WO2002099093A2 - Regulation de l'enzyme humaine hydroxymethyl-glutaryl-coa de type lyase - Google Patents

Regulation de l'enzyme humaine hydroxymethyl-glutaryl-coa de type lyase Download PDF

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WO2002099093A2
WO2002099093A2 PCT/EP2002/005991 EP0205991W WO02099093A2 WO 2002099093 A2 WO2002099093 A2 WO 2002099093A2 EP 0205991 W EP0205991 W EP 0205991W WO 02099093 A2 WO02099093 A2 WO 02099093A2
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enzyme
coa lyase
hydroxymethylglutaryl
human
polynucleotide
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PCT/EP2002/005991
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WO2002099093A3 (fr
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Rainer H. KÖHLER
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Bayer Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the regulation of human hydroxymethylglutaryl-CoA lyase- like enzyme.
  • 3-Hydroxy-3-methylglutaryl-coenzyme A lyase (EC 4.1.3.4) catalyzes the transformation of hydroxymethylglutaryl-CoA into acetyl-CoA and acetoacetate.
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 5;
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 6;
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 5;
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 6; and the amino acid sequence shown in SEQ ID NO: 6.
  • Binding between the test compound and the human hydroxyrnethylglutaryl-CoA lyase-like enzyme polypeptide is detected.
  • a test compound which binds to the human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the human hydroxymethylglutaryl-CoA lyase-like enzyme.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ;
  • nucleotide sequences which are at least about 50%) identical to the nucleotide sequence shown in SEQ ID NO: 4.
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the human hydroxymethylglutaryl-CoA lyase-like enzyme through interacting with the human hydroxymethylglutaryl-CoA lyase-like enzyme mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 5;
  • amino acid sequences which are at least about 68% identical to the amino acid sequence shown in SEQ ID NO: 6;
  • a human hydroxymethylglutaryl-CoA lyase-like enzyme activity of the polypeptide is detected.
  • a test compound which increases human hydroxymethylglutaryl-CoA lyase-like enzyme activity of the polypeptide relative to human hydroxy- methylglutaryl-CoA lyase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases human hydroxymethylglutaryl-CoA lyase-like enzyme activity of the polypeptide relative to human hydroxymethylglutaryl-CoA lyase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a human hydroxymethylglutaryl-CoA lyase-like enzyme product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4.
  • Binding of the test compound to the human hydroxymethylglutaryl-CoA lyase-like enzyme product is detected.
  • a test compound which binds to the human hydroxy- methylglutaryl-CoA lyase-like enzyme product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: l;and
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4.
  • the invention thus provides a human hydroxymethylglutaryl-CoA lyase-like enzyme that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.
  • Fig. 1 shows the DNA-sequence encoding a human hydroxymethylglutaryl-CoA lyase-like enzyme Polypeptide (SEQ ID NO: 1).
  • Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.l (SEQ ID NO: 2).
  • Fig. 3 shows the amino acid sequence of the protein identified swissnew
  • Fig. 4 shows the DNA-sequence encoding a human hydroxymethylglutaryl-CoA lyase-like enzyme Polypeptide (SEQ ID NO: 4).
  • Fig. 5 shows the amino acid sequence of a human hydroxymethylglutaryl-CoA lyase-like enzyme Polypeptide (SEQ ID NO: 5).
  • Fig. 6 shows the amino acid sequence of a human hydroxymethylglutaryl-CoA lyase-like enzyme Polypeptide (SEQ ID NO: 6).
  • Fig.7 shows the BLASTX - alignment of human hydroxymethylglutaryl-CoA lyase-like enzyme against swissnew
  • Fig. 4 shows the DNA-sequence encoding a human hydroxy
  • Fig. 8 shows the HMMPFAM - alignment of human hydroxymethylglutaryl-CoA lyase-like enzyme against pfam
  • Fig. 9 shows the exon-intron structure as determined by Genewise using the genomic sequence ALS 90406.4 with the human homologue
  • the invention relates to an isolated polynucleotide from the group consisting of:
  • a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a human hydroxymefhyl- glutaryl-CoA lyase-like enzyme polypeptide;
  • e a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme comprises the amino acid sequence shown in SEQ ID NO:2.
