WO2001068840A2 - REGULATION DE LA PROTEINE HUMAINE SEMBLABLE A ACYLE CoA/INHIBITEUR DE LIAISON DU DIAZEPAM - Google Patents

REGULATION DE LA PROTEINE HUMAINE SEMBLABLE A ACYLE CoA/INHIBITEUR DE LIAISON DU DIAZEPAM Download PDF

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WO2001068840A2
WO2001068840A2 PCT/EP2001/002780 EP0102780W WO0168840A2 WO 2001068840 A2 WO2001068840 A2 WO 2001068840A2 EP 0102780 W EP0102780 W EP 0102780W WO 0168840 A2 WO0168840 A2 WO 0168840A2
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acbp
dbi
protein
polypeptide
polynucleotide
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PCT/EP2001/002780
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WO2001068840A3 (fr
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Shyam Ramakrishnan
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Bayer Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the use of a novel human diazepam binding inhibitor/acyl Co A like protein and the regulation of its activity for therapeutic effect.
  • Diazepam binding inhibitor/acyl-CoA binding protein (DBI/ACBP) protein is a 10 kDa protein found in species ranging from yeast to mammals. It is expressed in a variety of organs and tissues. Originally, DBI was purified from rat brain based on its ability to displace diazepam from type A gamma-aminobutyrate (GAB A,*) receptors (1). An acyl-Coenzyme A (acyl-CoA) binding protein (ACBP) subsequently purified from liver was found to be identical to DBI (2, See also U.S. Patent No. 5,734,038). The protein was known as endozepine, DBI, or ACBP, but it is now generally referred to as DBI/ACBP. DBI/ACBP, and polypeptides derived from it, have been implicated in multiple biological processes, such as (1)
  • GABA ⁇ /benzodiazepam receptor modulation (2) acyl-CoA metabolism, (3) steroi- dogenesis, and (4) insulin secretion (3).
  • DBI/ACBP consists of four alpha helices (Al through A4) arranged in a left-handed anti-parallel bundle, with parallel helices Al and A4 anti -parallel to helices A2 and A3.
  • Helix A2 interacts with each of the other three helices in a structure reminiscent of a bowl.
  • the inner surface of the bowl has a patch of non-polar and uncharged residues at the interface between helices A2 and A3.
  • the rims of the bowl have mainly polar and charged groups which are contributed by the hydrophilic residues of the amphipathic helices.
  • the ligand binding site is located on the inner surface of the bowl, and it binds the aliphatic acyl chain of the fatty acyl-CoA ligand in a non-polar arrangement created partly by the protein and partly by the pantetheine and the adenosine-3 '-phosphate of CoA.
  • the pantetheine and CoA moieties likewise form a highly polar and charged surface, so that the surface together with the polar and charged rims of the protein bowl ensure the solubility of the entire complex (5).
  • the binding affinity of bovine DBI/ACBP towards acyl-CoA esters depends on the length of the acyl chain, where the highest affinity is for long-chain (C14 to C22) acyl-CoA esters.
  • the protein is very specific in binding acyl-CoA esters, binding neither free CoA nor free fatty acids (6).
  • DBI/ACBP sequesters bound long-chain fatty acyl-CoA, protects acyl-CoAs from hydrolysis, extracts acyl-CoAs from phos- phatidyl choline membranes, and mediates intermembrane acyl-CoA transport (7).
  • DBI/ACBP DBI/ACBP
  • glioblastomas glioblastomas and medullablastomas suggests that it may be involved in the regulation of high-energy acyl-CoA metabolism in rapidly growing neuronal cells (8).
  • DBI/ACBP also inhibits the binding of benzodiazepines to the GAB A,* receptor.
  • the GABA ⁇ receptor is a post-synaptic Cl " channel.
  • the Cl " ion channel opening burst, elicited by the inhibitory neurotransmitter GABA, is prolonged by benzodiazepines.
  • DBI/ACBP octadecaneneuropeptide
  • ODN DBI/ACBP amino acids 32-50
  • DBI/ACBP is also involved in the regulation of steroid biosynthesis in mitochondria (10).
  • DBI/ACBP stimulates mitochondrial steroidogenesis in the adrenal gland by facilitating cholesterol delivery to the inner mitochondrial membrane ( 3 and 11) suggest that DBI/ACBP may also scavenge fatty acyl-CoA esters produced from fatty acids released in the conversion of cholesterol to steroids.
  • Antisense oligonucleotides to DBI/ACBP inhibit hormone-stimulated steroid production in Leydig cells of rat testis (12).
  • DBI/ACBP has been found in all tissues tested; the highest amounts have been found in liver, kidney, brain, adrenal gland, intestine and salivary gland (3).
  • DBI/ACBP is selectively expressed in specialized cells within a given organ. Elevated levels of DBI/ACBP have been found in cells of the adrenal cortex and testis which produce steroids and in liver hepatocytes which are involved in steroid and fat metabolism. Elevated levels of DBI/ACBP have also been found in epithelial cells of kidney tubules, the upper intestinal tracts and large bronchioles, cells which are specialized for water and electrolyte absorption and secretion. In brain, high DBI/ACBP concentrations are found in choroid plexus and circumventricular organs, which are specialized for the control of secretion and osmolality of cerebrospinal fluid (13).
  • the selective modulation of the expression or receptor binding of a novel tissue-specific DBI/ACBP-like protein may allow the successful management of diseases or biochemical abnormalities relating to the tissues in which it is expressed.
