WO2000052173A2 - Homologues de sphingosine kinase humaine clones - Google Patents

Homologues de sphingosine kinase humaine clones Download PDF

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WO2000052173A2
WO2000052173A2 PCT/CA2000/000223 CA0000223W WO0052173A2 WO 2000052173 A2 WO2000052173 A2 WO 2000052173A2 CA 0000223 W CA0000223 W CA 0000223W WO 0052173 A2 WO0052173 A2 WO 0052173A2
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human
sequence
sphingosine kinase
human sphingosine
polypeptide
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PCT/CA2000/000223
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WO2000052173A3 (fr
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Donald Munroe
Ashwani Gupta
Germaine R. Falzone
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Nps Allelix Corp.
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Publication of WO2000052173A3 publication Critical patent/WO2000052173A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • the present invention is related to the field of molecular biology.
  • the present invention is related to newly identified and isolated polynucleotides and their polypeptides and their uses and in particular to newly identified and isolated polynucleotides and polypeptides of the sphingosine kinase family.
  • Sphingolipids are complex structural lipids which are found in membranes.
  • Sphingomyelin is hydrolysized by sphinogmyelinase to form ceramide which in turn is metabloized by ceramidase to form sphingosine.
  • Sphingosine kinase (SK) is the enzyme which phosphorylates sphingosine to form sphingosine 1 -phosphate (SIP) and thereby SK in effect controls SIP production.
  • SIP plays an intracellualar role in cell proliferation and inhibits and/or blocks apoptosis, among other things.
  • SIP also acts as an endogenous ligand for certain G-protein coupled receptors, including the edg-3 receptor.
  • SK inhibitors block thrombin signalling pathways and induce apoptosis.
  • SK inhibitors could be determined which inhibitors could be used in anti-proliferative diseases: cancer, psoriasis, reactive gliosis; or to surpress inappropriate cell survival and to block inflammatory SIP production in neurodegenerative dieseases, demyelinating diseases, asthma, and allergies Summary of the Invention
  • the present invention provides the isolated polynucleotides and polypeptides for the human SK homologues.
  • the present invention provides three isolated polynucleotides and polypeptides for the three human SK homologues: SKA; SKB; and SKC; and variants thereof.
  • isolated polypeptides for the human SK homologues SKA, SKB and SKC comprising the sequences as set out in Figures 3, 6 and 9, respectively, and variants thereof.
  • isolated polynucleotides of SKA, SKB and SKC comprising the sequences as illustrated in Figures 2, 5 and 8, respectively, and variants thereof.
  • isolated polynucleotides encoding human SKA, SKB and SKC, including mRNAs, cDNAs, genomic DNAs.
  • embodiments of the invention include diagnostic, prophylactic, clinical or therapeutical useful variants of these isolated nucleotide sequences SKA, SKB and SKC and compositions thereof. Also included in an aspect of the invention are natuarally occurring allelic variants of SKA, SKB and SKC and polypeptides encoded thereby.
  • polynucleotides that hybridize to SKA, SKB and SKC nucleotide sequences, particularly under stringent conditions.
  • methods for identifying compounds which interact with the polypeptide or polynucleotide of SKA, SKB or SKC are provided.
  • compositions comprising a polypeptide or polynucleotide of SKA, SKB or SKC for administration to a cell or to a multicellular organism.
  • Figure 1 is an illustration of the full length nucleotide sequence of the cDNA of human SKA.
  • Figure 2 is an illustration of the nucleotide sequence of the coding region of
  • Figure 3 is an illustration of the predicted amino acid sequence of SKA as illustrated in Figure 2.
  • Figure 4 is an illustration of the full length nucleotide sequence of the cDNA of human SKB.
  • Figure 5 is an illustration of the nucleotide sequence of the coding region of SKB.
  • Figure 6 is an illustration of the predicted amino acid sequence of SKB as illustrated in Figure 5.
  • Figure 7 is an illustration of the full length nucleotide sequence of the cDNA of human SKC.
  • Figure 8 is an illustration of the nucleotide sequence of the coding region of SKC.
  • Figure 9 is an illustration of the predicted amino acid sequence of SKC as illustrated in Figure 8.
