Identification of a new member of the cytochrome P450 3A (CYP3A): gene family: CYP3AX

Abstract

The present invention relates to polynucleotides encoding the CYP3AX protein and variants thereof. Further, the present invention also provides vectors comprising said polynucleotides, in particular vectors, wherein polynucleotides of the present invention are operatively linked to regulatory elements allowing expression in prokaryotic and/or eukaryotic host cells. In addition, the present invention relates to proteins encoded by said polynucleotides and antibodies specifically recognizing such proteins. The present invention also concerns transgenic non-human animals comprising the above-described polynucleotide or vectors. Moreover, the present invention relates to methods for identifying and obtaining drug candidates and inhibitors for therapy of disorders related to the malfunction of the CYP3AX genes as well as to methods of diagnosing the status of such disorders. The present invention also relates to methods for the identification of molecular variants of the CYP3AX polynucleotide or protein. The present invention furthermore provides pharmaceutical and diagnostic compositions comprising the above-described polynucleotides, vectors, proteins, antibodies, drugs and inhibitors obtainable by the above-described method. Said compositions are particularly useful for diagnosing and treating various diseases with drugs that are substrates, inhibitors or modulators of CYP3AX genes or their product.

What is claimed is:  
     
         1 . A polynucleotide encoding a cytochrome P450 (CYP) 3AX polypeptide or a biologically active fragment thereof, selected from the group consisting of: 
 (a) a polynucleotide encoding a polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8 10 or 12;    (b) a polynucleotide encoding a polypeptide, said polynucleotide comprising a coding sequence as depicted in any one of SEQ ID NOS: 1, 3, 5, 7, 9 or 11;    (c) a polynucleotide encoding a polypeptide derived from the polypeptide encoded by a polynucleotide of (a) or (b) by way of substitution, deletion or addition of one or more amino acids of the amino acid sequence encoded by the polynucleotide of (a) or (b);    (d) a polynucleotide the complementary strand of which hybridizes under moderately stringent conditions with a polynucleotide of any one of (a) to (c);    (e) a polynucleotide encoding a polypeptide the sequence of which has an identity of at least 80% to the amino acid sequence of the polypeptide encoded by a polynucleotide of any one of (a) to (d);    (f) a polynucleotide encoding a fragment or an epitope-bearing portion of a polypeptide encoded by a polynucleotide of any one of (a) to (e);    (g) a polynucleotide encoding an epitope-bearing portion of a CYP3AX polypeptide comprising amino acid residues from about 405 to about 425 in SEQ ID NO: 2;    (h) a polynucleotide comprising at least 15 nucleotides of a polynucleotide of any one of (a) to (g);    (i) a polynucleotide of any one of (a) to (d), wherein at least one nucleotide is deleted, added or substituted and wherein the nucleotide deletion, substitution and/or addition results in an altered expression or activity of the CYP3AX polypeptide;    (j) a polynucleotide encoding a molecular variant of the polypeptide encoded by the polynucleotide of (a) or (b);    (k) a polynucleotide of (j), wherein the methionine at position 275 in SEQ ID NO. 2 is mutated;    (l) a polynucleotide encoding a polypeptide that is immunospecifically recognized by an antibody that has been elicited by immunization with a polypeptide encoded by a polynucleotide of (a) or (b);    (m) a polynucleotide which hybridizes under stringent conditions with a probe having the sequence of the polynucleotide of (a) or (b), or a fragment thereof; and    (n) a polynucleotide the nucleotide sequence of which is a variant of the nucleotide sequence of a polynucleotide of any one of (a) to (m), due to genetic code degeneracy, or a complementary sequence thereto; 
 provided that the polynucleotide does not consist of the nucleotide sequence set forth in SEQ ID NO:35, in SEQ ID NO:36, in SEQ ID NO:37 or in SEQ ID NO:38.  
   
     
     
         2 . The polynucleotide of  claim 1  which is DNA.  
     
     
         3 . The polynucleotide of  claim 1  or  2  which is genomic DNA.  
     
     
         4 . The polynucleotide of  claim 1  or  2  which is RNA.  
     
     
         5 . The polynucleotide of any one of  claims 1  to  4 , wherein the polynucleotide is operatively linked to an expression control sequence.  
     
     
         6 . A vector comprising the polynucleotide of any one of  claims 1  to  5 .  
     
     
         7 . A host cell comprising the polynucleotide of any one of  claims 1  to  5  or the vector of  claim 6 .  
     
     
         8 . A method for producing a CYP3AX polypeptide or fragment thereof comprising 
 (a) culturing the host cell of  claim 7  under conditions and a time sufficient to permit expression of the polypeptide; and    (b) isolating said protein or fragment from the culture.    
     
     
         9 . A method for producing cells capable of expressing the CYP3AX polypeptide comprising genetically engineering cells with the polynucleotide of any one of  claims 1  to  5  or the vector of  claim 6 .  
     
     
         10 . A CYP3AX protein or fragment thereof selected from the group consisting of a polypeptide encoded by the polynucleotide of any one of  claims 1  to  4 , a polypeptide produced by the method of  claim 8 , and a polypeptide expressed by cells produced according to the method of  claim 9 .  
     