  • the binding properties of this small protein may be utilized in drug delivery ap- plications as a soluble carrier for otherwise insoluble therapeutic molecules.
  • One embodiment of the invention is a DBI/ACBP-like polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences which are at least about 70% identical to the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 2.
  • Yet another embodiment of the invention is a method of screening for agents which inhibit a human diazepam binding inhibitor/acyl CoA binding protein-like protein
  • DBI/ACBP-like protein activity A test compound is contacted with a DBI/ACBP- like polypeptide comprising an amino acid sequence selected from a group of amino acid sequences which are at least about 70% identical to the amino acid sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:2. Binding between the test compound and the DBI/ACBP-like polypeptide is detected.
  • a test compound which binds to the DBI/ACBP-like polypeptide is identified as a potential agent for inhibiting a DBI/ACBP-like protein activity.
  • the agent can work by decreasing the activity of the DBI/ACBP-like protein.
  • Another embodiment of the invention is a method of screening for agents which inhibit the activity of a DBI/ACBP-like protein.
  • a test compound is contacted with a polynucleotide encoding a DBI/ACBP-like polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of nucleotide sequences which are at least about 70% identical to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 1.
  • Binding of the test compound to the polynucleotide is detected.
  • a test compound which binds to the polynucleotide is identified as a potential agent for inhibiting the activity of a DBI/ACBP-like protein. The agent can work by decreasing the amount of the DBI/ACBP-like protein through interacting with the DBI/ACBP-like protein mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate the activity of a DBI/ACBP-like protein.
  • a test compound is contacted with a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences which are at least about 70% identical to the amino acid sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:
  • a DBI/ACBP-like protein activity of the polypeptide is detected.
  • a test compound which increases DBI/ACBP-like protein activity of the polypeptide relative to DBI/ACBP-like protein activity in the absence of the test compound is thereby identified as a potential agent for increasing the activity of a DBI/ACBP-like protein.
  • a test compound which decreases DBI/ACBP-like protein activity of the polypeptide relative to DBI/ACBP-like protein activity in the absence of the test compound is thereby identified as a potential agent for decreasing the activity of DBI/ACBP-like protein.
  • Another embodiment of the invention is a method of screening for agents which in- hibit a DBI/ACBP-like protein activity.
  • a test compound is contacted with a product of a polynucleotide which comprises a nucleotide sequence selected from a group of nucleotide sequences which are at least about 70% identical to the nucleotide sequence shown in SEQ ID NO:l and the nucleotide sequence shown in SEQ ID NO:l. Binding of the test compound to the product is detected.
  • a test compound which binds to the product is identified as a potential agent for inhibiting a DBI/ACBP-like protein activity.
  • Even another embodiment of the invention is a method of screening for agents which inhibit the activity of DBI/ACBP-like protein activity.
  • a test compound is contacted with a product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of nucleotide sequences which are at least about 70% identical to the nucleotide sequence shown in SEQ ID NO:l and the nucleotide sequence shown in SEQ ID NO:l. Binding of the test compound to the product is detected.
  • a test compound which binds to the product is thereby identified as a poten- tial agent for inhibiting the activity of DBI/ACBP-like protein.
  • Another embodiment of the invention is a method of inhibiting DBI/ACBP-like protein activity.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a DBI/ACBP-like polypeptide or the product encoded by a polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from a group of nucleotide sequences which are at least about 70% identical to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO:l. DBI/ACBP-like protein activity in the cell. Is thereby inhibited.
  • Still another embodiment of the invention is a method of increasing a DBI/ACBP- like protein activity in a cell.
  • An expression construct encoding a DBI/ACBP-like polypeptide is introduced into a cell. The cell thereby expresses the DBI/ACBP-like polypeptide.
  • the invention thus provides reagents and methods for regulating human DBI/ACBP- like protein.
  • Such reagents and methods can be used inter alia, to treat malignant cells and certain neurological disorders, to regulate steroid biosynthesis and to control insulin secretion.
  • Fig. 1 shows the DNA-sequence encoding a DBI/ACBP-like polypeptide.
  • Fig. 2 shows the amino acid sequence of a DBI/ACBP-like polypeptide.
  • the invention relates to an isolated polynucleotide encoding a DBI/ACBP-like polypeptide and being selected from the group consisting of:
  • DBI/ACBP-like protein can be regulated to affect steroid biosynthesis, to treat neurological disorders, to control insulin secretion, and to treat malignant cells.
  • Human DBI/ACBP-like protein has a binding activity that specifically targets GABA ⁇ re- ceptors and acyl-CoA esters.
  • Human DBI/ACBP-like protein can be used to develop treatments for various diseases, to develop diagnostic assays for these diseases, and to provide new tools for basic research especially in the fields of medicine and biology.
  • the pre- sent invention can be used to develop new drugs which will regulate the function of human DBI/ACBP-like protein.
  • Human DBI/ACBP-like protein and regulators of human DBI/ACBP-like protein thus can provide treatments for cancer, neurological disorders, diabetes, and abnormalities of steroid biosynthesis.
  • Human DBI/ACBP- like protein or its fragments also can be used to identify specific molecules which it sequesters or with which it interacts.
  • DBI/ACBP-like protein can be used to sequester therapeutic agents, specifically binding the agents so that the complex is water-insoluble and suitable for therapeutic delivery.
  • Human DBI/ACBP-like polypeptides according to the invention comprise an amino acid sequence as shown in SEQ ID NO:2, a portion of one of those amino acid sequences, or a biologically active variant of an amino acid sequence shown in SEQ ID NO:2, as defined below.
