WO2004003190A1 - Polypeptide identifie comme cytochrome p450 - Google Patents

Polypeptide identifie comme cytochrome p450 Download PDF

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Publication number
WO2004003190A1
WO2004003190A1 PCT/GB2003/002824 GB0302824W WO2004003190A1 WO 2004003190 A1 WO2004003190 A1 WO 2004003190A1 GB 0302824 W GB0302824 W GB 0302824W WO 2004003190 A1 WO2004003190 A1 WO 2004003190A1
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Prior art keywords
polypeptide
nucleic acid
disease
acid molecule
cytochrome
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PCT/GB2003/002824
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English (en)
Inventor
Christopher Benjamin Phelps
Richard Joseph Fagan
Ellie Louise James
Valerie Nathalie Pierron
Lindsey Reynolds
Sarah Helen Kamp
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Inpharmatica Limited
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Priority to AU2003236932A priority Critical patent/AU2003236932A1/en
Publication of WO2004003190A1 publication Critical patent/WO2004003190A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0071Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • This invention relates to a protein, termed hCP44020.1 herein identified as a novel Cytochrome P450 and to the use of this protein and nucleic acid sequence from the encoding gene in the diagnosis, prevention and treatment of disease. All publications, patents and patent applications cited herein are incorporated in full by reference.
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
  • This tool is a database system, termed the Biopendium search database, that is the subject of co-pending International Patent Application No. PCT/GBO 1/01105, published as WOOl/69507.
  • This database system consists of an integrated data resource created using proprietary technology and containing information generated from an all-by-all comparison of all available protein or nucleic acid sequences.
  • sequence data from separate data resources is to combine as much data as possible, relating both to the sequences themselves and to information relevant to each sequence, into one integrated resource. All the available data relating to each sequence, including data on the three-dimensional structure of the encoded protein, if this is available, are integrated together to make best use of the information that is known about each sequence and thus to allow the most educated predictions to be made from comparisons of these sequences.
  • the annotation that is generated in the database and which accompanies each sequence entry imparts a biologically relevant context to the sequence information.
  • P450s are a large superfamily of enzymes all of which use a heme bound iron atom to catalyse the insertion of an oxygen atom into a substrate.
  • the overall reaction of a P450 converts an organic substrate, molecular oxygen and NADPH to a hydroxylated organic substrate, water and NADP+.
  • the oxidation of NADPH is generally carried out by a separate enzyme or enzymes: P450 reductase or ferredoxin and ferredoxin reductase, which subsequently transfer the electrons to the P450. Examples have been found where P450 and P450 reductase have been fused however. Subsequent rearrangements and reactions of the hydroxylated product lead to P450s catalysing over 40 known reactions.
  • P450s catalyse the oxygenation of a large range of substrates: over 1000 are known to date and there may be 10 6 in total.
  • This broad range of biochemical functions gives P450s a similarly broad range biological functions: detoxification of harmful chemicals, activation (by modification) of beneficial drug precursors and hormones, activation of harmful chemicals (such as carcinogens), breakdown and synthesis of steroids, vitamins, fatty acids, pigments, pheromones, insecticides amongst other classes of biological molecule.
  • P450s are found in nearly all known organisms including plants, animals, fungi and bacteria. In mammals P450s are found in most tissues though their concentration is highest in the liver where the detoxification of many chemicals takes place.
  • P450 genes are also implicated in growth and differentiation of cells due to their tissue and developmental specific expression patterns.
  • the P450s' role in the metabolism (both activation and deactivation) of drugs and carcinogens has made them the subject of much medical interest.
  • the susceptibility of a drug to inactivation by P450s may make it biologically inactive.
  • drugs are administered in an inactive form and only become active when they have passed through the liver and been altered by P450s once and even twice.
  • P450s have been the subject of extensive site-directed mutagenesis experiments which have aimed to determine residues essential for substrate binding specificity.
  • Most P450 structures are of soluble bacterial enzymes though there have been efforts to homology model mammalian enzymes in order to aid understanding of variations in substrate binding specificities and to aid rational drug design efforts.
  • P450s are also of medical interest because of their potential as antibiotic targets. P450s catalyse the formation of many crucial biological compounds required by pathogens and inhibitors of P450 activity are usually strong antibiotics.
  • Inhibitors of P450s could stop inactivation of drugs and activation of tumour promoters.
  • the drug exemestane has been approved for use as an inhibitor of aromatase P450 in breast cancer.
  • Azole antifungals such as Nizoral and Diflucan inhibit the P450 lanosterol demethylase, which catalyses the synthesis of ergosterol, a major component of fungal plasma membranes.
  • Recent studies have also crystallised a Mycobacterium tuberculosis P450 in complex with two different azole inhibitors, 4-phenylimidazole (4-PI) and FLU, helping understanding of binding of these important antifungals.
  • novel P450s As these proteins are implicated in the diseases identified above, as well as in other disease states.
  • the identification of novel P450s in bacterial, fungal and human systems is therefore extremely relevant for the treatment and diagnosis of disease, particularly those identified above.
  • the invention is based on the discovery that the hCP44020.1 protein (P450G3) functions as a Cytochrome P450.
  • P450G3 functions as a Cytochrome P450.
  • hCP44020.1 protein it has been found that a region including residues 57 to 516 of this protein sequence adopts an equivalent fold to residues 1 to 446 of the Rabbit Cytochrome P450 2C5 (PDB code 1DT6:A).
  • Rabbit Cytochrome P450 2C5 is known to function as a Cytochrome P450. This relationship is not just to Rabbit Cytochrome P450 2C5, but rather to the Cytochrome P450 family as a whole.
