Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1996-08-01
1998-10-13
Degen, Nancy
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 691, 4352523, 43525411, 4353201, 435325, 530350, C07H 2104, C12N 115, C12P 2102, C07K 14395
Patent
active
058213538
DESCRIPTION:
BRIEF SUMMARY
SUMMARY OF THE INVENTION
DNA molecules encoding proteins involved in biosynthesis of 1,3-beta-D glucan are identified, cloned, expressed and used in in vitro assays to screen for antifungal compounds, including compounds that affect cell wall biosynthesis. The invention includes the purified DNA molecules, assays employing the DNA molecules, proteins encoded by the DNA molecules, cells expressing the DNA molecules and altered forms of the molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a restriction map of plasmid pFF119.
FIG. 2 is a restriction map of plasmid pFF334.
FIG. 3 is a restriction map of 11.0 kb EcoRI insert of pGS3.
FIG. 4 is a restriction map of 11.0 kb XbaI insert of pGS6. The bold line designates the part of the fksA gene that was sequenced. The insert of pGS15 is shown and its derivatives containing the nested deletions (pGS17-pGS21).
FIGS. 5A-5D show the DNA sequence and putative amino acid translation of part of the fksA gene. The DNA sequence depicted is SEQ. ID. NO.:5. The amino acid sequence depicted is SEQ. ID. NO.:6.
FIGS. 6A-6L show the FKS1 DNA sequence. The DNA sequence depicted is SEQ. ID. NO.:1.
FIGS. 7A-7C show the amino acid sequence of FKS1 protein. The amino acid sequence depicted is SEQ. ID. NO.:2.
FIGS. 8A-8K show the FKS2 DNA sequence. The DNA sequence depicted is SEQ. ID. NO.:3.
FIGS. 9A-9C show the amino acid sequence of FKS2 protein. The amino acid sequence depicted is SEQ. ID. NO.:4.
FIGS. 10A-10G show the DNA and amino acid sequences of fksA. The DNA sequence depicted is SEQ. ID. NO.:5. The amino acid sequence depicted is SEQ. ID. NO.:6.
FIGS. 11A-11D show the DNA sequence of an FKS1 homolog isolated from Candida albicans. The DNA sequence depicted is SEQ. ID. NO.:7.
FIGS. 12A-12D show the amino acid sequence of an FKS1 homolog of C. albicans. The amino acid sequence depicted is SEQ. ID. NO.:8.
FIG. 13 is a partial list of yeast strains.
BACKGROUND OF THE INVENTION
DNA molecules encoding proteins involved in biosynthesis of 1,3-beta-D glucan are identified, cloned, expressed and used in in vitro assays to screen for antifungal compounds, including compounds that affect cell wall biosynthesis. The invention includes but is not limited to the purified DNA molecules, assays employing the DNA molecules, proteins encoded by the DNA molecules, cells expressing the DNA molecules and altered forms of the molecule.
The present application is directed to purified DNA fragments that contain a gene which reverses the mutant phenotypes of several different strains of Saccharomyces cerevisiae. The gene is called FKS1, for FK506 sensitivity gene 1, and is also known as ETG1 (echinocandin target gene 1). Echinocandins are acyl-substituted cyclic hexapeptides that inhibit the synthesis of 1,3-beta-D-glucan in many fungi. FKS2 is a homolog of FKS1. FKS1 was cloned from a genomic library of Saccharomyces cerevisiae. The properties of FKS1 suggest that it encodes a subunit of 1,3-.beta.-D glucan synthase. Proteins encoded by FKS1 or homologs thereof represent possible targets for drug therapy for fungal disease. The invention includes homologs such as FKS2, which also encodes a target of the echinocandins, and closely-related genes from pathogenic fungi such as Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans.
The invention comprises a gene which reverses the drug-related phenotypes of distinct mutants of S. cerevisiae. Several mutant strains were identified by their altered sensitivity to specific classes of fungal cell wall inhibitors, while another mutant strain is hypersensitive to the immuno-suppressive compounds FK506 and cyclosporin A.
Understanding the mode of action of novel therapeutic compounds employs a variety of experimental approaches involving both biochemistry and genetics. One approach is to try to isolate organisms resistant or sensitive to test compounds. Such mutants can then sometimes be used to isolate genes encoding the drug targets. A general description of some of the relevant areas of yeast biology and the mutant organisms follo
REFERENCES:
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Breuder, et al., "Calcineurin is Essential in Cyclosporin A- and FK506-sensitive yeast strains", PNAS, vol. 91, pp. 5372-5376 (1994).
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Chrebet Gary L.
Clemas Joseph
Douglas Cameron M.
El-Sherbeini Mohammed
Foor Forrest
Coppola Joseph A.
Degen Nancy
Merck & Co. , Inc.
Tribble Jack L.
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