     
         11 . A gene encoding the protein of  claim 10 .  
     
     
         12 . An antibody which binds specifically to the protein of  claim 10 .  
     
     
         13 . A nucleic acid molecule comprising a nucleic acid sequence that is complementary to the polynucleotide of any one of  claims 1  to  4 .  
     
     
         14 . A nucleic acid molecule capable of specifically recognizing and cleaving the polynucleotide of any one of  claims 1  to  4 .  
     
     
         15 . A vector comprising the nucleic acid molecule of either  claim 13  or  14 .  
     
     
         16 . A transgenic non-human animal comprising at least one polynucleotide selected from the group consisting of the polynucleotide of any one of  claims 1  to  5 , the vector of  claim 6  and the gene of  claim 11 .  
     
     
         17 . The transgenic non-human animal of  claim 16  further comprising at least one inactivated wild type allele of the CYP3AX gene.  
     
     
         18 . The transgenic non-human animal of  claim 16  or  17 , which is a mouse or a rat.  
     
     
         19 . A method of identifying and obtaining a CYP3AX inhibitor or activator capable of modulating the activity of a CYP3AX gene or a gene product thereof, comprising the steps of: 
 (a) contacting, in the presence of at least one component capable of providing a detectable signal in response to drug metabolism, either 
 (i) the protein of  claim 10 , or  
 (ii) a cell expressing the gene of  claim 11  or comprising a polynucleotide of any one of  claims 1  to  4 ,  
   in the absence and presence of a candidate compound under conditions and for a time sufficient to permit CYP3AX-mediated drug, metabolism; and    (b) comparing a level of the detectable signal provided in the absence of the agent to a level of the detectable signal provided in the presence of the agent, wherein an altered signal level is indicative of an inhibitor or activator of a CYP3AX gene or of a CYP3AX gene product.    
     
     
         20 . The method of  claim 19  wherein said cell is selected from the group consisting of a cell according to  claim 7 , a cell obtained by the method of  claim 9 , and a cell derived from the transgenic non-human animal of any one of  claims 16  to  18 .  
     
     
         21 . A method of identifying and obtaining an CYP3AX inhibitor or activator capable of modulating the activity of the CYP3AX gene or the gene product thereof comprising the steps of 
 (a) contacting the CYP3AX protein of  claim 10  with a first molecule known to be bound by the protein to form a first complex of said protein and said first molecule;    (b) contacting said first complex with a candidate compound to be screened; and    (c) measuring whether said compound displaces said first molecule from said first complex.    
     
     
         22 . The method of  claim 21 , wherein said measuring step comprises measuring the formation of a second complex of said protein and said compound.  
     
     
         23 . The method of  claim 21  or  22 , wherein said measuring step comprises measuring the amount of said first molecule that is not bound to said protein.  
     
     
         24 . The method of any one of  claim 21  to  23  wherein said first molecule is cyclosporin, midazolam, lovastatin, nifedipin, diltiazem, erythromycin, lidocaine, amiodarone, or taxol.  
     
     
         25 . The method of any one of  claims 21  to  24  wherein said first molecule is detectably labeled.  
     
     
         26 . A method for identifying a molecular variant of CYP3AX comprising determining, in a sample from a subject, a level selected from the group consisting of 
 (a) a level of the polynucleotide of any one of  claims 1  to  4 ;    (b) a level of a CYP3AX protein or fragment thereof according to claim  10 ; and    (c) a level of the presence of a mutation in the polynucleotide of any one of  claims 1  to  4 .    
     
     
         27 . A method for determining, in a subject, a risk for having or being susceptible to, the presence of, or the prognosis of a disorder related to the expression of a molecular variant CYP3AX gene, comprising the steps of determining, in a sample from the subject, 
 (1) a level of drug metabolism; and    (2) a level selected from the group consisting of 
 (a) a level of the polynucleotide of any one of  claims 1  to  4 ;  
 (b) a level of a CYP3AX protein or fragment thereof according to claim  10 ; and  
 (c) a level of the presence of a mutation in the polynucleotide of any one of  claims 1  to  4 .  
   
     
     
         28 . The method of  claim 26  or  27 , wherein said disorder is cancer.  
     
     
         29 . The method of any one of  claims 26  to  28  wherein at least one level is determined by a method selected from the group consisting of PCR, ligase chain reaction, restriction nuclease digestion, direct nucleotide sequencing, a nucleic acid amplification technique, a nucleic acid hybridization technique and an immunoassay.  
     
     
         30 . A method for treating a disorder related to the expression of a molecular variant CYP3AX gene in a subject, comprising 
 (a) identifying the disorder according to the method of any one of  claims 26  to  29 ; and    (b) administering to the subject a medicament to abolish or alleviate said disorder.    
     
     
         31 . A method for the production of a pharmaceutical composition comprising 
 (a) identifying and obtaining a CYP3AX inhibitor or activator capable of modulating the activity of a CYP3AX gene or a gene product thereof according to the method of any one of  claims 19  to  25 ; and    (b) synthesizing, in a pharmaceutically acceptable form and as a drug or pro-drug, the CYP3AX inhibitor or activator identified and obtained in step (a), or a derivative thereof.    
     