  • a DBI/ACBP-like polypeptide can be a portion of a DBI/ACBP-like molecule, a full-length DBI/ACBP-like molecule, or a fusion protein comprising all or a portion of a DBI/ACBP-like molecule.
  • a DBI/ACBP-like polypeptide binds to DBI/ACBP or a DBI/ACBP derivative. DBI/ACBP-like binding activity can be measured, r ⁇ ter alia, as described in Examples 1 and 2.
  • One form of full length human DBI/ACBP-like protein has the amino acid sequence shown in SEQ ID NO:2.
  • DBI/ACBP-like variants which are biologically active, i.e., retain a DBI/ACBP-like binding activity, also are DBI/ACBP-like polypeptides.
  • naturally or non- naturally occurring DBI/ACBP-like variants have amino acid sequences which are at least about 70, preferably about 75, 90, 96, or 98% identical to an amino acid sequence shown in SEQ ID NO:2. Percent identity between a putative DBI/ACBP-like variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program.
  • 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 replace- ments are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a DBI/ACBP-like polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active DBI/ACBP-like polypeptide can readily be determined by assaying for binding of an acyl-Co A ester to the DBI/ACBP-like protein, as described, for example, in the specific examples below. Alternatively, inhibition of binding of labeled benzodiazepene to a GAB A receptor can be determined, as is known in the art.
  • Fusion proteins can comprise at least 5, 6, 8, 10, 25, 50, or 75 or more contiguous amino acids of an amino acid sequence shown in SEQ ID NO:2. Fusion proteins are useful for generating antibodies against DBI/ACBP-like amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to iden- tify proteins which interact with portions of a DBI/ACBP-like 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 DBI/ACBP-like fusion protein comprises two protein segments fused together by means of a peptide bond.
  • the first protein segment comprises at least 5, 6, 8, 10, 25, 50, or 75 or more contiguous amino acids of a DBI/ACBP-like polypeptide.
  • Contiguous amino acids for use in a fusion protein can be selected from the amino acid sequence shown in SEQ ID NO:2 or from a biologically active variant of those se- quences, such as those described above.
  • the first protein segment also can comprise full-length DBI/ACBP-like protein.
  • the second protein segment can be a full-length protein or a protein fragment or polypeptide.
  • Proteins commonly used in fusion protein construction include - ga- lactosidase, -glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), lu- ciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located between the DBI/ACBP-like polypeptide-encoding sequence and the heterologous protein sequence, so that the DBI/ACBP-like 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 protein segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO: 1 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology
  • Species homologs of human DBI/ACBP-like protein can be obtained using DBI/ACBP-like 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 DBI/ACBP-like pro- tein, and expressing the cDNAs as is known in the art.
  • a DBI/ACBP-like polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a DBI/ACBP-like polypeptide.
  • the complements of partial nucleotide sequences for DBI/ACBP-like polypeptides are shown in SEQ ID NO: 1.
  • nucleotide sequences encoding human DBI/ACBP-like polypeptides as well as homologous nucleotide sequences which are at least about 70, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ID NO:l also are DBI/ACBP-like 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 molecules molecules, species homologs, and variants of DBI/ACBP-like polynucleotides which encode biologically active DBI/ACBP-like polypeptides also are DBI/ACBP-like polynucleotides.
  • Variants and homologs of the DBI/ACBP-like polynucleotides described above also are DBI/ACBP-like polynucleotides.
  • homologous DBI/ACBP-like polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known DBI/ACBP-like polynucleotides under stringent conditions, as is known in the art.
  • 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%o basepair mismatches.
  • Species homologs of the DBI/ACBP-like 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 DBI/ACBP-like 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 DBI/ACBP-like polynucleotides or DBI/ACBP-like polynucleotides of other species can therefore be identified by hybridizing a putative homologous DBI/ACBP-like polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or the complements thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising DBI/ACBP-like 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 DBI/ACBP-like polynucleotides or their complements following stringent hybridization and/or wash conditions also are DBI/ACBP-like 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 DBI/ACBP-like polynucleotide having a nucleotide sequence shown in SEQ ID NO:l or the complements thereof and a polynucleotide sequence which is at least about 70, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be cal- culated, for example, using the equation of Bolton and McCarthy, Proc. Natl Acad.
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% form- amide, 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 naturally occurring DBI/ACBP-like 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 poly- merase 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 DBI/ACBP-like polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise DBI/ACBP-like nucleotide sequences.
  • Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • DBI/ACBP-like cDNA molecules can be made with standard molecular biology techniques, using DBI/ACBP-like mRNA as a template. DBI/ACBP-like 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 DBI/ACBP-like polynucleotides using either human genomic DNA or cDNA as a template.
  • DBI/ACBP-like polynucleotides can be synthesized.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a DBI/ACBP-like polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant of one of those sequences. Extending the Polynucleotide Sequence of DBI/ACBP -Like Protein
  • PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human DBI/ACBP-like protein to detect upstream sequences such as promoters and regulatory elements.
  • upstream sequences such as promoters and regulatory elements.
  • 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 sepa- ration, four different fluorescent dyes (one for each nucleotide) which 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 which might be present in limited amounts in a particular sample.