  • a coding sequence for human hydroxymethylglutaryl-CoA lyase-like enzyme is shown in SEQ ID NOS:l AND 4. This sequence is contained within the longer sequence shown in SEQ ID NO: 4, which is located on chromosome 6.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme is 67% identical over 303 amino acids to swissnew
  • HMGL-like region stretches from residues 22-268.
  • BLOCKS search identified a number of hydroxy- methylglutaryl-CoA lyase protein regions. There is a possible splice variant supported by one mRNA. The regions that is spliced out is underlined in FIG. 4.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme of the invention is expected to be useful for the same purposes as previously identified hydroxymethylglutaryl- CoA lyase - enzymes. Human hydroxymethylglutaryl-CoA lyase-like enzyme is believed to be useful in therapeutic methods to treat disorders such as cancer and
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme also can be used to screen for human hydroxymethylglutaryl-CoA lyase-like enzyme activators and inhibitors.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 340 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2, at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150,
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide of the invention therefore can be a portion of a hydroxymethylglutaryl- CoA lyase-like enzyme protein, a full-length hydroxymethylglutaryl-CoA lyase-like enzyme protein, or a fusion protein comprising all or a portion of a hydroxymethyl- glutaryl-CoA lyase-like enzyme protein.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide variants that are biologically active, e.g., retain a hydroxymethylglutaryl-CoA lyase activity, also are hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides.
  • naturally or non-naturally occurring hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide variants have amino acid sequences which are at least about 68, 70, 75, 80, 85, 90, 96, 96, 98, or 99% identical over 303 amino acids to the amino acid sequence shown in SEQ ID NO:2, 5, or 6 or a fragment thereof.
  • Percent identity between a putative hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide variant and an amino acid sequence of SEQ ID NO:2, 5, or 6 is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA SP:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992.
  • the "FASTA" similarity search algo- rithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant.
  • the FASTA algorithm is described by Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 55:2444(1988), and by Pearson, Meth. Enzymol 183:63 (1990).
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman- Wunsch- Sellers algorithm (Needleman & Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl Math.26:7%7 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence.
  • Fusion proteins are useful for generating antibodies against hydroxymethylglutaryl- CoA lyase-like enzyme polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length hydroxymethylglutaryl-CoA lyase-like enzyme protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • GST glutathione-S-transferase
  • luciferase horseradish peroxidase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide-encoding sequence and the heterologous protein sequence, so that the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NOs:l and 4 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega
  • Species homologs of human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be obtained using hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, and expressing the cDNAs as is known in the art.
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • a coding sequence for human hydroxymethylglutaryl-CoA lyase-like enzyme is shown in SEQ ID NOS :1 AND 4.
  • nucleotide sequences encoding human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO:l or 4 or its complement also are hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA
  • species homologs and variants of hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides that encode biologically active hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides also are hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of
  • SEQ ID NO:l or 4 or its complement also are hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides. Identification of Polynucleotide Variants and Homologs
  • Variants and homologs of the hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides described above also are hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides.
  • homologous hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions ⁇ 2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS,
  • each-homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25%) basepair mismatches, even more preferably 5-15%> basepair mismatches.
  • Species homologs of the hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1%» decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973).
  • Variants of human hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides or hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides of other species can therefore be identified by hybridizing a putative homologous hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or 4 or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides or their complements following stringent hybridization and/or wash conditions also are hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a hydroxy- methylglutaryl-CoA lyase-like enzyme polynucleotide having a nucleotide sequence shown in SEQ ID NO:l or 4 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated hydroxymethylglutaryl-
  • CoA lyase-like enzyme polynucleotides For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise hydroxy- methylglutaryl-CoA lyase-like enzyme nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme cDNA molecules can be made with standard molecular biology techniques, using hydroxymethylglutaryl-CoA lyase-like enzyme mRNA as a template. Human hydroxymethylglutaryl-CoA lyase-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988).
