WO2001057195A1 - La 25204, nouvelle deshydrogenase/reductase humaine a chaine courte - Google Patents

La 25204, nouvelle deshydrogenase/reductase humaine a chaine courte Download PDF

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WO2001057195A1
WO2001057195A1 PCT/US2001/003335 US0103335W WO0157195A1 WO 2001057195 A1 WO2001057195 A1 WO 2001057195A1 US 0103335 W US0103335 W US 0103335W WO 0157195 A1 WO0157195 A1 WO 0157195A1
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sdr
polypeptide
nucleic acid
seq
amino acid
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PCT/US2001/003335
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English (en)
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Rachel E. Meyers
Mark Williamson
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Millennium Pharmaceuticals, Inc.
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Priority to AU2001231281A priority Critical patent/AU2001231281A1/en
Publication of WO2001057195A1 publication Critical patent/WO2001057195A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)

Definitions

  • the present invention relates to newly identified human short chain dehydrogenase (SDR) belonging to the superfamily of mammalian dehydrogenases/reductases.
  • SDR human short chain dehydrogenase
  • the invention also relates to polynucleotides encoding the SDR.
  • the invention further relates to methods using the SDR polypeptides and polynucleotides as a target for diagnosis and treatment in SDR-mediated or -related disorders.
  • the invention further relates to drug-screening methods using the SDR polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment.
  • the invention further encompasses agonists and antagonists based on the SDR polypeptides and polynucleotides.
  • the invention further relates to procedures for producing the SDR polypeptides and polynucleotides.
  • Short-chain dehydrogenases/reductases constitute a large protein family. Members of the SDR family appear to have similar activities though they work via different mechanisms and structures.
  • the SDR superfamily comprises isomerases, lyases and oxidoreductases. The enzymes of this family cover a wide range of substrate specificities including steroids, alcohols, and aromatic compounds, however, most family members are known to be NAD + - or NADP + - dependent oxidoreductases. In the combined SDR superfamily, only a single tyrosine residue is strictly conserved and ascribed a critical enzymatic function. The extended SDR family therefore represents a remarkable spread of enzyme reactions covering EC numbers from three different enzyme classes.
  • SDR proteins function as dimers or tetramers and possess at least two domains: the first domain comprising the coenzyme binding site, and the second domain comprising the substrate binding site. This latter domain determines the substrate specificity and contains the amino acids involved in catalysis.
  • 2,4 dienoyl-CoA reductases are a subfamily of the short-chain dehydrogenase/reductase family and are auxiliary enzymes of fatty acid ⁇ -oxidation. Fatty acids represent a major source of energy and most tissues are able to degrade fatty acids to CO and H 0. In vertebrate cells degradation of fatty acids occurs in peroxisomes and in mitochondria.
  • Beta oxidation occurs via a series of sequential steps including dehydrogenation, hydration, a second dehydrogenation, followed by thiolytic cleavage. See, for example, Wanders et al. (1999) J. Inker. Metab. Dis. I . 442-487.
  • the beta oxidation reaction of mono- and polyunsaturated fatty acids requires auxiliary enzyme activities to remove the double bonds of the carbon chain.
  • One such auxiliary enzyme is 2,4- dienoyl-CoA reductase.
  • a peroxisome localized rat homologue of a 2,4-dienoyl-CoA reductase (Accession No. AF044574) having 42% identity to the yeast 2,4-dienoyl-CoA reductase has been identified.
  • the reductase contains a C-terminal tripeptide AKL sequence.
  • the AKL tripeptide is a known peroxisomal-matrix targeting signal (Gould et al. (1989) J Cell Biol. 108: 1657-1664) that targets the rat 2,4-dienoyl-CoA reductase to the peroxisome. Furthermore, when expressed in E.
  • a mouse peroxisomal 2,4-dienoyl-CoA reductase (known as PDCR) has also been identified (Accession No. AF155575) (Geibrecht et al. (1999) Journal of Biological Chem. 274:14814-15810, herein incorporated by reference).
  • This reductase also contains an AKL tripeptide C-terminal repeat.
  • the observed substrate preference for the mouse reductase was to 2 trans,4 trans-decadienoyl-CoA, however PDCR was also active on fatty acids of chain lengths of 22 carbons and multiple unsaturations.
  • 2,4-dienoyl-CoA reductase activity is required for complete oxidation of all 2,4-dienoyl-CoAs, as well as a portion of 2,5-dienoyl-CoAs
  • the 2,4,-dienoyl-CoA reductase represents a candidate disease gene for disorders involving impaired polyunsaturated fatty acid metabolism (Kunau et al. (1978) Eur. J. Biochem 91:533- 544). For instance, low concentrations of docosahexaenoic acid in the tissue of Zell- weger syndrome patients is regarded as reflecting impaired conversion of unsaturated fatty acids.
  • Enzymes involved in fatty acid synthesis are also implicated in tumor progression. Recent discoveries have indicated that many human cancer cells, including carcinoma of the colon, prostate, ovary, and breast express high levels of enzymes involved in fatty acid synthesis (for example, fatty acid synthase). High levels of fatty acid synthesis have been associated with clinically aggressive tumor behavior, and the fatty acid synthase inhibitor C75 has been shown to have significant anti-tumor activity.
  • short chain dehy drogenase/reductases are a major target for drug action and development. Therefore, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown SDRs.
  • the present invention advances the state of the art by providing a previously unidentified human SDR.
  • a specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of the novel SDR.
  • a further specific object of the invention is to provide compounds that modulate expression of the SDR for treatment and diagnosis of SDR -related disorders.
  • the invention is thus based on the identification of novel human alcohol dehydrogenases.
  • the amino acid sequence for SDR 25204 is shown in SEQ ID NO:2.
  • the nucleotide sequence for SDR 25204 is shown in SEQ ID NO: 1.
  • the coding sequence for SDR 25204 is shown in SEQ ID NO:3.
  • the invention provides isolated SDR polypeptides, including a polypeptide having the amino acid sequence shown in SEQ ID NO:2, or the amino acid sequence encoded by the cDNA deposited as ATCC No. on ("the deposited cDNA").
  • the invention also provides isolated SDR nucleic acid molecules having the sequences shown in SEQ ID NO:l, SEQ ID NO:3, or in the deposited cDNA.
  • the invention also provides variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequences shown in SEQ ID NO:2, or encoded by the deposited cDNA.
  • the invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequences shown in SEQ ID NO: 2 or in the deposited cDNA.
  • the invention also provides fragments of the polypeptides shown in SEQ ID NO:2, and nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:3, as well as substantially homologous fragments of the polypeptides or nucleic acids.
  • the invention further provides nucleic acid constructs comprising the nucleic acid molecules described herein.
  • the nucleic acid molecules of the invention are operatively linked to a regulatory sequence.
  • the invention also provides vectors and host cells for expressing the SDR nucleic acid molecules and polypeptides, and particularly recombinant vectors and host cells.
  • the invention also provides methods of making the vectors and host cells and methods for using them to produce the SDR nucleic acid molecules and polypeptides.
  • the invention also provides antibodies or antigen-binding fragments thereof that selectively bind the SDR polypeptides and fragments.
  • the invention also provides methods of screening for compounds that modulate expression or activity of the SDR polypeptides or nucleic acid (RNA or DNA).
  • the invention also provides a process for modulating SDR polypeptide or nucleic acid expression or activity, especially using the screened compounds. Modulation may be used to treat conditions related to aberrant activity or expression of the SDR polypeptides or nucleic acids.
  • the invention also provides assays for determining the activity of or the presence or absence of the SDR polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
  • the invention also provides assays for determining the presence of a mutation in the polypeptides or nucleic acid molecules, including for disease diagnosis.
  • the invention provides a computer readable means containing the nucleotide and/or amino acid sequences of the nucleic acids and polypeptides of the invention, respectively.
  • Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO:2) of the novel 25204 SDR.
  • the coding sequence of 25204 (nucleotides 103-981 of SEQ ID NO:l) is set forth in SEQ ID NO:3.
  • Figure 1 shows an analysis of the 25204 SDR amino acid sequence: ⁇ turn and coil regions; hydrophilicity; amphipathic regions; flexible regions; antigenic index; and surface probability plot.
  • Figure 3 shows a hydrophobicity plot of the 25204 SDR amino acid sequence (SEQ ID NO:2). Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line.
  • the cysteine residues (cys) and N glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace.
  • the numbers corresponding to the amino acid sequence (shown in SEQ ID NO:2) of human 25204 are indicated .
  • Polypeptides of the invention include fragments which include: all or a part of a hydrophobic sequence (a sequence above the dashed line); or all or part of a hydrophilic fragment (a sequence below the dashed line).
  • fragments include a cysteine residue or as N-glycosylation site. Also shown is the predicted transmembrane segment from about amino acid 30 to about amino acid 48. In addition, a graphical representation of the functional domain for the short chain dehydrogenase and the short chain dehydrogenase C-terminal domain is also shown.
  • Figure 4 shows an analysis of the 25204 SDR open reading frame (SEQ ID NO:2) for amino acids corresponding to specific functional sites.
  • Putative N- glycosylation sites are found from about amino acid 143 to about amino acid 146, from about amino acid 162 to about 165, and from about 241 to about 244.
  • a putative glycosaminoglycan attachment site is found from about amino acid 38 to about 41.
  • Putative protein kinase C phosphorylation sites are found from about amino acid 76 to about amino acid 78, from about amino acid 180 to about amino acid 182, from about amino acid 228 to about amino acid 230, and from about amino acid 289 to about amino acid 291.
  • Putative casein kinase II phosphorylation sites are found from about amino acid 132 to about amino acid 135, and from about amino 212 to about amino acid 215.
  • Putative N-myristoylation sites are found from about amino acid 35 to about amino acid 40, from about amino acid 158 to about amino acid 163, from about amino acid 179 to about amino acid 184, from about amino acid 221 to about amino acid 226, and from about amino acid 270 to about amino acid 275.
  • a putative amidation site is found from about amino acid 76 to about amino acid 79.
  • a microbodies C-terminal targeting signal is found from about amino acid 290 to about amino acid 292.
  • Figure 5 depicts an alignment of the short chain dehydrogenase domain of human 25204 with a consensus amino acid sequence derived from a hidden Markov model.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:4), while the lower amino acid sequence corresponds to amino acids 29 to 267 of SEQ ID NO:2.
  • Figure 6 depicts the expression of 25204 in the following human tissues: normal spinal chord (column 1 ), normal brain cortex (column 2), normal brain hypothalmus (column 3), astrocytes (column 4), glioblastoma (column 5), normal breast (column 6), tissue from invasive ductal carcinoma of the breast (column 7), normal ovary (column 8), ovary tumor tissue (column 9), pancreas (column 10), normal prostate (column 11), prostate tumor tissue (column 12), normal colon (column 13), colon tumor tissue (column 14), colon tissue from a subject with IBD (column 15), normal kidney (column 16), normal liver (column 17), fibrotic liver (column 18), normal fetal liver (column 19), normal lung (column 20), lung tumor (column 21), lung tissue from a subject with chronic obstructive pulmonary disease (column 22), normal spleen (
  • Figure 7 shows the expression of 25204 in the following human tissues: normal breast (columns 1-3), breast tumor (columns 4-9), normal ovary (columns 10- 12), ovary tumor (columns 13-20), normal lung (columns 21-24), and lung tumor (columns 25-32). Note that 25204 expression was elevated in breast and lung carcinomas. Expression was determined by quantitative RT-PCR as described in the legend for Figure 6.