  • results presented herein clearly indicate that the hCP44020.1 transcript is present at detectable levels in a variety of human tissues and cell lines. This confirms the relevance of the hCP44020.1 protein as an important target for further biochemical characterisation.
  • the particular tissues and cell lines identified herein as expressing the hCP44020.1 protein represent ideal targets for further studies of hCP44020.1 protein function in vivo. Such studies may, for example, make use of the ligands identified using the assays and screening methods disclosed herein to investigate the effects of inducing or inhibiting hCP44020.1 protein function.
  • the cloning of the hCP44020.1 protein allows for high-level expression, purification and characterisation of the polypeptides of the invention described herein.
  • the inventors have discovered that the mRNA for the hCP44020.1 protein is expressed at significant levels in the human brain.
  • P450s are involved in the synthesis and metabolism of various components of metabolic pathways including steroids, fatty acids, prostaglandins, leukotrienes, bile acids and retinoids.
  • the finding of a novel P450 that is expressed preferentially in the human brain is consistent with a role for this P450 in regulating metabolic pathways associated with inflammatory conditions in the brain such as multiple sclerosis and dementia.
  • neurosteroids have been shown to influence neurotransmission particularly in the field of receptors such as those for GABA and NMDA and Sigma receptors. Neurosteroids have been shown to play a neuroprotective role.
  • Therapeutic intervention through the development of agonists or antagonists to the hCP44020.1 protein may therefore have a role in treatment of neurodegenerative conditions such as dementia, Parkinson's disease and neurodegeneration following cerebrovascular disease such as infarction or haemorrhage (stroke) and trauma to the central nervous system and spinal cord.
  • neurodegenerative conditions such as dementia, Parkinson's disease and neurodegeneration following cerebrovascular disease such as infarction or haemorrhage (stroke) and trauma to the central nervous system and spinal cord.
  • neurosteroids have been shown to influence cognitive processing, spatial learning and memory, anxiety and behaviours such as craving which leads to addictive behaviour patterns.
  • Development of agonists and antagonists to the hCP44020.1 protein may therefore lead to therapeutic intervention to treat dementias, learning difficulties, anxiety and addictive behaviours including alcoholism, eating disorders and drug addiction.
  • the inventors have shown that the hCP44020.1 protein is not exclusively expressed in the brain. Significant levels of mRNA are also found in the testis and placenta. The testis and placenta are significant sites for the biosynthesis of steroids and the expression of the hCP44020.1 protein in these tissues is consistent with a role for P450G3 in steroid action.
  • the invention provides a polypeptide, which polypeptide:
  • (ii) is a fragment thereof having Cytochrome P450 activity or having an antigenic determinant in common with the polypeptides of (i); or
  • a polypeptide according to the first aspect of the invention consists of the amino acid sequence as recited in SEQ ID NO:2.
  • polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "the P450G3 polypeptide”.
  • polypeptides that comprise amino acid sequence or structural features that can be identified as conserved features within the polypeptides of the P450 family, such that the polypeptide's interaction with ligand is not substantially affected detrimentally in comparison to the function of the full length wild type polypeptide.
  • the polypeptide exhibits a characteristic P450 spectrum such that when the heme iron is complexed with carbon monoxide, the spectrum shows a Soret absorption maximum at around 450nm (Garfinkel, 1958, Arch. Biochem. Biophys. 77: 493-509; Klingenberg, 1958, Arch. Biochem. Biophys; Omuar & Sato, 1964, J Biol. Chem., 239:2370).
  • the polypeptides and functional equivalents according to the invention function as P450 enzymes.
  • functions as a P450 enzyme we mean that the polypeptide retains its ability to convert an organic substrate, NADPH and molecular oxygen to NADP+, a hydroxylated organic substrate and water.
  • the ability of a polypeptide to hydroxylate a substrate may be determined by using a suitable assay known in the art.
  • a preferred polypeptide fragment according to part ii) above includes the region of the P450G3 polypeptide that is predicted as that responsible for Cytochrome P450 activity (hereafter, the "P450G3 Cytochrome P450 region"), or is a variant thereof.
  • the P450G3 Cytochrome P450 region is considered to extend between residue 57 and residue 516 of the P450G3 polypeptide sequence.
  • This aspect of the invention also includes fusion proteins that incorporate polypeptide fragments and variants of these polypeptide fragments as defined above, provided that said fusion proteins possess activity as a Cytochrome P450.
  • the invention provides a purified nucleic acid molecule that encodes a polypeptide of the first aspect of the invention.
  • the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NO:l (encoding the P450G3 polypeptide), or is a redundant equivalent or fragment of this sequence.
  • a preferred nucleic acid fragment is one that encodes a polypeptide fragment according to part ii) above, preferably a polypeptide fragment that includes the P450G3 Cytochrome P450 region, or that encodes a variant of these fragments as this term is defined above.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the host cells of the invention may co-express a reductase protein which forms an active complex with the polypeptides of the first aspect of the invention and thus maximises the activity of the polypeptides of the invention in the cell.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the Cytochrome P450 activity of, a polypeptide of the first aspect of the invention.
  • Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods dislcosed herein.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the function of the region defined herein as the P450G3 Cytochrome P450 region of the P450G3 polypeptide allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of diseases in which Cytochrome P450s are implicated. Examples of suitable assays and screening methods are provided herein.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of disease.
  • These molecules may also be used in the manufacture of a medicament for the treatment of cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity; AIDS, renal disease, infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.
