transduction pathway for endpoint determination.
  • Activation can also be detected by a reporter gene containing an easily detectable coding region operably linked to a transcriptional regulatory sequence that is part of the native signal transduction pathway.
  • the invention provides competition binding assays designed to discover compounds that interact with the adenylate cyclase.
  • a compound is exposed to a adenylate cyclase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide.
  • Soluble adenylate cyclase polypeptide is also added to the mixture. If the test compound interacts with the soluble adenylate cyclase polypeptide, it decreases the amount of complex formed or activity from the adenylate cyclase target.
  • This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the adenylate cyclase.
  • the soluble polypeptide that competes with the target adenylate cyclase region is designed to contain peptide sequences corresponding to the region of interest.
  • Another type of competition-binding assay can be used to discover compounds that interact with specific functional sites.
  • calmodulin or one or more G- protein subunits and a candidate compound can be added to a sample of the adenylate cyclase.
  • Compounds that interact with the adenylate cyclase at the same site as these components will reduce the amount of complex formed between the adenylate cyclase and these components. Accordingly, it is possible to discover a compound that specifically prevents interaction between the adenylate cyclase and these components.
  • Another example involves adding a candidate compound to a sample of adenylate cyclase and ATP or GTP.
  • a compound that competes with ATP or GTP will reduce the amount of cyclization or binding of the ATP or GTP to the adenylate cyclase. Accordingly, compounds can be discovered that directly interact with the adenylate cyclase and compete with ATP or GTP. Such assays can involve any other component that interacts with the adenylate cyclase.
  • adenylate cyclase or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S- transferase/adenylate cyclase 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 cell lysates (e.g., S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes is dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS- PAGE, and the level of adenylate cyclase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art.
  • antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation.
  • Preparations of a adenylate cyclase-binding component, such as ATP or G-protein subunit, and a candidate compound are incubated in the adenylate cyclase-presenting wells and the amount of complex trapped in the well can be quantitated.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the adenylate cyclase target molecule, or which are reactive with adenylate cyclase and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • Modulators of adenylate cyclase level or activity identified according to these assays can be used to test the effects of modulation of expression of the enzyme on the outcome of clinically relevant disorders. This can be accomplished in vitro, in vivo, such as in human clinical trials, and in test models derived from other organisms, such as non- human transgenic subjects. Modulation in such subjects includes, but is not limited to, modulation of the cells, tissues, and disorders particularly disclosed herein.
  • Modulators of adenylate cyclase activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the adenylate cyclase pathway, by treating cells that express the adenylate cyclase, such as those disclosed herein, especially in Figures 5 and 6, as well as those disorders disclosed in the references cited herein above.
  • the cells that are treated are derived from prostate, skeletal muscle, brain, testis and aorta, and as such, modulation is particularly relevant to disorders involving these tissues.
  • modulation is in aortic tissue with intimal proliferations or in ischemic or myopathic heart tissue. Accordingly, disorders in which modulation is particularly relevant can include these tissues.
  • These methods of treatment include the steps of administering the modulators of adenylate cyclase activity in a pharmaceutical composition as described herein, to a subject in need of such treatment.
  • disorders involving the colon include, but are not limited to, congenital anomalies, such as atresia and stenosis, Meckel diverticulum, congenital aganglionic megacolon-Hirschsprung disease; enterocolitis, such as diarrhea and dysentery, infectious enterocolitis, including viral gastroenteritis, bacterial enterocolitis, necrotizing enterocolitis, antibiotic-associated colitis (pseudomembranous colitis), and collagenous and lymphocytic colitis, miscellaneous intestinal inflammatory disorders, including parasites and protozoa, acquired immunodeficiency syndrome, transplantation, drug-induced intestinal injury, radiation enterocolitis, neutropenic colitis (typhlitis), and diversion colitis; idiopathic inflammatory bowel disease, such as Crohn disease and ulcerative colitis; tumors of the colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and car
  • Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia— infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lac
  • subacute encephalitis vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and
  • Diseases of the skin include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors of the dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular
  • disorders involving the heart include but are not limited to, heart failure, including but not limited to, cardiac hypertrophy, left-sided heart failure, and right- sided heart failure; ischemic heart disease, including but not limited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not limited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not limited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration of the mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and end
  • vascular diseases involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease— the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurys
  • disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases of the kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) poly cystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis- associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heymann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathway
  • Testicular tumors including germ cell tumors that include, but are not limited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sex cord-gonadal stroma including, but not limited to, leydig (interstitial) cell tumors and sertoli cell tumors (androblastoma), and testicular lymphoma, and miscellaneous lesions of tunica vaginalis.
  • Disorders involving the prostate include, but are not limited to, inflammations, benign enlargement, for example, nodular hyperplasia (benign prostatic hypertrophy or hyperplasia), and tumors such as carcinoma.
  • Disorders involving the skeletal muscle include tumors such as rhabdomyosarcoma.
  • ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.
  • the invention thus provides methods for treating a disorder characterized by aberrant expression or activity of a adenylate cyclase.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity of the protein.
  • the method involves administering the adenylate cyclase as therapy to compensate for reduced or aberrant expression or activity of the protein.
  • Methods for treatment include but are not limited to the use of soluble adenylate cyclase or fragments of the adenylate cyclase protein that compete for ATP or GTP or G-protein. These adenylate cyclases or fragments can have a higher affinity for the target so as to provide effective competition.
  • Stimulation of activity is desirable in situations in which the protein is abnormally downregulated and/or in which increased activity is likely to have a beneficial effect.
  • inhibition of activity is desirable in situations in which the protein is abnormally upregulated and/or in which decreased activity is likely to have a beneficial effect.
  • a subject has a disorder characterized by aberrant development or cellular differentiation.
  • the subject has a proliferative disease (e.g., cancer) or a disorder characterized by an aberrant hematopoietic response.
  • it is desirable to achieve tissue regeneration in a subject e.g., where a subject has undergone brain or spinal cord injury and it is desirable to regenerate neuronal tissue in a regulated manner).
  • the invention also provides methods for diagnosing a disease or predisposition to disease mediated by the adenylate cyclase, including, but not limited to, diseases involving tissues in which the adenylate cyclases are expressed, as disclosed herein, and particularly in prostate, skeletal muscle, brain, testes, as well as aorta, aorta with intimal proliferations, internal mammary artery, kidney, and saphenous vein.
  • positive differential expression occurs in diseased heart tissue from patients with myopathy and ischemia.
  • these disorders are treated by modulating the level or activity of the adenylate cyclase gene in diseased hearts.
  • Treatment is therefore especially directed to these tissues and cells thereof.
  • diagnosis is directed to cells and tissues involved in these disorders.
  • treatment and diagnosis can be in human subjects in which the disease normally occurs and in model systems, both in vitro and in vivo, such as in transgenic animals.
  • methods are directed to detecting the presence, or levels of, the adenylate cyclase in a cell, tissue, or organism. The methods involve contacting a biological sample with a compound capable of interacting with the adenylate cyclase such that the interaction can be detected.
  • One agent for detecting adenylate cyclase is an antibody capable of selectively binding to adenylate cyclase.
  • a biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the invention also provides methods for diagnosing active disease, or predisposition to disease, in a patient having a variant adenylate cyclase.
  • adenylate cyclase can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in an aberrant protein. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification.
  • Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered adenylate cyclase activity in cell-based or cell-free assay, alteration in ATP or GTP binding or cyclization, G- protein subunit binding or calmodulin or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein in general or in a adenylate cyclase specifically.