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50%> or more, and to anneal to the target sequence at temperatures about 68-72 °C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATor, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of hydroxy- methylglutaryl-CoA lyase-like enzyme polynucleotides, or by direct chemical synthesis.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with hydroxymethylglutaryl-CoA lyase-like enzyme expression constructs.
  • a purified hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is separated from other compounds that normally associate with the hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art.
  • Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified hydroxymethylglutaryl- CoA lyase-like enzyme polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99%> pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et ah, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g.,
  • control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPorTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide. For example, when a large quantity of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • a sequence encoding the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pLN vectors Van Heeke & Schuster, J Biol. Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • sequences encoding hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et ah, EMBO J.
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRA HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).
  • An insect system also can be used to express a hydroxymethylglutaryl-CoA lyase- like enzyme polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • hydroxymefhylglutaryl- CoA lyase-like enzyme polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. fi-ugiperda cells or Trichoplusia larvae in which hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • a number of viral-based expression systems can be used to express, hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptides in mammalian host cells.
  • sequences encoding hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptide in infected host cells (Logan &
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides.
  • Such signals include the ATG initiation codon and adjacent sequences.
  • sequences encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • the efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed hydroxymethylglutaryl-CoA lyase- like enzyme polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the intro- duction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced hydroxy- methylglutaryl-CoA lyase-like enzyme sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986. Any number of selection systems can be used to recover transformed cell lines.
  • herpes simplex virus thymidine kinase include, but are not limited to, the herpes simplex virus thymidine kinase
  • dhfr confers resistance to methotrexate (Wigler et ah, Proc. Natl. Acad. Sci. 77, 3567-70, 1980)
  • npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et ah, J. Mol.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et ah, Methods Mol. Biol. 55, 121-131, 1995).
  • marker gene expression suggests that the hydroxy- methylglutaryl-CoA lyase-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the hydroxy- methylglutaryl-CoA lyase-like enzyme polynucleotide.
  • host cells which contain a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide and which express a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide to detect transformants that contain a hydroxymethylglutaryl-CoA lyase-like enzyme poly- nucleotide.
  • a variety of protocols for detecting and measuring the expression of a hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a hydroxymethyl- glutaryl-CoA lyase-like enzyme polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a hydroxymethylglutaryl- CoA lyase-like enzyme polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode hydroxymethylglutaryl- CoA lyase-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of soluble hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et ah, Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide from the fusion protein.
  • Vectors that contain fusion proteins are disclosed in Kroll et ah, DNA Cell Biol. 12, 441-453, 1993.
  • Sequences encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc.
  • Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • the composition of a synthetic hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein binding protein
  • An antibody which specifically binds to an epitope of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be used therapeutically, as well as in immuno- chemical assays, such as Western blots, ELISAs, radioimmunoassays, immuno- histochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immuno- radiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody which specifically binds to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immuno- chemical assay.
  • antibodies which specifically bind to hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide from solution.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies that specifically bind to a hydroxymethylglutaryl-CoA lyase- like enzyme polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256,
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies that specifically bind to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et ah, 1996, Eur. J. Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et ah, 1995, Int. J. Cancer 61, 497-501; Nicholls et ah, 1993, J. Immunol. Meth. 165, 81- 91).
  • Antibodies which specifically bind to hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et ah, Proc. Natl. Acad. Sci.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense Oligonucleotides can be affinity purified by passage over a column to which a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of hydroxymethylglutaryl-CoA lyase- like enzyme gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkyl- phosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et ah,
  • Modifications of hydroxymethylglutaryl-CoA lyase-like enzyme gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the hydroxymethylglutaryl-CoA lyase-like enzyme gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent hydroxymethylglutaryl-CoA lyase-like enzyme nucleotides, can provide sufficient targeting specificity for hydroxymethylglutaryl-CoA lyase-like enzyme mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et ah, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cecil, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515,
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a hydroxymethylglutaryl-CoA lyase-like enzyme poly- nucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).