  • DBI/ACBP-like polypeptides can be obtained, for example, by purification from human cell types, such as, liver, kidney, adrenal cortex, testis, upper intestinal tracts, large bronchioles, choroid plexus of the brain, and circumventricular organs, as well as from host cells which express DBI/ACBP-like polynucleotides. DBI/ACBP-like polypeptides also can be obtained by expression of DBI/ACBP-like polynucleotides, or by direct chemical synthesis. Protein Purification
  • DBI/ACBP-like polypeptides can be purified, for example, from human liver, kid- ney, adrenal cortex, testis, upper intestinal tracts, large bronchioles, choroid plexus of the brain, and circumventricular organs.
  • a purified DBI/ACBP-like polypeptide is separated from other compounds which normally associate with the DBI/ACBP-like 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 exclu- sion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • DBI/ACBP-like protein from rat brain is taught in (3).
  • a preparation of purified DBI/ACBP-like 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. Binding activity of the purified preparations can be assayed, for example, as described in the specific examples below.
  • a DBI/ACBP-like polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding DBI/ACBP-like 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.
  • microorganisms such as bacteria transformed with recombinant bacterio- phage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacterio- phage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or
  • 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 DBI/ACBP-like 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 a DBI/ACBP-like polypeptide. For example, when a large quantity of a DBI/ACBP-like 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). In a BLUESCRIPT vector, a sequence encoding a DBI/ACBP-like polypeptide can be ligated in frame with sequences for the amino-terminal Met and the subsequent 7 residues of -galactosidase so that a hybrid protein is produced.
  • BLUESCRIPT a sequence encoding a DBI/ACBP-like polypeptide can be ligated in frame with sequences for the amino-terminal Met and the subsequent 7 residues of -galactosidase so that a hybrid protein is produced.
  • pIN vectors Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989 or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by 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
  • DBI/ACBP-like polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671- 1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., Results Probl. Cell Differ. 17, 85-105, 1991).
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • pathogen-mediated transfection Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murry, in MCGRAW 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 DBI/ACBP-like 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 DBI/ACBP-like polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of
  • DBI/ACBP-like polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which DBI/ACBP-like 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 DBI/ACBP-like polypeptides in mammalian host cells.
  • sequences encoding DBI/ACBP-like polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a DBI/ACBP-like polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding DBI/ACBP-like polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a DBI/ACBP-like polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or transla- tional control signals may be needed. However, in cases where only coding se- quence, or a fragment thereof, is inserted, exogenous translational control signals
  • 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
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed DBI/ACBP-like 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 which have specific cellular machinery and characteristic mechanisms for post- translational activities are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express DBI/ACBP-like polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced DBI/ACBP-like sequences.
  • Resistant clones of stably trans- formed 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. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 , 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22, 817-23, 1980) genes which can be employed in tk ⁇ or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980)
  • npt confers resistance to the amino- glycosides neomycin and G-418 (Colbere-Garapin et al., J. Mol Biol. 150, 1-14,
  • trpB allows cells to utilize indole in place of trypto- phan
  • hisD 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 al., Methods Mol. Biol. 55, 121-131, 1995). De tec ting Expression of DBI/ACBP-like Polypeptides
  • marker gene expression suggests that a DBI/ACBP-like polynucleotide is also present, its presence and expression may need to be confirmed.
  • a sequence encoding a DBI/ACBP-like polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode the DBI/ACBP-like polypeptide can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding a DBI/ACBP-like polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of a DBI/ACBP-like polynucleotide.
  • host cells which contain a DBI/ACBP-like polynucleotide and which express a DBI/ACBP-like 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 which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a DBI/ACBP-like polypeptide can be detected by
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding the DBI/ACBP-like polypeptide to detect transformants which contain a DBI/ACBP-like polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a DBI/ACBP- like 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 Fluorescence Activated Cell Sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to poly- nucleotides encoding DBI/ACBP-like polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a DBI/ACBP-like polypeptide can be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6.
  • reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as sub- strates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a DBI/ACBP-like poly- peptide 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 DBI/ACBP-like polypeptides can be designed to con- tain signal sequences which direct secretion of DBI/ACBP-like polypeptides through a prokaryotic or eukaryotic cell membrane.
  • purification facili- tating domains include, but are not limited to, metal chelating peptides such as his- tidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the DBI/ACBP-like polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a DBI/ACBP-like polypeptide and 6 histidine residues preceding a thiore- doxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp.
  • enterokinase cleavage site provides a means for purifying the DBI/ACBP-like polypeptide from the fusion protein.
  • Vectors which contain fusion proteins are disclosed in Kroll et al., DNA Cell Biol. 12, 441-453, 1993.
  • Sequences encoding a DBI/ACBP-like 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-
  • a DBI/ACBP-like 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. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995). 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 (Per- kin Elmer).
  • fragments of DBI/ACBP-like 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 DBI/ACBP-like polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, su- pra). Additionally, any portion of the amino acid sequence of the DBI/ACBP-like 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 which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods gener- ally known in the art to alter DBI/ACBP-like 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 DBI/ACBP-like polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a DBI/ACBP-like polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipita- tions, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipita- tions, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
  • an antibody which specifically binds to a DBI/ACBP-like polypeptide provides a detection signal at least 5-, 10-, or 20- fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to DBI/ACBP-like polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a DBI/ACBP- like polypeptide from solution.
  • DBI/ACBP-like polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If de- sired, a DBI/ACBP-like polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such 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 which specifically bind to a DBI/ACBP-like polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al., J Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-
  • 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 suffi- ciently 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. Alternatively, humanized antibodies can be produced using recombinant methods, as described in
  • Antibodies which specifically bind to a DBI/ACBP-like 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 which specifically bind to DBI/ACBP-like 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 al., 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,
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant D ⁇ A methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int. J. Cancer 61, 497-501; ⁇ icholls et al., 1993, J Immunol. Meth. 165, 81- 91).