  • In vitro techniques for detection of adenylate cyclase include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • the protein can be detected in vivo in a subject by introducing into the subject a labeled anti-adenylate cyclase antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods, which detect the allelic variant of the adenylate cyclase expressed in a subject, and methods, which detect fragments of the adenylate cyclase in a sample.
  • the invention also provides methods of pharmacogenomic analysis including, but not limited to, in the cells, tissues and disorders disclosed herein in which expression of the adenylate cyclase either occurs or shows differential expression.
  • Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-17 ⁇ :983-985, and Linder, M.W. (1997) Clin. Chem. 43(2) :254-266.
  • the clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism.
  • the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes affects both the intensity and duration of drug action.
  • the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype.
  • the discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages.
  • Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the adenylate cyclase in which one or more of the adenylate cyclase functions in one population is different from those in another population.
  • polypeptides can be used as a target to ascertain a genetic predisposition that can affect treatment modality.
  • polymorphism may give rise to catalytic regions that are more or less active. Accordingly, dosage would necessarily be modified to maximize the therapeutic effect within a given population containing the polymorphism.
  • specific polymorphic polypeptides could be identified.
  • the invention also provides for monitoring therapeutic effects during clinical trials and other treatment.
  • the therapeutic effectiveness of an agent that is designed to increase or decrease gene expression, protein levels or adenylate cyclase activity can be monitored over the course of treatment using the adenylate cyclase polypeptides as an end-point target.
  • the monitoring can be, for example, as follows: (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression or activity of the protein in the pre- administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the protein in the post- administration samples; (v) comparing the level of expression or activity of the protein in the pre-administration sample with the protein in the post-administration sample or samples; and (vi) increasing or decreasing the administration of the agent to the subject accordingly.
  • the methods and uses herein disclosed can be based on polypeptide reagents and targets.
  • the invention is thus based on the discovery of a novel human adenylate cyclase.
  • an expressed sequence tag EST was selected based on homology to adenylate cyclase sequences. This EST was used to design primers based on sequences that it contains and used to identify a cDNA from a fetal testis cDNA library. Positive clones were sequenced and the overlapping fragments were assembled. Analysis of the assembled sequence revealed that the cloned cDNA molecule encodes an adenylate cyclase similar to a rat adenylate cyclase.
  • EST expressed sequence tag
  • the invention thus relates to a novel human adenylate cyclase and to the expression of the adenylate cyclase having the deduced amino acid sequence shown in Figure 1 (SEQ ID NO: 1) or having the amino acid sequence encoded by the deposited cDNA, ATCC Patent Deposit No. PTA- 1871.
  • the deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.
  • the deposits are provided as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the deposited sequences as well as the polypeptides encoded by the sequences, are incorporated herein by reference and control in the event of any conflict, such as a sequencing error, with description in this application.
  • Adenylate cyclase polypeptide or “adenylate cyclase protein” refers to the polypeptide in SEQ ID NO:l or encoded by the deposited cDNA.
  • Tissues and/or cells in which the adenylate cyclase is found include, but are not limited to those shown in Figures 5 and 6, and particularly in prostate, skeletal muscle, brain, testis and aorta.
  • the adenylate cyclase is expressed in diseased tissues, including but limited to, heart tissue derived from patients with myopathy or ischemia.
  • the present invention thus provides an isolated or purified adenylate cyclase polypeptide and variants and fragments thereof.
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material, when it is isolated from recombinant and non- recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell and still be considered “isolated” or “purified.”
  • the adenylate cyclase polypeptides can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful and considered to contain an isolated form of the polypeptide.
  • the critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components.
  • the invention encompasses various degrees of purity.
  • the language "substantially free of cellular material” includes preparations of the adenylate cyclase having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
  • culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the protein preparation.
  • an adenylate cyclase polypeptide is also considered to be isolated when it is part of a membrane preparation or is purified and then reconstituted with membrane vesicles or liposomes.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of the adenylate cyclase polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • the adenylate cyclase polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the invention also encompasses sequence variants that retain the adenylate cyclase activity of the amino acid sequence shown in SEQ ID NO: 1.
  • Variants include a substantially homologous protein encoded by the same genetic locus in an organism, i.e., an allelic variant.
  • Variants also encompass proteins derived from other genetic loci in an organism, but having substantial homology to the adenylate cyclase of SEQ ID NO: 1.
  • Variants also include proteins substantially homologous to the adenylate cyclase but derived from another organism, i.e., an ortholog.
  • Variants also include proteins that are substantially homologous to the adenylate cyclase that are produced by chemical synthesis. Variants also include proteins that are substantially homologous to the adenylate cyclase that are produced by recombinant methods. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.
  • two proteins are substantially homologous when the amino acid sequences are at least about 70-75%, typically at least about 80-85%, and most typically at least about 90-95% or more homologous.
  • a substantially homologous amino acid sequence will be encoded by a nucleic acid sequence hybridizing to the nucleic acid sequence, or portion thereof, of the sequence shown in SEQ ID NO:2 under stringent conditions as more fully described below.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% or more of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the adenylate cyclase. Similarity is determined by conserved amino acid substitution.
  • substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics.
  • Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) (J. Mol. Biol. 45:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux et al. (1984) Nucleic Acids Res.
  • a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.
  • Variant polypeptides can be fully functional or can lack function in one or more activities.
  • variations can affect the function, for example, of one or more of the regions corresponding to a catalytic region, regulatory region, targeting region, regions involved in membrane association, regions involved in enzyme activation, for example, by phosphorylation, and regions involved in interaction with components of the cyclic nucleotide-dependent signal transduction pathways, (e.g., ATP, GTP, G-protein, or calmodulin).
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions.
  • Functional variants can also contain substitution of similar amino acids, which results in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • variants can be naturally-occurring or can be made by recombinant means or chemical synthesis to provide useful and novel characteristics for the adenylate cyclase polypeptide. This includes preventing immunogenicity from pharmaceutical formulations by preventing protein aggregation.
  • Useful variations further include alteration of catalytic activity. For example, one embodiment involves a variation at the binding site that results in binding but not cyclization, or slower cyclization, of ATP or GTP. A further useful variation at the same site can result in altered affinity for ATP or GTP. Useful variation includes one that prevents activation by G-protein . Another useful variation provides a fusion protein in which one or more domains or subregions are operationally fused to one or more domains or subregions from another adenylate cyclase isoform or family.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis
  • the invention thus also includes polypeptide fragments of the adenylate cyclase. Fragments can be derived from the amino acid sequence shown in SEQ ID NO:l. However, the invention also encompasses fragments of the variants of the adenylate cyclase as described herein.
  • fragments to which the invention pertains are not to be construed as encompassing fragments per se that may have been disclosed prior to the invention (although the methods herein can pertain to known fragments). Accordingly, a fragment can comprise at least about 10, 15, 20, 25, 30, 35, 40,
  • Fragments can retain one or more of the biological activities of the protein, for example the ability to bind to or cyclize GTP or ATP, as well as fragments that can be used as an immunogen to generate adenylate cyclase antibodies.
  • Biologically active fragments peptides which are, for example, 5, 7, 10, 12, 15,
  • a domain or motif e.g., catalytic site, adenylate cyclase signature, and sites for glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, and N-myristoylation.
