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WO2003038083A1 - Regulation de l'adenylate cyclase humaine de type i - Google Patents

Regulation de l'adenylate cyclase humaine de type i Download PDF

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Publication number
WO2003038083A1
WO2003038083A1 PCT/EP2002/012059 EP0212059W WO03038083A1 WO 2003038083 A1 WO2003038083 A1 WO 2003038083A1 EP 0212059 W EP0212059 W EP 0212059W WO 03038083 A1 WO03038083 A1 WO 03038083A1
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adenylate cyclase
type
polynucleotide
polypeptide
amino acid
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PCT/EP2002/012059
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English (en)
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Zhimin Zhu
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Bayer Healthcare Ag
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Publication of WO2003038083A1 publication Critical patent/WO2003038083A1/fr

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    • 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)
    • 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.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the invention relates to the regulation of human type I adenylate cyclase.
  • Cyclases play important roles in the transduction of extracellular signals via their synthesis of "secondary messengers" such as adenosine 3', 5'-cyclic phosphate (cyclic adenosine monophosphate, cAMP) and guanosine 3', 5'- cyclic phosphate (cyclic guanosine monophosphate, cGMP).
  • Cell surface receptors mediate the transduction of an extracellular signal, such as the binding of a ligand to a receptor, into a signal that is transmitted internally within the cell.
  • the internal signal is carried by secondary messengers, which typically are produced in response to the binding of an external signal.
  • Cyclic AMP and cGMP play important roles in the regulation of a multitude of cellular activities.
  • cAMP responds to cellular signals through a specific protein kinase (cAMP-dependent protein kinase or protein kinase A) to phosphorylate target molecules, e.g., other protein kinases or proteins involved in transport or cellular morphology.
  • target molecules e.g., other protein kinases or proteins involved in transport or cellular morphology.
  • intracellular cAMP mediates many of the effects of hormones in the regulation of cellular metabolism and cell growth.
  • Cyclic GMP also acts as an intracellular messenger, for example, by activating cGMP-dependent kinases and regulating cGMP sensitive ion channels.
  • the role of cGMP as a secondary messenger has been well established in vascular smooth muscle relaxation and retinal phototransduction.
  • adenylate cyclase also referred to as adenylyl cyclase and adenyl cyclase.
  • adenylate cyclase activity may be affected by a factor/receptor binding event transmitted through an associated G protein. Interaction of several different external factors with their distinct receptors causes alterations in cAMP intracellular concentration (Broach et al., U.S. Patent 6,001,553). Different receptors are associated with their own particular G-protein intermediary, which itself is associated with adenylate cyclase.
  • adenylate cyclases types 1-9 At least nine distinct isoenzymes of mammalian adenylate cyclase have been identified and are designated as adenylate cyclases types 1-9 (Antoni et al., U.S. Patent 6,090,612). These adenylate cyclases have a general structure consisting of 12 transmembrane helices and two cytoplasmic, catalytic domains (Hurley, 1998, Curr. Opin. Struct. Biol. 8:770-77). Some of these enzymes have been analyzed functionally and appear to confer unique signal processing capacities to cells (Taussig et al., 1995, J. Biol. Chem. 270:1-4).
  • adenylate cyclase isozymes have distinct tissue distribution profiles (Iyengar, U.S. Patent 6,034,071). Localization studies using mRNA probes have been used to determine tissue distribution of the various adenylate cyclases (Pieroni et al, 1993, Curr. Opin. Neurobiol. 3: 345-351). Adenylate cyclase type 1 (AC 1) appears to be present only in neuronal tissue, whereas AC 2 has been found in brain and lung.
  • AC 3 has been localized in olfactory neurons as well as other neuronal and non- neuronal tissues (Glatt and Snyder, 1993, Nature 361 : 536-538; Xia et al, 1992, Neurosci. Lett. 144: 169-173).
  • AC 4 appears to be present at very low levels in brain, and throughout most tissues.
  • AC 5 and AC 6 have also been found to be widely distributed (Premont et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 9809-9813; Krupinski et al, 1992, J. Biol. Chem. 267: 24858- 24862) although AC 6 seems to be of very low abundance in the brain.
  • AC 5 is particularly abundant in the heart and in some regions of the brain.
  • AC 7 appears to be widely distributed, but may be scarce in the brain.
  • AC 8, like AC 1 seems to be abundant in the brain.
  • the distributions of AC 1, AC 2, AC 3, AC 5 and AC 8 show distinct regional patterns.
  • cyclases that have been identified thus far, together with their differing tissue distributions, demonstrate the importance of particular cellular cyclase profiles and their roles in signal transduction with respect to cellular function. Alterations in the levels of cyclic nucleotide intracellular (or secondary) messenger levels that result from alterations in particular cyclase activities have been implicated in a wide range of conditions and diseases, including cardiovascular disease, diseases of the central nervous system, intestinal conditions, retinal diseases, and shock. For instance, the high distribution of adenylate cyclases in brain has been correlated with a likely role for adenylate cyclases in neurological disorders such as Alzheimer's disease and Parkinson's disease. The high distribution of adenylate cyclase in the heart is likely to contribute to cardiovascular diseases, such as angina and hypertension.
  • One embodiment of the invention is an isolated polynucleotide encoding a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase.
  • the isolated polynucleotide is selected from the group consisting of: a polynucleotide encoding a protein that comprises amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 21 ; the amino acid sequence shown in SEQ ID NO:21; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 23; and the amino acid sequence shown in SEQ ID NO: 23.
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a type I adenylate cyclase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 21; the amino acid sequence shown in SEQ ID NO:21 ; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 23; and the amino acid sequence shown in SEQ ID NO: 23.
  • Binding between the test compound and the type I adenylate cyclase polypeptide is detected.