  • Figure 8 shows the expression of 25204 in the following human tissues: normal colon (columns 1-3), colon tumor (columns 4-1 1), metastatic liver tumor (columns 12-15), normal liver (columns 16-17), normal brain (18-21), astrocytes
  • column 22 brain tumor (columns 23-27), arresting human microvascular endothelial cells (column 28), proliferating human microvascular endothelial cells (column 29), placenta (column 30), fetal adrenal gland (columns 31-32), and fetal liver (columns 33-34). Expression was determined by quantitative RT-PCR as described in the legend for Figure 6.
  • Figure 9 shows the expression of 25204 in the following human clinical samples: normal colon (columns 1-3), colon tumor (columns 4-11), metastatic liver tumor (columns 12-15), and normal liver (columns 16 and 17). Note that 25204 expression was elevated in 5 of the 8 colon tumor samples. Expression was determined by quantitative RT-PCR as described in the legend for Figure 6.
  • FIG. 10 shows the expression of 25204 in human cell lines.
  • Breast cancer cell lines shown are MCF-7 (column 1), ZR75 (column 2), T47D (column 3), MDA231 (column 4), and MDA435 (column 5).
  • Colon cancer cell lines shown are DLD-1 (column 6), SW 480 (column 7), SW 620 (column 8), HCT116 (column 9), HT29 (column 10), and Colo 205 (column 11).
  • Lung cancer cell lines shown are NCIH 125 (column 12), NICH 67 (column 13), NCIH 322 (column 14), NCIH 460 (column 15), and A549 (column 16).
  • FIG. 11 shows the expression of 25204 in the following tissues: normal colon (samples shown are PIT 337, CHT 410, CHT 425, CHT 371, PIT 281, and NDR211 ; columns 1-6, respectively), colonic adenoma (samples shown are CHT 122 and CHT 887; columns 7 and 8, respectively), colonic adenocarcinoma B (samples shown are CHT 414, CHT 841, CHT 890, CHT 910, CHT 807, CHT 382, and CHT 377; columns 9-15, respectively), colonic adenocarcinoma C (samples shown are
  • the invention is based on the identification of novel human short chain dehydrogenase/reductase.
  • an expressed sequence tag (EST) was selected based on homology to the SDR sequence. This EST was used to design primers based on sequences that it contains and used to identify cDNAS from human primary osteoblast cDNA libraries. Positive clones were sequenced and the overlapping fragments were assembled. Analysis of each of the assembled sequences revealed that the cloned cDNA molecules encoded a SDR.
  • the invention thus relates to a novel SDR having the deduced amino acid sequence shown in Figure 1, or the amino acid sequences shown in SEQ ID NO:2, or the amino acid sequence encoded by the deposited cDNA as ATCC No. .
  • the deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.
  • the deposit is provided as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the deposited sequence, as well as the polypeptide encoded by the sequence, is incorporated herein by reference and controls in the event of any conflict, such as a sequencing error, with description in this application.
  • SDR polypeptide or “SDR protein” refers to the polypeptide in SEQ ID NO:2, or the polypeptide encoded by the deposited cDNA.
  • SDR protein or “SDR polypeptide”, however, further includes the numerous variants described herein, as well as fragments derived from the full-length SDR and variants.
  • the present invention thus provides isolated or purified polypeptides of the 25204 SDR and variants and fragments thereof.
  • a C-terminal motif AKL and an NADPH nucleotide binding domain in the N-terminal region have been identified in the putative rat 2,4, peroxisomal 2,4-dienoyl- CoA reductase from Rattus Norvegicus. Both of these features are also found in the 25204 SDR.
  • the NADPH binding site of 25204 SDR is from about amino acid 193 to about amino acid 228.
  • short chain dehydrogenase domain includes an amino acid sequence of about 80-300 amino acid residues in length and having a bit score for the alignment of the sequence to the short chain dehydrogenase domain (HMM) of at least 8.
  • a short chain dehydrogenase domain includes at least about 110-250 amino acids, more preferably about 160-220 amino acid residues, or about 185 amino acids and has a bit score for the alignment of the sequence to the short chain dehydrogenase domain (HMM) of at least 16 or greater.
  • the short chain dehydrogenase domain (HMM) has been assigned the PF AM Accession PFOO 106 (http;//pfam.wustl.edu/).
  • 25204 polypeptide or protein has a short chain dehydrogenase domain or a region which includes at least about 100-250 more preferably about 130-200 or 160-200 amino acid residues and has at least about 60%, 70%, 80%o, 90%, 95%o, 99%, or 100% sequence identity with a short change dehydrogenase domain," e.g., the short chain dehydrogenase domain of human 25204 (e.g., amino acid residues 29-267 of SEQ ID NO:2).
  • a short change dehydrogenase domain e.g., the short chain dehydrogenase domain of human 25204 (e.g., amino acid residues 29-267 of SEQ ID NO:2).
  • the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search).
  • HMMs e.g., the Pfam database, release 2.1
  • the default parameters http://www.sanger.ac.uk/Software/Pfam/HMM_search.
  • the hmmsf program which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit.
  • the threshold score for determining a hit can be lowered (e.g., to 8 bits).
  • a description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355- 4358; Krogh et al. (1994) J. Mol. Biol. 235: 1501-1531 ; and Stultz et al. (1993) Protein Sci.
  • a 25204 protein includes at least one transmembrane domain.
  • transmembrane domain includes an amino acid sequence of about 15 amino acid residues in length that spans a phospholipid membrane. More preferably, a transmembrane domain includes about at least 18, 20, or 22 amino acid residues and spans a phospholipid membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an ⁇ -helical structure.
  • At least 50%>, 60%, 70%, 80%, 90%>, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans.
  • a 25204 polypeptide or protein has at least one transmembrane domain or a region which includes at least 18, 20, or 22 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity with a "transmembrane domain,” e.g., at least one transmembrane domain of human 25204 (e.g., amino acid residues 30-48 of SEQ ID NO:2).
  • a 25204 protein includes at least one "non- transmembrane domain.”
  • non-transmembrane domains are domains that reside outside of the membrane. When referring to plasma membranes, non-transmembrane domains include extracellular domains (i.e., outside of the cell) and intracellular domains (i.e., within the cell).
  • non-transmembrane domains include those domains of the protein that reside in the cytosol (i.e., the cytoplasm), the lumen of the organelle, or the matrix or the intermembrane space (the latter two relate specifically to mitochondria organelles).
  • the C-terminal amino acid residue of a non- transmembrane domain is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally occurring 25204, or 25204-like protein.
  • a 25204 polypeptide or protein has a "non- transmembrane domain" or a region which includes at least about 1-400, preferably about 100-300, more preferably about 210-275, and even more preferably about 235- 255 amino acid residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%) sequence identity with a "non-transmembrane domain", e.g., a non- transmembrane domain of human 25204 (e.g., residues 1-29 or 49-292 of SEQ ID NO:2).
  • a non-transmembrane domain is capable of catalytic activity, for example, the beta oxidation of unsaturated fatty acids.
  • N-terminal non-transmembrane domain located at the N-terminus of a 25204 protein or polypeptide is referred to herein as an "N-terminal non-transmembrane domain.”
  • an "N-terminal non-transmembrane domain” includes an amino acid sequence having about 1-350, preferably about 30-325, more preferably about 50-320, or even more preferably about 80-310 amino acid residues in length and is located outside the boundaries of a membrane.
  • an N-terminal non- transmembrane domain is located at about amino acid residues 1-29 of SEQ ID NO:2.
  • a non-transmembrane domain located at the C-terminus of a 25204 protein or polypeptide is referred to herein as a "C-terminal non-transmembrane domain.”
  • an "C-terminal non-transmembrane domain” includes an amino acid sequence having about 1-300, preferably about 15-290, preferably about 20-270, more preferably about 25-255 amino acid residues in length and is located outside the boundaries of a membrane.
  • an C-terminal non- transmembrane domain is located at about amino acid residues 49-292 of SEQ ID NO:2.
  • a polypeptide is said to be "isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non- recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell and still be considered “isolated” or “purified.”
  • the SDR polypeptides can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful and considered to contain an isolated form of the polypeptide.
  • the critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity.
  • the language "substantially free of cellular material” includes preparations of the SDR having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 10% other proteins, less than about
  • polypeptide When the polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%, or less than about 5%> of the volume of the protein preparation.
  • culture medium represents less than about 20%, less than about 10%, or less than about 5%> of the volume of the protein preparation.
  • a SDR polypeptide is also considered to be isolated when it is part of a membrane preparation or is purified and then reconstituted with membrane vesicles or liposomes.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of the SDR polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • the SDR polypeptides comprise the amino acid sequences shown in SEQ ID NO:2.
  • the invention also encompasses sequence variants.
  • Variants include a substantially homologous protein encoded by the same genetic locus in an organism, i.e., an allelic variant.
  • the 25204 SDR has been mapped to human chromosome 16 (16pl3) with flanking markers D16S521 (5.1 cR) and WI-7742 (16.6 cR).
  • Mutations near this locus include, but are not limited to, the following: hemoglobin H-related mental retardation; microphthalmia-cataract; PKDTS, polycystic kidney disease, infantile severe, with tuberous sclerosis; and PXE, pseudoxanthoma elasticum, autosomal recessive. Genes near this locus include ARHGDID, MPG, RGS11, AXLN1, NME4, KIAA0665, HBA1,
  • NY-CO-7 MPF, HAGH, GFER, RPL3, TPS2, BAIAP3, CLCN7, UBE2I, ANX2,
  • variants proteins or polypeptides having an amino acid sequence that is at least about 45%, 55%, 65%, preferably about 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2.
  • variants also include polypeptides encoded by the cDNA insert of the plasmid deposited with ATCC as Patent Accession Number , or polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO:l or SEQ ID NO:3, or a complement thereof, under stringent conditions.
  • a variant of an isolated polypeptide of the present invention differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues from the sequence shown in SEQ ID NO:2. If alignment is needed for this comparison the sequences should be aligned for maximum identity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences. Such variants generally retain the functional activity of the short chain dehydrogenase proteins of the invention.
  • Variants include polypeptides that differ in amino acid sequence due to natural allelic variation or mutagenesis. Variants also encompass proteins derived from other genetic loci in an organism, but having substantial homology to the SDR of SEQ ID NO:2.
  • Variants also include proteins substantially homologous to the SDR but derived from another organism, i.e., an ortholog. Variants also include proteins that are substantially homologous to the SDR that are produced by chemical synthesis. Variants also include proteins that are substantially homologous to the SDR that are produced by recombinant methods. In some embodiments, the variants retain the biological activity (e.g. the short chain dehydrogenase activity) of the reference polypeptide, SEQ ID NO:2. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.
  • two proteins are substantially homologous when the amino acid sequences are at least about 70-75%>, typically at least about 80-85%), and most typically at least about 90-95%> or more homologous.