  • Further possible fragments include the catalytic site, sites important for cellular and subcellular targeting, sites functional for interacting with components of other cGMP or cAMP-dependent signal transduction pathways, and regulatory sites.
  • Such domains or motifs can be identified by means of routine computerized homology searching procedures. Fragments, for example, can extend in one or both directions from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100 amino acids. Further, fragments can include sub-fragments of the specific domains mentioned above, which sub- fragments retain the function of the domain from which they are derived.
  • the invention also provides fragments with immunogenic properties. These contain an epitope-bearing portion of the adenylate cyclase and variants. These epitope- bearing peptides are useful to raise antibodies that bind specifically to a adenylate cyclase polypeptide or region or fragment. These peptides can contain at least 10, 12, at least 14, or between at least about 15 to about 30 amino acids.
  • Non-limiting examples of antigenic polypeptides that can be used to generate antibodies include but are not limited to peptides derived from an extracellular site. Regions having a high antigenicity index are shown in Figure 3. However, intracellularly-made antibodies (“intrabodies”) are also encompassed, which would recognize intracellular peptide regions.
  • the epitope-bearing adenylate cyclase polypeptides may be produced by any conventional means (Houghten, R.A. (1985) Proc. Natl. Acad. Sci. USA 52:5131-5135). Simultaneous multiple peptide synthesis is described in U.S. Patent No. 4,631 ,211.
  • Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the adenylate cyclase fragment and an additional region fused to the carboxyl terminus of the fragment.
  • the invention thus provides chimeric or fusion proteins. These comprise a adenylate cyclase peptide sequence operatively linked to a heterologous peptide having an amino acid sequence not substantially homologous to the adenylate cyclase. "Operatively linked” indicates that the adenylate cyclase peptide and the heterologous peptide are fused in-frame.
  • the heterologous peptide can be fused to the N-terminus or
  • C-terminus of the adenylate cyclase or can be internally located.
  • the fusion protein does not affect adenylate cyclase function per se.
  • the fusion protein can be a GST-fusion protein in which the adenylate cyclase sequences are fused to the N- or C-terminus of the GST sequences.
  • fusion proteins include, but are not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions.
  • enzymatic fusion proteins for example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions.
  • Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant adenylate cyclase.
  • expression and/or secretion of a protein can be increased by using a heterologous signal sequence. Therefore, in another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus.
  • EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions.
  • the Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists (Bennett et al. (1995) J. Mol. Recog. 5:52-58 (1995) and Johanson et al. J. Biol. Chem. 270:9459-941 ).
  • this invention also encompasses soluble fusion proteins containing a adenylate cyclase polypeptide and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl , where fusion takes place at the hinge region.
  • the Fc part can be removed in a simple way by a cleavage sequence, which is also incorporated and can be cleaved with factor Xa.
  • a chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re- amplified to generate a chimeric gene sequence (see Ausubel et al. (1992) Current Protocols in Molecular Biology).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
  • An adenylate cyclase-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the adenylate cyclase.
  • a adenylate cyclase polypeptide is encompassed by the present invention in which one or more of the adenylate cyclase domains (or parts thereof) has been replaced by homologous domains (or parts thereof) from another adenylate cyclase family. Accordingly, various permutations are possible.
  • the aminoterminal regulatory domain, or subregion thereof can be replaced with the domain or subregion from another isoform or adenylate cyclase family.
  • the catalytic domain or parts thereof can be replaced; the carboxyterminal domain or subregion can be replaced.
  • chimeric adenylate cyclases can be formed in which one or more of the native domains or subregions has been replaced by another.
  • chimeric adenylate cyclase proteins can be produced in which one or more functional sites is derived from a different isoform, or from another adenylate cyclase family. It is understood, however, that sites could be derived from adenylate cyclase families that occur in the mammalian genome but which have not yet been discovered or characterized.
  • Such sites include but are not limited to a catalytic site, regulatory site, sites important for targeting to subcellular and cellular locations, sites functional for interaction with components of cyclic AMP- and cyclic GMP-dependent signal transduction pathways, phosphorylation sites, glycosylation sites, and other functional sites disclosed herein.
  • the isolated adenylate cyclase can be purified from cells that naturally express it, such as from those shown in Figures 5 and 6 and/or specifically disclosed herein above, among others, especially purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • the protein is produced by recombinant DNA techniques.
  • a nucleic acid molecule encoding the adenylate cyclase polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell.
  • the protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art.
  • polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included
  • the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.
  • Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides.
  • the aminoterminal residue of polypeptides made in E. coli, prior to proteolytic processing almost invariably will be N-formylmethionine.
  • the modifications can be a function of how the protein is made.
  • the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications.
  • the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.
  • the invention provides methods using antibodies that selectively bind to the adenylate cyclase and its variants and fragments.
  • An antibody is considered to selectively bind, even if it also binds to other proteins that are not substantially homologous with the adenylate cyclase. These other proteins share homology with a fragment or domain of the adenylate cyclase. This conservation in specific regions gives rise to antibodies that bind to both proteins by virtue of the homologous sequence. In this case, it would be understood that antibody binding to the adenylate cyclase is still selective.
  • the invention provides methods of using antibodies to isolate a adenylate cyclase by standard techniques, such as affinity chromatography or immunoprecipitation.
  • the antibodies can facilitate the purification of the adenylate cyclase from cells naturally expressing it and cells recombinantly producing it.
  • the antibodies can be used to detect the presence of adenylate cyclase in cells or tissues to determine the pattern of expression of the adenylate cyclase among various tissues in an organism and over the course of normal development.
  • the antibodies can be used to detect adenylate cyclase in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.
  • the antibodies can be used to assess abnormal tissue distribution or abnormal expression during development.
  • Antibody detection of circulating fragments of the full length adenylate cyclase can be used to identify adenylate cyclase turnover. Further, the antibodies can be used to assess adenylate cyclase expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to adenylate cyclase function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, or level of expression of the adenylate cyclase protein, the antibody can be prepared against the normal adenylate cyclase protein.
  • antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant adenylate cyclase.
  • intracellularly-made antibodies (“intrabodies”) are also encompassed, which would recognize intracellular adenylate cyclase peptide regions.
  • the antibodies can also be used to assess normal and aberrant subcellular localization in cells in the various tissues in an organism. Antibodies can be developed against the whole adenylate cyclase or portions of the adenylate cyclase.
  • the diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting adenylate cyclase expression level or the presence of aberrant adenylate cyclases and aberrant tissue distribution or developmental expression, antibodies directed against the adenylate cyclase or relevant fragments can be used to monitor therapeutic efficacy.
  • Antibodies accordingly can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic adenylate cyclase can be used to identify individuals that require modified treatment modalities.
  • Antibodies can also be used in diagnostic procedures as an immunological marker for aberrant adenylate cyclase analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.
  • the antibodies are also useful for tissue typing.
  • tissue typing where the adenylate cyclase is expressed in a specific tissue, antibodies that are specific for this adenylate cyclase can be used to identify the tissue type.
  • the antibodies are also useful in forensic identification. Accordingly, where an individual has been correlated with a specific genetic polymorphism resulting in a specific polymorphic protein, an antibody specific for the polymorphic protein can be used as an aid in identification.
  • the antibodies are also useful for inhibiting adenylate cyclase function, for example, blocking binding of GTP or ATP, G-protein, or the catalytic site.
  • An antibody can be used, for example, to block ATP or GTP binding.
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact adenylate cyclase. Completely human antibodies are particularly desirable for therapeutic treatment of human patients.
