Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1998-03-23
1999-08-24
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 6, 435 74, 435 76, C12Q 125, C12Q 168
Patent
active
059423862
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to the identification and cloning of the topoisomerase I gene (TOP1) from Candida albicans and the use of the gene in complementation assays to identify inhibitors of the C. albicans TOP1 while having no effect on the homologous human TOP1. The invention relates to compounds that selectively inhibit C. albicans TOP1 and the use of such compounds to kill fungi and in the treatment of individuals with fungal infections.
BACKGROUND OF THE INVENTION
Candida albicans is the most important fungal pathogen infecting humans. This fungal pathogen causes vaginal yeast infections, as well as oral infections and tissue invasion in immunocompromised patients. Oral infections are highly prevalent in AIDS patients and in cancer patients undergoing bone marrow replacement therapy. Only three types of anti-fungal drugs are currently approved for use in humans. Unfortunately, these anti-fungal drugs have serious side effects and have limited efficacy.
Yeast Saccharomyces cerevisiae strains that express DNA topoisomerase I and are permeable to the anti-tumor alkaloid camptothecin compounds are killed by the compound (Nitiss, et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 7501-7505). Yeast strains which are permeable to camptothecin but lack topoisomerase I can establish sensitivity to camptothecin by expression of human DNA topoisomerase I (Bjornsti, et al., Cancer Res., 1989, 49, 6318-6323). Thus, yeast cells lacking endogenous topoisomerase I are killed by camptothecin if they express human topoisomerase I. Camptothecin kills such yeast strains by stabilizing a covalent topoisomerase I-DNA conjugate which leaves a broken DNA strand. The broken single strand can be processed to a double-strand break during DNA replication. If this damage is not repaired by DNA recombination, it leads to cell death. Camptothecin, however, is not a candidate for human therapy for fungal-associated conditions due to its activity on human topoisomerase I.
There is a need for compounds which selectively inhibit C. albicans topoisomerase I activity but which do not inhibit human topoisomerase I activity. There is a need for kits and methods of identifying such compounds. There is a need for isolated C. albicans topoisomerase I protein, and for compositions and methods of producing and isolating C. albicans topoisomerase I protein. There is a need for methods of treating individuals that have fungal infections.
SUMMARY OF THE INVENTION
The present invention relates to substantially pure C. albicans topoisomerase I protein.
The present invention relates to substantially pure C. albicans topoisomerase I protein having the amino acid sequence of SEQ ID NO:2.
The present invention relates to nucleic acid molecules that encode C. albicans topoisomerase I protein.
The present invention relates to nucleic acid molecules encoding C. albicans topoisomerase I protein that consists of SEQ ID NO:1.
The present invention relates to recombinant expression vectors that comprise a nucleic acid sequence that encodes C. albicans topoisomerase I protein.
The present invention relates to host cells that comprise recombinant expression vectors that encode C. albicans topoisomerase I protein.
The present invention relates to fragments of nucleic acid molecules with sequences encoding C. albicans topoisomerase I protein that have at least 10 nucleotides.
The present invention relates to oligonucleotide molecules that comprise a nucleotide sequence complimentary to a nucleotide sequence of at least 10 nucleotides of SEQ ID NO:1.
The present invention relates to isolated antibodies which bind to an epitope on SEQ ID NO:2.
The present invention relates to host cells that have deficient or non-functional endogenous topoisomerase I proteins and comprise recombinant expression vectors that encode C. albicans topoisomerase I protein.
The present invention relates to methods of identifying inhibitors of C. albicans topoisomeerase I protein. The methods comprise contacting a first host cell which is deficient in a functional top
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Gerhold David L.
Kmiec Eric B.
Strauss Allyson Cole
Bugaisky Gabriele E.
Thomas Jefferson University
Wax Robert A.
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