  • Specific ribozyme cleavage sites within a hydroxymethylglutaryl-CoA lyase-like enzyme RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable.
  • RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Differentially Expressed Genes
  • genes whose products interact with human hydroxymethylglutaryl-CoA lyase-like enzyme may represent genes that are differentially expressed in disorders including, but not limited to, cancer and CNS disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human hydroxymethylglutaryl-CoA lyase-like enzyme gene or gene product may itself be tested for differential expression.
  • the degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques.
  • standard characterization techniques such as differential display techniques.
  • Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et ah, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et ah, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et ah,
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human hydroxymethylglutaryl-CoA lyase-like enzyme.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human hydroxymethylglutaryl-CoA lyase-like enzyme.
  • the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human hydroxymethylglutaryl-CoA lyase-like enzyme gene or gene product are up-regulated or down-regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide.
  • a test compound preferably binds to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or polynucleotide. More preferably, a test compound decreases or increases enzymatic activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to hydroxymethylglutaryl- CoA lyase-like enzyme polypeptides or polynucleotides or to affect hydroxy- methylglutaryl-CoA lyase-like enzyme activity or hydroxymethylglutaryl-CoA lyase- like enzyme gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et ah, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the hydroxymethylglutaryl-CoA lyase- like enzyme polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the hydroxymethylglutaryl-CoA lyase- like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • a micro- physiometer e.g., CytosensorTM
  • a micro- physiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS).
  • Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide (McConnell et ah, Science 257, 1906-1912, 1992).
  • BIA Bimolecular Interaction Analysis
  • a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et ah, Cell 72, 223-232, 1993; Madura et ah, J. Biol Chem.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the hydroxymethylglutaryl- CoA lyase-like enzyme polypeptide.
  • a reporter gene e.g., LacZ
  • either the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is a fusion protein comprising a domain that allows the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide to be bound to a solid support.
  • gluta- thione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptide or polynucleotide
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or polynucleotide can be used in a cell-based assay system. A hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease the enzymatic activity of a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide. Enzymatic activity can be measured, for example, as described in Hruz & Miziorko, Protein Sci 1992 Sep;l(9):l 144-53; Narisawa et ah, Clin Chim Acta 1989 Sep
  • Enzyme assays can be carried out after contacting either a purified hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound that decreases an enzymatic activity of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) is identified as a potential therapeutic agent for decreasing hydroxymethylglutaryl-CoA lyase-like enzyme activity.
  • a test compound which increases an enzymatic activity of a human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human hydroxymethylglutaryl-CoA lyase- like enzyme activity.
  • test compounds that increase or decrease hydroxy- methylglutaryl-CoA lyase-like enzyme gene expression are identified.
  • a hydroxy- methylglutaryl-CoA lyase-like enzyme polynucleotide is contacted with a test compound, and the expression of an NA or polypeptide product of the hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • the test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • the test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of hydroxymethylglutaryl-CoA lyase-like enzyme mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • polypeptide products of a hydroxymethylglutaryl-CoA lyase- like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide can be used in a cell-based assay system.
  • the hydroxymethylglutaryl- CoA lyase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention can comprise, for example, a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, hydroxymethylglutaryl-CoA lyase-like enzyme polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, or mimetics, activators, or inhibitors of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide activity.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • agent such as stabilizing compound
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2%) sucrose, and 2-7%) mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme can be regulated to treat cancer and CNS disorders.
  • Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins.
  • Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.
  • Pain that is associated with C ⁇ S disorders also can be treated by regulating the activity of human hydroxymethylglutaryl-CoA lyase-like enzyme. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • central nervous system disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • ⁇ on-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgia- radioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia.
  • RSD reflex sympathetic dystrophy
  • trigeminal neuralgia- radioculopathy e.g., post-surgical pain
  • HIV/AIDS related pain HIV/AIDS related pain
  • cancer pain e.g., cancer pain
  • metabolic neuropathies e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease
  • paraneoplastic polyneuropathy associated, for example
  • Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.
  • headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache for example, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a hydroxy- methylglutaryl-CoA lyase-like enzyme polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • a reagent which affects hydroxymethylglutaryl-CoA lyase-like enzyme activity can be administered to a human cell, either in vitro or in vivo, to reduce hydroxy- methylglutaryl-CoA lyase-like enzyme activity.
  • the reagent preferably binds to an expression product of a human hydroxymethylglutaryl-CoA lyase-like enzyme gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 s cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about IO 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about IO 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid compo- sition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • from about 0.1 ⁇ g to about 10 ⁇ g of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993);
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases hydroxymethylglutaryl-CoA lyase-like enzyme activity relative to the hydroxymethylglutaryl-CoA lyase-like enzyme activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in
  • LD 50 the dose lethal to 50%> of the population
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g ofDNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a hydroxymethylglutaryl-CoA lyase-like enzyme gene or the activity of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a hydroxymethylglutaryl-CoA lyase- like enzyme gene or the activity of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to hydroxymethylglutaryl-CoA lyase-like enzyme-specific mRNA, quantitative RT-PCR, immunologic detection of a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide, or measurement of hydroxymethylglutaryl-CoA lyase-like enzyme activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnostic Methods
  • Human hydroxymethylglutaryl-CoA lyase-like enzyme also can be. used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding hydroxymethylglutaryl-CoA lyase-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et ah, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et ah, Proc.
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of hydroxymethylglutaryl-CoA lyase-like enzyme also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays,
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-human hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide obtained is transfected into human embryonic kidney 293 cells.
  • hydroxymethylglutaryl-CoA lyase-like enzyme activity is measured spectrophotometrically in an assay using HMG-CoA as a substrate.
  • the fractions obtained by density centrifugation or from the digitonin permeabilization experiment are assayed for the activities of the following marker enzymes: catalase (peroxisomes); glutamate dehydrogenase (mitochondria), and phosphoglucose isomerase (cytosol).
  • catalase peroxisomes
  • glutamate dehydrogenase mitochondria
  • cytosol phosphoglucose isomerase
  • one unit of enzyme activity is defined as the amount of enzyme that converts 1 ⁇ mol of substrate/min
  • relative activity is the percentage of total cellular activity found in the subcellular fraction.
  • Proteins are assayed according to Bradford with bovine serum albumin (Sigma) as a standard. It is shown that the polypeptide of SEQ ID NO: 2 has a human hydroxy
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human hydroxymethylglutaryl-CoA lyase- like enzyme polypeptides in yeast.
  • the hydroxymethylglutaryl-CoA lyase-like enzyme-encoding DNA sequence is derived from SEQ ID NO:l or 4.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San
  • Purified hydroxymethylglutaryl-CoA lyase-like enzyme polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human hydroxymethylglutaryl- CoA lyase-like enzyme polypeptides comprise the amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells.
  • Binding of a test compound to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound that increases the fluorescence in a well by at least 15 > relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a hydroxymethylglutaryl-CoA lyase-like enzyme polypeptide.
  • test compound is administered to a culture of human cells transfected with a hydroxymethylglutaryl-CoA lyase-like enzyme expression construct and incubated at
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et ah, Biochem. 18,
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled hydroxymethylglutaryl-CoA lyase-like enzyme-specific probe at 65° C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:l or 4.
  • a test compound that decreases the hydroxymethylglutaryl-CoA lyase-like enzyme-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of hydroxymethylglutaryl-CoA lyase-like enzyme gene expression.
  • test compound is administered to a culture of human cells transfected with a hydroxymethylglutaryl-CoA lyase-like enzyme expression construct and incubated at 37°C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • Enzyme activity is measured using the method of Hruz & Miziorko, Protein Sci 1992 Sep; 1(9): 1144-53; Narisawa et ah, Clin Chim Acta 1989 Sep 15;184(l):57-64; Barash et ah, Prenat Diagn 1988 Nov;8(9):691; Gibson, Methods Enzymol 1988;166:219-25; or Gibson et ah, Clin Chim Acta 1982 Dec 9;126(2):171-
  • a test compound which decreases the hydroxymethylglutaryl-CoA lyase-like enzyme activity of the hydroxymethylglutaryl-CoA lyase-like enzyme relative to the hydroxymethylglutaryl-CoA lyase-like enzyme activity in the absence of the test compound is identified as an inhibitor of hydroxymethylglutaryl-CoA lyase-like enzyme activity.