  • cell proliferative disorders including neoplasm, melanoma, lung, colorectal, breast, pancreas
  • these molecules may be used in the manufacture of a medicament for the treatment of: diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); and diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviours such as alcoholism, eating disorders and drug addiction.
  • diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia
  • diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine)
  • diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and
  • moieties of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
  • the invention also provides kits that are useful in these methods for diagnosing disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • the disease diagnosed by a method of the ninth aspect of the invention is a disease in which Cytochrome P450s are implicated.
  • Diseases which may be diagnosed by a method according to the ninth aspect of the invention include treatment of cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity; AIDS, renal disease, infections including viral infection, bacterial infection, fungal infection and parasitic infection and other
  • diseases which may be diagnosed include: diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); and diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviours such as alcoholism, eating disorders and drug addiction.
  • the invention provides for the use of a polypeptide of the first aspect of the invention, or a fragment thereof, as a Cytochrome P450.
  • Suitable uses of the polypeptides of the invention as a P450 include use as a regulator of cellular growth, metabolism or differentiation, use as part of a receptor/ligand pair and use as a diagnostic marker for a physiological or pathological condition selected from the list given above.
  • the invention also provides use of the polypeptides of the invention to hydroxylate organic substrates, using assays described above.
  • the invention includes use of the polypeptides of the invention as drug metabolising enzymes.
  • the invention also provides for the use of a nucleic acid molecule according to the second or third aspects of the invention to express a protein that possesses Cytochrome P450 activity.
  • Such nucleic acid molecules are of utility in the production of the polypeptides of the invention, which polypeptides are useful in a variety of situations, as described above.
  • the invention also provides a method for effecting Cytochrome P450 activity, said method utilising a polypeptide of the first aspect of the invention, or a fragment thereof.
  • the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell according to a fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, such as cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's
  • a disease such as cell pro
  • diseases which may be treated include: diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); and diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviours such as alcoholism, eating disorders and drug addiction.
  • diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia
  • diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine)
  • diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviour
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • the disease is one in which P450s are implicated.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • a fusion protein may contain one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the P450G3 polypeptide. Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide.
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the P450G3 polypeptide.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group;
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the P450G3 polypeptide, or with active fragments thereof, of greater than 30%. More preferred polypeptides have degrees of identity of greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the P450G3 polypeptide, or with active fragments thereof.
  • preferred active fragments of the P450G3 polypeptide are those that include the P450G3 Cytochrome P450 region.
  • this aspect of the invention includes polypeptides that have degrees of identity of greater than 30%, preferably, greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively, with the Cytochrome P450 region of the P450G3 polypeptide.
  • the P450G3 Cytochrome P450 region is considered to extend between residue 57 and residue 516 of the P450G3 polypeptide sequence.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome ThreaderTM technology that forms one aspect of the search tools used to generate the Biopendium search database may be used (see co-pending International patent application PCT/GBO 1/01105 published as WOO 1/67507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the P450G3 polypeptide, are predicted to have Cytochrome P450 activity, by virtue of sharing significant structural homology with the P450G3 polypeptide sequence.
  • the Inpharmatica Genome ThreaderTM predicts two proteins, or protein regions, to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and ibove.
  • the certainty value of the Inpharmatica Genome ThreaderTM is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome Threader exclusively using sequences of known structure. Some of the comparisons were between proteins that were known to be related (on the basis of structure). A neural network was then trained on the basis that it needed to best distinguish between the known relationships and known not-relationships taken from the CATH structure classification (www.biochem.ucl.ac.uk/bsm/cath).
  • Structural homologues of P450G3 should share structural homology with the P450G3 Cytochrome P450 region. Such structural homologues are predicted to have Cytochrome P450 activity by virtue of sharing significant structural homology with this polypeptide sequence.
  • polypeptides of the first aspect of the invention also include fragments of the P450G3 polypeptide, functional equivalents of the fragments of the P450G3 polypeptide, and fragments of the functional equivalents of the P450G3 polypeptides, provided that those functional equivalents and fragments retain Cytochrome P450 activity or have an antigenic determinant in common with the P450G3 polypeptide.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the P450G3 polypeptides or one of its functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Preferred polypeptide fragments according to this aspect of the invention are fragments that include a region defined herein as the P450G3 Cytochrome P450 region of the P450G3 polypeptide. This region is the region that has been annotated as Cytochrome P450. For the P450G3 polypeptide, this region is considered to extend between residue 57 and residue 516. Variants of this fragment are included as embodiments of this aspect of the invention, provided that these variants possess activity as a Cytochrome P450.
  • variable is meant to include extended or truncated versions of this polypeptide fragment.
  • Cytochrome P450 region of the P450G3 polypeptide will fold correctly and show Cytochrome P450 activity if additional residues C terminal and/or N terminal of these boundaries in the P450G3 polypeptide sequence are included in the polypeptide fragment.
  • an additional 5, 10, 20, 30, 40 or even 50 or more amino acid residues from the P450G3 polypeptide sequence, or from a homologous sequence may be included at either or both the C terminal and/or N terminal of the boundaries of the Cytochrome P450 regions of the P450G3 polypeptide, without prejudicing the ability of the polypeptide fragment to fold correctly and exhibit Cytochrome P450 activity.
  • one or more amino acid residues may be deleted at either or both the C terminus or the N terminus of the Cytochrome P450 region of the P450G3 polypeptide.
  • variant includes homologues of the polypeptide fragments described above, that possess significant sequence homology with the Cytochrome P450 region of the P450G3 polypeptide provided that said variants retain activity as an Cytochrome P450.