  • this technology for producing human antibodies see Lonberg et al. (1995) Int. Rev. Immunol. 75:65-93.
  • this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661 ,016; and U.S. Patent 5,545,806.
  • kits for using antibodies to detect the presence of a adenylate cyclase protein in a biological sample can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting adenylate cyclase in a biological sample; means for determining the amount of adenylate cyclase in the sample; and means for comparing the amount of adenylate cyclase in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect adenylate cyclase.
  • the methods for using antibodies described above are based on the generation of antibodies that specifically bind to the adenylate cyclase or its variants or fragments.
  • an isolated adenylate cyclase polypeptide is used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Either the full-length protein or antigenic peptide fragment can be used. Regions having a high antigenicity index are shown in Figure 3.
  • Antibodies are preferably prepared from these regions or from discrete fragments in these regions. However, antibodies can be prepared from any region of the peptide as described herein. A preferred fragment produces an antibody that diminishes or completely prevents G-protein ATP or GTP binding.
  • Antibodies can be developed against the entire adenylate cyclase or domains of the adenylate cyclase as described herein. Antibodies can also be developed against specific functional sites as disclosed herein.
  • the antigenic peptide can comprise a contiguous sequence of at least 12, 14, 15, or 30 amino acid residues.
  • fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions. These fragments are not to be construed, however, as encompassing any fragments, which may be disclosed prior to the invention.
  • Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab') ) can be used.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I,
  • adenylate cyclase polynucleotide is particularly applicable to the cells, tissues, and disorders shown in Figures 5 and 6, and specifically discussed herein above.
  • nucleic acid fragments useful to practice the invention provide probes or primers in assays, such as those described herein.
  • Probes are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid. Such probes include polypeptide nucleic acids, as described in Nielsen et al. (1991) Science 254:1497-1500.
  • a probe comprises a region of nucleotide sequence that hybridizes under highly stringent conditions to at least about 15, typically about 20- 25, and more typically about 40, 50 or 75 consecutive nucleotides of the nucleic acid sequence shown in SEQ ID NO: 2 and the complements thereof. More typically, the probe further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
  • primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis using well- known methods (e.g., PCR, LCR) including, but not limited to those described herein.
  • the appropriate length of the primer depends on the particular use, but typically ranges from about 15 to 30 nucleotides.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the nucleic acid sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the sequence to be amplified.
  • the adenylate cyclase polynucleotides can be utilized as probes and primers in biological assays.
  • polynucleotides are used to assess adenylate cyclase properties or functions, such as in the assays described herein, all or less than all of the entire cDNA can be useful.
  • Assays specifically directed to adenylate cyclase functions such as assessing agonist or antagonist activity, encompass the use of known fragments.
  • diagnostic methods for assessing adenylate cyclase function can also be practiced with any fragment, including those fragments that may have been known prior to the invention.
  • all fragments are encompassed including those, which may have been known in the art.
  • the invention utilizes the adenylate cyclase polynucleotides as a hybridization probe for cDNA and genomic DNA to isolate a full-length cDNA and genomic clones encoding variant polypeptides and to isolate cDNA and genomic clones that correspond to variants producing the same polypeptides shown in SEQ ID NO: 1 or the other variants described herein.
  • Variants can be isolated from the same tissue and organism from which the polypeptide shown in SEQ ID NO:l was isolated, different tissues from the same organism, or from different organisms. This method is useful for isolating variant genes and cDNA that are developmentally controlled and therefore may be expressed in the same tissue or different tissues at different points in the development of an organism. This method is useful for isolating variant genes and cDNA that are expressed in the cells, tissues, and disorders disclosed herein.
  • the probe can correspond to any sequence along the entire length of the gene encoding the adenylate cyclase. Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3' noncoding regions.
  • the nucleic acid probe can be, for example, the full-length cDNA of SEQ ID NO: 1
  • fragments of the polynucleotides described herein can also be used to synthesize larger fragments or full-length polynucleotides described herein. For example, a fragment can be hybridized to any portion of an mRNA and a larger or full-length cDNA can be produced. Fragments can also be used to synthesize antisense molecules of desired length and sequence.
  • Antisense nucleic acids useful in treatment and diagnosis, can be designed using the nucleotide sequences of SEQ ID NO:2, and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 - methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • nucleic acid molecules useful to practice the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5).
  • peptide nucleic acids or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 95:14670.
  • PNAs can be further modified, e.g., to enhance their stability, specificity or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DN A chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • nucleic acid molecules and fragments useful to practice the invention can also include other appended groups such as peptides (e.g., for targeting host cell adenylate cyclases in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 56:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 54:648-652; PCT Publication No. WO 88/0918) or the blood brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell adenylate cyclases in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 56:6553-6556; Lemaitre
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
  • adenylate cyclase polynucleotides can also be used as primers for PCR to amplify any given region of a adenylate cyclase polynucleotide.
  • the adenylate cyclase polynucleotides can also be used to construct recombinant vectors.
  • Such vectors include expression vectors that express a portion of, or all of, the adenylate cyclase polypeptides.
  • Vectors also include insertion vectors, used to integrate into another polynucleotide sequence, such as into the cellular genome, to alter in situ expression of adenylate cyclase genes and gene products.
  • an endogenous adenylate cyclase coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.
  • the adenylate cyclase polynucleotides can also be used to express antigenic portions of the adenylate cyclase protein.
  • the adenylate cyclase polynucleotides can also be used as probes for determining the chromosomal positions of the adenylate cyclase polynucleotides by means of in situ hybridization methods, such as FISH.
  • FISH in situ hybridization methods
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with a specified gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations, that are visible from chromosome spreads, or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the adenylate cyclase polynucleotide probes can also be used to determine patterns of the presence of the gene encoding the adenylate cyclase with respect to tissue distribution, for example, whether gene duplication has occurred and whether the duplication occurs in all or only a subset of cells in a tissue.
  • the genes can be naturally occurring or can have been introduced into a cell, tissue, or organism exogenously.
  • the adenylate cyclase polynucleotides can also be used to design ribozymes corresponding to all, or a part, of the mRNA produced from genes encoding the polynucleotides described herein, the ribozymes being useful to treat or diagnose a disorder or otherwise modulate expression of the nucleic acid.
  • the adenylate cyclase polynucleotides can also be used to make vectors that express part, or all, of the adenylate cyclase polypeptides.
  • the adenylate cyclase polynucleotides can also be used to construct host cells expressing a part, or all, of the adenylate cyclase polynucleotides and polypeptides.
  • the adenylate cyclase polynucleotides can also be used to construct transgenic animals expressing all, or a part, of the adenylate cyclase polynucleotides and polypeptides.
  • the adenylate cyclase polynucleotides can also be used as hybridization probes to determine the level of adenylate cyclase nucleic acid expression. Accordingly, the probes can be used to detect the presence of, or to determine levels of, adenylate cyclase nucleic acid in cells, tissues, and in organisms. DNA or RNA level can be determined. Probes can be used to assess gene copy number in a given cell, tissue, or organism. This is particularly relevant in cases in which there has been an amplification of the adenylate cyclase gene.
  • the probe can be used in an in situ hybridization context to assess the position of extra copies of the adenylate cyclase gene, as on extrachromosomal elements or as integrated into chromosomes in which the adenylate cyclase gene is not normally found, for example, as a homogeneously staining region.
  • disorders include diseases of the heart, such as myopathy and ischemia.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant expression or activity of adenylate cyclase nucleic acid, in which a test sample is obtained from a subject and nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of the nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of the nucleic acid.