  • a test compound which binds to the type I adenylate cyclase polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the type I adenylate cyclase.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a type I adenylate cyclase polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; the nucleotide sequence shown in SEQ ID NO: 4; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence shown in SEQ ID NO: 20; nucleotide sequences which are at least about 50%) identical to the nucleotide sequence shown in SEQ ID NO: 22; and the nucleotide sequence shown in SEQ ID NO:22.
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the type I adenylate cyclase through interacting with the type I adenylate cyclase mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a type I adenylate cyclase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 21 ; the amino acid sequence shown in SEQ ID NO:21; amino acid sequences which are at least about 92% identical to the amino acid sequence shown in SEQ ID NO: 23; and the amino acid sequence shown in SEQ ID NO: 23.
  • a type I adenylate cyclase activity of the polypeptide is detected.
  • a test compound which increases type I adenylate cyclase activity of the polypeptide relative to type I adenylate cyclase activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases type I adenylate cyclase activity of the polypeptide relative to type I adenylate cyclase activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a type I adenylate cyclase product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; the nucleotide sequence shown in SEQ ID NO: 4; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence shown in SEQ ID NO: 20; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 22; and the nucleotide sequence shown in SEQ ID NO: 22.
  • Binding of the test compound to the type I adenylate cyclase product is detected.
  • a test compound which binds to the type I adenylate cyclase product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a type I adenylate cyclase polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ; the nucleotide sequence shown in SEQ ID NO: 1 ; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; the nucleotide sequence shown in SEQ ID NO: 4; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence shown in SEQ ID NO: 20; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown
  • Even another embodiment of the invention is an expression vector containing a polynucleotide that encodes a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase.
  • Yet another embodiment of the invention is a host cell containing an expression vector.
  • the expression vector contains a polynucleotide that encodes a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase.
  • Another embodiment of the invention is a substantially purified protein exhibiting biological properties of human type I adenylate cyclase.
  • Still another embodiment of the invention is a method for producing an isolated protein exhibiting biological properties of human type I adenylate cyclase.
  • a host cell containing an expression vector is cultured under conditions suitable for expression of a polypeptide encoded by a polynucleotide contained in the expression vector.
  • the polynucleotide encodes a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase.
  • the polypeptide is recovered from the host cell culture.
  • Another embodiment of the invention is a method for detection of polynucleotides encoding a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase in a biological sample.
  • a polynucleotide encoding a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase is hybridized to nucleic acid material of a biological sample, thereby forming a hybridization complex. The hybridization complex is detected.
  • Even another embodiment of the invention is a method for the detection of a polynucleotide encoding a human type I adenylate cyclase or a protein exhibiting biological properties of human type I adenylate cyclase.
  • a biological sample is contacted with a reagent which specifically interacts with the polynucleotide or protein. The interaction is detected.
  • Another embodiment of the invention is a diagnostic kit for detecting a polynucleotide encoding a human type I adenylate cyclase or a protein exhibiting biological properties of human type I adenylate cyclase in a biological sample.
  • Yet another embodiment of the invention is a method of modulating the activity of human type I adenylate cyclase.
  • a cell is contacted with a reagent that specifically binds to a polynucleotide encoding a human type I adenylate cyclase or a protein exhibiting biological properties of a human type I adenylate cyclase.
  • the activity of human type I adenylate cyclase is thereby reduced.
  • a further embodiment of the invention is a purified reagent that modulates the activity of a human type I adenylate cyclase polypeptide or polynucleotide.
  • Another embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a reagent that modulates the activity of a human type I adenylate cyclase and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising an expression vector comprising a polynucleotide encoding a human type I adenylate cyclase and a pharmaceutically acceptable carrier.
  • Still another embodiment of the invention is a method for treating disease.
  • An effective amount of a pharmaceutical composition comprising a reagent that modulates the activity of a human type I adenylate cyclase or an expression vector comprising a polynucleotide encoding a human type I adenylate cyclase and a pharmaceutically acceptable carrier is administered to a subject in need of such treatment.
  • the invention thus provides a human type I adenylate cyclase that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Human type I adenylate cyclase and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.
  • Fig. 1 shows the BLASTP - alignment of a portion of SEQ ID NO:2 against swiss
  • FIG. 2 shows the BLASTP - alignment of a portion of SEQ ID NO:2 against aageneseq
  • Fig. 3 shows the BLASTP - alignment of a portion of SEQ ID NO:5 against swiss
  • Fig. 5 shows the BLASTP - alignment of a portion of SEQ ID NO: 2 against swiss
  • FIG. 6 shows the BLASTP - alignment of a portion of SEQ ID NO:2 against swiss
  • Fig. 8 shows the BLASTP - alignment of a portion of SEQ ID NO:2 against pdb
  • Fig. 9 shows the HMMPFAM - alignment of a portion of SEQ ID NO: 2 against pfam
  • guanylate_cyc Adenylate and Guanylate cyclase catalyst. This hit is scoring at : 313.8; E 2e-90. Scoring matrix : BLOSUM62 (used to infer consensus pattern) .
  • Fig. 10 shows the HMMPFAM - alignment of a portion of SEQ ID NO:2 against pfam
  • guanylate_cyc Adenylate and Guanylate cyclase catalyst . This hit is scoring at : 334.3, E 1.4e-96 ; Scoring matrix : BLOSUM62 (used to infer consensus pattern).
  • Fig. 11 shows the TMHMM result.
  • Fig. 12 shows the relative expression of human type I adenylate cyclase (long splice variant) in various tissues of the body.
  • Fig. 13 shows the relative expression of human type I adenylate cyclase (short splice variant) in various tissues of the body.
  • Fig. 14 shows the multiple alignments of the orthologs of SEQ ID NOs: 2 and 5.