  • variant homologues of polypeptide fragments of this aspect of the invention have a degree of sequence identity with the P450G3 Cytochrome P450 region of the P450G3 polypeptides of greater than 40%.
  • variant polypeptides have degrees of identity of greater than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the P450G3 Cytochrome P450 region of the P450G3 polypeptides, provided that said variants retain activity as a Cytochrome P450.
  • variant polypeptides also include homologues of the truncated forms of the polypeptide fragments discussed above, provided that said variants retain activity as a Cytochrome P450.
  • polypeptide fragments of the first aspect of the invention may be polypeptide fragments that exhibit significant structural homology with the structure of the polypeptide fragment defined by the P450G3 Cytochrome P450 regions, of the P450G3 polypeptide sequence, for example, as identified by the Inpharmatica Genome ThreaderTM. Accordingly, polypeptide fragments that are structural homologues of the polypeptide fragments defined by the P450G3 Cytochrome P450 region of the P450G3 polypeptide sequence should adopt the same fold as that adopted by this polypeptide fragment, as this fold is defined above.
  • fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment of the invention When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region.
  • certain preferred embodiments relate to a fragment having a pre - and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
  • several fragments may be comprised within a single larger polypeptide.
  • polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.
  • substantially greater affinity we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known Nuclear Hormone Receptors.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 - fold, 10 -fold, 10 -fold or greater for a polypeptide of the invention than for known Nuclear Hormone Receptors.
  • a selected mammal such as a mouse, rabbit, goat or horse
  • a polypeptide of the first aspect of the invention may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically.
  • the polypeptide can be conjugated to a carrier protein.
  • Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaf inity chromatography.
  • Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Nail. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239: 1534 (1988); Kabat et al, J. Immunol., 147: 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci.
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode the polypeptide sequences recited in SEQ ID NO:2, and functionally equivalent polypeptides, including active fragments of the P450G3 polypeptide, such as a fragment including the P450G3 Cytochrome P450 region of the P450G3 polypeptide sequence, or a homologue thereof.
  • nucleic acid molecules encompassing these stretches of sequence form a preferred embodiment of this aspect of the invention. These nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
  • the nucleic acid molecules may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • nucleic acid molecule also includes analogues of DNA and RNA, such as those containing modified backbones ,and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2, or an active fragment thereof may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:l. These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes the polypeptide SEQ ID NO:2, or an active fragment of the P450G3 polypeptide, such as a fragment including the P450G3 Cytochrome P450 region, or a homologue thereof.
  • the P450G3 Cytochrome P450 region is considered to extend between residue 57 and 516 of the P450G3 polypeptide sequence.
  • the P450G3 Cytochrome P450 region is thus encoded by a nucleic acid molecule including nucleotide 169 to nucleotide 1548. Nucleic acid molecules encompassing this stretch of sequence, and homologues of this sequence, form a preferred embodiment of this aspect of the invention.
  • nucleic acid molecules that encode the polypeptide of SEQ ID NO:2 may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non- translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • the nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
  • a preferred fragment of the P450G3 polypeptide is a fragment including the P450G3 Cytochrome P450 region, or a homologue thereof.
  • the Cytochrome P450 region is encoded by a nucleic acid molecule including nucleotide 169 to nucleotide 1548 of SEQ ID NO:l.
  • nucleic acid molecules according to the invention may be naturally-occurring variants such as a naturally-occurring allelic variant, or the molecules may be a variant that is not known to occur naturally.
  • Such non-narurally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
  • the substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).
  • the inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al [supra]).
  • a substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507- 511).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the P450G3 polypeptide (SEQ ID NO:2), and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a preferred active fragment is a fragment that includes an P450G3 Cytochrome P450 region of the P450G3 polypeptide sequences, resepctively.
  • preferred nucleic acid molecules include those that are at least 10% identical over their entire length to a nucleic acid molecule encoding the Cytochrome P450 region of the P450G3 polypeptide sequence.
  • Percentage identity is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/).
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecule having the sequence given in SEQ ID NO:l, to a region including nucleotides 169-1548 of this sequence, or a nucleic acid molecule that is complementary to any one of these regions of nucleic acid.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the P450G3 polypeptide.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the P450G3 polypeptide and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the P450G3 polypeptide, particularly with an equivalent function to the P450G3 Cytochrome P450 region of the P450G3 polypeptide is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:l), particularly a region from nucleotides 169-1548 of SEQ ID NO:l, are particularly useful probes.
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al., Proc. Natl. Acad. Sci. USA (1988) 85: 8998-9002).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1: 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • nucleic acid molecules of the present invention may be used for chromosome localisation. In this technique, a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • mapping of relevant sequences to chromosomes is an important step in the confirmatory correlation of those sequences with the gene-associated disease.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
  • RNA interference (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell.
  • expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al, (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
  • Cell-free translation systems can also be employed to produce the polypeptides of the invention.
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • a control sequence such as a signal peptide or leader sequence
  • These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation.
  • Such regulatory sequences may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • stable expression is preferred.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
  • Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
  • Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells Any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt ⁇ cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression- of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may 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 bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al.
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • a vector for the production of an mRNA probe Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992) Prot. Exp. Purif.
  • polypeptide is to be expressed for use in screening assays, it may be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FACS fluorescence activated cell sorting
  • the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises: 'a) contacting a cell expressing on the surface thereof the polypeptide, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound.
  • polypeptide may, for example, be artificially anchored to the cell membrane, or form part of a chimeric receptor.
  • An alternative method may involve contacting a labelled or unlabeled compound with a polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • An example of a method for determining the level of a signal generated from the interaction of the compound with the polypeptide is using FRET detection of a ligand bound to the polypeptide in the presence of peptide co-activators (Norris et al, Science 285, 744, 1999).