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • colon COLO-205 (colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD- 1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.
  • hydroxymethylglutaryl-CoA lyase-like enzyme is involved in CNS disorders
  • tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in Higuchi et ah, BioTechnology 10, 413-17, 1992, and Higuchi et ah, BioTechnology
  • the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et ah, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et ah, Genome Res. 6, 986-94, 1996, and Gibson et ah, Genome Res. 6, 995-1001, 1996).
  • RNAs labeled "from autopsy” were, extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.
  • RNA is extracted once with 1 volume of phenol :chloro- formasoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophotometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is
  • Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers.
  • TaqMan quantitative analysis Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetra- methyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate. Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).
  • PDAR Pre-Developed TaqMan Assay Reagents
  • the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix
  • the experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA).
  • fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.
  • the cell line used for testing is the human colon cancer cell line HCT116.
  • Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37°C in a 95% air/5%CO 2 atmosphere.
  • Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamidite chemistry. A sequence of 24 bases complementary to the nucleotides at position 1 to 24 of SEQ ID NO:l is used as the test oligonucleotide. As a control, another (random) sequence is used: 5' -TCA ACT GAC TAG ATG TAC ATG GAC-3'. Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration.
  • oligonucleotides Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 ⁇ M once per day for seven days.
  • test oligonucleotide for seven days results in significantly reduced expression of human hydroxymethylglutaryl-CoA lyase-like enzyme as determined by Western blotting. This effect is not observed with the control oligonucleotide.
  • the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100% ⁇ ) is compared with the number of cells in cultures treated with the control oligonucleotide.
  • the number of cells in cultures treated with the test oligonucleotide is not more than 30%> of control, indicating that the inhibition of human hydroxymethylglutaryl-CoA lyase-like enzyme has an anti-proliferative effect on cancer cells.
  • This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus.
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c).
  • test compound p.o., i.p., i.v., i.m., or s.c
  • Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.
  • a biologic stimulus i.e., LHRH may be injected i.m.
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • specific readout assay protocol these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week).
  • animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded.
  • Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent.
  • Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol.
  • Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea.
  • Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet).
  • Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p ⁇ 0.05 as compared to the growth factor or cells only group. 2.3.2. Matrigel Angiogenesis
  • Matrigel containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05 as compared to the vehicle control group.
  • Tumor cells or fragments are implanted subcutaneously on Day 0.
  • Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1 , prior to the ability to measure the tumor burden.
  • Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day.
  • Anti- tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan- Meier curves from the times for individual tumors to attain the evaluation size. Significance is p ⁇ 0.05. 3.1.2. Intraperitoneal/Intracranial Tumor Models
  • Tumor cells are injected intraperitoneally or intracranially on Day 0.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long- term survivors is indicated separately. Survival times are used to generate Kaplan- Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Tumor cells or fragments, of mammary adenocarcinoma origin are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment. 3.3.2. Intraprostatic Assay
  • Tumor cells or fragments, of prostatic adenocarcinoma origin are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents.
  • the prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.
  • the successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed.
  • Hormones may also be administered to the rodents to support the growth of the tumors.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.
  • Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea.
  • the trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment. 3.3.4. Intracecal Assay
  • Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t- test to compare tumor sizes in the treated and control groups at the end of treatment.
  • Significance is p ⁇ 0.05 as compared to the control group.
  • This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined.
  • Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with signi- ficance determined at p ⁇ 0.05 compared to the control group in the experiment for both of these endpoints.
  • Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • the mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p ⁇ 0.05 compared to the vehicle control group in the experiment for both endpoints.
  • Acute pain is measured on a hot plate mainly in rats.
  • Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56 °C) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking.