  • SEQ ID NO:2, oo Len: 1134 Check: 9404 Weight: 0.1; SEQ ID NO:5, oo Len: 1134 Check: 3979 Weight: 56.0; swiss
  • Fig. 15 shows the Genewise analysis of embl
  • Fig. 16 shows the BLASTP - alignment of SEQ ID NO: 5 against swissnew
  • Fig. 17 shows the BLASTP - alignment of SEQ ID NO:2 against swissnew
  • Euteleostomi Mammalia; Eutheria; Primates; Catarrhini; Hominidae;
  • BTADC_l product "adenylyl cyclase Type I"; Bovine adenylyl cyclase Type I mRNA, complete cds. //:gp
  • the invention relates to an isolated polynucleotide from the group consisting of:
  • a novel human type I adenylate cyclase is a discovery of the present invention Furthermore, it has been discovered by the present applicant that atype I adenylate cyclase, particularly acyclase, can be used in therapeutic methods to treat CNS diseases, diabetes and COPD.
  • Human type I adenylate cyclase comprises the amino acid sequence shown in SEQ ID NO:2; a splice variant is shown in SEQ ID NO:5, further variants are shown in SEQ ID NOs: 21 and 23.
  • a coding sequence for the human type I adenylate cyclase of SEQ ID NO:2 is shown in SEQ ID NO:l; a coding sequence for SEQ ID
  • SEQ ID NO:5 is shown in SEQ ID NO:4
  • a coding sequence for SEQ ID NO:21 is shown in SEQ ID NO:20
  • a coding sequence for SEQ ID NO:23 is shown in SEQ ID NO: 22.
  • the coding sequences are located on chromosome 7.
  • Related ESTs (SEQ ID NOS: 9- 19) are expressed in brain, colon, heart, lung carcinoid, uterus adenocarcinoma, and melanotic melanoma.
  • FIGS. 1-10 Prosite identifies guanylate cyclase domains between amino acids 402-425 and 982-1005.
  • Human type I adenylate cyclase of the invention is expected to be useful for the same purposes as previously identified type I adenylate cyclase enzymes.
  • Human type I adenylate cyclase is believed to be useful in therapeutic methods to treat disorders such as CNS disorders, diabetes, and COPD.
  • Human type I adenylate cyclase also can be used to screen for human type I adenylate cyclase activators and inhibitors.
  • Human type I adenylate cyclase polypeptides comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, or 1119 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or at least or 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, or 563 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:5 or at least 6, 10, 15, 20, 25, 50, 75, 100,
  • a type I adenylate cyclase polypeptide of the invention therefore can be a portion of a type I adenylate cyclase protein, a full-length type I adenylate cyclase protein, or a fusion protein comprising all or a portion of a type I adenylate cyclase protein.
  • Human type I adenylate cyclase polypeptide variants that are biologically active, e.g., retain an enzymatic activity, also are type I adenylate cyclase polypeptides.
  • type I adenylate cyclase polypeptide variants Preferably, naturally or non-naturally occurring type I adenylate cyclase polypeptide variants have amino acid sequences which are at least about 92, 93, 94, 95, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO: 2, 5, 21 or 23 or a fragment thereof.
  • Percent identity between a putative type I adenylate cyclase polypeptide variant and an amino acid sequence of SEQ ID NO: 2, 5, 21 or 23 is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA #9:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "BLOSUM62" scoring matrix of Henikoff & Henikoff,
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman & Wunsch, J. Mol Biol.48:444 (1970); Sellers, SIAM J. Appl Math.26:7&7 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties.
  • conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids.
  • the invention additionally, encompasses type I adenylate cyclase polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.
  • the type I adenylate cyclase polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • the invention also provides chemically modified derivatives of type I adenylate cyclase polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337).
  • the chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like.
  • the polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.
  • Fusion proteins are useful for generating antibodies against type I adenylate cyclase polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a human type I adenylate cyclase polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a human type I adenylate cyclase polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, or 1119 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or at least or 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, or 563 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 5 or at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, or 1114 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:22 or at least 6, 10, 15, 20,
  • the first polypeptide segment also can comprise full-length type I adenylate cyclase protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ - galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), auto- fluorescent proteins, including blue fluorescent protein (BFP), glutathione-S- transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the type I adenylate cyclase polypeptide-encoding sequence and the heterologous protein sequence, so that the type I adenylate cyclase polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l, 4, 20 or 22 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL
  • Species homologs of human type I adenylate cyclase polypeptide can be obtained using type I adenylate cyclase polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of type I adenylate cyclase polypeptide, and expressing the cDNAs as is known in the art.
  • a human type I adenylate cyclase polynucleotide can be single- or double- stranded and comprises a coding sequence or the complement of a coding sequence for a type I adenylate cyclase polypeptide.
  • a coding sequence for human type I adenylate cyclase is shown in SEQ ID NOs:l. Variant are shown in SEQ ID NOs:4, 20 and 22.
  • nucleotide sequences encoding human type I adenylate cyclase polypeptides as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 1, 4, 20 or 22 or its complement also are type I adenylate cyclase polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA
  • species homologs, and variants of type I adenylate cyclase polynucleotides that encode biologically active type I adenylate cyclase polypeptides also are type I adenylate cyclase polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO: 1, 4, 20 or 22 its complement also are type I adenylate cyclase polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.
  • Variants and homologs of the type I adenylate cyclase polynucleotides described above also are type I adenylate cyclase polynucleotides.
  • homologous type I adenylate cyclase polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known type I adenylate cyclase polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions— 2X SSC (0.3 M
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
  • Species homologs of the type I adenylate cyclase polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of type I adenylate cyclase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol Biol. 81, 123 (1973).
  • Variants of human type I adenylate cyclase polynucleotides or type I adenylate cyclase polynucleotides of other species can therefore be identified by hybridizing a putative homologous type I adenylate cyclase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, 4, 20 or 22 or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to type I adenylate cyclase polynucleotides or their complements following stringent hybridization and/or wash conditions also are type I adenylate cyclase polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T m of the hybrid under study.
