WO2005075510A1 - Agents diagnostiques et therapeutiques pour des maladies associees au canal potassique, agents membres 9 de la sous-famille k (kcnk9) - Google Patents

Agents diagnostiques et therapeutiques pour des maladies associees au canal potassique, agents membres 9 de la sous-famille k (kcnk9) Download PDF

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WO2005075510A1
WO2005075510A1 PCT/EP2005/000612 EP2005000612W WO2005075510A1 WO 2005075510 A1 WO2005075510 A1 WO 2005075510A1 EP 2005000612 W EP2005000612 W EP 2005000612W WO 2005075510 A1 WO2005075510 A1 WO 2005075510A1
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diseases
kcnk9
polypeptide
cancer
inflammation
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PCT/EP2005/000612
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English (en)
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Stefan Golz
Ulf Brüggemeier
Andreas Geerts
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Bayer Healthcare Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human KCNK9 and its regulation for the treatment of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in mammals.
  • Ion channels are membrane-spanning proteins involved in the regulation of ion movements across biological membranes and play a crucial role in maintaining and modulating cellular homeostasis of excitable and non-excitable cells [[Kim, Y. (2000)], [Pei, L. (2003)], [Mu, D. (2003)], [Rajan, S (2002)], WO0177174, WO0166741, WO0053628, WO0053628]. Ion channels mediate many fundamental and medically significant biological signalling processes such as secretion of hormones and neurotransmitters, generation of electric signals in the cardiovascular system and the conversion of chemical and mechanical extracellular stimuli into electrical responses in the nervous system.
  • ion channels The broad functional diversity of ion channels is based on their specificity for ions (e.g. anions, cations, potassium, sodium, calcium, or chloride), their susceptibility to regu- lation and their molecular diversity.
  • Activation and/or opening of ion channels can be voltage- or ligand-dependent or a combination of both.
  • ligand-gated channels comprise nicotinic acetylcholine receptors, gamma-amino butyric acid (GABA) receptors, glycine receptors, and glutamate receptors.
  • GABA gamma-amino butyric acid
  • Examples for voltage-gated channels are the highly diversified families of calcium, potassium- and sodium channels.
  • Most of the ion channel proteins are complex multisubunit macromolecules with several membrane-spanning helical regions forming the channel pore and accessory subunits with regulatory functions.
  • the diseases to be addressed include, but are not limited to, infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HTV viruses, cancers, allergies including asthma, cardiovascular diseases including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, hematological diseases, genito-urinary diseases including urinary incontinence and benign prostate hyperplasia, osteoporosis, and peripheral and central nervous system disorders including pain, Alzheimer's disease and Parkinson's disease (Willumsen et al., 2003 Receptors and Channels 9,3-12).
  • infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HTV viruses, cancers, allergies including asthma, cardiovascular diseases including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, hematological diseases, genito-urinary diseases including urinary incontinence and benign prostate hyperplasia, osteoporosis, and peripheral and central
  • TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.
  • CT threshold cycle
  • Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action.
  • the TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue.
  • Expression profiling has been used in identifying genes, which are up- or downregulated in a variety of diseases.
  • An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drug treatment or in patients versus healthy individuals.
  • the premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets.
  • physiological or environmental stimuli e.g., drugs
  • the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.
  • the nucleotide sequence of KCNK9 is accessible in public databases by the accession number NM_016601 and is given in SEQ DD NO:l.
  • the amino acid sequence of KCNK9 is depicted in SEQ ID O:2.
  • Potassium channels are ubiquitous multisubunit membrane proteins that regulate membrane potential in numerous cell types.
  • One family of mammalian K+ channels is characterized by the presence of 4 transmembrane (TM) domains and 2 pore-forming P) domains per subunit. All of these subunits, including KCNK9, share a conserved P domain that is essential for providing K+ selectivity.
  • KCNK9 exhibited a time-independent, noninactivating K(+)-selective current when expressed in COS-7 cells.
  • the KCNK9 current was highly sensitive to changes in extracellular pH, a hallmark of the TASK family of K+ channels. Mutation of histidine at position 98 to aspartate abolished pH sensitivity.
  • KCNK9 was blocked by barium, quinidine, and lidocaine. Although the KCNK9 protein has multiple potential phosphorylation sites for protein kinases A and C, it is not regulated via phosphorylation by either kinase.
  • KCNK9 is published in [Kim, Y. (2000), Pei, L. (2003), Mu, D. (2003), Rajan, S (2002)] and patents O0177174, WO0166741, O0053628 and WO0053628.
  • the KCNK9 channel shows the highest homology (88 %) to the Cavia porcellus KCNK9 as shown in example 1.
  • the invention relates to novel disease associations of KCNK9 polypeptides and polynucleotides.
  • the invention also relates to novel methods of screening for therapeutic agents for the treatment of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal.
  • the invention also relates to pharmaceutical compositions for the treatment of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising a KCNK9 polypeptide, a KCNK9 polynucleotide, or regulators of KCNK9 or modulators of KCNK9 activity.
  • the invention further comprises methods of diagnosing cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal.
  • Fig. 1 shows the nucleotide sequence of a KCNK9 polynucleotide (SEQ ED NO:l).
  • Fig. 2 shows the amino acid sequence of a KCNK9 polypeptide (SEQ ID NO:2).
  • Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ DD NO:3).
  • Fig. 4 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:4).
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ ID NO:5).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligo- nucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • Chimeric molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following KCNK9 characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • KCNK9 refers to those forms, fragments, or domains of a KCNK9 polypeptide which retain the biological and/or antigenic activity of a KCNK9 polypeptide.
  • Frctional activity of ion channels refers to their ability to conduct and control ion movements across cellular membranes resulting in measurable changes in (1) the current, (2) the membrane potential, (3) the change in concentration of the transported ions and (4) any other measurable change exerted by KCNK9.
  • functional activity of said channel is determined by the activation state and gating properties of the channel which are influenced by parameters including but not limited to membrane potential, pH, temperature, physiological ligands, or a combination of two or more of these parameters and can be regulated by modulators such as peptides, toxins, antibodies or small compounds.
  • Non- occurring KCNK9 polypeptide refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • Oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists including small compound modulators of functional ion channel activity.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • domestic e.g., cats, dogs, etc.
  • agricultural e.g., cows, horses, sheep, etc.
  • test species e.g., mouse, rat, rabbit, etc.
  • KCNK9 polynucleotide within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ ID NO: 1,
  • nucleic acid molecules having the sequence of SEQ DD NO: 1 (iii) nucleic acid molecules having the sequence of SEQ DD NO: 1, (iv) nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and
  • nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code;
  • polypeptide encoded by said nucleic acid molecule has KCNK9 activity.
  • KCNK9 polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii);
  • polypeptide has KCNK9 activity.
  • nucleotide sequences encoding a KCNK9 have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligonucleotides for PCR, use for chromosome and gene mapping, use in the recombinant production of KCNK9, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding a KCNK9 disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • KCNK9 - encoding nucleotide sequences may be produced. Some of these will only bear minimal homology to the nucleotide sequence of the known and naturally occurring KCNK9.
  • the invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring KCNK9, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which encode a KCNK9, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring KCNK9 polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding KCNK9 polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a KCNK9 polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to KCNK9 polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for KCNK9-specif ⁇ c hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding KCNK9. Such probes may also be used for the detection of similar ion channel encoding sequences and should preferably show at least 40% nucleotide identity to KCNK9 polynucleotides.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ DD NO: 1 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such KCNK9 encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity.
  • Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human- derived probes.
  • Stringent conditions within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7 % (w/v) SDS.