  • a substantially homologous amino acid sequence, according to the present invention will be encoded by a nucleic acid sequence hybridizing to the nucleic acid sequence, or portion thereof, of the sequence shown in SEQ ID NO: 1 or SEQ ID NO:3 under stringent conditions as more fully described below.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%), more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%>, 80%, or 90%> of the length of the reference sequence (e.g., when aligning a second sequence to the amino acid sequences herein having 502 amino acid residues, at least 165, preferably at least 200, more preferably at least 250, even more preferably at least 300, and even more preferably at least 350, 400, 450, and 500 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989) CABIOS 4:11-17 which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215 :403 - 10.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402.
  • BLAST programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • the invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the SDR. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al, Science 2 7:1306-1310 (1990). TABLE 1. Conservative Amino Acid Substitutions.
  • a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.
  • variants of the SDR can have an altered developmental expression, temporal expression or tissue-preferred expression.
  • SDR variants can also have an altered interaction with cellular components, substrates, coenzymes, metal ions, or SDR subunits.
  • An altered interaction comprises either a higher or lower affinity of the SDR for the various cellular components, substrates, coenzymes, metal ions, or SDR subunits.
  • coenzyme is intended a molecule that is associated with the SDR and is essential for short chain dehydrogenase/reductase activity. Some coenzymes are covIERly linked to their enzyme while others are less tightly bound.
  • a covendingly linked coenzyme is referred to as a prosthetic group of the enzyme.
  • coenzyme is also intended the oxidized or reduced product of the coenzyme which is formed following the enzymatic reaction mediated by the SDR polypeptide.
  • a hydrogen ion is removed from the coenzyme NADPH to form the coenzyme product NADP + .
  • Coenzymes of SDRs include, but are not limited to, NAD + and NAD + analogues (Plapp et al. (1986) Biochemistry 25:5396-5402 and Yamazaki et al.
  • Coenzyme for the 2,4-dienoyl-CoA reductase family include, but are not limited to, NADP + and NADPH.
  • substrates include, but are not limited to, primary or secondary alcohols or hemiacetals, and cyclic secondary alcohols.
  • substrate is also intended the products resulting from the oxidation of the above mentioned substrates. Such products include, for example, various aldehydes and ketones.
  • substrates of the 2,4-dienoyl-CoA reductase subfamily include the 2-trans,4-cis dienoyl-CoA intermediates and the 2,5, dienoyl-CoA intermediates of the unsaturated fatty acids.
  • trans, trans-2,4,-decadienoyl-CoA trans, trans-2,4-hexadienoyl-CoA
  • Further substrates may also comprise complex, very long chain polyunsaturated fatty acids such as prostaglandins.
  • Variants of the SDR can also have an altered subunit interaction that affects the ability of the SDR to form an active multimeric structure.
  • Useful variants of SDR polypeptides further include alterations in catalytic activity.
  • the enzymatic reaction mediated by the SDR is reversible and comprises either the oxidation, i.e., removal of electrons, of the above mentioned substrates or their reduction, i.e., addition of electrons.
  • the catalytic reaction further comprises the oxidation or reduction of the coenzyme. Therefore, one embodiment involves a variant that results in binding of the substrate but results in slower oxidation/reduction or no oxidation/reduction of the substrate or coenzyme. Another variation can result in an increased rate of substrate or coenzyme oxidation/reduction. Other useful variation can include an altered binding affinity for a coenzyme or substrate.
  • an increased or decreased binding affinity of a coenzyme can alter the binding affinity of the SDR to the substrate and also alter the rate of substrate or coenzyme oxidation/reduction.
  • Another variation can prevent the SDR monomer from associating with other SDR subunits to form an active multimeric complex.
  • Another useful variation provides a fusion protein in which one or more domains or subregions are operationally fused to one or more domains or subregions from another SDR.
  • a domain or subregion can be introduced that alters the coenzyme or substrate specificities or the rate of the enzymatic reaction.
  • Fully functional variants typically contain only conservative variations or variations in non-critical residues or in non-critical regions.
  • Functional variants can also contain substitution of similar amino acids, which results in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • variants can be naturally-occurring or can be made by recombinant means or chemical synthesis to provide useful and novel characteristics for the SDR polypeptide. This includes preventing immunogenicity from pharmaceutical formulations by preventing protein aggregation.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al. (1985) Science 244:1081-1085). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as the binding affinity for the coenzyme or substrate or determining the catalytic constants for substrate or coenzyme oxidation/reduction. Sites that are critical for coenzyme and substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al (1992) J. Mol. Biol. 114:899-904; de Vos et al. (1992) Science 255:306-312).
  • the assays for SDR enzyme activity are well known in the art and can be found for example, in Zubay et al. (1988) Biochemistry 2 nd ed. Mac Millan Publishing Co, New York. These assays include, but are not limited to, determination of the Michaelis constants (K m ) or the dissociation constant for the SDR/substrate complex. Such analysis of enzyme activity may be performed spectrophotometrically by recording the change in absorbance of NADP + . The catalytic efficiency or k cat can also be measured. K cat is defined as the maximum number of molecules of substrate converted to product per active site per unit of time. The specificity constant (k cat /K m ) can also be used to measure the ability of the SDR to discriminate between competing substrates.
  • assays to measure 2,4-dienoyl-CoA reductase activity include, for example, metabolic studies using myocytes as described in Nada et al ((1995) Biochimica et Biophysica acta 1155: 244-250) which can be used to determine the rate of beta-oxidation of various fatty acid substrates. Metabolism of acylcarnitines fatty acids can be monitored using fast atom bombardment with tandem mass spectrometry to determine the fatty acid intermediate in a patient's blood and urine (Roe et al. (1990) J. Clin. Invest. 85:1103-1101).
  • This technique can be used to identify unusual acylcarnitine species in urine and blood and therefore can detect disorders involving both saturated and unsaturated fatty acid oxidation. Further assays can exploit the characteristic spectrum of 2,4, dienoyl-CoA.
  • the 2,4, dienoyl- Co A intermediate has major absorbance bands centered near 260 nm and 300 nm that are attributable to the coenzyme A and dienoyl thioester moieties, respectively.
  • Substantial homology can be to the entire nucleic acid or amino acid sequence or to fragments of these sequences.
  • the invention thus also includes polypeptide fragments of the SDR. Fragments can be derived from the amino acid sequence shown in SEQ ID NO:2. However, the invention also encompasses fragments of the variants of the SDR as described herein.
  • a fragment of the 25204 SDR can comprise fragments of 32, 35, 40, 45, 50, 55 or greater contiguous amino acids. Fragments can retain one or more of the biological activities of the protein, for example the ability to bind a coenzyme or substrate or the ability catalyze the oxidation/reduction of a substrate or coenzyme. Alternatively, fragments can be used as an immunogen to generate SDR antibodies. Biologically active fragments (peptides which are, for example, 5, 7, 10, 12, 15,
  • 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length can comprise, for example, a domain or motif, e.g., catalytic site, substrate binding site, coenzyme binding site, short-chain alcohol dehydrogenase signature, microbodies C-terminal targeting signals, and sites for glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, glycosaminoglycan attachment site, tyrosine kinase phosphorylation, and N-myristoylation. Further possible fragments include sites important for cellular and subcellular targeting.
  • a domain or motif e.g., catalytic site, substrate binding site, coenzyme binding site, short-chain alcohol dehydrogenase signature, microbodies C-terminal targeting signals, and sites for glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, glycosaminoglycan attachment
  • Such domains or motifs can be identified by means of routine computerized homology searching procedures. Fragments, for example, can extend in one or both directions from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100 amino acids. Further, fragments can include sub-fragments of the specific domains mentioned above, which sub- fragments retain the function of the domain from which they are derived.
  • the invention also provides fragments with immunogenic properties. These contain an epitope-bearing portion of the SDR or SDR variants. These epitope-bearing peptides are useful to raise antibodies that bind specifically to a SDR polypeptide or region or fragment. These peptides can contain at least 10, 12, at least 14, or between at least about 15 to about 30 amino acids.
  • Non-limiting examples of antigenic polypeptides that can be used to generate antibodies include but are not limited to peptides derived from an extracellular site. Regions having a high antigenicity index are shown in Figure 2 for the 25204 SDR.
  • intracellularly-made antibodies (“intrabodies”) are also encompassed, which would recognize intracellular peptide regions.
  • the epitope-bearing SDR polypeptides may be produced by any conventional means (Houghten, R.A. (1985) Proc. Natl Acad. Sci. USA 52:5131-5135). Simultaneous multiple peptide synthesis is described in U.S. Patent No. 4,631 ,211.
  • Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the SDR fragment and an additional region fused to the carboxyl terminus of the fragment.
  • the invention thus provides chimeric or fusion proteins. These comprise a SDR peptide sequence operatively linked to a heterologous peptide having an amino acid sequence not substantially homologous to the SDR. "Operatively linked” indicates that the SDR peptide and the heterologous peptide are fused in-frame.
  • the heterologous peptide can be fused to the N-terminus or C-terminus of the SDR or can be internally located.
  • the fusion protein does not affect SDR function per se.
  • the fusion protein can be a GST-fusion protein in which the SDR sequences are fused to the C-terminus of the GST sequences.
  • Other types of fusion proteins include, but are not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions.
  • Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant
  • the fusion protein contains a heterologous signal sequence at its N- terminus.
  • EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists (Bennett et al. (1995) J. Mol. Recog.
  • this invention also encompasses soluble fusion proteins containing an SDR polypeptide and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl , where fusion takes place at the hinge region.
  • the Fc part can be removed in a simple way by a cleavage sequence, which is also incorporated and can be cleaved with factor Xa.
  • a chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re- amplified to generate a chimeric gene sequence (see Ausubel et al. (1992) Current Protocols in Molecular Biology).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
  • a SDR -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SDR.
  • SDR polypeptide is encompassed by the present invention in which one or more of the SDR domains (or parts thereof) has been replaced by homologous domains (or parts thereof) from another SDR or a member of the 2,4-dienoyl-CoA reductase family. Accordingly, various permutations are possible.
  • the substrate binding domain, or subregion thereof can be replaced with the substrate binding domain or subregion from another SDR or a 2,4-dienoyl-CoA reductase family member.
  • the catalytic domain, or coenzyme binding domains or parts thereof can be replaced with the appropriate domain from another SDR or 2,4- dienoyl-CoA reductase family member.
  • chimeric SDRs can be formed in which one or more of the native domains or subregions has been replaced by another.
  • chimeric SDR proteins can be produced in which one or more functional sites is derived from a different SDR or a 2,4-dienoyl-CoA family member. It is understood however that sites could be derived from the SDR that occur in the mammalian genome but which have not yet been discovered or characterized. Such sites include but are not limited to the catalytic site, substrate binding site, coenzyme binding site, sites important for targeting to subcellular and cellular locations, sites functional for interaction with SDR subunits, protein kinase A phosphorylation sites, glycosylation sites, and other functional sites disclosed herein.
  • the isolated SDR can be purified from cells that naturally express it.
  • Tissues and cells that express high levels of the 25204 SDR include, but are not limited to, hippocampus, aortic endothelial cells, embryo, Jurkat T-cells, gall bladder, fetal liver, prostate, bone marrow, skin, B-cells, neuron, heart, and lung.
  • the 25204 SDR of the present invention can also be purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. In one embodiment, the protein is produced by recombinant DNA techniques.
  • a nucleic acid molecule encoding the SDR polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell.
  • the protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in polypeptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art.
  • polypeptides also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included
  • the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence for purification of the mature polypeptide or a pro-protein sequence.
  • Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine.
  • the modifications can be a function of how the protein is made. For recombinant polypeptides, for example, the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence.