  • T m of a hybrid between a type I adenylate cyclase polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1, 4, 20 or 22 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl Acad. Sci. U.S.A. 48,
  • Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.
  • a human type I adenylate cyclase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated type I adenylate cyclase polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise type I adenylate cyclase nucleotide sequences.
  • Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human type I adenylate cyclase cDNA molecules can be made with standard molecular biology techniques, using type I adenylate cyclase mRNA as a template. Human type I adenylate cyclase cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • the partial sequence disclosed herein can be used to identify the corresponding full-length gene from which it was derived.
  • the partial sequence can be nick-translated or end-labeled with P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al, eds., Elsevier Press, N.Y.,
  • a lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (1989, pg. 1.20).
  • pBluescript Stratagene Cloning Systems, La Jolla, Calif. 92037
  • Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis. [81] Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined , for example after exonuclease III digestion (McCombie et al., Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region.
  • amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • Type I adenylate cyclase polypeptides can be obtained, for example, by purification from human cells, by expression of type I adenylate cyclase polynucleotides, or by direct chemical synthesis.
  • Type I adenylate cyclase polypeptides can be purified from any human cell which expresses the receptor, including host cells which have been transfected with type I adenylate cyclase polynucleotides.
  • a purified type I adenylate cyclase polypeptide is separated from other compounds that normally associate with the type I adenylate cyclase polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromato- graphy, affinity chromatography, and preparative gel electrophoresis.
  • type I adenylate cyclase polypeptide can be conveniently isolated as a complex with its associated G protein, as described in the specific examples, below.
  • a preparation of purified type I adenylate cyclase polypeptides is at least 80%) pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • polynucleotides [91] To express a human type I adenylate cyclase polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding type I adenylate cyclase polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a human type I adenylate cyclase polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • the control elements or regulatory sequences are those non-translated regions of the vector ⁇ enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation.
  • transcription and translation elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a human type I adenylate cyclase polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • Bacterial and yeast expression systems In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the type I adenylate cyclase polypeptide. For example, when a large quantity of a human type I adenylate cyclase polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • a sequence encoding the type I adenylate cyclase polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pIN vectors Van Heeke & Schuster, J Biol. Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516-544, 1987.
  • sequences encoding type I adenylate cyclase polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311 , 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl Cell Differ. 17, 85-105, 1991).
  • These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g.,
  • An insect system also can be used to express a human type I adenylate cyclase polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding type I adenylate cyclase polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of type I adenylate cyclase polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which type I adenylate cyclase polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • Mammalian expression systems can be used to express type I adenylate cyclase polypeptides in mammalian host cells.
  • sequences encoding type I adenylate cyclase polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a human type I adenylate cyclase polypeptide in infected host cells (Logan & Shenk, Proc. Natl Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding type I adenylate cyclase polypeptides.
  • Such signals include the ATG initiation codon and adjacent sequences.
  • sequences encoding a human type I adenylate cyclase polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals (including the ATG initiation codon) should be provided.
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl Cell Differ. 20, 125-162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed type I adenylate cyclase polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express type I adenylate cyclase polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced type I adenylate cyclase sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I.
  • any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes that can be employed in tk ⁇ or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl Acad. Sci.
  • npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988).
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).
  • marker gene expression suggests that the type I adenylate cyclase polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a human type I adenylate cyclase polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an type I adenylate cyclase polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding an type I adenylate cyclase polypeptide under the control of a single promoter.
  • Expression of the marker gene in response to induction or selection usually indicates expression of the type I adenylate cyclase polynucleotide.
  • host cells which contain a human type I adenylate cyclase polynucleotide and which express a human type I adenylate cyclase polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding an type I adenylate cyclase polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a human type I adenylate cyclase polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding an type I adenylate cyclase polypeptide to detect transformants which contain an type I adenylate cyclase polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a human type I adenylate cyclase polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a human type I adenylate cyclase polypeptide can be used, or a competitive binding assay can be employed.
  • sequences encoding a human type I adenylate cyclase polypeptide can be cloned into a vector for the production of an mRNA probe.
  • vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
  • Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a human type I adenylate cyclase polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode type I adenylate cyclase polypeptides can be designed to contain signal sequences which direct secretion of soluble type I adenylate cyclase polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound type I adenylate cyclase polypeptide.
  • other constructions can be used to join a sequence encoding a human type I adenylate cyclase polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, Wash.
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the type I adenylate cyclase polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a human type I adenylate cyclase polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification by
  • IMAC immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp. Purif 3, 263-281, 1992
  • enterokinase cleavage site provides a means for purifying the type I adenylate cyclase polypeptide from the fusion protein.
  • Vectors that contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.
  • Sequences encoding a human type I adenylate cyclase polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).
  • a human type I adenylate cyclase polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc.
  • Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of type I adenylate cyclase polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • composition of a synthetic type I adenylate cyclase polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the type I adenylate cyclase polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. [112] As will be understood by those of skill in the art, it may be advantageous to produce type I adenylate cyclase polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter type I adenylate cyclase polypeptide- encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibodies Any type of antibody known in the art can be generated to bind specifically to an epitope of a human type I adenylate cyclase polypeptide.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a human type I adenylate cyclase polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a human type I adenylate cyclase polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody which specifically binds to a human type I adenylate cyclase polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to type I adenylate cyclase polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a human type I adenylate cyclase polypeptide from solution.
  • Human type I adenylate cyclase polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • a human type I adenylate cyclase polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g.
  • Monoclonal antibodies which specifically bind to a human type I adenylate cyclase polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030,
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies that specifically bind to a human type I adenylate cyclase polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al,
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant D ⁇ A methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-
  • Antibodies which specifically bind to type I adenylate cyclase polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a human type I adenylate cyclase polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences or nwhich are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of type I adenylate cyclase gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester intemucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.