  • Figure 10 is an illustration of the alignment of the amino acid sequences of human SKA, SKB and SKC.
  • Figure 11 is an illustration of the results of the phosphorylation assays exemplified in Example 4 for SKA, SKB and SKB.
  • Bioly Active refers to those forms, fragments, or domains of any sphingosine kinase polypeptide which retain at least some of the biological and/or antigenic activities of a naturally occurring sphingosine kinase.
  • “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 more than one) of the following characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • Derivative refers to those amino acid sequences and nucleotide sequences which have been chemically modified. Such techniques for polypeptide derivatives include: 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.
  • a nucleotide sequence derivative would encode an amino acid which retains its essential biological activity and characteristics of the natural molecule.
  • human sphingosine kinase refers to the isolated polypeptide or polynucleotide sequences of the different isoforms of human sphingosine kinase, including human SKA, human SKB and human SKC, in either naturally occurring or synthetic form.
  • human sphingosine kinase A or “human SKA” refers to the polynucleotide or polypeptide of an isoform of human sphingosine kinase as illustrated by the sequences of Figure 2 and 3, respectively, and by polypeptide sequences which preferably have at least 85% sequence identity with each other and Figure 3, and more preferably at least 90% sequence identity with each other and Figure 3, and most preferably at least 95% sequence identity with each other and Figure 3, or polynucleotide sequences which encode such polypeptide sequence identities.
  • human sphingosine kinase B or “human SKB” refers to the polyucleotide or polypeptide of an isoform of human sphingosine kinase as illustrated by the sequences of Figure 5 and 6, respectively, and to the polypeptide sequences which preferably have at least 85% sequence identity with each other and Figure 6, and more preferably at least 90% sequence identity with each other and Figure 6, and most preferably at least 95% sequence identity with each other and Figure 6 or polynucleotide sequences which encode such polypeptide sequence identities.
  • human sphingosine kinase C or “human SKC” refers to the polynucleotide or polypeptide of an isoform of human sphingosine kinase as illustrated by the sequences of Figure 8 and 9, respectively, and by the polypeptide sequences which preferably have at least 85% sequence identity with each other and Figure 9, and more preferably at least 90% sequence identity with each other and Figure 9, and most preferably at least 95% sequence identity with each other and Figure 9 or polynucleotide sequences which encode such polypeptide sequence identities.
  • “Inhibitor” is any substance which retards or prevents a biochemical, cellular or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids and do not result in a change in biological activity of the polypeptide. The variation allowed may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the human sphingosine kinase sequence using recombinant DNA techniques.
  • isolated means separated from nucleotide sequences s that encode other proteins or from other peptides.
  • a polypeptide or polynucleotide naturally present in a living organism is not “isolated” but when separated from the coexisting nucleotides/peptides it is “isolated”.
  • Nucleotide sequences as used herein are oligonucleotides, polynucleotides, and fragments or portions thereof, and are DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or complement or antisense strands.
  • oligonucleotide is a stretch of nucleotide residues, which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. It may be functionally equivalent to and the same length as (or considerably shorter than) a "fragment", "portion”, or “segment” of a polypeptide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino acids, typically at least about 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • purified refers to amino acid sequences that are removed from their natural environment, and are isolated or separated, and are at least 60% free, preferably at least 75 % free, and most preferably at least 90% free from other components with which they are naturally associated.
  • a "portion” or “fragment” of a nucleotide or nucleic acid sequence comprises all or any part of the sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • a portion or fragment can be used as a probe.
  • 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 spingosine kinase is present in a cell type, tissue, or organ.
  • Probes may be derived from naturally occurring, recombinant, or chemically synthesized single - or double - stranded nucleic acids or be chemically synthesized. They are useful in detecting the presence of identical or similar sequences.
  • Reporter molecules are those radionuclides , enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with, establish the presence of, and may allow quantification of a particular nucleotide or amino acid sequence.
  • a “signal 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.
  • substitutions are conservative in nature when they 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.
  • Standard 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.