  • nucleic acid e.g., mRNA, genomic DNA
  • One aspect of the invention relates to diagnostic assays for determining nucleic acid expression as well as activity in the context of a biological sample (e.g., blood, serum, cells, tissue) to determine whether an individual has a disease or disorder, or is at risk of developing a disease or disorder, associated with aberrant nucleic acid expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with expression or activity of the nucleic acid molecules.
  • In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.
  • Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express the adenylate cyclase, such as by measuring the level of a adenylate cyclase- encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if the adenylate cyclase gene has been mutated.
  • Nucleic acid expression assays are useful for drug screening to identify compounds that modulate adenylate cyclase nucleic acid expression (e.g., antisense, polypeptides, peptidomimetics, small molecules or other drugs).
  • a cell is contacted with a candidate compound and the expression of mRNA determined.
  • the level of expression of the mRNA in the presence of the candidate compound is compared to the level of expression of the mRNA in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression.
  • the modulator can bind to the nucleic acid or indirectly modulate expression, such as by interacting with other cellular components that affect nucleic acid expression.
  • Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the gene to a subject) in patients or in transgenic animals.
  • the invention thus provides a method for identifying a compound that can be used to treat a disorder associated with expression of the adenylate cyclase gene.
  • the method typically includes assaying the ability of the compound to modulate the expression of the adenylate cyclase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by excessive or deficient adenylate cyclase nucleic acid expression.
  • the assays can be performed in cell-based and cell-free systems, such as systems using the tissues described herein, in which the gene is expressed or in model systems for the disorders to which the invention pertains.
  • Cell-based assays include cells naturally expressing the adenylate cyclase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
  • candidate compounds can be assayed in vivo in patients or in transgenic animals.
  • the assay for adenylate cyclase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway (such as cAMP or cGMP turnover). Further, the expression of genes that are up- or down-regulated in response to the adenylate cyclase signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase. Thus, modulators of adenylate cyclase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined.
  • the level of expression of adenylate cyclase mRNA in the presence of the candidate compound is compared to the level of expression of adenylate cyclase mRNA in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression.
  • expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression.
  • nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
  • the invention provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate adenylate cyclase nucleic acid expression.
  • Modulation includes both up- regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or effects on nucleic acid activity (e.g. when nucleic acid is mutated or improperly modified).
  • Treatment is of disorders characterized by aberrant expression or activity of the nucleic acid.
  • the gene is particularly relevant for the treatment of disorders involving the tissues shown in Figures 5 and 6, particularly in prostate, skeletal muscle, brain, and testes, as well as tissues and cells involved in myopathy and ischemia.
  • a modulator for adenylate cyclase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the adenylate cyclase nucleic acid expression.
  • the adenylate cyclase polynucleotides are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the adenylate cyclase gene in clinical trials or in a treatment regimen.
  • the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance.
  • the gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant.
  • the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.
  • Monitoring can be, for example, as follows: (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a specified mRNA or genomic DNA of the invention in the pre- administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the mRNA or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the mRNA or genomic DNA in the pre-administration sample with the mRNA or genomic DNA in the post-administration sample or samples; and (vi) increasing or decreasing the administration of the agent to the subject accordingly.
  • the adenylate cyclase polynucleotides can be used in diagnostic assays for qualitative changes in adenylate cyclase nucleic acid, and particularly in qualitative changes that lead to pathology.
  • the polynucleotides can be used to detect mutations in adenylate cyclase genes and gene expression products such as mRNA.
  • the polynucleotides can be used as hybridization probes to detect naturally-occurring genetic mutations in the adenylate cyclase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation.
  • Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the adenylate cyclase gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a adenylate cyclase.
  • Mutations in the adenylate cyclase gene can be detected at the nucleic acid level by a variety of techniques. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In certain embodiments, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 247:1077-1080; and Nakazawa et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 57: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 56:1173-1 177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1 197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a adenylate cyclase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.
  • sequence-specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method.
  • sequence differences between a mutant adenylate cyclase gene and a wild-type gene can be determined by direct DNA sequencing.
  • a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 79:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 56:127-162; and Griffin et al. ( ⁇ 993) Appl. Biochem. Biotechnol. 55:147-159).
  • RNA/RNA or RNA/DNA duplexes Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science 230:1242); Cotton et al. (1988) PNAS 55:4397; Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al. (1989) PNAS 56:2766; Cotton et al. (1993) Mutat. Res. 255:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
  • RNA rather than DNA
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5). Examples of other techniques for detecting point mutations include, selective oligonucleotide hybridization, selective amplification, and selective primer extension.
  • genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759).
  • genetic mutations can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations.
  • This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • the adenylate cyclase polynucleotides can also be used for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality.
  • the polynucleotides can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship).
  • a mutation in the adenylate cyclase gene that results in altered affinity for ATP or GTP could result in an excessive or decreased drug effect with standard concentrations of ATP or GTP.
  • the adenylate cyclase polynucleotides described herein can be used to assess the mutation content of the gene in an individual in order to select an appropriate compound or dosage regimen for treatment.
  • polynucleotides displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.
  • the methods can involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting mRNA, or genomic DNA, such that the presence of mRNA or genomic DNA is detected in the biological sample, and comparing the presence of mRNA or genomic DNA in the control sample with the presence of mRNA or genomic DNA in the test sample.
  • the adenylate cyclase polynucleotides are also useful for chromosome identification when the sequence is identified with an individual chromosome and to a particular location on the chromosome.
  • the DNA sequence is matched to the chromosome by in situ or other chromosome-specific hybridization. Sequences can also be correlated to specific chromosomes by preparing PCR primers that can be used for PCR screening of somatic cell hybrids containing individual chromosomes from the desired species. Only hybrids containing the chromosome containing the gene homologous to the primer will yield an amplified fragment. Sublocalization can be achieved using chromosomal fragments.
  • mapping strategies include prescreening with labeled flow-sorted chromosomes and preselection by hybridization to chromosome- specific libraries.
  • Further mapping strategies include fluorescence in situ hybridization, which allows hybridization with probes shorter than those traditionally used.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on the chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • the adenylate cyclase polynucleotides can also be used to identify individuals from small biological samples. This can be done for example using restriction fragment- length polymorphism (RFLP) to identify an individual.
  • RFLP restriction fragment- length polymorphism
  • the polynucleotides described herein are useful as DNA markers for RFLP (See U.S. Patent No. 5,272,057).
  • the adenylate cyclase sequence can be used to provide an alternative technique, which determines the actual DNA sequence of selected fragments in the genome of an individual.
  • the adenylate cyclase sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify DNA from an individual for subsequent sequencing.
  • Panels of corresponding DNA sequences from individuals prepared in this manner can provide unique individual identifications, as each individual will have a unique set of such DNA sequences. It is estimated that allelic variation in humans occurs with a frequency of about once per each 500 bases. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions.
  • the adenylate cyclase sequences can be used to obtain such identification sequences from individuals and from tissue.
  • the sequences represent unique fragments of the human genome.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • a panel of reagents from the sequences is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification of the individual, living or dead can be made from extremely small tissue samples.
  • the adenylate cyclase polynucleotides can also be used in forensic identification procedures. PCR technology can be used to amplify DNA sequences taken from very small biological samples, such as a single hair follicle, body fluids (e.g. blood, saliva, or semen). The amplified sequence can then be compared to a standard allowing identification of the origin of the sample.