  • Nucleic acid molecules that will hybridize to KCNK9 polynucleotides under stringent conditions can be identified functionally.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express KCNK9; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of KCNK9; and detecting polymorphisms of KCNK9.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes KCNK9. Such probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both. Oligonucleotides may comprise discrete nucleotide sequences employed under optimized conditions for identification of KCNK9 in specific tissues or diagnostic use. The same two oligomers, a nested set of oligonucleotides, or even a degenerate pool of oligonucleotides may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • PCR primers i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with KCNK9.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence.
  • appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.
  • PCR methods for amplifying nucleic acid will utilize at least two primers.
  • One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications particularly under preferred stringent hybridization conditions, are well known.
  • KCNK9 Other means of producing specific hybridization probes for KCNK9 include the cloning of nucleic acid sequences encoding KCNK9 or KCNK9 derivatives into vectors for the production of mRNA probes.
  • vectors are known in the art, are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well known in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • KCNK9 polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • Chromosome-based techniques such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be narrowed further using loss of heterozygosity analysis (LOH), in which disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker.
  • LHO loss of heterozygosity analysis
  • RFLPs restriction fragment length polymorphisms [Johnson, (1989)]
  • hypervariable minisatelhte DNA Barnes, 2000].
  • a gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization.
  • This approach suffers from several drawbacks.
  • the method requires that each sample has equal input amounts of the nucleic acid and that the amplification efficiency between samples is identical until the time of analysis.
  • QC-PCR quantitative competitive PCR
  • An internal control competitor in each reaction [Piatak, (1993), BioTechniques].
  • the efficiency of each reaction is normalized to the internal competitor.
  • a known amount of internal competitor is typically added to each sample.
  • the unknown target PCR product is compared with the known competitor PCR product to obtain relative quantitation.
  • a difficulty with this general approach lies in developing an internal control that amplifies with the same efficiency than the target molecule.
  • Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product.
  • the basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently referred to in the literature simply as the "TaqMan method” [Piatak,(1993), Science; Heid, (1996); Gibson, (1996); Holland. (1991)].
  • the probe used in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes.
  • the 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well.
  • the probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter.
  • the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector.
  • a nucleic acid polymerase such as Taq polymerase
  • One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 or 4700 HT manufactured by Applied Biosystems, Inc. in Foster City, Calif.
  • the ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube arrangement.
  • the instrument includes a laser for exciting the labels and is capable of measuring the fluorescence spectra intensity from each tube with continuous monitoring during PCR amplification. Each tube is re-examined every 8.5 seconds.
  • Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification.
  • the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.
  • the fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.
  • amplification product which is double stranded
  • amplification product binds dye molecules in solution to form a complex.
  • dyes it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product.
  • certain dyes fluoresce only when bound to amplification product.
  • dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc.
  • These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair.
  • the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process.
  • the alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.
  • the upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. In the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe.
  • the labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.
  • the primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization.
  • the length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe.
  • the primer typically includes 15-30 nucleotides.
  • the length of the primer may be more or less depending on the complexity of the primer binding site and the factors listed above.
  • Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide.
  • the label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.
  • a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe.
  • the probe includes multiple labels, it is generally advisable to maintain spacing between the labels which is sufficient to permit separation of the labels during digestion of the probe through the 5'-3' nuclease activity of the nucleic acid polymerase.
  • a number of diseases are associated with changes in the copy number of a certain gene.
  • the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent 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.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, or he ⁇ es simplex virus (HSV) BP16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located adjacent to the KCNK9.
  • KCNK9 cDNA molecules can be made with standard molecular biology techniques, using KCNK9 mRNA as a template. KCNK9 cDNA molecules can thereafter be replicated using molecular biology techniques known in the art. 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.
  • KCNK9 polynucleotides
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode KCNK9 having, for example, an amino acid sequence shown in SEQ DD NO: 2 or a biologically active variant thereof.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • KCNK9 can be obtained, for example, by purification from human cells, by expression of KCNK9 polynucleotides, or by direct chemical synthesis.
  • KCNK9 polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding KCNK9 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 KCNK9. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
  • 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 KCNK9, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected.
  • vectors which direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • BLUESCRIPT a sequence encoding KCNK9 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 or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion 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.
  • KCNK9 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.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used.
  • These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • An insect system also can be used to express KCNK9.
  • Autographa californica nuclear polyhedrosis virus AcNPV
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • HACs Human artificial chromosomes
  • HACs also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding KCNK9. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding KCNK9, 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
  • 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.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the 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
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the aminoglycosides, neomycin and G-418 [Wigler, (1980)]
  • ⁇ ls and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981].
  • Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Visible markers such as anthocyanins, ⁇ - glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • KCNK9 polynucleotide is also present, its presence and expression may need to be confirmed.
  • a sequence encoding KCNK9 is inserted within a marker gene sequence, transformed cells containing sequences which encode KCNK9 can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding KCNK9 under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of KCNK9 polynucleotide.
  • host cells which contain a KCNK9 polynucleotide and which express KCNK9 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 which 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 KCNK9 can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding KCNK9.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding KCNK9 to detect transformants which contain a KCNK9 polynucleotide.
  • KCNK9 A variety of protocols for detecting and measuring the expression of KCNK9, 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 KCNK9 can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding KCNK9 include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding KCNK9 can be cloned into a vector for the production of an RNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
  • Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with KCNK9 polynucleotides 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 KCNK9 polynucleotides can be designed to contain signal sequences which direct secretion of soluble KCNK9 through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound KCNK9.
  • Sequences encoding KCNK9 can be synthesized, in whole or in part, using chemical methods well known in the art.
  • KCNK9 itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of KCNK9 can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic KCNK9 can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of KCNK9 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. Production of Altered Polypeptides
  • 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.
  • nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter KCNK9 polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or rnRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site- directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of KCNK9.
  • 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 KCNK9.
  • Fab fragments thereof
  • F(ab') 2 fragments thereof
  • Fv fragments thereof
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid.
  • An antibody which specifically binds to an epitope of KCNK9 can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radio- immunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radio- immunoassays, 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 which specifically binds to the KCNK9 immunogen.
  • an antibody which specifically binds to KCNK9 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 KCNK9 do not detect other proteins in immunochemical assays and can immunoprecipitate KCNK9 from solution.
  • KCNK9 can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • KCNK9 can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • 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., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • BCG Bacilli Calmette-Gueri ⁇
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to KCNK9 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 [Roberge, (1995)].
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to KCNK9 can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to KCNK9.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to KCNK9 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.
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of KCNK9 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 internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkyl- phosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of KCNK9 gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the KCNK9 gene. Oligo- nucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature [Nicholls, (1993)]. An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a KCNK9 polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent KCNK9 nucleotides, can provide sufficient targeting specificity for KCNK9 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 KCNK9 polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a KCNK9 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)]. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a KCNK9 polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a KCNK9 polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a KCNK9 RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate KCNK9 RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ DD NO: 1 and its complement provide sources of suitable hybridization region sequences.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease KCNK9 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • Antagonists of KCNK9 are molecules which, when bound to KCNK9, decrease the amount or the duration of the functional activity of KCNK9. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of KCNK9.
  • modulate refers to a change in the activity of KCNK9 polypeptide. For example, modulation may cause an increase or a decrease in functional activity, binding characteristics, or any other biological, functional, or immunological properties of KCNK9.
  • the invention provides methods (also referred to herein as “screening assays") for identifying compounds which can be used for the treatment of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • the methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) which bind to KCNK9 and/or have a stimulatory or inhibitory effect on the biological functional activity of KCNK9 or its expression and then determining which of these compounds have an effect on symptoms or diseases regarding cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in an in vivo assay.