  • Stringency conditions is used herein to mean conditions that allow for hybridization of substantially related nucleic acid sequences. . Such hybridization conditions are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989. Generally, stringency occurs within a range from about 5 °C below the melting temperature of the probe to about 20 °C - 25 °C below the melting temperature. As understood by ordinary skilled persons in the art, the stringency conditions may be altered in order to identify or detect identical or related nucleotide sequences.
  • Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.) and the concentration of the salts and other componenets (e.g. the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency.
  • Sequence Identity is known in the art, and is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences, particularly, as determined by the match between strings of such sequences.
  • Sequence identity can be readily calculated by known methods (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).
  • Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al, J. Molec. Biol. 215: 403-410 (1990)).
  • the BLASTX program is publicly available from NCBI (blast@ncbi.nlm.nih.gov) and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et ⁇ /., J Mol. Bio.
  • “Variants” are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively, but retain essential properties of the reference, preferably, in the case of polypeptides the variant retains the biological activity of the naturally occurring polypeptide .
  • a typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequences of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, insertions and deletions in the polypeptide encoded by the reference sequences, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and ,in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions and deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurrring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • the invention relates to novel polypeptides and polynucleotides for human sphingosine kinase as described in greater detail below.
  • the invention particularly relates to the three sphingosine kinase homologues: human SKA, human SKB and human SKC. More particularly, human SKA, SKB and SKC having the nucleotide sequences as set out in Figures 2, 5 and 8 for SKA, SKB and SKC, respectively, and variants thereof are provided for herein.
  • polypeptides of the invention include the polypeptides comprising the sequences as set out in Figures 3, 6 and 9 as well as variants of these polypeptides, particularly variants which retain the biological activity of the naturally occurring sphingosine kinase.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full length polypeptides of the invention.
  • polynucleotides comprising sequences encoding human SKA, SKB and SKC (or their complement) and variants thereof have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of SKA, SKB and SKC, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding SKA, SKB and SKC disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • nucleotide sequences which encode SKA, SKB and SKC, their derivatives or their variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring SKA, SKB and SKC, respectively, under stringent conditions, it may be advantageous to produce nucleotide sequences encoding SKA, SKB and SKC 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 human SKA, SKB and SKC may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques (Sambrook J et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY; or Ausubel FM et al (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York City).
  • Useful nucleotide sequences for joining to human SK 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.
  • Human SK specific hybridization probes are capable of hybridizing with naturally occurring nucleotide sequences encoding human SKA, SKB and SKC. Such probes may also be used for the detection of similar sequences and should preferably contain at least 60% nucleotide identity to SK sequence.
  • the hybridization probes of human SK may be derived from the nucleotide sequence presented in the Figures for the full length sequence for SKA, SKB and SKC, namely, Figures 1 , 4 and 7, respectively, or from genomic sequences including promoter, enhancers, introns or 3 '-untranslated regions of the native gene.
  • Hybridization probes may be labeled by a variety of reporter molecules using techniques well known in the art.
  • the hybridization probes incorporate at least 15 nucleotides, and preferably at least 25 nucleotides, of the SK protein.
  • Suitable hybridization probes would include: consensus fragments, for example, those regions of the human SK isoforms that are identical, as particularly exemplified in Figure 10.
  • nucleic acid sequences for human SK will be effective hybridization probes for human SK nucleic acid. Accordingly, the invention relates to nucleic acid sequences that hybridize with such SK encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions will generally allow hybridization of sequence with at least about 70% sequence identity, more preferably at least about 80-85% sequence identity, even more preferably at least about 90% sequence identity, and most 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.
  • Nucleic acid molecules that will hybridize to human SK encoding nucleic acid under stringent conditions can be identified functionally, using methods outlined above, or by using for example the hybridization rules reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.
  • hybridization probes include: histochemical uses such as identifying tissues that express human SK; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of human SK; and detecting polymorphisms in the human SK.
  • RNA hybridization procedures are described in Maniatis et al. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989). PCR as described US Patent No's. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes the human SK sequences of the invention.
  • Such probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of human SK in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNA's or RNA's. Rules for designing PCR primers are now established, as reviewed by PCR Protocols, Cold Spring Harbor Press, 1991.