  • Antibodies which specifically bind to DBI/ACBP-like polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349, 293-299, 1991).
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention.
  • 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
  • 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 DBI/ACBP-like 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 which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of DBI/ACBP-like 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 alkylphos- phonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkyl- phosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev. 90, 543-583, 1990.
  • Modifications of DBI/ACBP-like gene expression can be obtained by designing an- tisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a DBI/ACBP-like 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 DBI/ACBP-like polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent DBI/ACBP-like nucleotides, can provide sufficient targeting specificity for DBI/ACBP-like 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,
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a DBI/ACBP-like 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 al., Trends Biotechnol 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90, 543-
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236,
  • 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 DBI/ACBP-like polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the DBI/ACBP- like polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been de- veloped 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 DBI/ACBP-like RNA target can be identified by scanning the DBI/ACBP-like target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate
  • DBI/ACBP-like RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • the nucleotide sequences shown in SEQ ID NOS:l and 3 and their complements provide sources of suitable hybridization region sequences. Longer comple- mentary 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. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease DBI/ACBP-like expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which 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.
  • the invention provides methods for identifying modulators, i.e., test compounds which bind to DBI/ACBP-like polypeptides or polynucleotides and/or have a stimulatory or inhibitory effect on, for example, expression or activity of the DBI/ACBP- like polypeptide or polynucleotide.
  • Decreased DBI/ACBP-like protein activity is useful, for example, for decreasing cell proliferation and for treating brain tumors, encephalopathy, depression, and anxiety.
  • increased cell proliferation may be desired, for example, in developmental disorders characterized by inappropriately low levels of cell proliferation.
  • the invention provides assays for screening test compounds which bind to or modulate the receptor binding of a DBI/ACBP-like polypeptide or a DBI/ACBP-like poly- nucleotide.
  • a test compound preferably binds to a DBI/ACBP-like polypeptide or polynucleotide. More preferably, a test compound decreases a DBI/ACBP-like activity of a DBI/ACBP-like polypeptide or expression of a DBI/ACBP-like polynucleotide 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 com- pounds 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 deconvo- lution, 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, Antican- cer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to DBI/ACBP-like polypeptides or polynucleotides or to affect DBI/ACBP-like binding activity or DBI/ACBP- like 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 al., 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 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. Thereafter, 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 which binds to and occupies the active site of a DBI/ACBP-like polypeptide, thereby making the active site inaccessible to substrate 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 DBI/ACBP-like polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to the DBI/ACBP-like 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 DBI/ACBP-like polypeptide can be determined without labeling either of the interactants.
  • a microphysi- ometer can be used to detect binding of a test compound with a DBI/ACBP-like polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • Determining the ability of a test compound to bind to a DBI/ACBP-like polypeptide also can be accomplished using a technology such as real-time Bimolecular Inter- action Analysis (BIA) (Sjolander & Urbaniczky, Anal Chem. 63, 2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol 5, 699-705, 1995).
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • a DBI/ACBP-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al., Cell 72, 223-232, 1993; Madura et al., 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. For example, in one construct a polynucleotide encoding a DBI/ACBP-like polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g. , GAL-4). In the other construct 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.
  • a DNA sequence that encodes an unidentified protein "prey" or "sample”
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the DBI/ACBP-like polypeptide.
  • a reporter gene e.g., LacZ
  • either the DBI/ACBP-like 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 DBI/ACBP-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive abso ⁇ tion, or pairs of binding moieties attached respectively to the polypeptide or 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 DBI/ACBP-like 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.
  • a DBI/ACBP-like polypeptide is a fusion protein comprising a domain that allows the DBI/ACBP-like polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed DBI/ACBP-like polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • a DBI/ACBP-like polypeptide or polynucleotide
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated DBI/ACBP- like polypeptides, polynucleotides, or test compounds can be prepared from biotin- NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinyla- tion kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of strepta- vidin-coated 96 well plates (Pierce Chemical).
  • antibodies which spe- cifically bind to a DBI/ACBP-like polypeptide, polynucleotides, or a test compound, but which do not interfere with a desired binding site, such as the active site of the DBI/ACBP-like polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • GST-immobilized complexes include immunodetection of complexes using antibodies which specifically bind to a DBI/ACBP-like polypeptide or test compound, enzyme-linked assays which rely on detecting a DBI/ACBP-like activity of the DBI/ACBP-like polypeptide, and SDS gel electrophoresis under non-reducing con- ditions.
  • Screening for test compounds which bind to a DBI/ACBP-like polypeptide or polynucleotide also can be carried out in an intact cell.
  • Any cell which comprises a DBI/ACBP-like polynucleotide or polypeptide can be used in a cell-based assay system.
  • a DBI/ACBP-like 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 including neoplastic cell lines such as the colon cancer cell lines HCT116, DLD1, HT29, Caco2, SW837, SW480, and RKO, breast cancer cell lines 21-PT, 21-MT, MDA-468, MDA-231, SK-BR3, and BT-474, the A549 lung cancer cell line, and the H392 glioblastoma cell line, can be used.
  • An intact cell is contacted with a test compound.
  • Binding of the test compound to a DBI/ACBP-like polypeptide or polynucleotide is determined as described above, after lysing the cell to release the DBI/ACBP-like polypeptide-or polynucleotide-test compound complex.