  • a polypeptide when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications.
  • the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the SDR polypeptides are useful for producing antibodies specific for the SDR, regions, or fragments. Regions having a high antigenicity index score are shown in Figures 2 for the 25204 SDR.
  • the SDR polypeptides are useful for biological assays related to SDRs. Such assays involve any of the known SDR functions or activities or properties useful for diagnosis and treatment of SDR -related conditions.
  • Treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • Subject can refer to a mammal, e.g. a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g. a horse, cow, goat, or other domestic animal.
  • a therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • the SDR polypeptides are also useful in drug screening assays, in cell-based or cell-free systems.
  • Cell-based systems can be native, i.e., cells that normally express the SDR, such as a biopsy or expanded in cell culture. In one embodiment, however, cell- based assays involve recombinant host cells expressing the SDR.
  • Determining the ability of the test compound to interact with the SDR can also comprise determining the ability of the test compound to preferentially bind to the polypeptide as compared to the ability of a known binding molecule (e.g. a coenzyme or substrate) to bind to the polypeptide.
  • the polypeptides can be used to identify compounds that modulate SDR activity.
  • Such compounds can increase or decrease the affinity of the substrate or coenzyme for the SDR. Such compound can also increase or decrease the enzymatic activity of the SDR. Additionally, such compounds can also alter the interaction of SDR with a metal ion or alter the ability of the SDR polypeptide to form a multimeric structure.
  • Examples of compounds that modulate 2,4-dienoly-CoA reductase include, but are not limited to, iodacetic acid and N-ethylmaleimide (Mizugaki et al. (1982) J Biochem 91: 1453- 1456).
  • the SDR of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the SDR. These compounds can be further screened against a functional SDR to determine the effect of the compound on the SDR activity. Compounds can be identified that activate (agonist) or inactivate (antagonist) the SDR to a desired degree. Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the SDR polypeptides can be used to screen a compound for the ability to stimulate or inhibit interaction between the SDR protein and a target molecule that normally interacts with the SDR protein.
  • the target can be a coenzyme, metal ion, SDR substrate or another SDR subunit of the multimeric SDR enzyme.
  • the assay includes the steps of combining the SDR protein with a candidate compound under conditions that allow the SDR protein or fragment to interact with the target molecule, and to detect the formation of a complex between the SDR protein and the target or to detect the biochemical consequence of the interaction with the SDR and the target, such as the oxidation/reduction of the substrate or coenzyme. For example, the conversion of NADPH to NADP + or the conversion of a 2,4-dienoyl-CoA to a 3-enoyl-CoA.
  • Determining the ability of the SDR to bind to a target molecule can also be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA).
  • BiA Bimolecular Interaction Analysis
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries are limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 72:145).
  • Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 554:82-84; Houghten et al.
  • One candidate compound is a soluble full-length SDR or fragment that competes for substrate binding or cofactor binding, interferes with the SDR catalyzed reaction, or interferes with SDR subunit interactions.
  • Other candidate compounds include mutant SDR or appropriate fragments containing mutations that affect SDR function and thus compete for cofactor binding or substrate binding or interfere with the SDR catalyzed reaction or interferes with the SDR subunit interactions. Accordingly, a fragment that competes for substrate or coenzyme binding, for example with a higher affinity, or a fragment that binds substrate but does not catalyze its oxidation/reduction is encompassed by the invention.
  • the invention provides other end points to identify compounds that modulate (stimulate or inhibit) SDR activity.
  • the assays typically involve an assay of events that result from substrate or coenzyme oxidation/reduction that indicates SDR activity.
  • the expression of genes that are up- or down-regulated in response to the SDR enzyme can be assayed.
  • the regulatory region of such genes can be operably linked to a marker that is easily detectable, such as luciferase.
  • “Misexpression or aberrant expression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non- wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength
  • any of the biological or biochemical functions mediated by the SDR can be used as an endpoint assay. These include all of the biochemical or biological events described herein, in the references cited herein and incorporated by reference for assays and functions, and other SDR functions known to those of ordinary skill in the art.
  • end points can include an altered NADPH/NADP + or NADVNADH ratio.
  • ethanol oxidation results in an increased NADH/NAD + redox potential within the cytosol and mitochondria with subsequent alterations in several tissue metabolites.
  • Further endpoints will include an increase or decrease in oxidized/reduced substrate or coenzyme concentration. For example, an increase or decrease in the concentration of (poly)unsaturated fatty acids or their various intermediates.
  • Binding and/or activating compounds can also be screened by using chimeric SDR proteins in which one or more domains, sites, and the like, as disclosed herein, or parts thereof, can be replaced by their heterologous counterparts derived from other SDRs or of any other 2,4-dienoyl-CoA reductase family member.
  • a substrate binding region or coenzyme binding region can be used that interacts with a different substrate or coenzyme specificity and/or affinity than the native SDR. Accordingly, a different set of oxidized/reduced substrates or coenzymes is available as an end-point assay for activation.
  • a heterologous targeting sequence can replace the native targeting sequence.
  • sites that are responsible for developmental, temporal, or tissue specificity can be replaced by heterologous sites such that the SDR can be detected under conditions of specific developmental, temporal, or tissue-specific expression.
  • the SDR polypeptides are also useful in competition binding assays in methods designed to discover compounds that interact with the SDR.
  • a compound is exposed to a SDR polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide.
  • Soluble SDR polypeptide is also added to the mixture. If the test compound interacts with the soluble SDR polypeptide, it decreases the amount of complex formed or activity from the SDR target.
  • This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the SDR.
  • the soluble polypeptide that competes with the target SDR region is designed to contain peptide sequences corresponding to the region of interest.
  • Another type of competition-binding assay can be used to discover compounds that interacf with specific functional sites.
  • a substrate such as, a 2- trans,4-cis dienoyl-CoA intermediate and a candidate compound can be added to a sample of the SDR.
  • Compounds that interact with the SDR at the same site as the a 2- trans,4-cis dienoyl-CoA intermediate will reduce the amount of complex formed between the SDR and the 2-trans,4-cis dienoyl-CoA intermediate. Accordingly, it is possible to discover a compound that specifically prevents interaction between the SDR and the substrate, in this example an unsaturated fatty acid intermediate.
  • Another example involves adding a candidate compound to a sample of the SDR and a coenzyme, such as NADPH.
  • a coenzyme such as NADPH
  • a compound that competes with NADPH will reduce the coenzyme interaction with SDR and thereby prevent the subsequent interaction with a substrate and/or the oxidation/reduction of the substrate.
  • compounds can be discovered that directly interact with the SDR and compete with various coenzymes and substrates.
  • Such assays can involve any other component that interacts with the SDR.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S- transferase/ SDR fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the cell lysates e.g., S-labeled
  • the candidate compound e.g., glutathione derivatized microtitre plates
  • the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes is dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of SDR -binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art.
  • antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation.
  • Preparations of an SDR-binding target component, such as a coenzyme or a substrate , and a candidate compound are incubated in the SDR -presenting wells and the amount of complex trapped in the well can be quantitated.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the SDR target molecule, or which are reactive with SDR and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • Modulators of SDR activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by SDRs, by treating cells that express the SDR. These methods of treatment include the steps of administering the modulators of SDR activity in a pharmaceutical composition as described herein, to a subject in need of such treatment.
  • Tissues and/or cells in which the 25204 SDR is found include, but are not limited to, hippocampus, aortic endothelial cells, embryo, Jurkat T-cells, gall bladder, fetal liver, prostate, bone marrow, skin, B-cells, neuron, heart, and lung, as well as the tissues and cell lines shown in Figures 6-11.
  • the short chain dehydrogenase/reductase of the present invention is relevant to treating disorders involving these tissues.
  • disorders involving the lung include, but are not limited to, congenital anomalies; atelectasis; diseases of vascular origin, such as pulmonary congestion and edema, including hemodynamic pulmonary edema and edema caused by microvascular injury, adult respiratory distress syndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, and infarction, and pulmonary hypertension and vascular sclerosis; chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis; diffuse interstitial (infiltrative, restrictive) diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia (pulmonary infiltration with eosinophilia), Bronchiolitis obliterans-organizmg pneumonia, diffuse pulmonary
  • disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and cirrhosis, such as cirrhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn errors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, a ⁇ - antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as
  • Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states— global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacun
  • subacute encephalitis vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and
  • T-cells disorders involving T-cells include, but are not limited to, cell-mediated hypersensitivity, such as delayed type hypersensitivity and T-cell-mediated cytotoxicity, and transplant rejection; autoimmune diseases, such as systemic lupus eryfhematosus, Sj ⁇ gren syndrome, systemic sclerosis, inflammatory myopathies, mixed connective tissue disease, and polyarteritis nodosa and other vasculitides; immunologic deficiency syndromes, including but not limited to, primary immunodeficiencies, such as thymic hypoplasia, severe combined immunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory) proliferations of white cells, including but not limited to, leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecific lymphadenitis; neoplastic proliferations of white cells, including but not limited to lymphoid neoplasms, such as precursor T-cell neoplasms, such as acute lymphoblastic leukemia/
  • Diseases of the skin include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors of the dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular
  • the myelocytic series (polymorphoneuclear cells) make up approximately 60%) of the cellular elements, and the erythrocytic series, 20- 30%. Lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15% of normal adult marrow.
  • cell types are add mixed so that precursors of red blood cells (erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts) can be visible in one microscopic field.
  • stem cells exist for the different cell lineages, as well as a precursor stem cell for the committed progenitor cells of the different lineages.
  • the various types of cells and stages of each would be known to the person of ordinary skill in the art and are found, for example, on page 42 ( Figure 2-8) of Immunology, Imunopathology and Immunity, Fifth Edition, Sell et al. Simon and Schuster (1996), incorporated by reference for its teaching of cell types found in the bone marrow. According, the invention is directed to disorders arising from these cells.
  • disorders include but are not limited to the following: diseases involving hematopoeitic stem cells; committed lymphoid progenitor cells; lymphoid cells including B and T-cells; committed myeloid progenitors, including monocytes, granulocytes, and megakaryocytes; and committed erythroid progenitors.
  • leukemias include B-lymphoid leukemias, T- lymphoid leukemias, undifferentiated leukemias; erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias are encompassed with and without differentiation]; chronic and acute lymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronic and acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic and acute myeloblastic leukemia, chronic and acute myelogenous leukemia, chronic and acute promyelocytic leukemia, chronic and acute myelocytic leukemia, hematologic malignancies of monocyte-macrophage lineage, such as juvenile chronic myelogenous leukemia; secondary AML, antecedent hematological disorder; refractory anemia;
  • disorders involving the heart include but are not limited to, heart failure, including but not limited to, cardiac hypertrophy, left-sided heart failure, and right- sided heart failure; ischemic heart disease, including but not limited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not limited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not limited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration of the mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and end
  • vascular diseases involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease— the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurys
  • B-cells include, but are not limited to precursor B-cell neoplasms, such as lymphoblastic leukemia/lymphoma.
  • Peripheral B-cell neoplasms include, but are not limited to, chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and related entities, lymphoplasmacytic lymphoma (Waldenstr ⁇ m macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma (MALToma), and hairy cell leukemia.
  • Disorders involving the prostate include, but are not limited to, inflammations, benign enlargement, for example, nodular hyperplasia (benign prostatic hypertrophy or hyperplasia), and tumors such as carcinoma.