  • Degenerate 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 to human SK.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence. See, Froman et al., Proc. Natl. Acad. Sci. USA 85: 8998, 1988 and Loh et al., Science 243: 217, 1989.
  • 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.
  • PCR methods of 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. See, for example, PCR Protocols, Cold Spring Harbor Press, 1991.
  • 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.
  • the nucleotide sequence for human SK can be used in an assay to detect inflammation or disease associated with abnormal levels of SK expression.
  • the cDNA can be labeled by methods known in the art, added to a fluid, cell or tissue sample from a patient, and incubated under hybridizing conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains a reporter molecule. After the compatible fluid is rinsed off, the reporter molecule is quantitated and compared with a standard as previously defined.
  • a diagnostic test for aberrant expression of SK can accelerate diagnosis and proper treatment of abnormal conditions of SK activity.
  • New nucleotide sequences can be assigned to chromosomal subregions by physical mapping.
  • the mapping of new genes or nucleotide sequences provide useful landmarks for investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • a disease or syndrome such as ataxia telangiectasia (AT)
  • AT ataxia telangiectasia
  • any sequences mapping to that area may represent or reveal genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in gene sequence between normal and carrier or affected individuals.
  • Nucleotide sequences encoding human SK may be used to produce a purified oligo - or polypeptide using well known methods of recombinant DNA technology.
  • Goeddel (1990, Gene Expression Technology, Methods and Enzymology, Vol. 185, Academic Press, San Diego CA) is one among many publications which teach expression of an isolated nucleotide sequence.
  • 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.
  • Human SK produced by a recombinant cell may be secreted or may be contained intracellularly, depending on the particular genetic construction used. In general, it is more convenient to prepare recombinant proteins in secreted form. Purification steps vary with the production process and the particular protein produced. Often an oligopeptide can be produced from a chimeric nucleotide sequence.
  • fragments of human SK may be produced by direct peptide synthesis using solid-phase techniques (e.g. Stewart at al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco QA; Merrifield J (1963) J Am Chem. Soc. 85:2149-2154). Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Foster City, CA) in accordance with the instructions provided by the manufacturer. Additionally, a particular portion of human SK may be mutated during direct synthesis and combined with other parts of the peptide using chemical methods.
  • solid-phase techniques e.g. Stewart at al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco QA; Merrifield J (1963) J Am Chem. Soc. 85:2149-2154. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Foster City, CA) in
  • Human SK for antibody induction does not require biological activity: however, the protein must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. They should mimic a portion of the amino acid sequence of the protein and may contain the entire amino acid sequence.
  • An antigenic portion of human SK may be fused to another protein such as keyhole limpet hemocyanin, and the chimeric molecule used for antibody production.
  • Antibodies specific for human SK may be produced by inoculation of an appropriate animal with the polypeptide or an antigenic fragment.
  • An antibody is specific for human SK if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein.
  • Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous processes such as the production of synthetic antibodies, the screening of recombinant immunoglobulin libraries for specific- binding molecules (e.g. Orlandi R et al (1989) PNAS 86:3833-3837, or Huse WD et al (1989) Science 256:1275-1281 ) or the in vitro stimulation of lymphocyte populations.
  • Current technology (Winter G and Mistein C (1991) Nature 349:293-299) provides for a number of highly specific binding reagents based on the principles of antibody formation. These techniques may be adapted to produce molecules which specifically bind SK.
  • An additional embodiment of the subject invention is the use of human SK specific antibodies, inhibitors, ligands or their analogs as bioactive agents to treat inflammation or disease possibly including, but not limited to 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 kidney, lung, heart, lymphoid or tissues of the nervous system.
  • Bioactive compositions comprising agonists, antagonists, receptors or inhibitors of human SK may be administered in a suitable therapeutic dose determined by any of several methodologies including clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complexed with a variety of well established compounds or compositions which enhance stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems involving aberrant expression of the EDG-7 gene.