  • candidate or test compounds or agents e.g., peptides, peptidomimetics, small molecules or other molecules
  • Candidate or test compounds or agents which bind to KCNK9 and/or have a stimulatory or inhibitory effect on the functional activity or the expression of KCNK9 are identified either in assays that employ cells which express KCNK9 on the cell surface (cell-based assays) or in assays with isolated KCNK9 (cell-free assays).
  • the various assays can employ a variety of variants of KCNK9 (e.g., full-length KCNK9, a biologically active fragment of KCNK9, or a fusion protein which includes all or a portion of KCNK9).
  • KCNK9 can be derived from any suitable mammalian species (e.g., human KCNK9, rat KCNK9 or murine KCNK9).
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known KCNK9 ligand to KCNK9.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of KCNK9.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of KCNK9 mRNA or KCNK9 protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the functional activity of a membrane-bound (cell surface expressed) form of KCNK9.
  • assays can employ full-length KCNK9, a biologically active fragment of KCNK9, or a fusion protein which includes all or a portion of KCNK9.
  • the test compound can be obtained by any suitable means, e.g., from conventional compound libraries.
  • test compound can be enzymatically labelled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of KCNK9 (e.g., full-length KCNK9, a biologically active fragment of KCNK9, or a fusion protein which includes all or a portion of KCNK9) expressed on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the functional activity of the membrane-bound form of KCNK9.
  • a membrane-bound form of KCNK9 e.g., full-length KCNK9, a biologically active fragment of KCNK9, or a fusion protein which includes all or a portion of KCNK9
  • Determining the ability of the test compound to modulate the functional activity of the membrane-bound form of KCNK9 can be accomplished by any method suitable for measuring the functional activity of KCNK9, e.g., any method suitable for measuring the activity of any ion channel or other electrogenic target protein (described in greater detail below).
  • the activity of an electrogenic target can be measured in a number of ways, not all of which are suitable for any given target protein. Among the measures of activity are: alteration in ionconcentrations (e.g. Terstappen, 1999 Analyt. Biochem.
  • the assay includes contacting KCNK9 with a known compound which carries a detectable lable and which binds KCNK9 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with KCNK9, wherein determining the ability of the test compound to interact with KCNK9 comprises determining the ability of the test compound to preferentially bind to KCNK9 as compared to the known compound.
  • the cell-free assays of the present invention are amenable to use of either a membrane-bound form of KCNK9 or a soluble fragment thereof.
  • a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • KCNK9 or a KCNK9 target molecule
  • binding of a test compound to KCNK9, or interaction of KCNK9 with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or KCNK9, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of KCNK9 can be determined using standard techniques.
  • KCNK9 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, HI.), and immobilized in the wells of streptavidin-coated plates (Pierce Chemical).
  • antibodies reactive with KCNK9 or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with KCNK9 or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with KCNK9 or target molecule.
  • the screening assay can also involve monitoring the expression of KCNK9.
  • regulators of expression of KCNK9 can be identified in a method in which a cell is contacted with a candidate compound and the expression of KCNK9 protein or mRNA in the cell is determined. The level of expression of KCNK9 protein or mRNA the presence of the candidate compound is compared to the level of expression of KCNK9 protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of KCNK9 based on this comparison.
  • the candidate compound when expression of KCNK9 protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of KCNK9 protein or mRNA expression.
  • the candidate compound when expression of KCNK9 protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of KCNK9 protein or mRNA expression.
  • the level of KCNK9 protein or mRNA expression in the cells can be determined by methods described below.
  • either the test compound or the KCNK9 polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to KCNK9 polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a KCNK9 polypeptide can be determined without labeling either of the interactants.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • BIA Bimolecular Interaction Analysis
  • the assay includes contacting KCNK9 with a known compound which carries a detectable lable and which is coupled to a detectable lable and which binds KCNK9 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with KCNK9, wherein determining the ability of the test compound to interact with KCNK9 comprises determining the ability of the test compound to preferentially bind to KCNK9 as compared to the known compound.
  • a KCNK9-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with KCNK9 and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding KCNK9 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.
  • 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 which interacts with KCNK9.
  • a reporter gene e.g., LacZ
  • either the KCNK9 (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 KCNK9-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive abso ⁇ tion, 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 KCNK9 (or a polynucleotide encoding for KCNK9) can be accomplished in any vessel suitable for containing the reactants Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • KCNK9 is a fusion protein comprising a domain that allows binding of KCNK9 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 KCNK9; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • KCNK9 or a polynucleotide encoding KCNK9
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated KCNK9 (or a polynucleotide encoding biotinylated KCNK9) 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 plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, 111.
  • streptavidin-coated plates Piereptavidin-coated plates
  • antibodies which specifically bind to KCNK9, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of KCNK9 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 KCNK9 polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of KCNK9 polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a KCNK9 polypeptide or polynucleotide can be used in a cell-based assay system.
  • a KCNK9 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 KCNK9 or a polynucleotide encoding KCNK9 is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease KCNK9 functional activity of a KCNK9 polypeptide.
  • the KCNK9 activity can be measured, for example, using methods described in the specific examples, below.
  • KCNK9 functional activity can be measured after contacting an intact cell having functional ion channel activity with a test compound.
  • a test compound which decreases KCNK9 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing KCNK9 activity.
  • a test compound which increases KCNK9 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing KCNK9 activity.
  • KCNK9 for example, transfected CHO cells
  • a second messenger response e.g., signal transduction or pH changes
  • Another such screening technique involves introducing RNA or DNA encoding KCNK9 into Xenopus oocytes to transiently express the channel.
  • Another screening technique involves expressing KCNK9 in cells in which the channel is linked to a phospholipase C or D.
  • Such cells include endothelial cells, smooth muscle cells, embryonic kidney cells, etc.
  • the screening may be accomplished as described above by quantifying the degree of activation of the channel from changes in the phospholipase activity.
  • microarray refers to an array of distinct polynucleotides or oligonucleotides arrayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • a KCNK9 polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of KCNK9 polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a regulator of expression based on this comparison.
  • 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.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses KCNK9 polynucleotide can be used in a cell-based assay system.
  • the KCNK9 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate KCNK9 expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites, such as the interaction domain of the ligand with KCNK9.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
  • the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • KCNK9 is expressed in various human tissues.
  • CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • CNS disorders include, but are not limited to brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.
  • Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkmsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld- Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoffs psychosis, within the meaning of the definition are also considered to be CNS disorders.
  • CNS disorders such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities are also considered to be CNS disorders.
  • Pain within the meaning of this definition, is also considered to be a CNS disorder. Pain can be associated with CNS disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • CNS disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • Non-central neuropathic pain includes that associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/ADDS 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-he ⁇ etic neuralgia. Pain associated with peripheral nerve damage, central pain (i.e.
  • Visceral pain such as pancreatits, intestinal cystitis, dysmenorrhea, irritable Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction and pain syndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders.
  • the expression in brain tissues and in particular the differential expression between diseased tissue Alzheimer brain and healthy tissue brain, between diseased tissue Alzheimer cerebral cortex and healthy tissue cerebral cortex, between diseased tissue Alzheimer brain frontal lobe and healthy tissue frontal lobe demonstrates that the human KCNK9 or mRNA can be utilized to diagnose nervous system diseases. Additionally the activity of the human KCNK9 can be modulated to treat nervous system diseases.
  • Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low- output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included as well as the acute treatment of MI and the prevention of complications.
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others.
  • the genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.
  • the atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.
  • the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.
  • Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.
  • the risk to develop atherosclerosis and coronary artery or carotid artery disease (and thus the risk of having a heart attack or stroke) increases with the total cholesterol level increasing. Nevertheless, extremely low cholesterol levels may not be healthy.