  • Disorders involving precursor T-cell neoplasms include precursor T lymphoblastic leukemia/lymphoma.
  • T-cell chronic lymphocytic leukemia large granular lymphocytic leukemia, mycosis fungoides and Sezary syndrome
  • peripheral T-cell lymphoma unspecified, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-cell lymphoma 43 ), intestinal T-cell lymphoma, adult T-cell leukemia/lymphoma, and anaplastic large cell lymphoma.
  • Disorders of the gall bladder include, but are not limited to, cholelithiasis, cholecystitis, inflammatory polyps, adenomyosis, and carcinoma.
  • Disorders of neurons include, but are not limited to, acute neruonal injury, neuronal atrophy, aconal reaction, neuronal inclusions, neurofibrillary tangles of alzheimer disease, Lewy bodies of Parkinson disease, amyotrophic lateral sclerosis, bulbospinal atrophy, spinal muscular atrophy, neuronal storage disease, leukodystrophies, Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and canavan disease.
  • disorders in which the SDR expression is relevant include, but are not limited to, disorders resulting from the disruption of the reductase dependant pathway of beta- oxidation of unsaturated fatty acids.
  • Disorders in fatty acid oxidation can be characterized by hypoketoic hypoglycaemia after a period of prolonged fasting. Liver disease with hyperammonaemia and Reye-like syndrome may also develop. Chronic disruption of muscle function may also develop with myopathy and/or cardiomyopathy with symptoms such as muscle weakness, hypotonia, congestive heart failure, arrhythmia, and heart block. See, for example, Wanders et al. (1999) J. Inker. Metab. Dis. 22:442-487, herein incorporated by reference.
  • SDR polypeptides are thus useful for treating an SDR -associated disorder characterized by aberrant expression or activity of a SDR.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity of the protein.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering the SDR as therapy to compensate for reduced or aberrant expression or activity of the protein.
  • Methods for treatment include but are not limited to the use of soluble SDR or fragments of the SDR protein that compete for substrate or coenzyme binding, interfere with subunit interaction, or interfere with the reaction mediated by the SDR polypeptide. These SDR or fragments can have a higher affinity for the target so as to provide effective competition.
  • Stimulation of activity is desirable in situations in which the protein is abnormally downregulated and/or in which increased activity is likely to have a beneficial effect.
  • inhibition of activity is desirable in situations in which the protein is abnormally upregulated and/or in which decreased activity is likely to have a beneficial effect.
  • a subject has a disorder characterized by aberrant development or cellular differentiation.
  • the subject having a disruption in the fatty acid oxidation pathway.
  • the proteins of the invention can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 71:113-131; Madura et al. (1993) J. Biol Chem. 265:12046-12054; Bartel et al. (1993) Biotechniques 14:910-914; Iwabuchi et al. (1993) Oncogene 5:1693-1696; and Brent WO 94/10300), to identify other proteins (captured proteins) which bind to or interact with the proteins of the invention and modulate their activity.
  • the SDR polypeptides also are useful to provide a target for diagnosing a disease or predisposition to disease mediated by the SDR, including, but not limited to, diseases involving tissues in which the SDR is expressed as disclosed herein. Accordingly, methods are provided for detecting the presence, or levels of, the SDR in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the SDR such that the interaction can be detected.
  • One agent for detecting SDR is an antibody capable of selectively binding to SDR.
  • a biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the SDR also provides a target for diagnosing active disease, or predisposition to disease, in a patient having a variant SDR.
  • SDR can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in an aberrant protein. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification.
  • Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered SDR activity in cell-based or cell-free assay, alteration in substrate or coenzyme binding, altered interaction with SDR subunits, altered rate of substrate oxidation/reduction, altered antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein in general or in an SDR specifically.
  • In vitro techniques for detection of SDR include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • the protein can be detected in vivo in a subject by introducing into the subject a labeled anti- SDR antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • Particularly useful are methods, which detect the allelic variant of the SDR expressed in a subject, and methods, which detect fragments of the SDR in a sample.
  • the SDR polypeptides are also useful in pharmacogenomic analysis.
  • Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp Pharmacol. Physiol. 13(10-11):983-985, and Linder, M.W. (1997) Clin. Chem. 43(1):154-166.
  • the clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism.
  • the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound.
  • the activity of drug metabolizing enzymes affects both the intensity and duration of drug action.
  • the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype.
  • the discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the SDR in which one or more of the SDR functions in one population is different from those in another population.
  • polypeptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality.
  • polymorphism may give rise to catalytic regions that are more or less active. Accordingly, dosage would necessarily be modified to maximize the therapeutic effect within a given population containing the polymorphism.
  • specific polymorphic polypeptides could be identified.
  • the SDR polypeptides are also useful for monitoring therapeutic effects during clinical trials and other treatment. Thus, the therapeutic effectiveness of an agent that is designed to increase or decrease gene expression, protein levels or SDR activity can be monitored over the course of treatment using the SDR polypeptides as an end-point target.
  • the monitoring can be, for example, as follows: (i) obtaining a pre- administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression or activity of the protein in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the protein in the post-administration samples; (v) comparing the level of expression or activity of the protein in the pre- administration sample with the protein in the post-administration sample or samples; and (vi) increasing or decreasing the administration of the agent to the subject accordingly.
  • the invention also provides antibodies that selectively bind to the SDR and its variants and fragments.
  • An antibody is considered to selectively bind, even if it also binds to other proteins that are not substantially homologous with the SDR. These other proteins share homology with a fragment or domain of the SDR. This conservation in specific regions gives rise to antibodies that bind to both proteins by virtue of the homologous sequence. In this case, it would be understood that antibody binding to the SDR is still selective.
  • an isolated SDR polypeptide is used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Either the full-length protein or antigenic peptide fragment can be used. Regions having a high antigenicity index are shown in Figures 2.
  • Antibodies are preferably prepared from these regions or from discrete fragments in these regions. However, antibodies can be prepared from any region of the peptide as described herein. A preferred fragment produces an antibody that diminishes or completely prevents substrate or coenzyme binding or prevents the oxidation/reduction of substrate. Antibodies can be developed against the entire SDR or domains of the SDR as described herein. Antibodies can also be developed against specific functional sites as disclosed herein.
  • the antigenic peptide can comprise a contiguous sequence of at least 12, 14, 15, or 30 amino acid residues. In one embodiment, fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions. These fragments are not to be construed, however, as encompassing any fragments, which may be disclosed prior to the invention.
  • Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab') ) can be used.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I,
  • An appropriate immunogenic preparation can be derived from native, recombinantly expressed, or chemically synthesized peptides.
  • the antibodies can be used to isolate a SDR by standard techniques, such as affinity chromatography or immunoprecipitation.
  • the antibodies can facilitate the purification of the natural SDR from cells and recombinantly produced SDR expressed in host cells.
  • the antibodies are useful to detect the presence of SDR in cells or tissues to determine the pattern of expression of the SDR among various tissues in an organism and over the course of normal development.
  • the antibodies can be used to detect SDR in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.
  • the antibodies can be used to assess abnormal tissue distribution or abnormal expression during development. Antibody detection of circulating fragments of the full length SDR can be used to identify SDR turnover.
  • the antibodies can be used to assess SDR expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to SDR function.
  • a disorder is caused by an inappropriate tissue distribution, developmental expression, or level of expression of the SDR protein
  • the antibody can be prepared against the normal SDR protein. If a disorder is characterized by a specific mutation in the SDR, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant SDR.
  • intracellularly-made antibodies (“intrabodies”) are also encompassed, which would recognize intracellular SDR peptide regions.
  • the antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism.
  • Antibodies can be developed against the whole SDR or portions of the SDR.
  • the diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting SDR expression level or the presence of aberrant SDR and aberrant tissue distribution or developmental expression, antibodies directed against the SDR or relevant fragments can be used to monitor therapeutic efficacy.
  • Antibodies accordingly can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • antibodies are useful in pharmacogenomic analysis.
  • antibodies prepared against polymorphic SDR can be used to identify individuals that require modified treatment modalities.
  • the antibodies are also useful as diagnostic tools as an immunological marker for aberrant SDR analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.
  • the antibodies are also useful for tissue typing.
  • a specific SDR has been correlated with expression in a specific tissue
  • antibodies that are specific for this SDR can be used to identify a tissue type.
  • the antibodies are also useful in forensic identification. Accordingly, where an individual has been correlated with a specific genetic polymorphism resulting in a specific polymorphic protein, an antibody specific for the polymorphic protein can be used as an aid in identification.
  • the antibodies are also useful for inhibiting SDR function, for example, blocking substrate or coenzyme binding or disrupting the oxidation/reduction of substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting SDR function.
  • An antibody can be used, for example, to block coenzyme or substrate binding.
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact SDR associated with a cell.
  • kits for using antibodies to detect the presence of a SDR protein in a biological sample can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting SDR in a biological sample; means for determining the amount of SDR in the sample; and means for comparing the amount of SDR in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect SDR.
  • polynucleotides The nucleotide sequence in SEQ ID NO: 1 was obtained by sequencing the deposited human cDNA. Accordingly, the sequence of the deposited clones are controlling as to any discrepancies between the two and any reference to the sequences of SEQ ID NO:l includes reference to the sequences of the deposited cDNA.
  • the specifically disclosed cDNAs comprise the coding region and 5' and 3' untranslated sequences in SEQ ID NO:l.
  • the invention provides isolated polynucleotides encoding the novel SDR.
  • SDR polynucleotide or “SDR nucleic acid” refers to the sequence shown in SEQ ID NO: 1 or in the deposited cDNA.
  • SDR polynucleotide or “SDR nucleic acid” further includes variants and fragments of the SDR polynucleotide.
  • an “isolated” SDR nucleic acid is one that is separated from other nucleic acid present in the natural source of the SDR nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the SDR nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • flanking nucleotide sequences for example up to about 5KB.
  • SDR nucleic acid is isolated from flanking sequences such that it can be subjected to the specific manipulations described herein, such as recombinant expression, preparation of probes and primers, and other uses specific to the SDR nucleic acid sequences.
  • an "isolated" nucleic acid molecule such as a cDNA or RNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.
  • an isolated nucleic acid comprises at least about 50, 80 or 90 %> (on a molar basis) of all macromolecular species present.
  • recombinant DNA molecules contained in a vector are considered isolated.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • the isolated material will form part of a composition (or example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.
  • an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
  • the SDR polynucleotides can encode the mature protein plus additional amino or carboxyterminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • the SDR polynucleotides include, but are not limited to, the sequence encoding the mature polypeptide alone, the sequence encoding the mature polypeptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature polypeptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA.
  • the polynucleotide may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.
  • SDR polynucleotides can be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
  • the nucleic acid, especially DNA can be double-stranded or single-stranded.
  • Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).
  • SDR nucleic acid can comprise the nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:3, corresponding to human the 25204 SDR cDNA.
  • the SDR nucleic acid comprises only the coding region.
  • the invention further provides variant SDR polynucleotides, and fragments thereof, that differ from the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:3.
  • the invention also provides SDR nucleic acid molecules encoding the variant polypeptides described herein.
  • Such polynucleotides may be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis.
  • variants may be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions.
  • variants typically have a substantial identity with the nucleic acid molecule of SEQ ID NO: 1 or SEQ ID NO:3 and the complements thereof. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.