  • PCR was conducted under the conditions as set out below on the templates from the following sources:
  • Fibroblasts WI-38 (Origene Technologies Inc., Cat. No. DLH-102), Human Liver (Origene Technologies Inc., Cat. No. DLH-100), cultured human Jurkat T-cells (Origene Technologies Inc., Cat. No. DLH-115), HeLa cultured cells (Origene Technologies Inc., Cat. No. DLH-103), Human kidney proximal tubules (ATCC), HeLa cultured cells (Invitrogen, Cat. No. A550-26), Human Lung (Clonetech, Cat. No.7114-1), HeLa cultured cells (Clonetech, Cat. No. HL5013a), human small intestine (Clonetech, Cat. No.HLl 133a).
  • Each template was amplified with each pair of primers under the following condition of PCR amplification by using Expand TM PCR kit of Boehringer Mannheim (Catalogue no. 1681 -842).
  • Each reaction contained the following reagents:
  • a 200 bp (approximately) DNA fragment was amplified from all templates except Origene' s HeLa cDNA library and Clonetech' s small intestine cDNA library.
  • the cDNA library from cultured HeLa cells appeared to contain PSKA clones.
  • 10 000 (10K) clone pools from cDNA library form cultured HeLa cells (Invitrogen, Cat. No. A550-26): Approximately, ten thousand clones were grown on an agar plate, scraped and re-suspended in one ml of 2X YT + 20% glycerol. Overall, 610 pools (10K) were prepared. Equal proportions of twelve 10K pools were mixed to prepare 120K pools. In all, there were fifty one 120K pools. All pools are kept as frozen stocks at 80°C. For PCR screening, a small portion of frozen stock was resuspended in 100 ul of 2X YT + 20% glycerol and used as template.
  • the bacterial colonies were grown from the positive 10K pool #403 on the agar plate. Plugs containing 300 - 1000 bacterial colonies were lifted from the agar plate. The bacterial colonies were re-suspended into 500 ul of 2XYT + 20% glycerol. The bacterial re-suspensions were used as template to amplify with the following pair of primers.
  • Sub-pool #403-42 was used further to isolate PSKA clone.
  • the filters were washed 2 times in 2X SSPE and 0.1% SDS at room temperature for 30 minutes each, then 2 times in 2XSSPE and 0.1 % SDS at 50°C for 20 minutes each and finally two times in 0.1XSSPE and 0.1% SDS.
  • PSKA#403-1 The plasmid DNA was prepared using midi-plasmid preparation kit (Qiagen, catalogue no. 12245) to use for sequencing and transfections.
  • midi-plasmid preparation kit Qiagen, catalogue no. 12245
  • 10K bacterial pool #532 was plated on agar plates. 100-500 colonies were scraped in sub-pool and re-suspended in 100 ⁇ l of 2XYT + 20% glycerol. The bacterial re-suspensions were used as template for PCR screening.
  • the positive sub-pool of 100-500 bacterial colonies was plated on agar plates. 20- 50 colonies were scraped in sub-pool and re-suspended in 100 ⁇ l of 2XYT + 20% glycerol. The bacterial re-suspensions were used as template for PCR screening. 3. The positive sub-pool of 20-50 bacterial colonies was plated on agar plates. The individual bacterial colonies were scraped and re-suspended in 100 ⁇ l of 2XYT + 20% glycerol. The bacterial re-suspensions were used as template for PCR screening.
  • the PCR screening was done by using Expand TM PCR system from Boehringer Mannheim (Catalogue no. 1681-842) with the following pair of primers.
  • a DNA fragment of approximately 250bp was amplified from ten 120K bacterial pools.
  • 10K pools of the four positive 120K pools were amplified under the following condition of PCR amplification by using Expand TM PCR system from Boehringer Mannheim (Catalogue no. 1681-842) with the following pair of primers.
  • 10K bacterial pool #330 was plated on agar plates. 100-500 colonies were scraped in sub-pool and re-suspended in 100 ⁇ l of 2XYT + 20% glycerol. The bacterial re-suspensions were used as template for PCR screening. 5. The positive sub-pool of 100-500 bacterial colonies was plated on agar plates. 20- 50 colonies were scraped in sub-pool and re-suspended in 100 ⁇ l of 2XYT + 20% glycerol. The bacterial re-suspensions were used as template for PCR screening.
  • the bacterial re-suspensions were used as template for PCR screening.
  • PCR screening were done by using Expand TM PCR system from Boehringer Mannheim (Catalogue no. 1681-842) with the following pair of primers.