  • the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
  • the method for identifying agonist or antagonist of a polypeptide of the present invention comprises : determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, or to any other solid support such as those described above, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be a competitor which may act as an agonist or antagonist.
  • the ligand is labelled.
  • a method of screening for a polypeptide antagonist or agonist compound comprises the steps of: (a) incubating a labelled ligand with a whole cell expressing a polypeptide according to the invention on the cell surface, or a cell membrane containing a polypeptide of the invention, or a solid support to which the polypeptide is bound,
  • step (b) measuring the amount of labelled ligand bound to the polypeptide on the solid support, whole cell or the cell membrane; (c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
  • step (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • the P450G3 polypeptides of the present invention may promote the metabolism of drugs.
  • the ability of the P450G3 polypeptides to promote metabolism of particular drugs can be examined and the methods described above can be used to identify agonists and antagonists of the drug metabolising effect of the P450G3 polypeptides.
  • assays for identifying substrates and antagonists of the P450G3 polypeptides are conducted using a system in which activity of the P450G3 polypeptides is maximised. This may be achieved by co-expressing the P450G3 polypeptide with a reductase protein that produces an active complex to screen for antagonists. Alternatively, the P450G3 protein and the reductase protein may be produced separately and introduced into the assay system.
  • Microsomal cytochromes occur on the membrane of the ER and require NADPH cytochrome reductase and a flavoprotein for activity, whereas mitochondrial cytochromes occur on the inner membrane and ferredoxin and NADPH ferredoxin reductase for activity (Beckman, M., and DeLuca, H. (1997) Methods in Enzymol. 282, 200-223; Armbrecht, H.J., Okuda, K., Wongsurawat, N., Nemani, R., Chen, M., and Boltz, M. (1992) J. Steroid Biochem. Molec. Biol. 43, 1073-1081.
  • human NADPH CYP-reductase may be used to maximise the activity of a P450G3 polypeptide of the invention in assays screening for antagonists.
  • polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose-dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • compositions comprising a polypeptide, nucleic acid, vector, host cell, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, vector, host cell, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targetted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also 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.
  • an effective amount for a human subject will depend upon 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. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg.
  • Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • an inhibitor compound as described above
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered.
  • polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered.
  • Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • 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 regulatory molecules.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection see, for example, www.powderject.com
  • jet injection may also be useful in the formulation of vaccine compositions.
  • This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques. Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291 -294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and, c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Such diagnostics are particularly useful for prenatal and even neonatal testing.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al., Genomics, 5, 874-879
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. Further, point mutations and other sequence variations, such as polymorphisms, can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
  • FISH Fluorescence in situ hybridization
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996) 274: 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al).
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • reporter molecules A wide variety of reporter molecules known in the art may be used, several of which are described above. Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise: (a) a nucleic acid molecule of the present invention
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease, particularly cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity; AIDS, renal disease, infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.
  • cell proliferative disorders including neoplasm, melanoma, lung, colorectal, breast, pancre
  • diseases which may be diagnosed include: diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); and diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviours such as alcoholism, eating disorders and drug addiction.
  • diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia
  • diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine)
  • diseases associated with neurosteroid synthesis including dementia, Parkinson's diseases, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviour
  • Figure 1 Front page of the BiopendiumTM. Search initiated using 1DT6:A.
  • Figure 2A Inpharmatica Genome ThreaderTM results of search using 1DT6:A.
  • the arrow points to P33261, Human Cytochrome P450 2C19, a typical Cytochrome P450 family member.
  • Figure 2B Inpharmatica Genome ThreaderTM results of search using 1DT6:A.
  • the arrow points to the hCP44020.1 (P450G3) protein.
  • Figure 2C Inpharmatica PSI-Blast results from search using 1DT6:A.
  • the arrow points to P24903 , human Cytochrome P450 2F 1 , a typical Cytochrome P450 family member.
  • Figure 2D Inpharmatica PSI-Blast results of search using 1DT6:A.
  • the arrow points to the hCP44020.1 (P450G3) protein.
  • FIG. 3 Celera PANTHER annotation display page for hCP44020.1 (P450G3).
  • Figure 4A InterPro search results for hCP44020.1 (P450G3).
  • Figure 4B NCBI conserveed Domain Database search results for hCP44020.1 (P450G3).
  • Figure 5 A Graphical view of NCBI PSI-Blast (10 iterations) results for hCP44020.1 (P450G3).
  • Figure 5B List of NCBI PSI-Blast (10 iterations) results for hCP44020.1 (P450G3).
  • Figure 6A Top NCBI non-redundent database Blastp result from search using hCP44020.1 (P450G3) as the query sequence.
  • Figure 6B NCBI protein report for AAH14952.1, a public domain sequence which is equivalent to hCP44020.1 (P450G3).
  • Figure 6C NCBI protein report for CAC09368.1, a public domain sequence which is equivalent to hCP44020.1 (P450G3).
  • Figure 6D PubMed abstract for entry relating to TH1 drosophila homolog protein.
  • Figure 7A Inpharmatica Genome ThreaderTM results of search using hCP44020.1 (P450G3) as the query sequence.
  • the arrow points to 1DT6:A, the structure of Rabbit Cytochrome P450 2C5.
  • Figure 7B Inpharmatica PSI-Blast results from search using hCP44020.1 (P450G3) as the query sequence.
  • the arrow points to Cytochrome P450 52A5, a known member of the Cytochrome P450 family.