  • hyperlipidemia abnormally high levels of fats (cholesterol, triglycerides, or both) in the blood, may be caused by family history of hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to high alcohol consumption, cigarette smoking, poorly controlled diabetes, and an underactive thyroid gland), hereditary hyperlipidemias (type I hyperlipoproteinemia (familial hyperchylomicronemia), type II hyperlipoproteinemia (familial hypercholesterolemia), type HI hyperlipoproteinemia, type IN hyperlipoproteinemia, or type V hyperlipoproteinemia), hypolipo- proteinemia, lipidoses (caused by abnormalities in the enzymes that metabolize fats), Gaucher's disease, ⁇ iemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.
  • hyperlipidemia abnormally high levels of fats (cholesterol,
  • Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.
  • the human KC ⁇ K9 is highly expressed in the following cardiovascular related tissues: pericardium, heart atrium (left), heart ventricle (left), aorta, artery, vein, adrenal gland, thrombocytes, kidney, kidney tumor. Expression in the above mentioned tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of cardiovascular diseases. Additionally the activity of the human KCNK9 can be modulated to treat cardiovascular diseases.
  • the human KCNK9 is highly expressed in kidney tissues : kidney, kidney tumor. Expression in kidney tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of blood pressure disorders as an cardiovascular disorder. Additionally the activity of the human KCNK9 can be modulated to treat - but not limited to - blood pressure disorders as hypertension or hypotension.
  • the human KCNK9 is highly expressed in adrenal gland. Expression in adrenal gland tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of blood pressure disorders as an cardiovascular disorder. Additionally the activity of the human KCNK9 can be modulated to treat - but not limited to - blood pressure disorders as hypertension or hypotension.
  • Hematological disorders comprise diseases of the blood and all its constituents as well as diseases of organs and tissues involved in the generation or degradation of all the constituents of the blood. They include but are not limited to 1) Anemias, 2) Myeloproliferative Disorders, 3) Hemorrhagic Disorders, 4) Leukopenia, 5) Eosinophilic Disorders, 6) Leukemias, 7) Lymphomas, 8) Plasma Cell Dyscrasias, 9) Disorders of the Spleen in the course of hematological disorders. Disorders according to 1) include, but are not limited to anemias due to defective or deficient hem synthesis, deficient erythropoiesis.
  • Disorders according to 2) include, but are not limited to polycythemia vera, tumor-associated erythrocytosis, myelofibrosis, thrombocythemia.
  • Disorders according to 3) include, but are not limited to vasculitis, thrombocytopenia, heparin-induced thrombocytopenia, thrombotic thrombocytopenic pu ⁇ ura, hemolytic-uremic syndrome, hereditary and acquired disorders of platelet function, hereditary coagulation disorders.
  • Disorders according to 4) include, but are not limited to neutropenia, lymphocytopenia.
  • Disorders according to 5) include, but are not limited to hypereosinophilia, idiopathic hypereosinophilic syndrome.
  • Disorders according to 6) include, but are not limited to acute myeloic leukemia, acute lymphoblastic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome.
  • Disorders according to 7) include, but are not limited to Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, mycosis fungoides cutaneous T-cell lymphoma.
  • Disorders according to 8) include, but are not limited to multiple myeloma, macroglobulinemia, heavy chain diseases.
  • iron deficiency anemia In extension of the preceding idiopathic thrombocytopenic pu ⁇ ura, iron deficiency anemia, megaloblastic anemia (vitamin B12 deficiency), aplastic anemia, thalassemia, malignant lymphoma bone marrow invasion, malignant lymphoma skin invasion, hemolytic uremic syndrome, giant platelet disease are considered to be hematological diseases too.
  • the human KCNK9 is highly expressed in the following tissues of the hematological system: erythrocytes, lymphnode, thrombocytes, bone marrow CD71+ cells.
  • the expression in the above mentioned tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of hematological diseases. Additionally the activity of the human KCNK9 can be modulated to treat hematological disorders. Gastrointestinal and Liver Diseases
  • Gastrointestinal diseases comprise primary or secondary, acute or chronic diseases of the organs of the gastrointestinal tract which may be acquired or inherited, benign or malignant or metaplastic, and which may affect the organs of the gastrointestinal tract or the body as a whole. They comprise but are not limited to 1) disorders of the esophagus like achalasia, vigoruos achalasia, dysphagia, cricopharyngeal incoordination, pre-esophageal dysphagia, diffuse esophageal spasm, globus sensation, Barrett's metaplasia, gastroesophageal reflux, 2) disorders of the stomach and duodenum like functional dyspepsia, inflammation of the gastric mucosa, gastritis, stress gastritis, chronic erosive gastritis, atrophy of gastric glands, metaplasia of gastric tissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3) disorders of the pancreas like acute
  • Liver diseases comprise primary or secondary, acute or chronic diseases or injury of the liver which may be acquired or inherited, benign or malignant, and which may affect the liver or the body as a whole. They comprise but are not limited to disorders of the bilirubin metabolism, jaundice, syndroms of Gilbert's, Crigler-Najjar, Dubin- Johnson and Rotor; intrahepatic cholestasis, hepatomegaly, portal hypertension, ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fatty liver, steatosis, Reye's syndrome, liver diseases due to alcohol, alcoholic hepatitis or cirrhosis, fibrosis and cirrhosis, f ⁇ brosis and cirrhosis of the liver due to inborn errors of metabolism or exogenous substances, storage diseases, syndromes of Gaucher's, Zellweger's, Wilson's - disease, acute or chronic hepatitis, viral hepatitis and its variants,
  • the human KCNK9 is highly expressed in the following tissues of the gastroenterological system: esophagus, esophagus tumor, colon, colon tumor, ileum, ileum tumor, ileum chronic inflammation, rectum.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue ileum chronic inflammation and healthy tissue ileum demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of gastroenterological disorders. Additionally the activity of the human KCNK9 can be modulated to treat gastroenterological disorders.
  • the endocrine system consists of a group of organs whose main function is to produce and secrete hormones directly into the bloodstream.
  • the major organs of the endocrine system are the hypothalamus, the pituitary gland, thyroid gland, the parathyroid glands, the islets of the pancreas, the adrenal glands, the testes, and the ovaries.
  • the pituitary gland coordinates many functions of the other endocrine glands, but some pituitary hormones have direct effects.
  • the insulin-secreting cells of the pancreas respond to glucose and fatty acids.
  • Parathyroid cells respond to calcium and phosphate.
  • the adrenal medulla (part of the adrenal gland) responds to direct stimulation by the parasympathetic nervous system.
  • Diabetes mellitus is a disorder in which blood levels of glucose are abnormally high because the body doesn't release or use insulin adequately.
  • Type I diabetes mellitus insulin-dependent diabetes
  • type I diabetes people with type I diabetes mellitus (insulin-dependent diabetes) produce little or no insulin at all.
  • type I diabetes more than 90 percent of the insulin-producing cells (beta cells) of the pancreas are permanently destroyed. The resulting insulin deficiency is severe, and to survive, a person with type I diabetes must regularly inject insulin.
  • pancreas In type II diabetes mellitus (non-insulin-dependent diabetes) the body develops resistance to insulin effects, resulting in a relative insulin deficiency.
  • the pancreas has two major functions: to secrete fluid containing digestive enzymes into the duodenum and to secrete the hormones insulin and glucagon.
  • Chronic pancreatitis is a longstanding inflammation of the pancreas.
  • An insulinoma is a rare type of pancreatic tumor that secretes insulin. The symptoms of an insulinoma result from low blood glucose levels.
  • a gastrinoma is a pancreatic tumor that produces excessive levels of the hormone gastrin, which stimulates the stomach to secrete acid and enzymes, causing peptic ulcers. The excess gastrin secreted by the gastrinoma causes symptoms, called the Zollinger-Ellison syndrome.