     
         32 . A method for the preparation of a pharmaceutical composition for treating a disorder related to the expression of a molecular variant CYP3AX gene, comprising formulating a CYP3AX inhibitor or activator drug or pro-drug in a form suitable for therapeutic application to a subject diagnosed according to the method of either  claim 27  or  claim 28 .  
     
     
         33 . The method of either  claim 31  or  claim 32  wherein said CYP3AX inhibitor or activator drug or pro-drug is a derivative of a medicament as defined in  claim 30 .  
     
     
         34 . An inhibitor or activator identified or obtainable by the method of any one of  claims 19  to  25 .  
     
     
         35 . The inhibitor or activator of  claim 34  which binds specifically to the protein of  claim 10 .  
     
     
         36 . A method for the detection of a polynucleotide selected from the group consisting of (i) a polynucleotide of any one of  claims 1  to  4 , (ii) a CYP3AX gene allele and (iii) a polynucleotide that is a CYP3AX variant, comprising contacting a sample with a probe selected from the group consisting of a CYP3AX-specific oligonucleotide and a CYP3AX-specific polynucleotide.  
     
     
         37 . The method of  claim 36  wherein said CYP3AX-specific polynucleotide is selected from the group consisting of (a) a polynucleotide according to any one of  claims 1  to  4 ; and (b) a nucleic acid molecule according to any one of  claim 13  or  14 .  
     
     
         38 . The method of  claim 36  wherein said CYP3AX-specific oligonucleotide is about 15 to 50 nucleotides in length and comprises a nucleotide sequence selected from the group consisting of a portion of SEQ ID NO: 1 and a complementary sequence thereto.  
     
     
         39 . A CYP3AX-specific primer or probe consisting of an oligonucleotide that is about 15 to 50 nucleotides in length and that comprises a nucleotide sequence selected from the group consisting of a portion of SEQ ID NO: 1 and a complementary sequence thereto.  
     
     
         40 . A method for detecting the CYP3AX protein or fragment thereof of  claim 10 , comprising 
 (a) contacting a sample comprising a CYP3AX protein or fragment thereof with at least one binding molecule selected from the group consisting of the antibody of  claim 11 , the antibody of  claim 12  and a substance capable of binding specifically to a CYP3AX gene product, under conditions and for a time sufficient for binding of the binding molecule to the CYP3AX gene product to occur; and    (b) detecting binding of said binding molecule to said CYP3AX protein or fragment thereof.    
     
     
         41 . A method for detecting the expression of a CYP3AX gene that comprises a polynucleotide of any one of  claims 1  to  4 , comprising: 
 (a) contacting a sample comprising an expressed CYP3AX gene product that is a CYP3AX protein or fragment thereof with at least one binding molecule selected from the group consisting of the antibody of  claim 11 , the antibody of  claim 12  and a substance capable of binding specifically to a CYP3AX gene product, under conditions and for a time sufficient for binding of the binding molecule to the CYP3AX gene product to occur; and  
 (b) detecting binding of said binding molecule to said CYP3AX protein or fragment thereof.  
 
     
     
         42 . A method for distinguishing one or more CYP3AX alleles, each of said alleles comprising a polynucleotide or a molecular variant thereof according to any one of  claims 1  to  4 , comprising: 
 (a) contacting a sample comprising an expressed CYP3AX allelic gene product that is a CYP3AX protein or fragment thereof with at least one binding molecule selected from the group consisting of the antibody of  claim 11 , the antibody of  claim 12  and a substance capable of binding specifically to a CYP3AX gene product, under conditions and for a time sufficient for binding of the binding molecule to the CYP3AX gene product to occur; and  
 (b) detecting binding of said binding molecule to said CYP3AX protein or fragment thereof.  
 
     
     
         43 . A composition comprising at least one member selected from the group consisting of (a) the polynucleotide of any one of  claims 1  to  4 , (b) the vector of either  claim 5  or  claim 6 , (c) either the host cell of  claim 7  or a cell produced according to the method of  claim 9 , (d) the protein of  claim 10 , (e) the antibody of either  claim 11  or  12 , (f) the nucleic acid molecule of either  claim 13  or  14 , (g) the vector of  claim 15 , (h) the inhibitor or activator identified or obtained according to the method of any one of claims  19 ,  21  or  22 , and (i) the primer or probe of  claim 39 .  
     
     
         44 . The composition of  claim 43  which is a diagnostic or a pharmaceutical composition.  
     
     
         45 . A method for treating a subject having a disorder related to the presence, in the genome of said subject, of a variant CYP3AX gene comprising a polynucleotide according to any one of  claims 1  to  4 , said method comprising administering to the subject a pharmaceutical composition comprising an effective dose of a drug or prodrug for the alteration of variant CYP3AX gene activity.  
     
     
         46 . The method of  claim 45  wherein the alteration of variant CYP3AX gene activity is selected from the group consisting of increased CYP3AX gene expression, decreased CYP3AX gene expression and altered conformation of at least one CYP3AX gene product.  
     
     
         47 . The use of  45  wherein said disorder is cancer.