  • Modifications of type I adenylate cyclase gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the type I adenylate cyclase gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an type I adenylate cyclase polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent type I adenylate cyclase nucleotides, can provide sufficient targeting specificity for type I adenylate cyclase mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular type
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a human type I adenylate cyclase polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a human type I adenylate cyclase polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the type I adenylate cyclase polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et ⁇ /., EP 321,201).
  • Specific ribozyme cleavage sites within a human type I adenylate cyclase RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable.
  • Suitability of candidate type I adenylate cyclase RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct.
  • ribozyme-containing DNA construct can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease type I adenylate cyclase expression.
  • the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • genes whose products interact with human type I adenylate cyclase may represent genes that are differentially expressed in disorders including, but not limited to, CNS disorders, diabetes, and COPD. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human type I adenylate cyclase gene or gene product may itself be tested for differential expression.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human type I adenylate cyclase.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human type I adenylate cyclase.
  • the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human type I adenylate cyclase gene or gene product are up- regulated or down-regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a human type I adenylate cyclase polypeptide or a human type I adenylate cyclase polynucleotide.
  • a test compound preferably binds to a human type I adenylate cyclase polypeptide or polynucleotide.
  • a test compound decreases or increases enzymatic activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory.
  • test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, 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.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997. [143] Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993;
  • Test compounds can be screened for the ability to bind to type I adenylate cyclase polypeptides or polynucleotides or to affect type I adenylate cyclase activity or type I adenylate cyclase gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • "free format assays" or assays that have no physical barrier between samples can be used. For example, an assay using pigment cells
  • Patent 5,976,813. In this method, test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the type I adenylate cyclase polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the type I adenylate cyclase polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the type I adenylate cyclase polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a human type I adenylate cyclase polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a human type I adenylate cyclase polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a human type I adenylate cyclase polypeptide (McConnell et al, Science
  • 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.
  • a human type I adenylate cyclase polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et o/., J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8,
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a human type I adenylate cyclase polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey” or “sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey” proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g.,
  • LacZ which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the type I adenylate cyclase polypeptide.
  • either the type I adenylate cyclase polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a human type I adenylate cyclase polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the type I adenylate cyclase polypeptide is a fusion protein comprising a domain that allows the type I adenylate cyclase polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed type I adenylate cyclase polypeptide; the mixture is then incubated under conditions conducive to complex formation
  • binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • Biotinylated type I adenylate cyclase polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a type I adenylate cyclase polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the type I adenylate cyclase polypeptide can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the type I adenylate cyclase polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the type I adenylate cyclase polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a human type I adenylate cyclase polypeptide or polynucleotide also can be carried out in an intact cell.
  • Any cell which comprises a type I adenylate cyclase polypeptide or polynucleotide can be used in a cell-based assay system.
  • a type I adenylate cyclase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a type I adenylate cyclase polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease the enzymatic activity of a human type I adenylate cyclase polypeptide. Enzymatic activity can be measured, for example, as described in U.S. Patent
  • Enzyme assays can be carried out after contacting either a purified type I adenylate cyclase polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound that decreases enzymatic activity of a human type I adenylate cyclase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing type I adenylate cyclase activity.
  • test compound which increases enzymatic activity of a human type I adenylate cyclase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) is identified as a potential therapeutic agent for increasing human type I adenylate cyclase activity.
  • test compounds that increase or decrease type I adenylate cyclase gene expression are identified.
  • a type I adenylate cyclase polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the type I adenylate cyclase polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • the test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of type I adenylate cyclase mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • polypeptide products of a human type I adenylate cyclase polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human type I adenylate cyclase polypeptide.
  • screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a human type I adenylate cyclase polynucleotide can be used in a cell-based assay system.
  • the type I adenylate cyclase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect.
  • Pharmaceutical compositions of the invention can comprise, for example, a human type I adenylate cyclase polypeptide, type I adenylate cyclase polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a type I adenylate cyclase polypeptide, or mimetics, activators, or inhibitors of a human type I adenylate cyclase polypeptide activity.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • a filler or binders such as lactose or starches
  • lubricants such as talc or magnesium stearate
  • stabilizers optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • Human type I adenylate cyclase can be regulated to treat CNS disorders, diabetes, and COPD.
  • Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.
  • Pain that is associated with CNS disorders also can be treated. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • central nervous system disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.
  • headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache tension-type like headache, cluster headache, and chronic par
  • Adenylyl Cyclase I (ACl) antagonists for treatment of pain [176] Adenylyl Cyclases (AC) catalyze the production of the second messenger cAMP from ATP, which in turn is responsible for activation of Protein
  • PKA Kinase A
  • AC/cAMP/PKA-signalling cascade induces hyperalgesia and allodynia, whereas its inhibition is analgetic (O.A. Kochuvelikakam et al.
  • the Ca 2+ /Calmodulin-regulated ACl and AC8 acts as a coincidence detector between stimulation-dependent Ca2+-influx and cAMP/PKA-signalling pathway in particular in the CNS. These pathways are important for many brain functions in particular drug addiction, such as morphine resistance and dependence, and persistent pain (Z. Xia et al. (1997); Cur. Opin. Neurobiol.
  • ACl activity is strictly regulated by the binding to Ca2+/Calmodulin and stimulating Ga proteins (Z. Xia et al. (1997); Cur. Opin. Neurobiol. 7: 391; S. Poser (2001); Int. J. Devi. Neurosci. 19: 387; T. Avidor-Reiss et al. (1997); J. Biol. Chem. 8: 5040). Accordingly, it has been shown that upon neuronal stimulation and subsequent Ca 2+" influx increased cAMP production is regulated by enhancement of enzymatic activity rather than on the transcriptional level (N. A. Parkinson et al. (2001); Mol. Brain. Res. 91 : 43; N.