  • Figure 7C Inpharmatica PSI-Blast results of search using hCP44020.1 (P450G3) as the query sequence.
  • the arrow points to 1DT6:A, Rabbit Cytochrome P4502C9.
  • Figure 7D Genome ThreaderTM alignment of hCP44020.1 (P450G3) and 1DT6:A.
  • the structure chosen is Rabbit Cytochrome P450 2C5, PDB code 1DT6:A ( Figure 1).
  • a search of the BiopendiumTM for homologues of 1DT6:A takes place and returns 4114 Inpharmatica Genome ThreaderTM results (selection given in Figure 2 A and 2B) and 2436 Inpharmatica PSI-Blast results (selection in Figure 2C and 2D).
  • the 4114 Genome ThreaderTM results include examples of other Cytochrome P450 family members, such as P33261, Human Cytochrome P450 2C19 ( Figure 2A, arrow).
  • hCP44020.1 P450G3, Figure 2B, arrow).
  • the Inpharmatica Genome ThreaderTM has identified residues 57-516 of a sequence, hCP44020.1 (P450G3), as having an equivalent structure to residues 1-446 of Rabbit Cytochrome P450 2C5 (PDB code: 1DT6:A). Note that due to a different numbering scheme, residues 57-516 of hCP44020.1 (P450G3) appear as 66-525 in Figure 2B. Having a structure equivalent to 1DT6:A suggests that hCP44020.1 (P450G3) is a protein that functions as a Cytochrome P450. The Inpharmatica Genome Threader identifies this with 100% confidence.
  • the 2436 Inpharmatica PSI-Blast results include examples of known Cytochrome P450s, such as P24903, Cytochrome P4502F1 ( Figure 2C, arrow). Forward iterations of Inpharmatica PSI-Blast are unable to identify the relationship between 1DT6:A and hCP44020.1 (P450G3). It is only in negative iterations that Inpharmatica PSI- Blast can identify Rabbit Cytochrome P450 2C5 (PDB code: 1DT6:A) as having a sequence relationship to residues 57-516 of hCP44020.1 (P450G3) (appears as residues 66-525 in Figure 2D).
  • the ability to identify relationships via negative iterations of PSI-Blast is a product of the all-by-all sequence comparison (reverse-maximisation) that underlies the Biopendium and is unique to Inpharmatica.
  • the identification of a relationship between 1DT6:A and hCP44020.1 (P450G3) in Inpharmatica PSI-Blast iteration -12 at a highly significant E-value of 9.0E-78 strongly supports the Genome Threader annotation of hCP44020.1 (P450G3) being a Cytochrome P450.
  • the Celera PANTHER annotation page is viewed ( Figure 3).
  • PANTHER annotates hCP44020.1 (P450G3) as Gene name HSPC130, Sub-Family ID SF1 and Sub-Family name TH1 Protein-Related.
  • Celera PANTHER does not annotate hCP44020.1 (P450G3) as having a Cytochrome P450 domain.
  • Celera PANTHER does not annotate hCP44020.1 as a Cytochrome P450.
  • hCP44020.1 In order to view what is known in the public domain secondary databases about hCP44020.1 (P450G3), the InterPro database is queried with hCP44020.1 (P450G3; Figure 4A). InterPro returns zero hits (no matches) to hCP44020.1 (P450G3). Returning zero hits means that InterPro does not identify any region of hCP44020.1 (P450G3) as containing Cytochrome P450 identity. Thus hCP44020.1 (P450G3) is unidentifiable as a Cytochrome P450 family member using InterPro.
  • NCBI conserveed Domain Database (CDD) is queried with hCP44020.1 (P450G3; Figure 4B). CDD returns zero hits to hCP44020.1 (P450G3). Returning zero hits means that CDD does not identify any region of hCP44020.1 (P450G3) as containing a Cytochrome P450 domain. Returning zero hits from CDD means that hCP44020.1 (P450G3) is unidentifiable as a Cytochrome P450 family member using CDD. NCBI provides a public domain PSI-Blast server.
  • Figure 5 A shows the graphical display of NCBI PSI-Blast results for hCP44020.1 (P450G3).
  • the horizontal axis corresponds to N-terminal to C-terminal residue numbering along the hCP44020.1 (P450G3) protein.
  • the accession codes of the sequences hit in NCBI PSI-Blast are listed in Figure 5B.
  • NCBI PSI-Blast does not annotate any region of hCP44020.1 (P450G3) as having a relationship to any known Cytochrome P450.
  • the sequence hCP44020.1 (P450G3) is not unique to the Celera dataset of human predicted proteins. Two equivalent sequences can be found at the National Centre for Biotechnology Information (NCBI) GenBank protein database, AAH14952.1 and CAC09368.1 ( Figure 6A).
  • NCBI National Centre for Biotechnology Information
  • the NCBI reports do not annotate the hCP44020.1 (P450G3) equivalent sequences AAH14952.1 and CAC09368.1 as containing a Cytochrome P450 domain.
  • the NCBI report does not annotate hCP44020.1 (P450G3) equivalent sequences AAH14952.1 and CAC09368.1 as being a Cytochrome P450 ( Figure 6B and 6C).
  • hCP44020.1 There is no further annotation for hCP44020.1.
  • the public domain information for this protein does not annotate it as containing a Cytochrome P450 domain .
  • the public domain does not annotate hCP44020.1 (P450G3) as being a Cytochrome P450.
  • Celera PANTHER annotates hCP44020.1 (P450G3) as being TH1 protein-related ( Figure 3).