  • Test compounds can be tested for the ability to increase or decrease a DBI/ACBP- like activity of a DBI/ACBP-like polypeptide.
  • Binding activity of a DBIJACBP-like protein can be measured, for example, after contacting either a purified DBI/ACBP- like polypeptide, a cell extract, or an intact cell with a test compound.
  • a test compound which decreases DBI/ACBP-like binding activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing cell proliferation.
  • a test compound which increases DBI/ACBP-like binding activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing cell proliferation.
  • test compounds which increase or decrease DBI/ACBP-like gene expression are identified.
  • a DBI/ACBP-like polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the DBI/ACBP-like polynucleotide is determined.
  • the level of expression of DBI/ACBP-like mRNA or polypeptide in the presence of the test compound is compared to the level of expression of DBI/ACBP-like 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.
  • test compound when expression of DBI/ACBP-like mRNA or polypeptide is greater in the presence of the test com- pound than in its absence, the test compound is identified as a stimulator or enhancer of DBI/ACBP-like mRNA or polypeptide expression.
  • test compound when expression of DBI/ACBP-like 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 DBI/ACBP-like mRNA or polypeptide expression.
  • the level of DBI/ACBP-like mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptides. Either qualitative or quantitative methods can be used.
  • polypeptide products of a DBI/ACBP-like 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 inco ⁇ oration of labeled amino acids into a DBI/ACBP-like polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses a DBI/ACBP-like polynucleotide can be used in a cell-based assay system.
  • the DBI/ACBP-like 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.
  • Particular anti-DBI/ACBP-like protein antibodies are useful for the diagnosis of conditions or diseases characterized by expression of DBI/ACBP-like protein or in assays to monitor patients being treated with DBI/ACBP-like protein, agonists or inhibitors.
  • Diagnostic assays for DBI/ACBP-like protein include methods utilizing an antibody and a label to detect DBI/ACBP-like protein in human body fluids or extracts of cells or tissues.
  • the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, polypeptides and/or antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. Many suitable reporter molecules are known in the art.
  • DBI/ACBP-like protein A variety of protocols for measuring DBI/ACBP-like protein, using either polyclonal or monoclonal antibodies which specifically bind to DBI/ACBP-like protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). For ex- ample, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on DBI ACBP-like protein can be used. Alternatively, a competitive binding assay may be employed. These assays are described, for example, in Maddox et al. (J. Exp. Med. 158, 1211, 1983).
  • DBI/ACBP-like protein expression To provide a basis for diagnosis, normal or standard values for DBI/ACBP-like protein expression must be established. This can be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with an antibody to DBI/ACBP-like protein under conditions suitable for complex formation which are well known in the art. The amount of standard complex formation can be quantified by comparing various artificial membranes containing known quantities of
  • DBI/ACBP-like protein with both control and disease samples from biopsied tissues. Then, standard values obtained from normal samples can be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
  • a polynucleotide encoding a DBI/ACBP-like polypeptide can be used for diagnostic pu ⁇ oses.
  • a DBI/ACBP-like polypeptide can be used to detect and quantitate gene expression in biopsied tissues in which it is desired to detect expression of DBI/ACBP-like protein.
  • the diagnostic assay is useful to distinguish between absence, presence, and excess expression of DBI/ACBP-like protein and to monitor regulation of DBI/ACBP-like protein levels during therapeutic intervention.
  • hybridization or PCR probes which can detect polynucleotide sequences encoding DBI/ACBP-like protein or closely related molecules can be used diagnosti- cally.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' region, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring DBI/ACBP-like protein, alleles, or related sequences.
  • Probes also can be used for the detection of related sequences and should preferably contain at least 50% of the nucleotides from a DBI/ACBP-like protein encoding sequence.
  • Hybridization probes can be derived from the nucleotide sequence of SEQ ID NO:l or from genomic sequence including promoter, enhancer elements and in- trons of the naturally occurring DBI/ACBP-like protein.
  • Hybridization probes can be labeled by a variety of reporter groups, including radionuclides such as 32 P or 32 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
  • DBI/ACBP-like polynucleotides can be used to diagnose of conditions or diseases associated with altered expression of DBI/ACBP-like protein.
  • DBI/ACBP-like protein For example,
  • DBI/ACBP-like polynucleotides can be used in hybridization or PCR assays of fluids or tissues from biopsies to detect DBI/ACBP-like protein expression. Detection can be qualitative or quantitative and can be carried out using Southern or Northern analysis, dot blot or other membrane-based technologies, PCR technologies, or dip stick, pin, chip, or ELISA technologies. All of these techniques are well known in the art and are the basis of many commercially available diagnostic kits.
  • the DBI/ACBP-like protein-encoding sequence shown in SEQ ID NO:l provides the basis for assays that detect activation or induction associated with inflammation or disease.
  • DBI/ACBP-like polynucleotides can be labeled by methods known in the art and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After an incubation period, the sample is washed with a compatible fluid which optionally contains a dye (or other label requiring a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed off, the dye is quantitated and compared with a standard.
  • the nucleotide sequence has hybridized with nucleotide sequences in the sample, and the presence of elevated levels of DBI/ACBP-like polynucleotide sequences in the sample indicates the presence of the associated inflammation and/or disease.