  • a glucagonoma is a tumor that produces the hormone glucagon, which raises the level of glucose in the blood and produces a distinctive rash.
  • Diabetes insipidus is a disorder in which insufficient levels of antidiuretic hormone cause excessive thirst (polydipsia) and excessive production of very dilute urine (polyuria). Diabetes insipidus results from the decreased production of antidiuretic hormone (vasopressin).
  • the body has two adrenal glands.
  • the medulla of the adrenal glands secretes hormones such as adrenaline (epinephrine) that affect blood pressure, heart rate, sweating, and other activities also regulated by the sympathetic nervous system.
  • the cortex secretes many different hormones, including corticosteroids (cortisone-like hormones), androgens (male hormones), and mineral- ocorticoids, which control blood pressure and the levels of salt and potassium in the body.
  • Adrenal Glands Several disorders are characterized by overactive Adrenal Glands.
  • the causes can be changes in the adrenal glands themselves or overstimulation by the pituitary gland. Examples of these diseases are listed in the following.
  • the thyroid is a small gland located under the Adam's apple. It secretes thyroid hormones, which control the metabolic rate. The thyroid gland traps iodine and processes it into thyroid hormones. The euthyroid sick syndrome is characterized by lack of conversion of the T4 form of thyroid hormone to the T3 form. Hyperthyroidism (overactive thyroid gland, production of too much hormone) may have several causes. Thyroiditis (an inflammation of the thyroid gland), typically leads to a phase of hyperthyroidism. The inflammation may damage the thyroid gland, so that in later stages the disease is characterized by transient or permanent underactivity (hypothyroidism). Toxic thyroid nodules (adenomas) often produce thyroid hormone in large quantities.
  • Toxic multinodular goiter is a disorder in which there are many nodules. Graves' disease (toxic diffuse goiter) is believed to be caused by an antibody that stimulates the thyroid to produce too much thyroid hormone. In toxic nodular goiter, one or more nodules in the thyroid produce too much thyroid hormone and aren't under the control of thyroid-stimulating hormone. Secondary hyperthyroidism may (rarely) be caused by a pituitary tumor that secretes too much thyroid-stimulating hormone, by resistance of the pituitary to thyroid hormone, which results in the pituitary gland secreting too much thyroid-stimulating hormone, or by a hydatidiform mole in women. Thyroid storm is a sudden extreme overactivity of the thyroid gland is a life-threatening emergency requiring prompt treatment.
  • hypothyroidism is a condition in which the thyroid gland is underactive and produces too little thyroid hormone. Very severe hypothyroidism is called myxedema. In Hashimoto's thyroiditis (autoimmune thyroiditis) the thyroid gland is often enlarged, and hypothyroidism results because the gland's functioning areas are gradually destroyed. Rarer causes of hypothyroidism include some inherited disorders which are caused by abnormalities of the enzymes in thyroid cells. In other rare disorders, either the hypothalamus or the pituitary gland fails to secrete enough of the hormone needed to stimulate normal thyroid function.
  • Thyroiditis are silent lymphocytic thyroiditis, Hashimoto's thyroiditis, or subacute granulomatous thyroiditis.
  • Thyroid cancer is any one of four main types of malignancy of the thyroid: papillary, follicular, anaplastic, or medullary.
  • the pituitary is a pea-sized gland that sits in a bony structure (sella turcica) at the base of the brain.
  • the sella turcica protects the pituitary but allows very little room for expansion. If the pituitary enlarges, it tends to push upward, often pressing on the areas of the brain that carry signals from the eyes, possibly resulting in headaches or impaired vision.
  • the pituitary gland has two distinct parts: the anterior (front) and the posterior (back) lobes.
  • the anterior lobe produces (secretes) hormones that ultimately control the function of the thyroid gland, adrenal glands, and reproductive organs (ovaries and testes); milk production (lactation) in the breasts; and overall body growth. It also produces hormones that cause the skin to darken and that inhibit pain sensations.
  • the posterior lobe produces hormones that regulate water balance, stimulate the letdown of milk from the breasts in lactating women, and stimulate contractions of the uterus.
  • Examples for disorders of the pituitary gland are Empty Sella Syndrome; hypopituitarism (an underactive pituitary gland); acromegaly, which is excessive growth caused by oversecretion of growth hormone, which is almost always caused by a benign pituitary tumor (adenoma); galactorrhea, which is the production of breast milk in men or in women who aren't breastfeeding, in both sexes, the most common cause of galactorrhea is a prolactin-producing tumor (prolactinoma) in the pituitary gland.
  • prolactin-producing tumor prolactinoma
  • the human KCNK9 is highly expressed in the following tissues of the endocrinological system: adrenal gland.
  • the expression in the above mentioned tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of endocrinological disorders. Additionally the activity of the human KCNK9 can be modulated to treat endocrinological disorders.
  • Cancer disorders within the scope of this definition comprise any disease of an organ or tissue in mammals characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole.
  • Cancer diseases within the scope of the definition comprise benign neoplasms, dysplasias, hype ⁇ lasias as well as neoplasms showing metastatic growth or any other transformations like e.g. leukoplakias which often precede a breakout of cancer.
  • Cells and tissues are cancerous when they grow more rapidly than normal cells, displacing or spreading into the surrounding healthy tissue or any other tissues of the body described as metastatic growth, assume abnormal shapes and sizes, show changes in their nucleocytoplasmatic ratio, nuclear polychromasia, and finally may cease.
  • Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cancer occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results.
  • the ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease.
  • Cancers are generally said to be in one of three stages of growth: early, or localized, when a tumor is still confined to the tissue of origin, or primary site; direct extension, where cancer cells from the tumour have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, in which cancer cells have migrated to distant parts of the body from the primary site, via the blood or lymph systems, and have established secondary sites of infection.
  • Cancer is said to be malignant because of its tendency to cause death if not treated. Benign tumors usually do not cause death, although they may if they interfere with a normal body function by virtue of their location, size, or paraneoplastic side effects. Hence benign tumors fall under the definition of cancer within the scope of this definition as well.
  • cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue.
  • Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers of the breast and prostate, for example, are considered dependent on specific hormones for their existence.
  • cancer under the scope of the definition is not limited to simple benign neoplasia but comprises any other benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Cancers of the blood-forming tissues, 5) tumors of nerve tissues including the brain, 6) cancer of skin cells.
  • Cancer according to 1) occurs in epithelial tissues, which cover the outer body (the skin) and line mucous membranes and the inner cavitary structures of organs e.g. such as the breast, lung, the respiratory and gastrointestinal tracts, the endocrine glands, and the genitourinary system.
  • Ductal or glandular elements may persist in epithelial tumors, as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • Cancers of the pavement-cell epithelium of the skin and of certain mucous membranes, such as e.g. cancers of the tongue, lip, larynx, urinary bladder, uterine cervix, or penis, may be termed epidermoid or squamous-cell carcinomas of the respective tissues and are in the scope of the definition of cancer as well.
  • Cancer according to 2) develops in connective tissues, including fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma.
  • Cancer according to 3) is cancer that develops in both epithelial and connective tissue.
  • Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion.
  • Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal.
  • malignant osteogenic sarcoma benign osteoma, cartilage tumors; like malignant chondrosarcoma or benign chondroma; bone marrow tumors like malignant myeloma or benign eosinophilic granuloma, as well as metastatic tumors from bone tissues at other locations of the body;
  • the human KC ⁇ K9 is highly expressed in the following cancer tissues: esophagus tumor, colon tumor, ileum tumor, lung tumor, ovary tumor, breast tumor, kidney tumor.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue esophagus tumor and healthy tissue esophagus, between diseased tissue colon tumor and healthy tissue colon, between diseased tissue ileum tumor and healthy tissue ileum, between diseased tissue lung tumor and healthy tissue lung, between diseased tissue ovary tumor and healthy tissue , between diseased tissue breast tumor and healthy tissue breast, between diseased tissue kidney tumor and healthy tissue kidney demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of cancer. Additionally the activity of the human KCNK9 can be modulated to treat cancer.