  • PSKC-F2 5' TTAACA TAG ACAAATACG ACG GCA TCG 3' PSKC-R1 5' ACACAT CCA TGG CCA GCGAGT CC 3'
  • Two colonies (330 - P1G3 - P1B8 - P2A9 and 330 - P1G3 - P4E10- P1B12) were found positive. They were given ID of pc3-PSKC#330-l and pc3-PSKC#330-2.
  • the plasmid DNA was prepared using mini-plasmid preparation kit (Qiagen, catalogue no. 12245) to use for sequencing and transfections.
  • sphingosine 1 -phosphate and sphingosine standards were visualized using a KMnO 4 stain (100 ml H 2 O: 4g KMnO 4 , 4g NaHCO 3 ). TLC plates were exposed to a phosphor screen overnight.
  • DNA/Plus/Lipofectamine mixture was added to each plate of 293-EBNA cells. The plates were left for 3 hr at 37°C in a 5% CO 2 incubator.
  • the phosphorylation assay was performed as outlined above, except 5 ⁇ l of Triton X-
  • Enzyme preparations of SKA and pcDNA3 were used in phosphorylation assays.
  • the protocol was the same as mentioned above in A.3-7 with a few exceptions: i) 5 ⁇ l of Triton X-100 was added to each reaction tube and ii) 33 P-ATP was not used, only "cold" ATP was used in each reaction (10 mM in 100 mM MgCl 2 ) .
  • SKA was shown to be involved in phosphorylating sphingosine whereas the tests did not exemplify phosphorylation by the cloned SKB and SKC genes.
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of SK are used either in vitro or in vivo to inhibit expression 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 morphology, etc.).
  • Expression of human SK is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into analogous expression hosts for example 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 Met and the subsequent 7 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.
  • the human SK cDNA is shuttled into other vectors known to be useful for expression of protein 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 bacteria 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; tip, 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 human SK are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • human SK can be expressibly cloned into the expression vector pcDNA3.
  • 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 catalog no. 18324-020) mediated gene transfer.
  • Human SK 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, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle WA).
  • the inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen) between the purification domain and the human SK sequence is useful to facilitate expression of human SK.
  • 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 micrograms 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 human SK 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. Analysis to select appropriate epitopes is described by Ausubel FM et al (supra).
  • 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-hydroxysuccinimide ester (MBS; Ausubel FM et al, supra). 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
  • MVS M-maleimidobenzoyl-N-hydroxysuccinimide ester
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine sewm 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 human SK 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 lg) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% BSA, washed and incubated with supematants from hybridomas. After washing the wells are incubated with labeled human SK at 1 mg/ml. Supematants with specific antibodies bind more labeled human SK than is detectable in the background.
  • clones producing specific antibodies are expanded and subjected to two cycles of cloning at limiting dilution.
  • Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from mouse ascetic fluid by affinity chromatography on Protein A.
  • Monoclonal antibodies with affinities of at least 10 8 M- 1 , preferably 10 9 to 10 10 or stronger, are typically made by standard procedures as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, New York City, both incorporated herein by reference.
  • Particular Human SK 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 human SK or downstream products of an active signaling cascade.
  • Diagnostic tests for human SK include methods utilizing antibody and a label to detect human SK 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. Patents teaching the use of such labels include US Patent No's.
  • recombinant immunoglobulins may be produced as shown in US Patent No.4,816,567, Incorporated herein by reference.
  • a variety of protocols for measuring soluble or membrane-bound human SK, 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 human SK is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, DE et al (1983, J Exp. Med. 158:121 If).
  • Native or recombinant human SK is purified by immunoaffinity chromatography using antibodies specific for human SK.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway NJ).
  • 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).
  • 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 instmctions.
  • Such immunoaffinity columns are utilized in the purification of human SK by preparing a fraction from cells containing human SK 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 human SK containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • This invention is particularly useful for screening therapeutic compounds by using human SK or binding fragments thereof in any of a variety of dmg screening techniques.
  • human SK activity can be measured using any of a variety of appropriate functional assays in which activation of the kinase results in an observable change in the level of a particular produc.