  • Such assays also can be used to evaluate the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for DBI/ACBP-like protein expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with DBI/ACBP-like protein, or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained for normal subjects with a dilution series of DBI/ACBP-like protein run in the same experiment where a known amount of substantially purified DBI/ACBP-like protein is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients afflicted with DBI/ACBP-like protein-associated diseases. Deviation between standard and subject values is used to establish the presence of disease.
  • a therapeutic agent is administered and a treatment profile is generated.
  • Such assays may be repeated on a regular basis to evaluate whether the values in the profile progress toward or return to the normal or standard pattern.
  • Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • compositions of the invention can comprise, for example, a DBI/ACBP-like polypeptide, DBI/ACBP-like polynucleotide, antibodies which specifically bind to DBI/ACBP-like protein, or mimetics, agonists, antagonists, or inhibitors of DBI/ACBP-like binding activity.
  • the 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, bio- compatible pharmaceutical carrier, including, but not limited to, saline, buffered sa- line, dextrose, and water.
  • 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.
  • Pharmaceutical preparations 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, hydroxy- propylmethyl-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 which 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 poly- ethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspen- sions 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 sus- pension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suitable stabilizers or agents which 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% manni- tol, 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.
  • Peripheral benzodiazepine receptors for which DBI/ACBP-like protein is a ligand, are upregulated in several different types of cancers (8, 16-18).
  • the human DBI/ACBP-like gene and protein provides a therapeutic target for decreasing cell proliferation, in particular for treating cancers. Upregulation of PBR and DBI/ACBP-like protein are tightly correlated to increased tumor cell proliferation (16). Suppression of DBI/ACBP-like protein can therefore be used to suppress tu- mor cell proliferation for treatment of various cancers.
  • Cancers whose proliferation can be suppressed according to the invention include cancers of the brain (including astrocytomas, gliobastomas, and medulloblastomas), breast, liver, kidney, adrenal gland, intestine, salivary gland, and testis.
  • the human DBI/ACBP-like gene product may be used in the diagnosis and treatment of conditions, disorders or diseases associated with abnormal function of paraganglia, including paragangliomas.
  • the clinical features and morbidity of paragangliomas are due predominantly to the abnormal release of catecholamines. Hypertension is the most common manifestation.
  • Paraganglioma is a correctable cause of high blood pressure. Indeed, it is rarely fatal if properly diagnosed and treated.
  • DBI/ACBP-like protein may be useful in the regulation of the biosynthesis or metabolism of biological molecules such as catecholamines in paraganglia. Molecules associated with paraganglial function, or their precursors or metabolic products, may bind to DBI/ACBP-like protein in a manner analogous to that of fatty acyl-CoAs to
  • DBI/ACBP DBI/ACBP-like protein may sequester and protect the bound ligand from unwanted side-reactions such as hydrolysis or oxidation, or present the bound ligand to a receptor molecule.
  • DBI/ACBP-like protein or a fragment thereof may act as a neuromodulator of catecholamine-induced responses in para- ganglia.
  • DBI/ACBP octadecane- neuropeptide
  • ODN octadecane- neuropeptide
  • DBI/ACBP octadecane- neuropeptide
  • Benzodiazepines enhance GABA-mediated synaptic inhibitory responses and reduce pathological anxiety.
  • DBI/ACBP inhibits binding of benzodiazepines to the GABA ⁇ receptor.
  • regulation of DBI/ACBP-like protein could be used to treat various neurological disorders.
  • DBI/ACBP-like protein has been shown to suppress glucose-induced insulin release from pancreatic islet cells (3). Therefore, DBI/ACBP-like protein could be used in the treatment of autoimmune disorders such as diabetes in which insulin regulation is affected.
  • DBI/ACBP-like protein also allows its use in delivery of other therapeutic molecules.
  • DBI/ACBP-like protein may bind therapeutic agents or drugs which are ordinarily insoluble or only slightly soluble in water.
  • the high charge density of the DBI/ACBP-like protein molecule renders the protein-ligand complex soluble in an aqueous environment.
  • the specificity and binding affinities of DBI/ACBP-like protein for therapeutic ligands may be manipulated by protein engineering techniques known to those skilled in the art.
  • 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 polypeptide-bind- ing partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of ac- tion 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 DBI/ACBP-like protein activity can be administered to a human cell, either in vitro or in vivo, to reduce DBI/ACBP-like protein activity.
  • the reagent preferably binds to an expression product of a DBI/ACBP-like gene. If the expression product is a polypeptide, for example, the reagent can be an antibody or a small chemical compound.
  • a reagent can be added to a preparation of stem cells which 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 or liver.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 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 composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell 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 which 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); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988);
  • a polynucleotide 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 phos- phate-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 of DNA.
  • the reagent is preferably an antisense oligo- nucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a DBI/ACBP-like gene or the activity of a DBI/ACBP-like 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 DBI/ACBP-like gene or the activity of a DBI/ACBP-like polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to DBI/ACBP-like- specific mRNA, quantitative RT-PCR, immunologic detection of a DBI/ACBP-like polypeptide, or measurement of DBI/ACBP-like activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeu- tic 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. Determination of a Therapeutically Effective Dose
  • a therapeutically effective dose refers to that amount of ac- tive ingredient which increases or decreases DBI/ACBP-like activity relative to
  • 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 which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to pro- vide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clear- ance rate of the particular formulation.
  • Normal dosage amounts of any particular reagent can vary from 0.1 to 100,000 mi- crograms, 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 litera- ture and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for polypeptides or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • 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.