  • Inflammatory diseases comprise diseases triggered by cellular or non-cellular mediators of the immune system or tissues causing the inflammation of body tissues and subsequently producing an acute or chronic inflammatory condition.
  • hypersensitivity reactions of type I - TV for example but not limited to hypersensitivity diseases of the lung including asthma, atopic diseases, allergic rhinitis or conjunctivitis, angioedema of the lids, hereditary angioedema, antireceptor hypersensitivity reactions and autoimmune diseases, Hashimoto's thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, myasthenia gravis, Grave's and Raynaud's disease, type B insulin-resistant diabetes, rheumatoid arthritis, psoriasis, Crohn's disease, scleroderma, mixed connective tissue disease, polymyositis, sarcoidosis, glomerulonephritis, acute or chronic host versus
  • the human KCNK9 is highly expressed in the following tissues of the immune system and tissues responsive to components of the immune system as well as in the following tissues responsive to mediators of inflammation: ileum chronic inflammation, lung COPD.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue ileum chronic inflammation and healthy tissue ileum, between diseased tissue lung COPD and healthy tissue lung demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of inflammatory diseases. Additionally the activity of the human KCNK9 can be modulated to treat inflammatory diseases.
  • Asthma is thought to arise as a result of interactions between multiple genetic and environmental factors and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness, and 3) airway inflammation.
  • Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE. These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to tissue destruction related to the disorder.
  • Other resident cells such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to its pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic and disabling disorder requiring long-term management.
  • COPD chronic obstructive pulmonary (or airways) disease
  • COPD chronic obstructive pulmonary (or airways) disease
  • Emphysema is characterised 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. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does also occur in non-smokers.
  • the human KCNK9 is highly expressed in the following tissues of the respiratory system: lung, lung tumor, lung COPD.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue lung COPD and healthy tissue lung demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of respiratory diseases. Additionally the activity of the human KCNK9 can be modulated to treat those diseases.
  • Genitourinary disorders comprise benign and malign disorders of the organs constituting the genitourinary system of female and male, renal diseases like acute or chronic renal failure, immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hype ⁇ lasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or overflow incontinence, pelvic pain, and erectile dysfunction.
  • renal diseases like acute or chronic renal failure
  • immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hype ⁇ lasia (BPH)
  • neurogenic bladder syndrome urinary incontinence like urge-, stress-, or overflow in
  • the human KCNK9 is highly expressed in the following urological tissues: dorsal root ganglia, spinal cord, penis, co ⁇ us cavernosum, kidney, kidney tumor.
  • the expression in the above mentioned tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of urological disorders. Additionally the activity of the human KCNK9 can be modulated to treat urological disorders.
  • the human KCNK9 is highly expressed in dorsal-root ganglia tissue. Expression in dorsal root ganglia demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of incontinence as an urological disorder. The dorsal root ganglia are involved in the neuronal regulation of the urological system. Additionally the activity of the human KCNK9 can be modulated to treat - but not limited to - incontinence.
  • the human KCNK9 is highly expressed in spinal cord tissues: spinal cord. Expression in spinal cord tissues demonstrates that the human KCNK9 or mRNA can be utilized to diagnose of incontinence as an urological disorder. The spinal cord tissues are involved in the neuronal regulation of the urological system. Additionally the activity of the human KCNK9 can be modulated to treat - but not limited to - incontinence.
  • the present invention provides for both prophylactic and therapeutic methods for cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • the regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of KCNK9.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or any small molecule.
  • the agent stimulates one or more of the biological activities of KCNK9. Examples of such stimulatory agents include the active KCNK9 and nucleic acid molecules encoding a portion of KCNK9.
  • the agent inhibits one or more of the biological activities of KCNK9. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of KCNK9 or a protein in the KCNK9 signaling pathway.
  • the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of KCNK9 or of any protein in the KCNK9 signaling pathway.
  • the method involves administering a regulator of KCNK9 as therapy to compensate for reduced or undesirably low expression or activity of KCNK9 or a protein in the KCNK9 signaling pathway.
  • Stimulation of activity or expression of KCNK9 is desirable in situations in which activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity or expression of KCNK9 is desirable in situations in which activity or expression of KCNK9 is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • compositions suitable for administration can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of KCNK9 expression or activity (and/or a regulator of the activity or expression of a protein in the KCNK9 signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier. Also within the invention are pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use. For regulators that are antagonists of KCNK9 activity or which reduce KCNK9 expression, the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases .
  • the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • An antagonist of KCNK9 may be produced using methods which are generally known in the art.
  • purified KCNK9 may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind KCNK9.
  • Antibodies to KCNK9 may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially preferred for therapeutic use.
  • the polynucleotides encoding KCNK9, or any fragment or complement thereof may be used for therapeutic pu ⁇ oses.
  • the complement of the polynucleotide encoding KCNK9 may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding KCNK9.
  • complementary molecules or fragments may be used to modulate KCNK9 activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding KCNK9.
  • Expression vectors derived from retroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding KCNK9. These techniques are described, for example, in [Scott and Smith (1990) Science 249:386-390].
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing KCNK9 in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of KCNK9, antibodies to KCNK9, and mimetics, agonists, antagonists, or inhibitors of KCNK9.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Ixi all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chloro- butanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • instructions for administration will specify use of the composition for cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • compositions which include an agonist of KCNK9 activity, a compound which increases expression of KCNK9, or a compound which increases expression or activity of a protein in the KCNK9 signaling pathway or any combination thereof
  • the instructions for administration will specify use of the composition cardiovascular diseases, endocrinological diseases, gastroentero- logical diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • antibodies which specifically bind KCNK9 may be used for the diagnosis of disorders characterized by the expression of KCNK9, or in assays to monitor patients being treated with KCNK9 or agonists, antagonists, and inhibitors of KCNK9.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as those described above for therapeutics.
  • Diagnostic assays for KCNK9 include methods which utilize the antibody and a label to detect KCNK9 in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • KCNK9 A variety of protocols for measuring KCNK9, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of KCNK9 expression.
  • Normal or standard values for KCNK9 expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to KCNK9 under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, preferably by photometric means. Quantities of KCNK9 expressed in subject samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding KCNK9 may be used for diagnostic pu ⁇ oses.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of KCNK9 may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of KCNK9, and to monitor regulation of KCNK9 levels during therapeutic intervention.
  • Polynucleotide sequences encoding KCNK9 may be used for the diagnosis of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hemato- logical diseases, neurological diseases, urological diseases and respiratory diseases associated with expression of KCNK9.
  • the polynucleotide sequences encoding KCNK9 may be used in Southern, Northern, or dot-blot analysis, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patient biopsies to detect altered KCNK9 expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding KCNK9 may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding KCNK9 may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding KCNK9 in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding KCNK9, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with KCNK9, or fragments thereof, and washed.
  • Bound KCNK9 is then detected by methods well known in the art.
  • Purified KCNK9 can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases KCNK9 activity relative to KCNK9 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 can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • 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, electro- poration, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of KCNK9 gene or the activity of KCNK9 by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of KCNK9 gene or the activity of KCNK9 can be assessed using methods well known in the art, such as hybridization of nucleotide probes to KCNK9-specific mRNA, quantitative RT-PCR, immunologic detection of KCNK9, or measurement of KCNK9 activity.
  • 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.
  • Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ DD NO: 2, (ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 1, (iii) nucleic acid molecules having the sequence of SEQ DD NO: 1, (iv)nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, wherein the polypeptide encoded by said nucleic acid molecule has KCNK9 activity.
  • Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ DD NO: 2, (ii) polypeptides comprising the sequence of SEQ DD NO: 2, (iii) polypeptides encoded by nucleic acid molecules of the invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii), wherein said purified polypeptide has KCNK9 activity.
  • An object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrino- logical diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising the steps of (i) contacting a test compound with a KCNK9 polypeptide, (ii) detect binding of said test compound to said KCNK9 polypeptide.
  • a test compound with a KCNK9 polypeptide
  • detect binding of said test compound to said KCNK9 polypeptide are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising the steps of (i) determining the activity of a KCNK9 polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound.
  • compounds that lead to a difference in the activity of the KCNK9 polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treat- ment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising the steps of (i) determining the activity of a KCNK9 polypeptide at a certain concentration of a test compound, (ii) determining the activity of a KCNK9 polypeptide at the presence of a compound known to be a regulator of a KCNK9 polypeptide.
  • compounds that show similar effects on the activity of the KCNK9 polypeptide in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treat- ment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising i) cell expressing KCNK9 having functional activity, ii) contacting said cell with the test compounds, iii) measuring functional activity.
  • Another object of the invention is the method of the above, where the cell is expressing endogenous KCNK9.
  • Another object of the invention are the methods of the above, where the cell is expressing recombinant KCNK9.
  • Another object of the invention are the methods of the above, where the measured functional activity is the KCNK9 current.
  • Another object of the invention are the methods of the above, where the functional activity is a change in membrane potential.
  • Another object of the invention are the methods of the above, where the functional activity is a change in ion concentration.
  • test compound displaces a ligand which is first bound to the polypeptide.
  • the ligand to be displaced is coupled to a detectable label.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising the steps of (i) contacting a test compound with a KCNK9 polynucleotide, (ii) detect binding of said test compound to said KCNK9 polynucleotide.
  • Compounds that, e.g., bind to the KCNK9 polynucleotide are potential therapeutic agents for the treatment of such diseases.
  • Another object of the invention is the method of the above, wherein the nucleic acid molecule is RNA.
  • Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.
  • Another object of the invention is a method of the above, wherein the contacting step is in a cell- free system.
  • Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.
  • Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.
  • Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising the steps of (i) determining the amount of a KCNK9 polynucleotide in a sample taken from said mammal, (ii) determining the amount of KCNK9 polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in the amount of KCNK9 polynucleotide in said test mammal as compared to a diseased mammal.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising a therapeutic agent which binds to a KCNK9 polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising a therapeutic agent which regulates the activity of a KCNK9 polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising a therapeutic agent which regulates the activity of a KCNK9 polypeptide, wherein said therapeutic agent is (i) a small molecule, (ii) an RNA molecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising a KCNK9 polynucleotide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising a KCNK9 polypeptide.
  • Another object of the invention is the use of regulators of a KCNK9 for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal.
  • Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal comprising the steps of (i) identifying a regulator of KCNK9, (ii) determining whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical carrier.
  • Another object of the invention is the use of a regulator of KCNK9 for the regulation of KCNK9 activity in a mammal having a disease comprised in a group of diseases consisting of cardiovascular diseases, endocrinological diseases, gastroenterological diseases, cancer, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • human kcnk9 in cancer and cardiovascular related tissues suggests a particular, but not limited to, utilization of kcnk9 for diagnosis and modulation of cancer and cardiovascular disorders. Furthermore the above described expression suggest a, but not limited to, utilization of kcnk9 to endocrinological diseases, gastroenterological diseases, inflammation, hematological diseases, neurological diseases, urological diseases and respiratory diseases.
  • the degree of homology can readily be calculated by known methods. Preferred methods to determine homology are designed to give the largest match between the sequences tested. Methods to determine homology are codified in publicly available computer programs such as BestFit, BLASTP, BLASTN, and FASTA. The BLAST programs are publicly available from NCBI and other sources in the internet.
  • NCBI non-redundant database
  • DERWENT patent database Geneseq
  • TWIK-related acid-sensitive K+ channel (TASK) mRNA >gb
  • RNA from each cell or tissue source was first reverse transcribed. 85 ⁇ g of total RNA was reverse transcribed using 1 ⁇ mole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Jnvitrogen, Groningen, Netherlands) in a final volume of 680 ⁇ l.
  • the first strand synthesis buffer and Omniscript reverse transcriptase (2 u/ ⁇ l) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 ⁇ l with water, yielding a final concentration of 12.5 ng/ ⁇ l of starting RNA.
  • KCNK9 For relative quantitation of the distribution of KCNK9 mRNA in cells and tissues the Applied Biosystems 7900 HT Sequence Detection system or Biorad iCycler was used according to the manufacturer's specifications and protocols. PCR reactions were set up to quantitate KCNK9 and the housekeeping genes HPRT (hypoxanthine phosphoribosyltransferase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ⁇ -actin, and others. Forward and reverse primers and probes for KCNK9 were designed using the Perkin Elmer ABI Primer ExpressTM software and were synthesized by TibMolBiol (Berlin, Germany).
  • the KCNK9 forward primer sequence was: Primerl (SEQ DD NO: 3).
  • the KCNK9 reverse primer sequence was Primer2 (SEQ DD NO: 4).
  • Probel SEQ DD NO: 5
  • FAM carboxyfluorescein succinimidyl ester
  • TAMRA carboxytetramethylrhodamine
  • the following reagents were prepared in a total of 25 ⁇ l : lx TaqMan buffer A, 5.5 mM MgCl 2 , 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/ ⁇ l AmpliTaq GoldTM, 0.01 U/ ⁇ l AmpErase and Probel (SEQ DD NO: 5), KCNK9 forward and reverse primers each at 200 nM, 200 nM KCNK9 FAMTAMRA-labelled probe, and 5 ⁇ l of template cDNA.
  • Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min.
  • the CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section.
  • the CF-value (factor for threshold cycle correction) is calculated as follows :
  • PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.
  • CTn KG -values were calculated as described in the "Quantitative determination of nucleic acids" section.
  • CT panneI mean value (CT mean value of all HKG in all tested cDNAs)
  • CT CDNA - n CT value of the tested gene for the cDNA n
  • KCNK9 The expression of KCNK9 was investigated in the tissues listed in table 1.
  • Table 1 Relative expression ofKCNK9 in various human tissues.
  • esophagus 468 esophagus tumor 10 stomach 74 stomach tumor 94 colon 12 colon tumor 156 small intestine 10 ileum 272 ileum tumor 9 ileum chronic inflammation 750 rectum 755 salivary gland 13 fetal liver 2 liver 2 liver liver cirrhosis 367 liver tumor 29
  • HeLa cells (cervix tumor) 2 placenta 2 uterus 44 uterus tumor 11 ovary tumor 1687 breast 929 breast tumor 153
  • MDA MB 231 cells (breast tumor) 59 mammary gland 4
  • cDNA sequence coding for KCNK9 enables its use as a tool for antisense technology in the investigation of gene function.
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of a polynucleotide coding for KCNK9 are used either in vitro or in vivo to inhibit translation of the mRNA.
  • antisense molecules can be designed at various locations along the nucleotide sequences.
  • the gene of interest is effectively turned off.
  • the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (e.g., lethality, loss of differentiated function, changes in mo ⁇ hology, etc.).
  • modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to trans-acting regulatory genes.
  • Expression of KCNK9 is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli.
  • the vector is engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of ⁇ -galactosidase.
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • Induction of the isolated, transfected bacterial strain with Isopropyl- ⁇ -D-thiogalactopyranoside (EPTG) using standard methods produces a fusion protein corresponding to the first seven residues of ⁇ -galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone inserts are generated by an essentially random process, there is probability of 33% that the included cDNA will lie in the correct reading frame for proper translation.