  • the present invention provides methods of screening for drugs or any other agents which are affect by humand SK.
  • the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of dmg screening utilizes eukaryotic or prokaryotic host cells (or membrane preparations therefrom) which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Dmgs are screened against such transformed cells. One measures, for example, the formation of a phosphorylated product and compares that with a control.
  • the goal of rational dmg design is to produce stmctural analogs of biologically active lipids of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors.
  • 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 stmctural information is used to design efficient inhibitors.
  • Useful examples of rational dmg design includes molecules which have improved activity or stability as shown by Braxton S and Wells JA (1992, Biochemistry 31 :7796-7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda SB et al (1993 J Biochem 113:742-46), incorporated herein by reference.
  • 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, half-life and antigenicity/immunogenicity. These and other characteristics aid in defining an effective carrier.
  • 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 dmgs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.
  • Normal dosage amounts vary from 0.1 to 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; see US Patent 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 humans SK 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.

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Abstract

L'invention concerne des polynucléotides nouvellement identifiés et isolés, les polypeptides de ces polynucléotides, de même que les utilisations de ceux-ci. Elle concerne notamment des polynucléotides et polypeptides nouvellement identifiés et isolés, faisant partie de la famille des sphingosines kinases. Trois polynucléotides et polypeptides isolés de trois homologues SK humains sont décrits: SKA, SKB et SKC.
PCT/CA2000/000223 1999-03-02 2000-03-02 Homologues de sphingosine kinase humaine clones WO2000052173A2 (fr)

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AU28999/00A AU2899900A (en) 1999-03-02 2000-03-02 Cloned human sphingosine kinase homologues

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WO2001060990A2 (fr) * 2000-02-14 2001-08-23 Curagen Corporation Nouvelles kinases de sphingosine
WO2001096575A1 (fr) * 2000-06-14 2001-12-20 Sankyo Company, Limited Ceramide kinase et adn la codant
EP1192247A1 (fr) * 1999-05-13 2002-04-03 Johnson & Johnson Research Pty Limited Enzyme sphingosine kinase
WO2002028906A2 (fr) * 2000-10-06 2002-04-11 Bayer Aktiengesellschaft Regulation d'une proteine humaine de type sphingosine kinase
EP1268509A1 (fr) * 2000-04-03 2003-01-02 Sankyo Company, Limited Isoformes de la sphingosine kinase mammalienne de type 2, clonage, expression et methodes associees
WO2003031628A1 (fr) * 2001-09-28 2003-04-17 Hokkaido Technology Licensing Office Co., Ltd. Polypeptides secretoires a activite sphingosine kinase et genes de sphingosine kinase codant pour ces polypeptides
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US9274130B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Prevention and treatment of pain using antibodies to lysophosphatidic acid
EP3791869A4 (fr) * 2018-03-21 2022-03-09 Kyungpook National University Industry-Academic Cooperation Foundation Composition pharmaceutique de prévention ou de traitement de maladies neurodégénératives comprenant l'agent d'acétylation cox2 comme ingrédient actif

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Cited By (33)

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US6730480B1 (en) 1999-05-13 2004-05-04 Johnson & Johnson Pharmaceutical Research And Development Llc Sphingosine kinase enzyme
EP1192247A1 (fr) * 1999-05-13 2002-04-03 Johnson & Johnson Research Pty Limited Enzyme sphingosine kinase
US7112427B2 (en) 1999-05-13 2006-09-26 Johnson & Johnson Pharmaceutical Research And Development Llc Sphingosine kinase enzyme