  • ACl is involved in induction and retention of long-term potentiation (LTP) by acting downstream and sensitizing glutamate-gated channels of the N- methyl-D-aspartate (NMD A) receptor type (Xia et al, 1997; Poser 2001; Sandkuhler (2000); Pain 88: 113).
  • LTP long-term potentiation
  • NMD A N- methyl-D-aspartate receptor type
  • the invention provides a method of using adenylyl cyclase I (ACl) inhibitors (e.g., antagonists) for treatment of pain and the use of adenylyl cyclase I (ACl) inhibitors (e.g., antagonists) in the preparation of a medicament for the treatment of pain.
  • ACl adenylyl cyclase I
  • Diabetes mellitus is a common metabolic disorder characterized by an abnormal elevation in blood glucose, alterations in lipids and abnormalities (complications) in the cardiovascular system, eye, kidney and nervous system. Diabetes is divided into two separate diseases: type 1 diabetes (juvenile onset), which results from a loss of cells which make and secrete insulin, and type 2 diabetes (adult onset), which is caused by a defect in insulin secretion and a defect in insulin action.
  • type 1 diabetes is initiated by an autoimmune reaction that attacks the insulin secreting cells (beta cells) in the pancreatic islets. Agents that prevent this reaction from occurring or that stop the reaction before destruction of the beta cells has been accomplished are potential therapies for this disease. Other agents that induce beta cell proliferation and regeneration also are potential therapies.
  • Type II diabetes is the most common of the two diabetic conditions (6% of the population).
  • the defect in insulin secretion is an important cause of the diabetic condition and results from an inability of the beta cell to properly detect and respond to rises in blood glucose levels with insulin release.
  • Therapies that increase the response by the beta cell to glucose would offer an important new treatment for this disease.
  • the defect in insulin action in Type II diabetic subjects is another target for therapeutic intervention.
  • Agents that increase the activity of the insulin receptor in muscle, liver, and fat will cause a decrease in blood glucose and a normalization of plasma lipids.
  • the receptor activity can be increased by agents that directly stimulate the receptor or that increase the intracellular signals from the receptor.
  • Other therapies can directly activate the cellular end process, i.e. glucose transport or various enzyme systems, to generate an insulin-like effect and therefore a produce beneficial outcome. Because overweight subjects have a greater susceptibility to Type II diabetes, any agent that reduces body weight is a possible therapy.
  • COPD chronic obstructive pulmonary (or airways) disease
  • Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung.
  • Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years.
  • airflow obstruction is usually progressive and is only partially reversible.
  • Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998).
  • the inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8 + lymphocytes.
  • Inhaled irritants such as cigarette smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic factors.
  • chemokines e.g., interleukin-8
  • These chemotactic factors act to increase the neutrophil/monocyte trafficking from the blood into the lung tissue and airways.
  • Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species.
  • Matrix degradation and emphysema along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion, all are potential sequelae of this inflammatory
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a human type I adenylate cyclase polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • a reagent which affects type I adenylate cyclase activity can be administered to a human cell, either in vitro or in vivo, to reduce type I adenylate cyclase activity.
  • the reagent preferably binds to an expression product of a human type I adenylate cyclase gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 " nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol 11,
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases enzymatic activity relative to the enzymatic activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio,
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • [200] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • the reagent is a single-chain antibody
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin- polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation,
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a human type I adenylate cyclase gene or the activity of a type I adenylate cyclase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Human type I adenylate cyclase also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding type I adenylate cyclase in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis.
  • DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of type I adenylate cyclase also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • Detection of type I adenylate cyclase activity [212] The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-adenylate cyclase polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and the type I adenylate cyclase activity is meassured in the following assay: A volume of 1.0 microliter of either H 2 O, NaF, guanylyl-5'-imidodiphosphate (GppNHp), isoproterenol, or isoproterenol + GppNHp are added to each of five reaction tubes and maintained at 0 C. Next, 25 microliter of reaction mixture A (Tris Acetate
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human type I adenylate cyclase polypeptides in yeast.
  • the type I adenylate cyclase-encoding DNA sequence is derived from SEQ ID NO: 1, 4, 20 or 22. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human type I adenylate cyclase polypeptide is obtained.
  • test compounds that bind to type I adenylate cyclase polypeptides
  • Purified type I adenylate cyclase polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human type I adenylate cyclase polypeptides comprise the amino acid sequence shown in SEQ ID NO: 2, 5, 22 or 23.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a human type I adenylate cyclase polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a human type I adenylate cyclase polypeptide.
  • test compound is administered to a culture of human cells transfected with a type I adenylate cyclase expression construct and incubated at 37 °C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979).
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled type I adenylate cyclase-specific probe at 65 ° C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1, 4, 20 or 22.
  • a test compound that decreases the type I adenylate cyclase- specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of type I adenylate cyclase gene expression.
  • EXAMPLE 5 Identification of a test compound which decreases type I adenylate cyclase activity [219] A test compound is administered to a culture of human cells transfected with a type I adenylate cyclase expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control. Enzymatic activity is measured using the method of U.S. Patent 5,795,756.
  • a test compound which decreases the enzymatic activity of the type I adenylate cyclase relative to the enzymatic activity in the absence of the test compound is identified as an inhibitor of type I adenylate cyclase activity.
  • Tissue-specific expression of type I adenylate cyclase [221] The qualitative expression pattem of type I adenylate cyclase in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
  • the initial expression panel consists of RNA samples from respiratory tissues and inflammatory cells relevant to COPD: lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cultured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara- like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like).
  • Body map profiling also is carried out, using total RNA panels purchased from Clontech.
  • the tissues are adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, trachea, thyroid, and uterus.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis” firstly described in Higuchi et al,
  • RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.