  • TH1 protein is of "unknown function" as decribed in a recently published paper by Bonthron et al, 2000 ( Figure 6D). Only the Inpharmatica Genome ThreaderTM and Inpharmatica PSI-Blast are able to annotate this protein as a Cytochrome P450 family member.
  • hCP44020.1 (P450G3) is now used as the query sequence in the BiopendiumTM.
  • the Inpharmatica Genome ThreaderTM identifies 312 hits ( Figure 7A) while Inpharmatica PSI-Blast returns 2366 hits ( Figure 7B).
  • the Inpharmatica Genome ThreaderTM ( Figure 7A, arrow) identifies residues 57-516 of hCP44020.1 (P450G3) as having a structure the same as Rabbit Cytochrome P450 2C5 (PDB code: 1DT6:A) with ' 100% confidence. Note that due to a different numbering scheme, residues 57-516 of hCP44020.1 (P450G3) appear as residues 66-525 in Figure 7A.
  • hCP44020.1 a region from residue 57 to residue 516 of hCP44020.1 (P450G3) has been identified as adopting an equivalent fold to Rabbit Cytochrome P450 2C5.
  • Inpharmatica PSI-Blast also identifies the same region of hCP44020.1 (P450G3) as having a relationship with known Cytochrome P450s by the sixth positive iteration.
  • Figure 7B shows a selection of Inpharmatica PSI-Blast results and it can be seen that the sequence P24458, Candida maltosa (Yeast) Cytochrome P450 52A5 has a highly significant relationship to hCP44020.1 (P450G3), being found in the sixth positive iteration with an E- value of 7.0E-90.
  • residues 44-502 of hCP44020.1 are related to residues 35-514 of P24458, the Yeast Cytochrome P450 52A5.
  • Residues 44-502 includes almost all of the hCP44020.1 (P450G3) Cytochrome P450 region identified by Genome ThreaderTM (residues 57-516), and matches them to a region of P24458, Yeast Cytochrome P450 52A5 which contains a known Cytochrome P450 domain (residues 90-504, as determined by PFAM).
  • Inpharmatica PSI-Blast is in strong agreement with Inpharmatica Genome ThreaderTM at annotating a region between residues 57 to 516 of hCP44020.1 (P450G3) as being a Cytochrome P450. This is in contrast to public domain NCBI PSI-Blast which fails to identify any relationship between hCP44020.1 (P450G3) and known Cytochrome P450s ( Figures 5A and 5B). Only Inpharmatica Genome ThreaderTM and Inpharmatica PSI-Blast are able to identify hCP44020.1 (P450G3) as being a Cytochrome P450.
  • inpharmatica PSI-Blast also identifies a relationship between hCP44020.1 (P450G3) and the original query structure 1DT6:A (Rabbit Cytochrome P450 2C5), Figure 7C arrow.
  • the relationship between hCP44020.1 (P450G3) and 1DT6:A is found in the twelth positive iteration and has a significant E-value of 9.0E-78. This further consolidates the Genome Threader annotation of hCP44020.1 (P450G3) as being a Cytochrome P450.
  • the I.M.A.G.E. Consortium ClonelD #4860894 ("The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression" Lennon G, Auffray C, Polymeropoulos M, Soares MB. (1996) Genomics. 33(1): 151-2) was used as a cDNA source for the P450G3 sequence.
  • Primers P450G3 F and P450G3 R were used to amplify the proposed P450 encompassing amino acids 1-590 of the P450G3 from the human cDNA clone I.M.A.G.E. Consortium ClonelD #4860894.
  • additional flanking sequences are added to either side of the cytochrome P450 region as defined by Genome Threader.
  • P450G3 full-length cDNA (1-590) was cloned. PCR was carried out using the Roche Expand Polymerase (Roche Diagnostics Ltd, Lewes, UK) in 1.5 mM MgCl 2 .
  • the primer sequences were: P450G3 F
  • P450G3 R CGGGATCCTCAATGATGATGATGATGATGTTAGTTCACCATGATGAAGTTAG
  • the resulting PCR product was then cloned into the vector pGEMTEasy (Promega UK Ltd, Southampton, UK) and verified by sequence analysis. Sequences were identical to the proposed sequence of P450G3. Inserts were then cloned into the vector pET-3b (CN Biosciences, Nottingham, UK) by restriction digest with the enzymes Nde I and BamHI. The construct can then be expressed with both N- and C- terminal His Tags.
  • Taqman RT-PCR quantitation was used.
  • the TaqMan 3'- 5' exonuclease assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a doublelabeled fluorogenic probe that hybridises to the target template at a site between the two primer recognition sequences (cf. U. S. Patent 5,876,930).
  • the ABI Prism 7700 automates the detection and quantitative measurement of these signals, which are stoichiometrically related to the quantities of amplicons produced, during each cycle of amplification. In addition to providing substantial reductions in the time and labour requirements for PCR analyses, this technology permits simplified and potentially highly accurate quantification of target sequences in the reactions.
  • Figure 8 shows normalised expression of P450G3 in 22 normal human tissues.
  • Taqman RT-PCR was carried out using 15ng of the indicated cDNA using primers/probes specific for P450G3 and 18s rRNA as described in the detailed description.
  • a standard curve for target and internal control was also carried out, using between 25ng to 0.39ng of cDNA template of a typical tissue sample.
  • Cycle threshold (Ct) determinations i.e. non- integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were performed by the instrument for each reaction using default parameters. Using linear regression analysis of the standard curves, the Ct values were used to calculate the amount of actual starting target or 18s cDNA in each test sample.
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, ovary.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in ovary.
  • the expression levels of P450G3 were then normalised to the expression levels of 18s.