EP1192247A4 (fr) * 1999-05-13 2003-05-28 Johnson & Johnson Res Pty Ltd Enzyme sphingosine kinase
WO2001031029A3 (fr) * 1999-10-28 2002-02-28 Warner Lambert Co Gene de la sphingosine kinase humaine
WO2001031029A2 (fr) * 1999-10-28 2001-05-03 Warner-Lambert Company Gene de la sphingosine kinase humaine
WO2001060990A2 (fr) * 2000-02-14 2001-08-23 Curagen Corporation Nouvelles kinases de sphingosine
WO2001060990A3 (fr) * 2000-02-14 2002-03-21 Curagen Corp Nouvelles kinases de sphingosine
EP2110379A3 (fr) * 2000-04-03 2010-07-28 Sankyo Company Limited Isoformes de la sphingosine kinase mammalienne de type 2, et utilisation
EP1268509A1 (fr) * 2000-04-03 2003-01-02 Sankyo Company, Limited Isoformes de la sphingosine kinase mammalienne de type 2, clonage, expression et methodes associees
EP1268509A4 (fr) * 2000-04-03 2004-09-22 Sankyo Co Isoformes de la sphingosine kinase mammalienne de type 2, clonage, expression et methodes associees
WO2001096575A1 (fr) * 2000-06-14 2001-12-20 Sankyo Company, Limited Ceramide kinase et adn la codant
US7037700B2 (en) 2000-10-06 2006-05-02 Bayer Aktiengesellschaft Regulation of human ceramide kinase
WO2002028906A3 (fr) * 2000-10-06 2002-11-14 Bayer Ag Regulation d'une proteine humaine de type sphingosine kinase
WO2002028906A2 (fr) * 2000-10-06 2002-04-11 Bayer Aktiengesellschaft Regulation d'une proteine humaine de type sphingosine kinase
US7901682B2 (en) 2000-12-22 2011-03-08 Lpath, Inc. Compositions and methods for the treatment and prevention of cancer, angiogenesis, and inflammation
US6858383B2 (en) 2000-12-22 2005-02-22 Medlyte, Inc. Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
US6881546B2 (en) 2000-12-22 2005-04-19 Medlyte, Inc., Sdsu Heart Institute Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
US7169390B2 (en) 2000-12-22 2007-01-30 Lpath Therapeutics, Inc. Compositions and methods for the treatment and prevention of cancer, angiogenesis, and inflammation
US7040185B2 (en) 2000-12-23 2006-05-09 Robert Bosch Gmbh Device for driving an output mechanism
WO2003031628A1 (fr) * 2001-09-28 2003-04-17 Hokkaido Technology Licensing Office Co., Ltd. Polypeptides secretoires a activite sphingosine kinase et genes de sphingosine kinase codant pour ces polypeptides
WO2003031627A1 (fr) * 2001-09-28 2003-04-17 Hokkaido Technology Licensing Office Co., Ltd. Polypeptides a origine plaquettaire a activite sphingosine kinase et genes de sphingosine kinase codant pour ces polypeptides
US9217749B2 (en) 2006-05-31 2015-12-22 Lpath, Inc. Immune-derived moieties reactive against lysophosphatidic acid
US9274129B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Methods and reagents for detecting bioactive lipids
US9274130B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Prevention and treatment of pain using antibodies to lysophosphatidic acid
US7956173B2 (en) 2006-10-27 2011-06-07 Lpath, Inc. Nucleic acids coding for humanized antibodies for binding sphingosine-1-phosphate
US8025877B2 (en) 2006-10-27 2011-09-27 Lpath, Inc. Methods of using humanized antibodies and compositions for binding sphingosine-1-phosphate
US8026342B2 (en) 2006-10-27 2011-09-27 Lpath, Inc. Compositions and methods for binding sphingosine-1-phosphate
US8067549B2 (en) 2006-10-27 2011-11-29 Lpath, Inc. Humanized antibodies and compositions for binding sphingosine-1-phosphate
US7829674B2 (en) 2006-10-27 2010-11-09 Lpath, Inc. Compositions and methods for binding sphingosine-1-phosphate
US8871202B2 (en) 2008-10-24 2014-10-28 Lpath, Inc. Prevention and treatment of pain using antibodies to sphingosine-1-phosphate
EP3791869A4 (fr) * 2018-03-21 2022-03-09 Kyungpook National University Industry-Academic Cooperation Foundation Composition pharmaceutique de prévention ou de traitement de maladies neurodégénératives comprenant l'agent d'acétylation cox2 comme ingrédient actif
US11899025B2 (en) 2018-03-21 2024-02-13 Kyungpook National University Industry-Academic Cooperation Foundation Pharmaceutical composition for preventing or treating neurodegenerative diseases comprising COX2 acetylating agent as active ingredient

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