  • Orthologs, homologs, and allelic variants can be identified using methods well known in the art. These variants comprise a nucleotide sequence encoding an SDR that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more homologous to the nucleotide sequence shown in SEQ LD NO:l, SEQ LD NO:3 or a fragment of these sequences.
  • Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ LD NO: 1 or SEQ LD NO:3 or a fragment of the sequence.
  • stringent hybridization does not indicate substantial homology where it is due to general homology, such as poly A sequences, or sequences common to all or most proteins, all SDRs, or all 2-4-dienoyl-CoA reductase. Moreover, it is understood that variants do not include any of the nucleic acid sequences that may have been disclosed prior to the invention.
  • the term "hybridizes under stringent conditions” describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • a preferred, example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50°C.
  • Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C.
  • a further example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1%) SDS at 60°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • Particularly preferred stringency conditions are 0.5M Sodium Phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID 1, or SEQ ID NO:3, corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the present invention also provides isolated nucleic acids that contain a single or double stranded fragment or portion that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:l or SEQ ID NO: 3 or the complements thereof.
  • the nucleic acid consists of a portion of the nucleotide sequence of SEQ ID NO : 1 , SEQ ID NO : 3 , or the complements thereof.
  • isolated fragments include any contiguous sequence not disclosed prior to the invention as well as sequences that are substantially the same and which are not disclosed. Accordingly, if a fragment is disclosed prior to the present invention, that fragment is not intended to be encompassed by the invention.
  • an isolated nucleic acid fragment is at least about 12, preferably at least about 15, 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 600, 700, 800, 900, 100 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic proteins or polypeptides described herein are useful.
  • a nucleic acid molecule that is a fragment of a 25204 nucleotide sequence of the present invention comprises a nucleotide sequence consisting of nucleotides 1-100, 101-200, 201-300, 301 -400, 401-500, 501-600, 601- 700, 701-800, 801-900, 901-1000, or 1001-1097 of SEQ ID NO:l.
  • nucleotide sequences from about nucleotide 1 to about nucleotide 60 are not disclosed prior to the present invention.
  • nucleotide sequence from about nucleotide 1 to about nucleotide 1097 encompasses fragments greater than 1037, 1045, 1050, 1060, 1070, 1080 or 1090 nucleotides.
  • the invention provides polynucleotides that comprise a fragment of the full-length SDR polynucleotides.
  • the fragment can be single or double-stranded and can comprise DNA or RNA.
  • the fragment can be derived from either the coding or the non-coding sequence.
  • an isolated SDR nucleic acid encodes the entire coding region.
  • Other fragments include nucleotide sequences encoding the amino acid fragments described herein.
  • SDR nucleic acid fragments further include sequences corresponding to the domains described herein, subregions also described, and specific functional sites. SDR nucleic acid fragments also include combinations of the domains, segments, and other functional sites described above. A person of ordinary skill in the art would be aware of the many permutations that are possible.
  • a SDR fragment includes any nucleic acid sequence that does not include the entire gene.
  • the invention also provides SDR nucleic acid fragments that encode epitope bearing regions of the SDR proteins described herein.
  • Nucleic acid fragments are not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
  • nucleic acid fragments of the invention provide probes or primers in assays such as those described below.
  • Probes are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid. Such probes include polypeptide nucleic acids, as described in Nielsen et al. (1991) Science
  • a probe comprises a region of nucleotide sequence that hybridizes under highly stringent conditions to at least about 15, typically about 20- 25, and more typically about 40, 50 or 75 consecutive nucleotides of the nucleic acid sequence shown in SEQ ID NO: 1 , SEQ ID NO:3, or the complements thereof. More typically, the probe further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
  • a label e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
  • primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis using well- known methods (e.g., PCR, LCR) including, but not limited to those described herein.
  • the appropriate length of the primer depends on the particular use, but typically ranges from about 15 to 30 nucleotides.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the nucleic acid sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the sequence to be amplified.
  • the SDR polynucleotides are thus useful for probes, primers, and in biological assays.
  • polynucleotides are used to assess SDR properties or functions, such as in the assays described herein, all or less than all of the entire cDNA can be useful.
  • Assays specifically directed to SDR functions such as assessing agonist or antagonist activity, encompass the use of known fragments. Further, diagnostic methods for assessing SDR function can also be practiced with any fragment, including those fragments that may have been known prior to the invention. Similarly, in methods involving treatment of SDR dysfunction, all fragments are encompassed including those, which may have been known in the art.
  • the SDR polynucleotides are useful as a hybridization probe for cDNA and genomic DNA to isolate a full-length cDNA and genomic clones encoding the polypeptide described in SEQ ID NO:2 and to isolate cDNA and genomic clones that correspond to variants producing the same polypeptide shown in SEQ ID NO:2 or the other variants described herein.
  • Variants can be isolated from the same tissue and organism from which the polypeptides shown in SEQ ID NO:2 was isolated, different tissues from the same organism, or from different organisms. This method is useful for isolating genes and cDNA that are developmentally-controlled and therefore may be expressed in the same tissue or different tissues at different points in the development of an organism.
  • the probe can correspond to any sequence along the entire length of the gene encoding the SDR. Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3' noncoding regions.
  • the nucleic acid probe can be, for example, the full-length cDNA of SEQ ID NO:l, SEQ ID NO:3, or a fragment thereof, such as an oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA or DNA. Fragments of the polynucleotides described herein are also useful to synthesize larger fragments or full-length polynucleotides described herein. For example, a fragment can be hybridized to any portion of an mRNA and a larger or full-length cDNA can be produced.
  • the fragments are also useful to synthesize antisense molecules of desired length and sequence.
  • Antisense nucleic acids of the invention can be designed using the nucleotide sequence of SEQ ID NO:l or SEQ ID NO: 3 and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-mefhylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5).
  • the terms "peptide nucleic acids” or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14610.
  • PNAs can be further modified, e.g., to enhance their stability, specificity or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • nucleic acid molecules and fragments of the invention can also include other appended groups such as peptides (e.g., for targeting host cell SDRs in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 54:648-652; PCT Publication No. WO 88/0918) or the blood brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell SDRs in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-916) or intercalating agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
  • the SDR polynucleotides are also useful as primers for PCR to amplify any given region of an SDR polynucleotide.
  • the SDR polynucleotides are also useful for constructing recombinant vectors.
  • Such vectors include expression vectors that express a portion of, or all of, the SDR polypeptides.
  • Vectors also include insertion vectors, used to integrate into another polynucleotide sequence, such as into the cellular genome, to alter in situ expression of SDR genes and gene products.
  • an endogenous SDR coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.
  • the SDR polynucleotides are also useful for expressing antigenic portions of the SDR proteins.
  • the SDR polynucleotides are also useful as probes for determining the chromosomal positions of the SDR polynucleotides by means of in situ hybridization methods, such as FISH.
  • FISH in situ hybridization methods
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations, that are visible from chromosome spreads, or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the SDR polynucleotide probes are also useful to determine patterns of the presence of the gene encoding the SDRs and their variants with respect to tissue distribution, for example, whether gene duplication has occurred and whether the duplication occurs in all or only a subset of tissues.
  • the genes can be naturally occurring or can have been introduced into a cell, tissue, or organism exogenously.
  • the SDR polynucleotides are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from genes encoding the polynucleotides described herein.
  • the SDR polynucleotides are also useful for constructing host cells expressing a part, or all, of the SDR polynucleotides and polypeptides.
  • the SDR polynucleotides are also useful for constructing transgenic animals expressing all, or a part, of the SDR polynucleotides and polypeptides.
  • the SDR polynucleotides are also useful for making vectors that express part, or all, of the SDR polypeptides.
  • the SDR polynucleotides are also useful as hybridization probes for determining the level of SDR nucleic acid expression. Accordingly, the probes can be used to detect the presence of, or to determine levels of, SDR nucleic acid in cells, tissues, and in organisms.
  • the nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the polypeptides described herein can be used to assess gene copy number in a given cell, tissue, or organism. This is particularly relevant in cases in which there has been an amplification of the SDR genes.
  • the probe can be used in an in situ hybridization context to assess the position of extra copies of the SDR genes, as on extrachromosomal elements or as integrated into chromosomes in which the SDR gene is not normally found, for example as a homogeneously staining region.
  • Tissues and/or cells in which the 25204 SDR is expressed include, but are not limited to, those described above herein. As such, the gene is particularly relevant for the treatment of disorders involving these tissues.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant expression or activity of SDR nucleic acid, in which a test sample is obtained from a subject and nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of the nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of the nucleic acid.
  • nucleic acid e.g., mRNA, genomic DNA
  • One aspect of the invention relates to diagnostic assays for determining nucleic acid expression as well as activity in the context of a biological sample (e.g., blood, serum, cells, tissue) to determine whether an individual has a disease or disorder, or is at risk of developing a disease or disorder, associated with aberrant nucleic acid expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with expression or activity of the nucleic acid molecules.
  • In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.
  • Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express the SDR, such as by measuring the level of a SDR -encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if the SDR gene has been mutated.
  • Nucleic acid expression assays are useful for drug screening to identify compounds that modulate SDR nucleic acid expression (e.g., antisense, polypeptides, peptidomimetics, small molecules or other drugs).
  • a cell is contacted with a candidate compound and the expression of mRNA determined.
  • the level of expression of the mRNA in the presence of the candidate compound is compared to the level of expression of the mRNA in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression.
  • the modulator can bind to the nucleic acid or indirectly modulate expression, such as by interacting with other cellular components that affect nucleic acid expression.
  • Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the gent to a subject) in patients or in transgenic animals.
  • the invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the SDR gene.
  • the method typically includes assaying the ability of the compound to modulate the expression of the SDR nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired SDR nucleic acid expression.
  • the assays can be performed in cell-based and cell-free systems.
  • Cell-based assays include cells naturally expressing the SDR nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
  • candidate compounds can be assayed in vivo in patients or in transgenic animals.
  • the assay for SDR nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the SDR catalyzed reaction (such as oxidized/reduced products or the NADPVNADPH ratio). Further, the expression of genes that are up- or down-regulated in response to the SDR can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase. Thus, modulators of SDR gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined.
  • the level of expression of SDR mRNA in the presence of the candidate compound is compared to the level of expression of SDR mRNA in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression.
  • expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression.
  • nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
  • the invention provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate SDR nucleic acid expression.
  • Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or effects on nucleic acid activity (e.g. when nucleic acid is mutated or improperly modified).
  • Treatment is of disorders characterized by aberrant expression or activity of the nucleic acid. Disorders that the gene is particularly relevant for treating have been disclosed herein above.
  • a modulator for SDR nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the SDR nucleic acid expression.
  • the SDR polynucleotides are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the SDR gene in clinical trials or in a treatment regimen.
  • the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance.
  • the gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.
  • Monitoring can be, for example, as follows: (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a specified mRNA or genomic DNA of the invention in the pre- administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the mRNA or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the mRNA or genomic DNA in the pre-administration sample with the mRNA or genomic DNA in the post-administration sample or samples; and (vi) increasing or decreasing the administration of the agent to the subject accordingly.
  • the SDR polynucleotides are also useful in diagnostic assays for qualitative changes in SDR nucleic acid, and particularly in qualitative changes that lead to pathology.