  • the other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t, Lev., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t, Lev., s.c, intradermal, transdermal
  • Persistent pain is measured with the formalin or capsaicin test, mainly in rats.
  • a solution of 1 to 5% formalin or 10 to 100 ⁇ g capsaicin is injected into one hind paw of the experimental animal.
  • the animals show nocifensive reactions like flinching, licking and biting of the affected paw.
  • the number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t, ev., s.c, intradermal, transdermal) prior to formalin or capsaicin administration.
  • application routes i.v., i.p., p.o., i.t, ev., s.c, intradermal, transdermal
  • Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats.
  • the operation is performed under anesthesia.
  • the first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve.
  • the second variant is the tight ligation of about the half of the diameter of the common sciatic nerve.
  • a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only.
  • the fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.
  • the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Horby, Sweden).
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10 °C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity.
  • a further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb.
  • Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt, Universitat zu Koln, Cologne, Germany), and by scoring differences in gait
  • Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw.
  • the animals develop an edema with mechanical allodynia as well as thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer,
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).
  • the animals are sacrificed and the affected hindpaws sectioned and weighed.
  • the second method comprises differences in paw volume by measuring water displacement in a plethysmometer (Ugo Basile, Comerio, Italy).
  • Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal
  • Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal
  • Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).
  • MFB medium forebrain bundle
  • mice Male Wistar rats (Harlan Winkelmann, Germany), weighing 200 ⁇ 250 g at the beginning of the experiment, are used. The rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions. The following in vivo protocols are approved by the governmental authorities. All efforts are made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.
  • the left medial fore-brain bundle are injected into the left medial fore-brain bundle at a rate of 1 ⁇ l/min (2.4 mm anterior, 1.49 mm lateral, -2.7 mm ventral to Bregma and the skull surface).
  • the needle is left in place an additional 5 min to allow diffusion to occur.
  • Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol.
  • the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface.
  • One paw is touching the table, and is then moved slowly sideways (5 s for
  • Balance adjustments following postural challenge are also measured during the stepping test sessions.
  • the rats are held in the same position as described in the stepping test and, instead of being moved sideways, tilted by the experimenter towards the side of the paw touching the table. This maneuver results in loss of balance and the ability of the rats to regain balance by forelimb movements is scored on a scale ranging from 0 to 3. Score 0 is given for a normal forelimb placement. When the forelimb movement is delayed but recovery of postural balance detected, score 1 is given. Score 2 represents a clear, yet insufficient, forelimb reaction, as evidenced by muscle contraction, but lack of success in recovering balance, and score
  • test 3 is given for no reaction of movement.
  • the test is repeated three times a day on each side for three consecutive days after an initial training period of three days prior to the first testing.
  • a modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement.
  • Plexiglass test boxes with a central platform and a removable staircase on each side are used.
  • the apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use.
  • For each test the animals are left in the test boxes for 15 min.
  • the double staircase is filled with 7 3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side.
  • MPTP neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydro-pyridine
  • DAergic mesencephalic dopaminergic
  • MPTP leads to a marked decrease in the levels of dopamine and its metabolites, and in the number of dopaminergic terminals in the striatum as well as severe loss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in the substantia nigra, pars compacta.
  • TH tyrosine hydroxylase
  • mice are perfused transcardially with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). The mice are perfused transcardially with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). The mice are perfused transcardially with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). The mice are perfused transcardially with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma).
  • 25 ⁇ m sections are taken from the genu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm) and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored in assorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.
  • TH free-floating tyrosine hydroxylase
  • the system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded.
  • the system also allows a weak current to be passed through the base grid, to aid training.
  • the object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents.
  • a rat is placed in an open field, in which two identical objects are present.
  • the rats inspects both objects during the first trial of the object recognition task.
  • a second trial after a retention interval of for example 24 hours, one of the two objects used in the first trial, the 'familiar' object, and a novel object are placed in the open field.
  • the inspection time at each of the objects is registered.
  • the basic measures in the or task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the 'familiar' object.
  • Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes.
  • Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.
  • the passive avoidance task assesses memory performance in rats and mice.