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-DBI/ACBP-like polypeptide obtained is trans- fected into human embryonic kidney 293 cells. DBI/ACBP-like polypeptide is expressed, isolated and used in an acyl-CoA competition binding assay as follows:
  • binding buffer 10 mM potassium phosphate buffer, pH 7,4.
  • DBI/ACBP- like polypeptide (0,05 pmol) is added in 25 ⁇ l of binding buffer.
  • the samples are mixed and incubated at 37°C for 30 min, chilled on ice for 10 min, and mixed with 0,2 ml of an ice-cold 50% slurry of Lipidex 1000 (Canberra Packard) in binding buffer. After 100 min on ice, the samples are centrifuged at 12000 x g for 5 min at 0
  • the radioactivity in 100 ⁇ l of the resulting supernatant is determined by scintillation counting.
  • the assay is carried out in triplicate, and controls with bovine ACBP are performed. DBI/ACBP-like protein activity of the polypeptide with the amino acid sequence of SEQ ID NO: 2 is shown.
  • DBI/ACBP-like 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.
  • DBI/ACBP-like polypeptides comprise an amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag.
  • the sam- pies 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 an DBI/ACBP-like polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound was not incubated is identified as a compound which binds to an DBI/ACBP-like polypeptide.
  • the binding of a test compound to DBI/ACBP-like protein is assayed by monitoring the resulting changes in enthalpy (heat production or abso ⁇ tion) in an isothermal titration microcalorimeter (Micro-Cal Inc, Northampton Mass.). Titration micro- calorimetry measurements do not require labeling of the test compound or DBI/ACBP-like protein molecules; detection is based solely on the intrinsic change in the heat of enthalpy upon binding.
  • Multiple computer-controlled injections of a known volume of test compound solution are directed into a thermally-controlled chamber containing DBI/ACBP-like protein. The change in enthalpy after each injection is plotted against the number of injections, producing a binding isotherm.
  • test compound is administered to a culture of paraganglioma cells and incubated at
  • RNA is isolated from the two cultures as described in Chirgwin et al., Biochem. 18, 5294-99, 1979).
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled DBI/ACBP-like protein-specific probe at 65 ° C in Ex- press-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from SEQ ID NO:l.
  • a test compound which decreases the DBI/ACBP-like protein-specific signal relative to the signal obtained in the absence of the test com- pound is identified as an inhibitor of DBI/ACBP-like gene expression.
  • oligonucleotides comprising at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:l are synthesized on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al., Chem. Rev. 90, 534-83, 1990). Following assembly and deprotec- tion, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electrophoreses and ion exchange HPLC.
  • PBS phosphate-buffered saline
  • Endo- toxin levels in the oligonucleotide preparation are determined using the Limulus Amebocyte Assay (Bang, Biol. Bull (Woods Hole, Mass.) 105, 361-362, 1953).
  • An aqueous composition containing the antisense oligonucleotides at a concentration of 0.1-100 ⁇ M is injected directly into an astrocytoma with a needle. The needle is placed in the tumors and withdrawn while expressing the aqueous composition within the astrocytoma.
  • the astrocytoma is monitored over a period of days or weeks. Additional injections of the antisense oligonucleotides can be given during that time. The size of the astrocytoma is decreased due to decreased DBI/ACBP-like activity.
  • ACBP Adyl-CoA-binding protein
  • Venturini I. et al., "Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver" Life Sci 65(21):2223-31 (1999).

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Abstract

On peut utiliser des réactifs régulant l'activité de liaison de la protéine humaine semblable à DBI/ACBP et des réactifs se liant à des produits génétiques de protéine semblable à DBI/ACBP afin de réguler la prolifération cellulaire. Cette régulation est particulièrement utile pour traiter des cellules malignes, des maladies neurologiques et s'applique également à la biosynthèse des stéroïdes.
PCT/EP2001/002780 2000-03-14 2001-03-13 REGULATION DE LA PROTEINE HUMAINE SEMBLABLE A ACYLE CoA/INHIBITEUR DE LIAISON DU DIAZEPAM WO2001068840A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020160183A1 (fr) * 2019-01-29 2020-08-06 Holobiome, Inc. Procédés et compositions pour traiter et prévenir des troubles du système nerveux central et d'autres états provoqués par une dysbiose microbienne intestinale

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006847A1 (fr) * 1996-08-16 1998-02-19 Incyte Pharmaceuticals, Inc. Proteine semblable a la dbi/acbp humaine
WO2001025436A2 (fr) * 1999-10-05 2001-04-12 Curagen Corporation Polypeptides de type endozepine et polynucleotides codant ces derniers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006847A1 (fr) * 1996-08-16 1998-02-19 Incyte Pharmaceuticals, Inc. Proteine semblable a la dbi/acbp humaine
WO2001025436A2 (fr) * 1999-10-05 2001-04-12 Curagen Corporation Polypeptides de type endozepine et polynucleotides codant ces derniers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EM_HUM [Online] EMBL Heidelberg, Germany; AC AC010999, 6 November 1999 (1999-11-06) ROWEN L ET AL.: "Sequencing of human chromosome 15 D15S146-D15S117 region" XP002182180 *
LIHRMANN I ET AL: "Frog diazepam-binding inhibitor: Peptide sequence, cDNA cloning, and expression in the brain." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 91, no. 15, 1994, pages 6899-6903, XP002182179 1994 ISSN: 0027-8424 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020160183A1 (fr) * 2019-01-29 2020-08-06 Holobiome, Inc. Procédés et compositions pour traiter et prévenir des troubles du système nerveux central et d'autres états provoqués par une dysbiose microbienne intestinale

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