  • RNA is extracted once with 1 volume of phenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M sodium acetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophotometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/ ⁇ L. Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers. [232] TaqMan quantitative analysis.
  • Probes and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
  • Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).
  • PDAR Pre-Developed TaqMan Assay Reagents
  • the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S
  • RNA from 20X stock
  • 300 nM forward primer from 20X stock
  • 900 nM reverse primer from 300 nM forward primer
  • 200 nM probe from 10 ng cDNA
  • water to 25 ⁇ l.
  • Glucose Production Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.
  • Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant.
  • the animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group.
  • Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined.
  • Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load.
  • compounds are administered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice.
  • an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined.
  • Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose.
  • mice When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will decrease glucose levels and the area-under- the glucose curve when compared to the vehicle-treated group given only glucose. Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load.
  • test compounds which regulate type I adenylate cyclase are administered by different routes (p.o., i.p., s.c, or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load
  • Plasma insulin levels are determined. Test compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60, and 90 minutes and plasma glucose levels determined. Test compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.
  • RNA is reverse transcribed, and the resultant cDNA is used in a realtime polymerase chain reaction (PCR).
  • the cDNA is added to a solution containing the sense and anti-sense primers and the 6-carboxy-tetramethyl- rhodamine labeled probe of the type I adenylate cyclase gene.
  • Cyclophilin is used as the housekeeping gene.
  • the expression of the type I adenylate cyclase gene is measured using the TaqMan real-time PCR system that generates an amplification curve for each sample. From this curve a threshold cycle value is calculated: the fractional cycle number at which the amount of amplified target reaches a fixed threshold.
  • a sample containing many copies of the type I adenylate cyclase gene will reach this threshold earlier than a sample containing fewer copies.
  • the threshold is set at 0.2, and the threshold cycle Cj / is calculated from the amplification curve.
  • the Cf value for the type I adenylate cyclase gene is normalized using the C value for the housekeeping gene. [243] Expression of the type I adenylate cyclase gene is increased by at least 3-fold between 10 minutes and 3 hours post tobacco smoke exposure compared to air exposed control animals.
  • Test compounds are evaluated as follows. Animals are pre-treated with a test compound between 5 minutes and 1 hour prior to the tobacco smoke exposure and they are then sacrificed up to 3 hours after the tobacco smoke exposure has been completed. Control animals are pre-treated with the vehicle of the test compound via the route of administration chosen for the test compound. A test compound that reduces the tobacco smoke induced upregulation of type I adenylate cyclase gene relative to the expression seen in vehicle treated tobacco smoke exposed animals is identified as an inhibitor of type I adenylate cyclase gene expression.
  • RNA used for Taqman quantitative analysis were either purchased (Clontech,CA) or extracted from tissues using TRIzol reagent (Life Technologies, Inc.).
  • RNA samples were treated with DNase I using RNase free- DNase (Qiagen, CA) for use with RNeasy or QiaAmp columns.
  • Ribogreen Molecular Probes Inc., OR
  • each sample was reverse transcribed using the GibcoBRL Superscript II First Strand Synthesis System for RT-PCR according to vendor protocol (Life Technologies, MD). The final concentration of RNA in the reaction mix was 50ng/ ⁇ L. Reverse transcription was performed with 50 ng of random hexamers.
  • the assay reaction mix was as follows: final TaqMan S YBR Green PCR Master Mix (2x) 1 x
  • Acute pain is measured on a hot plate mainly in rats.
  • Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56°C) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking.
  • the other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold.
  • Compounds are tested against a vehicle treated control group.
  • Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.e. v., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.e. v., s.c, intradermal, transdermal
  • Persistent pain Persistent pain is measured with the formalin or capsaicin test, mainly in rats. A solution of 1 to 5% formalin or 10 to 100 ⁇ g capsaicin is injected into one hind paw of the experimental animal. After formalin or capsaicin application the animals show nocifensive reactions like flinching, licking and biting of the affected paw. The number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to formalin or capsaicin administration.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats. The operation is performed under anesthesia. The first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve. The second variant is the tight ligation of about the half of the diameter of the common sciatic nerve. In the next variant, a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only.
  • the fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured.
  • Control animals are treated with a sham operation.
  • the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10 °C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity.
  • a further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb.
  • Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt, Universitat zu K ⁇ ln, Cologne, Germany), and by scoring differences in gait
  • Compounds are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • Inflammatory Pain Inflammatory Pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw.
  • the animals develop an edema with mechanical allodynia as well as thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc. -Life Science Instruments, Woodland Hills, SA, USA).
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).
  • edema measurement two methods are being used. In the first method, the animals are sacrificed and the affected hindpaws sectioned and weighed. The second method comprises differences in paw volume by measuring water displacement in a plethysmometer (Ugo Basile, Comerio, Italy).
  • Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • 6-Hydroxydopamine (6-OH-DA) Lesion Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).
  • MFB medium forebrain bundle
  • Male Wistar rats Male Wistar rats (Harlan Winkelmann, Germany), weighing 200 ⁇ 250 g at the beginning of the experiment, are used. The rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions.
  • Stepping Test Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol. In brief, the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface. One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction.
  • the number of adjusting steps is counted for both paws in the backhand and forehand direction of movement.
  • the sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions.
  • the test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing.
  • Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.
  • Staircase Test (Paw Reaching).
  • a modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement.
  • Plexiglass test boxes with a central platform and a removable staircase on each side are used.
  • the apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use.
  • the animals are left in the test boxes for 15 min.
  • the double staircase is filled with 7 x 3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side.
