Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2000-05-30
2003-11-11
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S252300, C435S320100, C435S006120, C435S071100, C536S023200, C536S023100
Reexamination Certificate
active
06645745
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the cytochrome P-450 (CYP) family of heme proteins, which mediate metabolic processes. More specifically, the present invention relates to polynucleotides encoding a novel CYP family member, the CYP3AX protein, and variants thereof. Further, the present invention relates to methods for identifying and obtaining drug candidates and inhibitors for therapy of disorders related to the malfunction of CYP3AX encoding genes, as well as to methods of diagnosing the status of such disorders.
BACKGROUND OF THE INVENTION
Members of the cytochrome P-450 (CYP) family of hemoproteins metabolize a wide variety of endogenous substrates and xenobiotics including carcinogens, toxins and drugs (Daly,
Toxicol. Lett.
102-103 (1998), 143-7; Touw,
Drug Metabol. Drug Interact.
14 (1997), 55-82). Of the human CYP proteins, members of the CYP3A subfamily are of major importance, since collectively they form the largest portion of all human CYP isoforms. The human CYP3A subfamily consists of three homologous proteins encoded by distinct genes (CYP3A4, CYP3A5 and CYP3A7) (Thummel,
Annu. Rev. Pharmacol. Toxicol.
38 (1998), 389-430). The pharmacological significance of CYP3A is due to its expression in all major organs contributing to drug disposition (gastrointestinal tract, liver, kidney) and to its remarkably broad substrate spectrum. Based on the available experimental data it is estimated that between 45% and 60% of currently used drugs are substrates for CYP3A (Li,
Toxicology
104 (1995), 1-8; Evans,
Science
286 (1999), 487-91). The substrates of CYP3A include substances as diverse as steroids, antidepressants, benzodiazepines, immunosuppressive agents, imidazole antimycotics, macrolide antibiotics and toxins. The high homology among the CYP3A proteins and the available experimental data have led to the assumption that the three CYP3A isoforms have similar substrate spectra; however, some studies indicate the possibility of differences (Thummel,
Annu. Rev. Pharmacol. Toxicol.
38 (1998), 389-430).
A considerable variation in the content and catalytic activity of CYP3A has been described in the general population. For example, the activities of the CYP3A4 protein in liver biopsies often vary up to 40-fold (Westlind,
Biochem. Biophys. Res. Commun.
259 (1999), 201-5; Shimada,
J. Pharmacol. Exp. Ther.
270 (1994), 414-23). Human in vivo studies have also indicated considerable interindividual variability in CYP3A4 activity, but its extent has been smaller. The reason for this discrepancy is not clear, but it could reflect the poor CYP3A isozyme specificity of the substrates used (Thummel,
Annu. Rev. Pharmacol. Toxicol.
38 (1998), 389-430). CYP3A5 exhibits a similar variability of expression. In adult Caucasians, the CYP3A5 mRNA and protein were detected in the liver of 10 to 30% of samples, while the protein was found in the kidney and intestine of 70% subjects (Jounaidi,
Biochem. Biophys. Res. Commun.
221 (1996), 466-70) and references therein). CYP3A7, the third CYP3A isoform, was originally isolated from fetal liver, and was subsequently found in 54% of liver samples in adults (Schuetz,
Pharmacogenetics
4 (1994), 11-20).
The variability of CYP3A expression, coupled with the broad spectrum of drugs that are metabolized by CYP3A proteins, creates a potential for potentially harmful drug interactions involving these isozymes in patients undergoing therapies with multiple drugs (Thummel,
Annu. Rev. Pharmacol. Toxicol.
38 (1998), 389-430). In addition, the interindividual variation in the CYP3A activity could also influence the individual predisposition to cancers caused by environmental carcinogens. For example, CYP3A proteins metabolize aflatoxin B1 (Wang,
Biochemistry
37 (1998), 12536-45), a mycotoxin strongly implicated in the etiology of liver cancer, which is a major cause of premature death in many areas of Africa and Asia (Henry,
Science
286 (1999), 2453-4). Forrester et al. (
Proc. Natl. Acad. Sci. U S A
87 (1990), 8306-10) found that the rates of metabolic activation of aflatoxin B1 correlated with the level of CYP3A proteins in microsomes. It has also been proposed that high levels of CYP3A in humans could predispose an individual to cancer risk from bioactivated tobacco-smoke procarcinogens (Paolini,
Nature
398 (1999), 760-1).
Clearly, there is a need for a better understanding of the factors underlying the variability of CYP3A expression and its effects on drug metabolism and drug efficacy. Such improved understanding of CYP3A family member structure, function and expression should lead to an optimization of therapies with drugs, for example in cancer treatment. The present invention fulfills this need and provides other related advantages.
SUMMARY OF THE INVENTION
It is an aspect of the present invention to provide a polynucleotide encoding a cytochrome P450 (CYP) 3AX polypeptide or a biologically active fragment thereof, that is (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, the 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; (1) 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; or (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, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38.
In certain embodiments the polynucleotide is DNA, in certain embodiments it is genomic DNA and in certain embodiments it is RNA. In certain embodiments the polynucleotide is operatively linked to an expression control sequence. In certain other embodiments the invention relates to a vector comprising any one of the just described polynucleotides, and in certain other embodiments the invention relates to a host cell comprising any one of the just described polynucleotides or the just described vector.
It is another aspect of the invention to provide a method for producing a CYP3AX polypeptide or fragment thereof comprising (a) culturing the above described host cell under con
Eiselt Regina
Gellner Klaus
Wojnowski Leszek
Achutamurthy Ponnathapu
Epidauros Biotechnologie AG
Fish & Neave
Gunnison Jane T.
Haley Jr. James F.
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