  • the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease HI or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the KCNK9 cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; t ⁇ , lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced KCNK9 are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • KCNK9 can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofectamine (Gibco BRL catolog no. 18324-020) mediated gene transfer.
  • KCNK9 is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ ., Seattle, Washington).
  • the inclusion of a cleavable linker sequence such as Factor Xa or enterokinase (Invitrogen, Groningen, The Netherlands) between the purification domain and the KCNK9 sequence is useful to facilitate expression of KCNK9.
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 ⁇ g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B- cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate KCNK9 domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies.
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) by reaction with M-maleimidobenzoyl-N-hydroxy- succinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled KCNK9 to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton- Dickinson, Palo Alto, CA
  • affinity purified, specific rabbit anti- mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% bovine serum albumin, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled KCNK9 at 1 mg/ml.
  • BSA bovine serum albumin
  • 10 8 M **1 preferably 10 9 to 10 10 M "1 or stronger, are typically made by standard procedures.
  • Particular KCNK9 antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of KCNK9 or downstream products of an active signaling cascade.
  • Diagnostic tests for KCNK9 include methods utilizing antibody and a label to detect KCNK9 in human body fluids, membranes, cells, tissues or extracts of such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like.
  • KCNK9 A variety of protocols for measuring soluble or membrane-bound KCNK9, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on KCNK9 is preferred, but a competitive binding assay may be employed.
  • Native or recombinant KCNK9 is purified by immunoaffinity chromatography using antibodies specific for KCNK9.
  • Ixi general, an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology,
  • monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology).
  • Such immunoaffinity columns are utilized in the purification of KCNK9 by preparing a fraction from cells containing KCNK9 in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble KCNK9 containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • a soluble KCNK9-contaming preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of KCNK9 (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and KCNK9 is collected.
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
  • KCNK9 is an electrogenic target any of the methods commonly used in the art may potentially be used to identify KCNK9 ligands modulating functional activity of the channel.
  • the functional activity of ion channels such as KCNK9 can be measured using any of a variety of appropriate functional assays in which modulation of the channel results in an observable change in membrane potential, ion current, ion concentration across the membrane, or any other measurable change exerted by KCNK9.
  • the polypeptide or fragment can be employed in a binding assay either free in solution, affixed to a solid support or expressed on a cell surface.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, are used for standard binding assays.
  • the present invention provides methods of screening for drug canditates, drugs, or any other agents which affects functional activity by binding to the channel protein. These methods, well known in the art, comprise contacting such an agent with KCNK9 polypeptide or a fragment thereof and assaying (i) for the presence of a complex between the agent and KCNK9 polypeptide or fragment, or (ii) for the presence of a complex between KCNK9 polypeptide or fragment and the cell.
  • the KCNK9 polypeptide or fragment is typically labeled. After suitable incubation, free KCNK9 polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to KCNK9 or to interfere with the KCNK9-agent complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to KCNK9 polypeptides. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with KCNK9 polypeptide and washed. Bound KCNK9 polypeptide is then detected by methods well known in the art. Purified KCNK9 are also coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies are used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding KCNK9 specifically compete with a test compound for binding to KCNK9 polypeptides or fragments thereof. Jxi this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic determinants with KCNK9.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating ami-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • Example 11 Use and Administration of Antibodies, Inhibitors, or Antagonists
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, its half-life and anti- genicity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are preferred as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formu- lations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.
  • Normal dosage amounts vary from 0.1 to 10 5 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art employ different formulations for different LSTs.
  • Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells.
  • abnormal signal transduction, trauma, or diseases which trigger KCNK9 activity are treatable with LSTs. These conditions or diseases are specifically diagnosed by the tests discussed above, and such testing should be performed in suspected cases of viral, bacterial or fungal infections, allergic responses, mechanical injury associated with trauma, hereditary diseases, lymphoma or carcinoma, or other conditions which activate the genes of lymphoid or neuronal tissues.
  • Animal model systems which elucidate the physiological and behavioral roles of the KCNK9 are produced by creating nonhuman transgenic animals in which the activity of the KCNK9 is either increased or decreased, or the amino acid sequence of the expressed KCNK9 is altered, by a variety of techniques.
  • these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a KCNK9, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these KCNK9 sequences.
  • the technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and hence is useful for producing an animal that cannot express native KCNK9s but does express, for example, an inserted mutant KCNK9, which has replaced the native KCNK9 in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added KCNK9, resulting in overexpression of the KCNK9.
  • transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding KCNK9 is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • microinjection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected is put in a depression slide.
  • the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transferred into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term.
  • microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary pu ⁇ oses.

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Abstract

La présente invention a trait à un gène KCNK9 humain qui est associé aux maladies cardio-vasculaires, aux maladies endocrinologiques, aux maladies gastroentérologiques, au cancer, à l'inflammation, aux maladies hématologiques, aux maladies neurologiques, aux maladies urologiques et aux maladies respiratoires. L'invention a également trait à des dosages pour l'identification de composés utiles dans le traitement ou la prévention des maladies cardio-vasculaires, des maladies endocrinologiques, des maladies gastroentérologiques, du cancer, de l'inflammation, des maladies hématologiques, des maladies neurologiques, des maladies urologiques et des maladies respiratoires. L'invention a trait en outre à des composés de liaison et/ou d'activation ou d'inhibition de l'activité du gène KCNK9 ainsi qu'à des compositions pharmaceutiques comportant de tels composés.
PCT/EP2005/000612 2004-02-04 2005-01-22 Agents diagnostiques et therapeutiques pour des maladies associees au canal potassique, agents membres 9 de la sous-famille k (kcnk9) WO2005075510A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012057421A1 (fr) * 2010-10-28 2012-05-03 경상대학교 산학협력단 Suppression de métastases cancéreuses au moyen d'une expression accrue d'un canal potassique task-3
WO2016149621A1 (fr) 2015-03-18 2016-09-22 The Johns Hopkins University Nouveaux inhibiteurs d'anticorps monoclonaux ciblant le canal potassique kcnk9

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053628A2 (fr) * 1999-03-05 2000-09-14 Smithkline Beecham Plc Nouveaux composes
WO2001066741A2 (fr) * 2000-03-03 2001-09-13 Tularik Inc. Kcnb: une nouvelle proteine des canaux a potassium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053628A2 (fr) * 1999-03-05 2000-09-14 Smithkline Beecham Plc Nouveaux composes
WO2001066741A2 (fr) * 2000-03-03 2001-09-13 Tularik Inc. Kcnb: une nouvelle proteine des canaux a potassium

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM Y ET AL: "TASK-3, a new member of the tandem pore K+ channel family", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 275, no. 13, 31 March 2000 (2000-03-31), pages 9340 - 9347, XP002970106, ISSN: 0021-9258 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012057421A1 (fr) * 2010-10-28 2012-05-03 경상대학교 산학협력단 Suppression de métastases cancéreuses au moyen d'une expression accrue d'un canal potassique task-3
WO2016149621A1 (fr) 2015-03-18 2016-09-22 The Johns Hopkins University Nouveaux inhibiteurs d'anticorps monoclonaux ciblant le canal potassique kcnk9
US20180092977A1 (en) * 2015-03-18 2018-04-05 The Johns Hopkins University Novel monoclonal antibody inhibitors targeting potassium channel kcnk9
CN108368169A (zh) * 2015-03-18 2018-08-03 约翰霍普金斯大学 靶向钾通道kcnk9的新的单克隆抗体抑制剂
EP3271397A4 (fr) * 2015-03-18 2018-10-31 The Johns Hopkins University Nouveaux inhibiteurs d'anticorps monoclonaux ciblant le canal potassique kcnk9

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