  • MPTP treatment The neurotoxin l-methyl-4-phenyl- 1,2,3, 6-tetrahydro- pyridine (MPTP) causes degeneration of mesencephalic dopaminergic
  • MPTP tyrosine hydroxylase
  • PBS fetal bovine serum
  • 0.005% ⁇ H 2 O 2 serves as chromogen in the subsequent visualization reaction. Sections are mounted on to gelatin-coated slides, left to dry overnight, counter-stained with hematoxylin dehydrated in ascending alcohol concentrations and cleared in butylacetate. Coverslips are mounted on entellan.
  • Rotarod Test We use a modification of the procedure described by Rozas and Labandeira-Garcia (1997), with a CR-1 Rotamex system (Columbus Instruments, Columbus, OH) comprising an IBM-compatible personal computer, a CIO-24 data acquisition card, a control unit, and a four-lane rotarod unit.
  • the rotarod unit consists of a rotating spindle (diameter 7.3 cm) and individual compartments for each mouse.
  • the system software allows preprogramming of session protocols with varying rotational speeds (0-80 rpm). Infrared beams are used to detect when a mouse has fallen onto the base grid beneath the rotarod.
  • the system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded.
  • the system also allows a weak current to be passed through the base grid, to aid training.
  • the object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents.
  • a rat is placed in an open field, in which two identical objects are present.
  • the rats inspects both objects during the first trial of the object recognition task.
  • a second trial after a retention interval of for example 24 hours, one of the two objects used in the first trial, the 'familiar' object, and a novel object are placed in the open field.
  • the inspection time at each of the objects is registered.
  • the basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the 'familiar' object.
  • the passive avoidance task assesses memory performance in rats and mice.
  • the inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment. The two compartments are separated by a guillotine door that can be operated by the experimenter. A threshold of 2 cm separates the two compartments when the guillotine door is raised. When the door is open, the illumination in the dark compartment is about 2 lux.
  • the light intensity is about 500 lux at the center of the floor of the light compartment.
  • test compound increases the enter latency compared with the scopolamine-treated controls, is likely to possess cognition enhancing potential.
  • the Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice.
  • the performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank.
  • Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly.
  • the animals receive four trials during five daily acquisition sessions. A trial is started by placing an animal into the pool, facing the wall of the tank.
  • Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized.
  • the escape platform is always in the same position.
  • a trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first.
  • the animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds.
  • an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds. In the probe trial, all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
  • the T-maze spontaneous alternation task assesses the spatial memory performance in mice.
  • the start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter.
  • a mouse is put into the start arm at the beginning of training.
  • the guillotine door is closed.
  • the 'forced trial' either the left or right goal arm is blocked by lowering the guillotine door.
  • the mouse After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door.
  • the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 'free choice' trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.
  • Scopolamine reduced the per-cent alternations to chance level, or below.
  • a cognition enhancer which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate.

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Abstract

La présente invention concerne des réactifs qui régulent l'adénylate cyclase humaine de type 1 et des réactifs qui lient des produits géniques de l'adénylate cyclase humaine de type 1, lesquels réactifs peuvent jouer un rôle dans la prévention, l'amélioration ou la correction de dysfonctionnements ou maladies parmi lesquels, de façon non limitative, les troubles du système nerveux central, le diabète et la bronchopneumopathie chronique obstructive.
PCT/EP2002/012059 2001-10-29 2002-10-29 Regulation de l'adenylate cyclase humaine de type i WO2003038083A1 (fr)

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US33071001P 2001-10-29 2001-10-29
US60/330,710 2001-10-29
US35001902P 2002-01-23 2002-01-23
US60/350,019 2002-01-23
US38953802P 2002-06-19 2002-06-19
US60/389,538 2002-06-19
US41359702P 2002-09-26 2002-09-26
US60/413,597 2002-09-26

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EP3099319A2 (fr) * 2014-01-31 2016-12-07 Cytomx Therapeutics Inc. Substrats pour matriptase et activateur u-plasminogène et autres fractions clivables, et leurs procédés d'utilisation
US11046759B2 (en) 2015-01-20 2021-06-29 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof
US11814410B2 (en) 2013-09-25 2023-11-14 Cytomx Therapeutics, Inc. Matrix metalloproteinase substrates and other cleavable moieties and methods of use thereof
US12049505B2 (en) 2018-12-06 2024-07-30 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine or cysteine protease-cleavable substrates and methods of use thereof

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11814410B2 (en) 2013-09-25 2023-11-14 Cytomx Therapeutics, Inc. Matrix metalloproteinase substrates and other cleavable moieties and methods of use thereof
EP3099319A2 (fr) * 2014-01-31 2016-12-07 Cytomx Therapeutics Inc. Substrats pour matriptase et activateur u-plasminogène et autres fractions clivables, et leurs procédés d'utilisation
US10138272B2 (en) 2014-01-31 2018-11-27 Cytomx Therapeutics, Inc. Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof
EP3628328A1 (fr) * 2014-01-31 2020-04-01 Cytomx Therapeutics Inc. Substrats d'activateur de matriptase et du plasminogène u et d'autres fractions clivables et leurs procédés d'utilisation
US11028126B2 (en) 2014-01-31 2021-06-08 Cytomx Therapeutics, Inc. Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof
EP4176896A1 (fr) * 2014-01-31 2023-05-10 Cytomx Therapeutics Inc. Substrats d'activateur de matriptase et du plasminogène u et d'autres fractions clivables et leurs procédés d'utilisation
US11884746B2 (en) 2014-01-31 2024-01-30 Cytomx Therapeutics, Inc. Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof
US11046759B2 (en) 2015-01-20 2021-06-29 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof
US11472875B1 (en) 2015-01-20 2022-10-18 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof
US11548944B2 (en) 2015-01-20 2023-01-10 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof
US11548943B2 (en) 2015-01-20 2023-01-10 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof
US12049505B2 (en) 2018-12-06 2024-07-30 Cytomx Therapeutics, Inc. Matrix metalloprotease-cleavable and serine or cysteine protease-cleavable substrates and methods of use thereof

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