  • the polynucleotides can be used to detect mutations in SDR genes and gene expression products such as mRNA.
  • the polynucleotides can be used as hybridization probes to detect naturally-occurring genetic mutations in the SDR gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the SDR gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of an SDR.
  • Mutations in the SDR gene can be detected at the nucleic acid level by a variety of techniques. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In certain embodiments, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 141:1011-1080; and Nakazawa et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication
  • mutations in a SDR gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.
  • sequence-specific ribozymes U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method.
  • sequence differences between a mutant SDR gene and a wild-type gene can be determined by direct DNA sequencing.
  • a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 79:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:111-161; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 55:147-159).
  • Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA RNA or RNA/DNA duplexes (Myers etal.
  • RNA rather than DNA
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5). Examples of other techniques for detecting point mutations include, selective oligonucleotide hybridization, selective amplification, and selective primer extension.
  • genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759).
  • genetic mutations can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations.
  • This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • the SDR polynucleotides are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality.
  • the polynucleotides can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship).
  • a mutation in the SDR gene that results in altered affinity for a coenzyme could result in an excessive or decreased drug effect with standard concentrations of the coenzyme that activates the SDR.
  • the SDR polynucleotides described herein can be used to assess the mutation content of the gene in an individual in order to select an appropriate compound or dosage regimen for treatment.
  • polynucleotides displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.
  • the methods can involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting mRNA, or genomic DNA, such that the presence of mRNA or genomic DNA is detected in the biological sample, and comparing the presence of mRNA or genomic DNA in the control sample with the presence of mRNA or genomic DNA in the test sample.
  • the SDR polynucleotides are also useful for chromosome identification when the sequence is identified with an individual chromosome and to a particular location on the chromosome.
  • the DNA sequence is matched to the chromosome by in situ or other chromosome-specific hybridization. Sequences can also be correlated to specific chromosomes by preparing PCR primers that can be used for PCR screening of somatic cell hybrids containing individual chromosomes from the desired species. Only hybrids containing the chromosome containing the gene homologous to the primer will yield an amplified fragment. Sublocalization can be achieved using chromosomal fragments.
  • mapping strategies include prescreening with labeled flow-sorted chromosomes and preselection by hybridization to chromosome-specific libraries.
  • Further mapping strategies include fluorescence in situ hybridization, which allows hybridization with probes shorter than those traditionally used.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on the chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • the SDR polynucleotides can also be used to identify individuals from small biological samples. This can be done for example using restriction fragment-length polymorphism (RFLP) to identify an individual.
  • RFLP restriction fragment-length polymorphism
  • the polynucleotides described herein are useful as DNA markers for RFLP (See U.S. Patent No. 5,272,057).
  • the SDR sequence can be used to provide an alternative technique, which determines the actual DNA sequence of selected fragments in the genome of an individual.
  • the SDR sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify DNA from an individual for subsequent sequencing.
  • Panels of corresponding DNA sequences from individuals prepared in this manner can provide unique individual identifications, as each individual will have a unique set of such DNA sequences. It is estimated that allelic variation in humans occurs with a frequency of about once per each 500 bases. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions.
  • the SDR sequences can be used to obtain such identification sequences from individuals and from tissue.
  • the sequences represent unique fragments of the human genome.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • a panel of reagents from the sequences is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification of the individual, living or dead can be made from extremely small tissue samples.
  • the SDR polynucleotides can also be used in forensic identification procedures. PCR technology can be used to amplify DNA sequences taken from very small biological samples, such as a single hair follicle, body fluids (e.g. blood, saliva, or semen). The amplified sequence can then be compared to a standard allowing identification of the origin of the sample.
  • the SDR polynucleotides can thus be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to the noncoding region are particularly useful since greater polymorphism occurs in the noncoding regions, making it easier to differentiate individuals using this technique.
  • the SDR polynucleotides can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This is useful in cases in which a forensic pathologist is presented with a tissue of unknown origin. Panels of SDR probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This is useful in cases in which a forensic pathologist is presented with a tissue of unknown origin.
  • Panels of SDR probes can be used to identify tissue by species and/or by organ type.
  • these primers and probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • the SDR polynucleotides can be used directly to block transcription or translation of SDR gene sequences by means of antisense or ribozyme constructs.
  • nucleic acids can be directly used for treatment.
  • the SDR polynucleotides are thus useful as antisense constructs to control SDR gene expression in cells, tissues, and organisms.
  • a DNA antisense polynucleotide is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of SDR protein. An antisense RNA or DNA polynucleotide would hybridize to the mRNA and thus block translation of mRNA into SDR protein.
  • antisense molecules useful to inhibit nucleic acid expression include antisense molecules complementary to a fragment of the 5' untranslated region of SEQ ID NO:l which also includes the start codon and antisense molecules which are complementary to a fragment of the 3' untranslated region of SEQ ID NO: 1.
  • a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of a SDR nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired SDR nucleic acid expression.
  • This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the SDR protein.
  • the SDR polynucleotides also provide vectors for gene therapy in patients containing cells that are aberrant in SDR gene expression.
  • recombinant cells which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired SDR protein to treat the individual.
  • kits for detecting the presence of a SDR nucleic acid in a biological sample can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting SDR nucleic acid in a biological sample; means for determining the amount of SDR nucleic acid in the sample; and means for comparing the amount of SDR nucleic acid in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect SDR mRNA or DNA.
  • nucleotide or amino acid sequences of the invention are also provided in a variety of mediums to facilitate use thereof.
  • "provided” refers to a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a nucleotide or amino acid sequence of the present invention.
  • Such a manufacture provides the nucleotide or amino acid sequences, or a subset thereof (e.g., a subset of open reading frames (ORFs)) in a form which allows a skilled artisan to examine the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form.
  • ORFs open reading frames
  • a nucleotide or amino acid sequence of the present invention can be recorded on computer readable media.
  • computer readable media refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • the skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide or amino acid sequence information of the present invention.
  • sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • DB2, Sybase, Oracle a database application
  • the skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
  • nucleotide or amino acid sequences of the invention can routinely access the sequence information for a variety of purposes.
  • one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
  • Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
  • a "target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids.
  • a skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.
  • the most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues.
  • commercially important fragments such as sequence fragments involved in gene expression and protein processing, may be of shorter length.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzyme active sites and signal sequences.
  • Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
  • Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences.
  • a variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
  • ORFs open reading frames
  • Such ORFs are protein encoding fragments and are useful in producing commercially important proteins such as enzymes used in various reactions and in the production of commercially useful metabolites.
  • the invention also provides vectors containing the SDR polynucleotides.
  • the term "vector” refers to a vehicle, preferably a nucleic acid molecule that can transport the SDR polynucleotides.
  • the vector is a nucleic acid molecule, the SDR polynucleotides are covIERly linked to the vector nucleic acid.
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the SDR polynucleotides.
  • the vector may integrate into the host cell genome and produce additional copies of the SDR polynucleotides when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the SDR polynucleotides.
  • the vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the SDR polynucleotides such that transcription of the polynucleotides is allowed in a host cell.
  • the polynucleotides can be introduced into the host cell with a separate polynucleotide capable of affecting transcription.
  • the second polynucleotide may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the SDR polynucleotides from the vector.
  • a trans-acting factor may be supplied by the host cell.
  • a transacting factor can be produced from the vector itself.
  • transcription and/or translation of the SDR polynucleotides can occur in a cell-free system.
  • the regulatory sequence to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adeno virus early and late promoters, and retro virus long-terminal repeats.
  • expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers.
  • regions that modulate transcription include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation.
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals.
  • the person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • a variety of expression vectors can be used to express a SDR polynucleotide.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.
  • Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • host cells i.e. tissue specific
  • inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • a variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • the SDR polynucleotides can be inserted into the vector nucleic acid by well- known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together.
  • Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
  • nucleic acid sequences of the invention can be altered to contain codons, which are preferred, or non preferred, for a particular expression system.
  • the nucleic acid can be one in which at least one altered codon, and preferably at least 10%, or 20% of the codons have been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells. Methods for determining such codon usage are well known in the art.
  • the invention provides fusion vectors that allow for the production of the SDR polypeptides.
  • Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification.
  • a proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired polypeptide can ultimately be separated from the fusion moiety.
  • Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Smith et al.
  • E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pET 1 Id (Studier et al. (1990) Gene Expression Technology: Methods in Enzymology 755:60-89).
  • Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein.
  • the sequence of the polynucleotide of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli.
  • the SDR polynucleotides can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S.
  • cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J 6:229-234 ), pMFa (Kurjan et al. (1982) Cell 50:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • the SDR polynucleotides can also be expressed in insect cells using, for example, baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 5:2156-2165) and the pVL series (Lucklow et al. (1989) Virology 770:31-39).
  • the polynucleotides described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 529:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the SDR polynucleotides.
  • the person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the polynucleotides described herein. These are found for example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, of the polynucleotide sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • the invention also relates to recombinant host cells containing the vectors described herein.
  • Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • the recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Id ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Host cells can contain more than one vector.
  • different nucleotide sequences can be introduced on different vectors of the same cell.
  • the SDR polynucleotides can be introduced either alone or with other polynucleotides that are not related to the SDR polynucleotides such as those providing trans-acting factors for expression vectors.
  • the vectors can be introduced independently, co-introduced or joined to the SDR polynucleotide vector.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction.
  • Viral vectors can be replication-competent or replication-defective.
  • Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs.
  • the marker can be contained in the same vector that contains the polynucleotides described herein or may be on a separate vector.
  • Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
  • RNA derived from the DNA constructs described herein can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
  • secretion signals are incorporated into the vector.
  • the signal sequence can be endogenous to the SDR polypeptides or heterologous to these polypeptides.
  • the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like.
  • the polypeptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.
  • polypeptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria.
  • polypeptides may include an initial modified methionine in some cases as a result of a host-mediated process.
  • host cells and “recombinant host cells” refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a "purified preparation of cells”, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10%) and more preferably 50%> of the subject cells.
  • the host cells expressing the polypeptides described herein, and particularly recombinant host cells have a variety of uses. First, the cells are useful for producing SDR proteins or polypeptides that can be further purified to produce desired amounts of SDR protein or fragments. Thus, host cells containing expression vectors are useful for polypeptide production. Host cells are also useful for conducting cell-based assays involving the SDR or
  • a recombinant host cell expressing a native SDR is useful to assay for compounds that stimulate or inhibit SDR function. This includes gene expression at the level of transcription or translation, interactions with coenzymes, substrates or SDR subunits, and catalysis of substrate oxidation/reduction.
  • Host cells are also useful for identifying SDR mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant SDR (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native SDR.
  • a desired effect on the mutant SDR for example, stimulating or inhibiting function
  • Recombinant host cells are also useful for expressing the chimeric polypeptides described herein to assess compounds that activate or suppress activation by means of a heterologous domain, segment, site, and the like, as disclosed herein.
  • mutant SDRs can be designed in which one or more of the various functions is engineered to be increased or decreased (e.g., coenzyme, substrate, or SDR subunits) and used to augment or replace SDR proteins in an individual.
  • host cells can provide a therapeutic benefit by replacing an aberrant SDR or providing an aberrant SDR that provides a therapeutic result.
  • the cells provide SDRs that are abnormally active.
  • the cells provide SDR that are abnormally inactive. These SDRs can compete with endogenous SDRs in the individual.