  • Figure 8 represents the fold expression of normalised target sequence relative to the level of expression in stomach cDNA, which is set arbitrarily to 1. Each sample was quantitated in 2 individual experiments. Figure 8 shows the mean ⁇ SEM for the multiple experiments. rlaterials and Methods
  • RNA prepared from non-diseased organs was purchased from either Ambion Europe (Huntingdon, UK) or Clontech (BD, Franklin Lakes, NJ). B. Oligo Design
  • Oligonucleotide primers and probes were designed using Primer Express software (Applied Biosystems, Foster City CA) with a GC-content of 40-60%, no G-nucleotide at the 5'-end of the probe, and no more than 4 contiguous Gs.
  • the sequence of the primers and probes were: P450G3 Fwd ACCGTCCTGTTCAAGATGTTCA
  • P450G3 Probe AAGCATGGACCCTCCTCCGGTTG
  • FAM fluorescent reporter dye
  • TAMRA fluorescent quencher dye
  • Mem 582nm
  • Primers were obtained from Sigma Genosys, UK and probes were obtained from Eurogentec, Belgium. Primer/probe concentrations were titrated in the range of 50nM to 900nM and optimal concentrations for efficient PCR reactions are determined.
  • Optimal primer and probe concentrations vary in between lOOnM and 900nM depending on the target gene amplified.
  • C. cDNA reaction cDNA was prepared using components from Applied Biosystems, Foster City CA. 50 ⁇ l reactions are prepared in 0.5ml RNase free tubes. Reactions contain 500ng total RNA; lx reverse transcriptase buffer; 5.5mM MgC12; ImM dNTP's; 2.5 ⁇ l random hexamers; 20U RNase inhibitor; and 62.5U reverse transcriptase.
  • reaction 25 ⁇ l reactions were prepared in 0.5 ml thin-walled, optical grade PCR 96 well plates (Applied Biosystems, Foster City CA). Reactions contain: lx final concentration of TaqMan Universal Master Mix (a proprietary mixture of AmpliTaq Gold DNA polymerase, AmpEraseX UNG, dNTPs with UTP, passive reference dye and optimised buffer components, Applied Biosystems, Foster City CA); lOOnM Taqman probe; 900nM forward primer; 900nM reverse primer and 15ng of cDNA template.
  • TaqMan Universal Master Mix a proprietary mixture of AmpliTaq Gold DNA polymerase, AmpEraseX UNG, dNTPs with UTP, passive reference dye and optimised buffer components, Applied Biosystems, Foster City CA
  • lOOnM Taqman probe 900nM forward primer
  • 900nM reverse primer 15ng of cDNA template.
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample.
  • the levels of internal control cDNA in each sample were normalised to the level of expression of internal control in a comparative sample.
  • the data was then represented as fold expression of normalised target sequence relative to the level of expression in the comparative sample, which is set arbitrarily to 1.
  • P450 function can be tested for the constructs using a reduced CO difference spectum.
  • the absorption between 500 and 400nm is recorded for the purified protein.
  • the sample is treated by bubbling through carbon monoxide and reduced by the addition of sodium dithionite.
  • the CO-difference spectrum between the sample and the blank can then be recorded. If the sample contains an active P450 enzyme a characteristic Soret peak at 450nm is observed. (Omura & Sato JBC 239, 2370, 1964).
  • Mass spectrometry analysis of heme binding Purified protein can be analysed using mass spectrometry. This method utilises measurements of the mass to charge ratio of a sample to identify groups contained within the sample. The heme group of P450s can be identified by its characteristic charge to mass ratio. Trypsin digestion of the protein into peptide fragments and analysis of the fragments via MS/MS spectrometry also provides confirmation of the protein sequence and any possible post-translational modifications.
  • P450 protein can be expressed in a variety of expression systems including bacteria, yeast, baculovirus and mammalian cells. In order to obtain maximum P450 activity from this enzyme the system must be re-constituted or co-expressed with a reductase protein. This produces an active complex which can be used to determine the enzyme substrate and screen for inhibitors. Examples of these experiments can be found in Pritchard MP, Glancey MJ, Blake JA, Gilham DE, Burchell B, Wolf CR, Friedberg T (1998) Pharmacogenetics 8, 33; Nakajima M, Kobayashi K, Oshima K, Shimada N, Tokudome S, Chiba K, Yokoi T, (1999) Xenobiotica, 29, 885-898.
  • SEQ ID NO:l Nucleotide coding sequence for HCP44020.1 (P450G3) protein

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Abstract

L'invention se rapporte à une protéine, appelée hCP44020.1, identifiée en tant que Cytochrome P450, ainsi que l'utilisation de cette protéine et de la séquence d'acides nucléiques du gène la codant, dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2003/002824 2002-07-01 2003-07-01 Polypeptide identifie comme cytochrome p450 WO2004003190A1 (fr)

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WO2021173847A1 (fr) * 2020-02-27 2021-09-02 The George Washington University, A Congressionally Chartered Not-For-Profit Corporation Cobra1/nelf-b utilisé en tant que renforçateur d'efficacité d'une thérapie à base de lymphocytes t cd8+

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 1 October 2001 (2001-10-01), STRAUSBERG, R.: "Homo sapiens clone MGC:22971, mRNA, complete cds.", XP002254896, retrieved from EBI Database accession no. BC014952 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021173847A1 (fr) * 2020-02-27 2021-09-02 The George Washington University, A Congressionally Chartered Not-For-Profit Corporation Cobra1/nelf-b utilisé en tant que renforçateur d'efficacité d'une thérapie à base de lymphocytes t cd8+

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