  • the adenylate cyclase polynucleotides can thus be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an identification marker i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to the noncoding region are particularly useful since greater polymorphism occurs in the noncoding regions, making it easier to differentiate individuals using this technique.
  • the adenylate cyclase polynucleotides can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This is useful in cases in which a forensic pathologist is presented with a tissue of unknown origin. Panels of adenylate cyclase probes can be used to identify tissue by species and/or by organ type. In a similar fashion, these primers and probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This is useful in cases in which a forensic pathologist is presented with a tissue of unknown origin.
  • the adenylate cyclase polynucleotides can be used directly to block transcription or translation of adenylate cyclase gene sequences by means of antisense or ribozyme constructs.
  • nucleic acids can be directly used for treatment.
  • the adenylate cyclase polynucleotides are thus useful as antisense constructs to control adenylate cyclase gene expression in cells, tissues, and organisms.
  • a DNA antisense polynucleotide is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of adenylate cyclase protein.
  • An antisense RNA or DNA polynucleotide would hybridize to the mRNA and thus block translation of mRNA into adenylate cyclase protein.
  • antisense molecules useful to inhibit nucleic acid expression include antisense molecules complementary to a fragment of the 5' untranslated region of SEQ ID NO:2 which also includes the start codon and antisense molecules which are complementary to a fragment of the 3' untranslated region of SEQ ID NO:2.
  • a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of adenylate cyclase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired adenylate cyclase nucleic acid expression.
  • This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the adenylate cyclase protein.
  • the adenylate cyclase polynucleotides also provide vectors for gene therapy in patients containing cells that are aberrant in adenylate cyclase gene expression.
  • recombinant cells which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired adenylate cyclase protein to treat the individual.
  • kits for detecting the presence of a adenylate cyclase nucleic acid in a biological sample can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting adenylate cyclase nucleic acid in a biological sample; means for determining the amount of adenylate cyclase nucleic acid in the sample; and means for comparing the amount of adenylate cyclase nucleic acid in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect adenylate cyclase mRNA or DNA.
  • the nucleotide sequence in SEQ ID NO:2 was obtained by sequencing the deposited human cDNA. Accordingly, the sequence of the deposited clone is controlling as to any discrepancies between the two and any reference to the sequence of SEQ ID NO:2 includes reference to the sequence of the deposited cDNA.
  • the specifically disclosed cDNA comprises the coding region and 5' and 3' untranslated sequences in SEQ ID NO:2.
  • the invention provides isolated polynucleotides encoding the adenylate cyclase.
  • adenylate cyclase polynucleotide or “adenylate cyclase nucleic acid” refers to the sequence shown in SEQ ID NO:2 or in the deposited cDNA.
  • adenylate cyclase polynucleotide or “adenylate cyclase nucleic acid” further includes variants and fragments of the adenylate cyclase polynucleotides.
  • the methods and uses described herein can be based on the adenylate cyclase polynucleotide as a reagent or as a target.
  • an "isolated" adenylate cyclase nucleic acid is one that is separated from other nucleic acid present in the natural source of the adenylate cyclase nucleic acid.
  • an "isolated” nucleic acid is free of sequences which naturally flank the adenylate cyclase nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • flanking nucleotide sequences for example up to about 5KB.
  • adenylate cyclase nucleic acid is isolated from flanking sequences such that it can be subjected to the specific manipulations described herein, such as recombinant expression, preparation of probes and primers, and other uses specific to the adenylate cyclase nucleic acid sequences.
  • an "isolated" nucleic acid molecule such as a cDNA or RNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.
  • an isolated nucleic acid comprises at least about 50, 80 or 90 % (on a molar basis) of all macromolecular species present.
  • recombinant DNA molecules contained in a vector are considered isolated.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • the isolated material will form part of a composition (or example, a crude extract containing other substances), buffer system or reagent mix.
  • the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.
  • an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
  • the adenylate cyclase polynucleotides can encode the mature protein plus additional amino or carboxyterminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • the adenylate cyclase polynucleotides include, but are not limited to, the sequence encoding the mature polypeptide alone, the sequence encoding the mature polypeptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature polypeptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non- translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA.
  • the polynucleotide may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.
  • Adenylate cyclase polynucleotides can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
  • the nucleic acid, especially DNA can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).
  • the adenylate cyclase nucleic acid comprises only the coding region.
  • the invention further provides variant adenylate cyclase polynucleotides, and fragments thereof, that differ from the nucleotide sequence shown in SEQ ID NO: 2 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence shown in SEQ ID NO:2.
  • the invention also provides adenylate cyclase nucleic acid molecules encoding the variant polypeptides described herein.
  • Such polynucleotides may be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis.
  • variants may be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions.
  • variants typically have a substantial identity with a nucleic acid molecule of SEQ ID NO:2 and the complements thereof. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non- conservative amino acid substitutions.
  • Orthologs, homologs, and allelic variants can be identified using methods well known in the art. These variants comprise a nucleotide sequence encoding a adenylate cyclase that is at least about 60-65%, 65-70%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more homologous to the nucleotide sequence shown in SEQ ID NO:2 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO:2 or a fragment of the sequence.
  • stringent hybridization does not indicate substantial homology where it is due to general homology, such as poly A sequences, or sequences common to all or most proteins, or all adenylate cyclases.
  • variants per se do not include any nucleic acid (or amino acid) sequence disclosed prior to the present invention, although the methods herein can encompass such variants.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a polypeptide at least about 60-65% homologous to each other typically remain hybridized to each other.
  • the conditions can be such that sequences at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95% or more identical to each other remain hybridized to one another.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • nucleic acid molecules are allowed to hybridize in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more low stringency washes in 0.2X SSC/0.1% SDS at room temperature, or by one or more moderate stringency washes in 0.2X SSC/0.1% SDS at 42°C, or washed in 0.2X SSC/0.1% SDS at 65°C for high stringency.
  • an isolated nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or SEQ ID NO:3 corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally- occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the exact conditions can be determined empirically and depend on ionic strength, temperature and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS. Other factors considered in determining the desired hybridization conditions include the length of the nucleic acid sequences, base composition, percent mismatch between the hybridizing sequences and the frequency of occurrence of subsets of the sequences within other non-identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules.
  • the present invention also provides isolated nucleic acids that contain a single or double stranded fragment or portion that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:2 or the complement of SEQ ID NO:2.
  • the nucleic acid consists of a portion of the nucleotide sequence of SEQ ID NO:2 and the complement of SEQ ID NO:2.
  • the nucleic acid fragments of the invention are at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic proteins or polypeptides described herein are useful.
  • the invention provides polynucleotides that comprise a fragment of the full-length adenylate cyclase polynucleotide.
  • the fragment can be single or double-stranded and can comprise DNA or RNA.
  • the fragment can be derived from either the coding or the non-coding sequence.
  • an isolated adenylate cyclase nucleic acid encodes the entire coding region. In another embodiment the isolated adenylate cyclase nucleic acid encodes a sequence corresponding to the mature protein that may be from about amino acid 6 to the last amino acid. Other fragments include nucleotide sequences encoding the amino acid fragments described herein.
  • adenylate cyclase nucleic acid fragments further include sequences corresponding to the domains described herein, subregions also described, and specific functional sites.
  • Adenylate cyclase nucleic acid fragments also include combinations of the domains, segments, and other functional sites described above.