  • cells expressing SDRs that are not catalytically active are introduced into an individual in order to compete with endogenous SDRs for substrate, coenzymes or SDR subunits.
  • endogenous SDRs for substrate, coenzymes or SDR subunits.
  • a SDR substrate is part of a treatment modality
  • Providing cells that compete for the molecule , but which cannot be affected by SDR activation would be beneficial.
  • Homologously recombinant host cells can also be produced that allow the in situ alteration of endogenous SDR polynucleotide sequences in a host cell genome.
  • the host cell includes, but is not limited to, a stable cell line, cell in vivo, or cloned microorganism. This technology is more fully described in WO 93/09222, WO 91/12650, WO 91/06667, U.S. 5,272,071, and U.S. 5,641,670. Briefly, specific polynucleotide sequences corresponding to the SDR polynucleotides or sequences proximal or distal to an SDR gene are allowed to integrate into a host cell genome by homologous recombination where expression of the gene can be affected. In one embodiment, regulatory sequences are introduced that either increase or decrease expression of an endogenous sequence.
  • a SDR protein can be produced in a cell not normally producing it.
  • increased expression of SDR protein can be effected in a cell normally producing the protein at a specific level.
  • expression can be decreased or eliminated by introducing a specific regulatory sequence.
  • the regulatory sequence can be heterologous to the SDR protein sequence or can be a homologous sequence with a desired mutation that affects expression. Alternatively, the entire gene can be deleted.
  • the regulatory sequence can be specific to the host cell or capable of functioning in more than one cell type.
  • specific mutations can be introduced into any desired region of the gene to produce mutant SDR proteins. Such mutations could be introduced, for example, into the specific functional regions such as the substrate- or coenzyme-binding site.
  • the host cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal containing the altered SDR gene.
  • the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See also Thomas et al, Cell 57:503 (1987) for a description of homologous recombination vectors.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous SDR gene is selected (see e.g., Li, E. etal. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • the genetically engineered host cells can be used to produce non-human transgenic animals.
  • a transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a SDR protein and identifying and evaluating modulators of SDR protein activity.
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
  • a host cell is a fertilized oocyte or an embryonic stem cell into which SDR polynucleotide sequences have been introduced.
  • a transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retro viral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Any of the SDR nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.
  • Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the SDR protein to particular cells.
  • Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals.
  • a transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
  • transgenic non-human animals can be produced which contain selected systems, which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991) Science 257:1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 555:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to a pseudopregnant female foster animal.
  • the offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • Transgenic animals containing recombinant cells that express the polypeptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could affect substrate and coenzyme binding, and oxidation of the substrate may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non- human transgenic animals to assay in vivo SDR function, including substrate and coenzyme interactions and substrate oxidation. Similar methods could be used to determine the effect of specific mutant SDRs and the effect of chimeric SDRs on such enzyme functions. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more SDR functions.
  • methods for producing transgenic animals include introducing a nucleic acid sequence according to the present invention, the nucleic acid sequence capable of expressing the SDR protein in a transgenic animal, into a cell in culture or in vivo.
  • the nucleic acid is introduced into an intact organism such that one or more cell types and, accordingly, one or more tissue types, express the nucleic acid encoding the SDR protein.
  • the nucleic acid can be introduced into virtually all cells in an organism by transfecting a cell in culture, such as an embryonic stem cell, as described herein for the production of transgenic animals, and this cell can be used to produce an entire transgenic organism.
  • the host cell can be a fertilized oocyte. Such cells are then allowed to develop in a female foster animal to produce the transgenic organism.
  • SDR nucleic acid molecules protein (such as an extracellular loop), modulators of the protein, and antibodies (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human.
  • Such compositions typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier.
  • administer is used in its broadest sense and includes any method of introducing the compositions of the present invention into a subject. This includes producing polypeptides or polynucleotides in vivo as by transcription or translation, in vivo, of polynucleotides that have been exogenously introduced into a subject. Thus, polypeptides or nucleic acids produced in the subject from the exogenous compositions are encompassed in the term "administer.”
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycer
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a SDR protein or anti- SDR antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a SDR protein or anti- SDR antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a 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
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 97:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • the invention features, a method of analyzing a plurality of capture probes.
  • the method can be used, e.g., to analyze gene expression.
  • the method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence; contacting the array with a 25204, preferably purified, nucleic acid, preferably purified, polypeptide, preferably purified, or antibody, and thereby evaluating the plurality of capture probes.
  • Binding e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the 25204 nucleic acid, polypeptide, or antibody.
  • the capture probes can be a set of nucleic acids from a selected sample, e.g., a sample of nucleic acids derived from a control or non-stimulated tissue or cell.
  • the method can include contacting the 25204 nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second array having a different plurality of capture probes.
  • the results of each hybridization can be compared, e.g., to analyze differences in expression between a first and second sample.
  • the first plurality of capture probes can be from a control sample, e.g., a wild type, normal, or non-diseased, non-stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
  • the second plurality of capture probes can be from an experimental sample, e.g., a mutant type, at risk, disease-state or disorder-state, or stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
  • the plurality of capture probes can be a plurality of nucleic acid probes each of which specifically hybridizes, with an allele of 25204.
  • Such methods can be used to diagnose a subject, e.g., to evaluate risk for a disease or disorder, to evaluate suitability of a selected treatment for a subject, to evaluate whether a subject has a disease or disorder.
  • 25204 is associated with short chain dehydrogenase activity, thus it is useful for disorders associated with abnormal lipid metabolism.
  • the method can be used to detect SNPs, as described above.
  • the invention features, a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression.
  • the method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express or misexpress 25204 or from a cell or subject in which a 25204 mediated response has been elicited, e.g., by contact of the cell with 25204 nucleic acid or protein, or administration to the cell or subject 25204 nucleic acid or protein; contacting the array with one or more inquiry probe, wherein an inquiry probe can be a nucleic acid, polypeptide, or antibody (which is preferably other than 25204 nucleic acid, polypeptide, or antibody); providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which
  • Binding e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.
  • the invention features, a method of analyzing 25204, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences.
  • the method includes: providing a 25204 nucleic acid or amino acid sequence; comparing the 25204 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 25204.
  • Preferred databases include GenBankTM.
  • the method can include evaluating the sequence identity between a 25204 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the internet.
  • the invention features, a set of oligonucleotides, useful, e.g., for identifying SNP's, or identifying specific alleles of 25204.
  • the set includes a plurality of oligonucleotides, each of which has a different nucleotide at an interrogation position, e.g., an SNP or the site of a mutation.
  • the oligonucleotides can be provided with different labels, such that an oligonucleotides which hybridizes to one allele provides a signal that is distinguishable from an oligonucleotides which hybridizes to a second allele.
  • the human 25204 sequence ( Figure 1; SEQ ID NO:l), which is approximately [1097] nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about [879] nucleotides (nucleotides 103-981 of SEQ ID NO: 1 ; SEQ ID NO:3).
  • the coding sequence encodes a 292 amino acid protein (SEQ ID NO:2).
  • Example 2 Expression Profiling of 25204 mRNA
  • 25204 expression was determined in clinical samples by in situ hybridization. Detectable 25204 expression was observed in 0 or 4 normal colon samples, 5 of 6 colon tumors, and 5 of 5 colon metastases. 25204 expression was assayed in one normal breast tissue sample and one breast tumor sample, and was detectable in the breast tumor sample but not in the normal breast tissue.
  • 25204 expression in HCT1 16 cells was determined by detecting 25204 message in cells sorted by florescence activated cell sorting (FACS) following synchronization. 25204 expression levels were regulated in the G /M-G 0 /G ! phase of synchronized HCT116 cells.
  • Tissues and cell lines in which 25204 expression has been identified by electronic northern include human hippocampus, aortic endothelial cells, embryo, Jurkat T-cells, gall bladder, fetal liver, prostate, bone marrow, skin, B-cells, neurons, heart, and lung.
  • Northern blot hybridizations with various RNA samples are performed under standard conditions and washed under stringent conditions, i.e., 0.2 X SSC at 65°C.
  • a DNA probe corresponding to all or a portion of the 25024 cDNA can be used.
  • the DNA is radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, CA) according to the instructions of the supplier.
  • Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, CA) are probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.
  • 25204 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 25204 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-25204 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.
  • GST glutathione-S-transferase
  • the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire 25204 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
  • the 25204 DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the 25204 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 25204 coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the 25204 gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 ⁇ , SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are subsequently transfected with the 25204-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression of the 25204 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • DNA containing the 25204 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 25204 polypeptide is detected by radiolabelling and immunoprecipitation using a 25204 specific monoclonal antibody.

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Abstract

L'invention porte sur une déshydrogénase/réductase humaine à chaîne courte (SDR) nouvellement identifiées appartenant à la super famille des déshydrogénases/réductases de mammifères présentant une forte homologie avec les 2,4,-péroxysomal 2,4-diénoyl-CoA réductases putatives du rat et de la souris. L'invention porte également: sur des polynucléotides codant pour ladite SDR: sur des procédés d'utilisation des polypeptides et polynucléotides SDR, comme cible pour le diagnostic et le traitement de troubles induits par la SDR ou lui étant associés; sur des procédés de criblage de médicaments utilisant les polypeptides et polynucléotides SDR pour identifier des agonistes et antagonistes à des fins de diagnostic et de traitement; sur des agonistes et antagonistes basés sur les polypeptides et polynucléotides SDR; et sur des procédés de production des polypeptides et polynucléotides SDR.
PCT/US2001/003335 2000-02-01 2001-02-01 La 25204, nouvelle deshydrogenase/reductase humaine a chaine courte WO2001057195A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000004135A2 (fr) * 1998-07-16 2000-01-27 Incyte Pharmaceuticals, Inc. Molecules apparentees a l'alcool-deshydrogenase chaine courte de type humain, scrm-1 et scrm-2
US6057140A (en) * 1998-06-30 2000-05-02 Incyte Pharmaceuticals, Inc. Human scad family molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6057140A (en) * 1998-06-30 2000-05-02 Incyte Pharmaceuticals, Inc. Human scad family molecules
WO2000004135A2 (fr) * 1998-07-16 2000-01-27 Incyte Pharmaceuticals, Inc. Molecules apparentees a l'alcool-deshydrogenase chaine courte de type humain, scrm-1 et scrm-2

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. GURVITZ ET AL.,: "Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta3-Delta2-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids", BIOCHEMJ., vol. 344, no. 3, 15 December 1999 (1999-12-15), pages 903 - 914, XP002167523 *
B.V. GEISBRECHT ET AL.,: "The mouse gene PDCR encodes a peroxisomal 2,4-dienoyl-CoA reductase", J. BIOL. CHEM., vol. 274, no. 36, 3 September 1999 (1999-09-03), pages 25814 - 25820, XP002167522 *
FRANSEN M ET AL: "IDENTIFICATION OF PEROXISOMAL PROTEINS BY USING M13 PHAGE PROTEIN VI PHAGE DISPLAY: MOLECULAR EVIDENCE THAT MAMMALIAN PEROXISOMES CONTAIN A 2,4-DIENOYL-COA REDUCTASE", BIOCHEMICAL JOURNAL,GB,PORTLAND PRESS, LONDON, vol. 340, no. PART 02, June 1999 (1999-06-01), pages 561 - 568, XP000862955, ISSN: 0264-6021 *

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