  • the inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment.
  • the two compartments are separated by a guillotine door that can be operated by the experimenter.
  • a threshold of 2 cm separates the two compartments when the guillotine door is raised.
  • the illumination in the dark compartment is about 2 lux.
  • the light intensity is about 500 lux at the center of the floor of the light compartment.
  • Two habituation sessions, one shock session, and a retention session are given, separated by inter-session intervals of 24 hours.
  • the rat In the habituation sessions and the retention session the rat is allowed to explore the apparatus for 300 sec. The rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds.
  • the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 1 mA footshock is administered for 2 sec. The rat is removed from the apparatus and put back into its home cage. The procedure during the retention session is identical to that of the habituation sessions.
  • the step-through latency that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is.
  • Scopolamine impairs the memory performance during the retention session 24 hours later. If the test compound increases the enter latency compared with the scopolamine-treated controls, is likely to possess cognition enhancing potential.
  • the Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice.
  • the performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank.
  • Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly.
  • the animals receive four trials during five daily acquisition sessions.
  • a trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized.
  • the escape platform is always in the same position.
  • a trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds.
  • an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds.
  • the probe trial all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
  • rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal learning, or aged animals which suffer from cognitive deficits, are used.
  • the T-maze spontaneous alternation task assesses the spatial memory performance in mice.
  • the start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter.
  • a mouse is put into the start arm at the beginning of training.
  • the guillotine door is closed.
  • the 'forced trial' either the left or right goal arm is blocked by lowering the guillotine door.
  • the mouse After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door.
  • the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 'free choice' trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.
  • the percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials
  • Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the per-cent alternations to chance level, or below.
  • a cognition enhancer which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate.

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Abstract

L'invention concerne des réactifs qui régulent l'enzyme humaine hydroxyméthylglutaryl-CoA de type lyase et des réactifs se liant à des produits géniques de cette enzyme, qui peuvent jouer un rôle de prévention, d'amélioration ou de correction dans le cas de dysfonctionnements ou de maladies, y compris, mais pas seulement, le cancer et les troubles du système nerveux central.
PCT/EP2002/005991 2001-06-01 2002-05-31 Regulation de l'enzyme humaine hydroxymethyl-glutaryl-coa de type lyase WO2002099093A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002304656A AU2002304656A1 (en) 2001-06-01 2002-05-31 Provision of a putative human hydroxymethyl-glutaryl-coa lyase-like enzyme

Applications Claiming Priority (2)

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US29458501P 2001-06-01 2001-06-01
US60/294,585 2001-06-01

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WO2002099093A2 true WO2002099093A2 (fr) 2002-12-12
WO2002099093A3 WO2002099093A3 (fr) 2003-04-10

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023547A1 (fr) * 1999-09-27 2001-04-05 Human Genome Sciences, Inc. 26 proteines secretees humaines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023547A1 (fr) * 1999-09-27 2001-04-05 Human Genome Sciences, Inc. 26 proteines secretees humaines

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] 1 June 1994 (1994-06-01) retrieved from EBI Database accession no. P35915 XP002222054 *
DATABASE EMBL [Online] 1 May 1999 (1999-05-01) retrieved from EBI Database accession no. O95896 XP002222055 *
DATABASE EMBL [Online] 15 March 1999 (1999-03-15) retrieved from EBI Database accession no. AF131827 XP002222058 *
DATABASE EMBL [Online] 26 February 2002 (2002-02-26) retrieved from EBI Database accession no. BC024194 XP002222059 *
DATABASE EMBL [Online] 7 September 1993 (1993-09-07) "EST07131 Infant Brain, Bento Soares Homo sapiens cDNA clone HIBBR10 5' end similar to Hydroxymethylglutaryl-CoA lyase, mRNA sequence." retrieved from EBI Database accession no. T09238 XP002222056 *
DATABASE GENBANK [Online] 31 May 2001 (2001-05-31) retrieved from NCBI XP002222057 *

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AU2002304656A1 (en) 2002-12-16

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