  • a adenylate cyclase fragment includes any nucleic acid sequence that does not include the entire gene.
  • the invention also provides adenylate cyclase nucleic acid fragments that encode epitope bearing regions of the adenylate cyclase proteins described herein.
  • nucleotide or amino acid sequences of the invention are also provided in a variety of mediums to facilitate use thereof.
  • "provided” refers to a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a nucleotide or amino acid sequence of the present invention.
  • Such a manufacture provides the nucleotide or amino acid sequences, or a subset thereof (e.g., a subset of open reading frames (ORFs)) in a form which allows a skilled artisan to examine the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form.
  • ORFs open reading frames
  • a nucleotide or amino acid sequence of the present invention can be recorded on computer readable media.
  • computer readable media refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • the skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide or amino acid sequence information of the present invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention.
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.
  • the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • the skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
  • nucleotide or amino acid sequences of the invention can routinely access the sequence information for a variety of purposes.
  • one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
  • Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
  • a "target sequence" can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.
  • sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues.
  • sequence fragments involved in gene expression and protein processing may be of shorter length.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzyme active sites and signal sequences.
  • Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
  • Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences.
  • a variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
  • ORFs open reading frames
  • Such ORFs are protein encoding fragments and are useful in producing commercially important proteins such as enzymes used in various reactions and in the production of commercially useful metabolites.
  • vectors and host cells are particularly relevant where vectors are expressed in the cells, tissues, and disorders shown in Figures 5 and 6, and otherwise discussed herein, or where the host cells are those that naturally express the gene, as shown in these figures and which may be the native or a recombinant cell expressing the gene.
  • host cells and “recombinant host cells” refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the host cells expressing the polypeptides described herein, and particularly recombinant host cells have a variety of uses. First, the cells are useful for producing adenylate cyclase proteins or polypeptides that can be further purified to produce desired amounts of adenylate cyclase protein or fragments. Thus, host cells containing expression vectors are useful for polypeptide production, as well as cells producing significant amounts of the polypeptide, for example, the high-expressers shown in Figure 6, in other words, testes, prostate, skeletal muscle and brain.
  • Host cells are also useful for conducting cell-based assays involving the adenylate cyclase or adenylate cyclase fragments.
  • a recombinant host cell expressing a native adenylate cyclase is useful to assay for compounds that stimulate or inhibit adenylate cyclase function. This includes ATP or GTP binding, gene expression at the level of transcription or translation, G-protein interaction, and components of the signal transduction pathway.
  • Host cells are also useful for identifying adenylate cyclase mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant adenylate cyclase (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native adenylate cyclase.
  • a desired effect on the mutant adenylate cyclase for example, stimulating or inhibiting function
  • Recombinant host cells are also useful for expressing the chimeric polypeptides described herein to assess compounds that activate or suppress activation by means of a heterologous domain, segment, site, and the like, as disclosed herein.
  • mutant adenylate cyclases can be designed in which one or more of the various functions is engineered to be increased or decreased (e.g., ATP binding or G- protein binding) and used to augment or replace adenylate cyclase proteins in an individual.
  • host cells can provide a therapeutic benefit by replacing an aberrant adenylate cyclase or providing an aberrant adenylate cyclase that provides a therapeutic result.
  • the cells provide adenylate cyclases that are abnormally active.
  • the cells provide a adenylate cyclase that is abnormally inactive.
  • This adenylate cyclase can compete with endogenous adenylate cyclase in the individual.
  • cells expressing adenylate cyclases that cannot be activated are introduced into an individual in order to compete with endogenous adenylate cyclase for ATP.
  • endogenous adenylate cyclase for ATP.
  • Homologously recombinant host cells can also be produced that allow the in situ alteration of endogenous adenylate cyclase polynucleotide sequences in a host cell genome.
  • the host cell includes, but is not limited to, a stable cell line, cell in vivo, or cloned microorganism. This technology is more fully described in WO 93/09222, WO 91/12650, WO 91/06667, U.S. 5,272,071, and U.S. 5,641,670.
  • adenylate cyclase polynucleotides or sequences proximal or distal to a adenylate cyclase gene are allowed to integrate into a host cell genome by homologous recombination where expression of the gene can be affected.
  • regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence. Accordingly, a adenylate cyclase protein can be produced in a cell not normally producing it. Alternatively, increased expression of adenylate cyclase protein can be effected in a cell normally producing the protein at a specific level. Further, expression can be decreased or eliminated by introducing a specific regulatory sequence.
  • the regulatory sequence can be heterologous to the adenylate cyclase protein sequence or can be a homologous sequence with a desired mutation that affects expression. Alternatively, the entire gene can be deleted.
  • the regulatory sequence can be specific to the host cell or capable of functioning in more than one cell type. Still further, specific mutations can be introduced into any desired region of the gene to produce mutant adenylate cyclase proteins. Such mutations could be introduced, for example, into the specific functional regions such as the nucleotide triphosphate site.
  • the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the altered adenylate cyclase gene.
  • the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See also Thomas et al., Cell 57:503 (1987) for a description of homologous recombination vectors.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous adenylate cyclase gene is selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • a transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a adenylate cyclase protein and identifying and evaluating modulators of adenylate cyclase protein activity.
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
  • a host cell is a fertilized oocyte or an embryonic stem cell into which adenylate cyclase polynucleotide sequences have been introduced.
  • a transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Any of the adenylate cyclase nucleotide sequences can be introduced as a transgene into the genome of a non- human animal, such as a mouse.
  • any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the adenylate cyclase protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes.
  • a transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
  • transgenic non-human animals can be produced which contain selected systems, which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991) Science 257:1351-1355.
  • cre/loxP recombinase system is used to regulate expression of the transgene
  • animals containing transgenes encoding both the Cre recombinase and a selected protein is required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 555:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to a pseudopregnant female foster animal.
  • the offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • Transgenic animals containing recombinant cells that express the polypeptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could affect cAMP binding, adenylate cyclase activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo adenylate cyclase function, including ATP interaction, the effect of specific mutant adenylate cyclases on adenylate cyclase function and ATP interaction, and the effect of chimeric adenylate cyclases. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more adenylate cyclase functions.
  • methods for producing transgenic animals include introducing a nucleic acid sequence according to the present invention, the nucleic acid sequence capable of expressing the protein in a transgenic animal, into a cell in culture or in vivo.
  • the nucleic acid is introduced into an intact organism such that one or more cell types and, accordingly, one or more tissue types, express the nucleic acid encoding the protein.
  • the nucleic acid can be introduced into virtually all cells in an organism by transfecting a cell in culture, such as an embryonic stem cell, as described herein for the production of transgenic animals, and this cell can be used to produce an entire transgenic organism.
  • the host cell can be a fertilized oocyte. Such cells are then allowed to develop in a female foster animal to produce the transgenic organism.
  • the methods using the vectors and host cells discussed above are based on the vectors and host cells including, but not limited to, those described below.
  • the invention also provides methods using vectors containing the adenylate cyclase polynucleotides.
  • the term "vector” refers to a vehicle, preferably a nucleic acid molecule that can transport the adenylate cyclase polynucleotides.
  • the vector is a nucleic acid molecule, the adenylate cyclase polynucleotides are covalently linked to the vector nucleic acid.
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the adenylate cyclase polynucleotides.
  • the vector may integrate into the host cell genome and produce additional copies of the adenylate cyclase polynucleotides when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the adenylate cyclase polynucleotides.
  • the vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the adenylate cyclase polynucleotides such that transcription of the polynucleotides is allowed in a host cell.
  • the polynucleotides can be introduced into the host cell with a separate polynucleotide capable of affecting transcription.
  • the second polynucleotide may provide a trans-acting factor interacting with the cis- regulatory control region to allow transcription of the adenylate cyclase polynucleotides from the vector.
  • a trans-acting factor may be supplied by the host cell.
  • a trans-acting factor can be produced from the vector itself.
  • transcription and/or translation of the adenylate cyclase polynucleotides can occur in a cell-free system.
  • the regulatory sequence to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
  • expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers.
  • regions that modulate transcription include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation.
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals.
  • the person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • a variety of expression vectors can be used to express a adenylate cyclase polynucleotide.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.
  • Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e., tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • tissue specific i.e., tissue specific
  • exogenous factor such as a hormone or other ligand.
  • vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • the adenylate cyclase polynucleotides can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.
  • the vector containing the appropriate polynucleotide can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells. As described herein, it may be desirable to express the polypeptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the adenylate cyclase polypeptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired polypeptide can ultimately be separated from the fusion moiety.
  • Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Smith et al. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • suitable inducible non- fusion E. coli expression vectors include pTrc (Amann et al.
  • the adenylate cyclase polynucleotides can also be expressed by expression vectors that are operative in yeast.
  • yeast e.g., S. cerevisiae
  • vectors for expression in yeast include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234 ), pMFa (Kurjan et al. (1982) Cell 50:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Co ⁇ oration, San Diego, CA).
  • the adenylate cyclase polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 5:2156-2165) and the pVL series (Lucklow et al. (1989) Virology 770:31-39).
  • the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 529:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the adenylate cyclase polynucleotides.
  • the person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the polynucleotides described herein. These are found for example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, of the polynucleotide sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • the invention also relates to recombinant host cells containing the vectors described herein.
  • Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • the recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Host cells can contain more than one vector.
  • different nucleotide sequences can be introduced on different vectors of the same cell.
  • the adenylate cyclase polynucleotides can be introduced either alone or with other polynucleotides that are not related to the adenylate cyclase polynucleotides such as those providing trans-acting factors for expression vectors.
  • the vectors can be introduced independently, co-introduced or joined to the adenylate cyclase polynucleotide vector.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction.
  • Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
  • Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs.
  • the marker can be contained in the same vector that contains the polynucleotides described herein or may be on a separate vector.
  • Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
  • RNA derived from the DNA constructs described herein can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences.
  • cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
  • appropriate secretion signals are incorporated into the vector.
  • the signal sequence can be endogenous to the adenylate cyclase polypeptides or heterologous to these polypeptides.
  • the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like.
  • the polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.
  • polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria.
  • polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process.
  • the invention encompasses use of the polypeptides, nucleic acids, and other agents in pharmaceutical compositions to administer to the cells in which expression of the adenylate cyclase is relevant and in disorders as disclosed herein. Uses are both diagnostic and therapeutic.
  • the adenylate cyclase nucleic acid molecules, protein, modulators of the protein, and antibodies can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human.
  • Such compositions typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier. It is understood however, that administration can also be to cells in vitro as well as to in vivo model systems such as non-human transgenic animals.
  • administer is used in its broadest sense and includes any method of introducing the compositions of the present invention into a subject. This includes producing polypeptides or polynucleotides in vivo as by transcription or translation, in vivo, of polynucleotides that have been exogenously introduced into a subject. Thus, polypeptides or nucleic acids produced in the subject from the exogenous compositions are encompassed in the term "administer.”
  • 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, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (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.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycer
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), 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 which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a adenylate cyclase protein or anti- adenylate cyclase antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a adenylate cyclase protein or anti- adenylate cyclase antibody
  • 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.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired 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.
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods.
  • 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.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like 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 suitable propellant 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.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, 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 Pharmaceuticals, Inc.
  • Liposomal suspensions 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. Patent No. 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 calculated 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 nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 97:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • organic or inorganic compounds i.e., including heteroorganic and organometallic compounds
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • Page 28 line 10; page 85, lines 7, 12, 19, 21 , 25 and 31 ; page 86, lines 8, 12, 25, 27 and 29; page 87, line 8

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Abstract

L'invention porte sur une adénylate cyclase humaine récemment identifiée; sur les polynucléotides codant pour elle; sur un procédé d'utilisation des polypeptides et polynucléotides de l'adénylate cyclase comme cibles pour diagnostiquer et traiter les troubles induits par ou associés à l'adénylate cyclase; sur des procédés de criblage recourant aux polypeptides et polynucléotides de l'adénylate cyclase pour identifier des agonistes et antagonistes à des fins de diagnostique et de traitement; sur des agonistes et antagonistes basés sur les polypeptides et polynucléotides de l'adénylate cyclase; et sur des agonistes et antagonistes identifiés par des procédés de criblage de médicaments utilisant comme cibles les polypeptides et polynucléotides de l'adénylate cyclase.
PCT/US2000/033797 1999-12-14 2000-12-13 La 25678, nouvelle adenylate cyclase humaine WO2001044453A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002038747A2 (fr) * 2000-11-13 2002-05-16 Bayer Aktiengesellschaft Regulation de la adenylate cyclase humaine
WO2003102175A1 (fr) * 2002-05-31 2003-12-11 Bayer Healthcare Ag Regulation de l'adenylate cyclase humaine, type ii

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996008260A1 (fr) * 1994-09-16 1996-03-21 Mount Sinai School Of Medicine Of The City University Of New York Gsα* ACTIVEE PAR MUTATION ET ADENYLYLE CYCLASE 2 UTILISABLES EN TANT QU'AGENTS THERAPEUTIQUES

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996008260A1 (fr) * 1994-09-16 1996-03-21 Mount Sinai School Of Medicine Of The City University Of New York Gsα* ACTIVEE PAR MUTATION ET ADENYLYLE CYCLASE 2 UTILISABLES EN TANT QU'AGENTS THERAPEUTIQUES

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FEINSTEIN, P.G. ET AL.: "Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain", PROC. NATL. ACAD. SCI., vol. 88, 1991, pages 10173 - 10177, XP000942286 *
KIKUNO R ET AL: "PREDICTION OF THE CODING SEQUENCE OF UNIDENTIFIED HUMAN GENES. XIV. THE COMPLETE SEQUENCES OF 100 NEW CDNA CLONES FROM BRAIN WHICH CODE FOR LARGE PROTEINS IN VITRO", DNA RESEARCH,UNIVERSAL ACADEMY PRESS,JP, vol. 6, 1999, pages 197 - 205, XP000930058, ISSN: 1340-2838 *
STENGEL,D. ET AL.: "Different chromosomal localization of two adenylyl cyclase genes expressed in human brain", HUM. GENET., vol. 90, 1992, pages 126 - 130, XP000986358 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002038747A2 (fr) * 2000-11-13 2002-05-16 Bayer Aktiengesellschaft Regulation de la adenylate cyclase humaine
WO2002038747A3 (fr) * 2000-11-13 2003-02-27 Bayer Ag Regulation de la adenylate cyclase humaine
US7205135B2 (en) 2000-11-13 2007-04-17 Bayer Healthcare Ag Regulation of human adenylate cyclase
WO2003102175A1 (fr) * 2002-05-31 2003-12-11 Bayer Healthcare Ag Regulation de l'adenylate cyclase humaine, type ii

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