Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1995-09-05
2002-05-14
Murthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S069100, C435S189000, C435S254100, C435S254110
Reexamination Certificate
active
06387647
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to a screening method for the identification of potential fungicides, based upon use of yeast strains defective in ergosterol biosynthesis.
BACKGROUND OF THE INVENTION
Ergosterol is the principal membrane sterol of fungi. It is structurally similar to its animal counterpart, cholesterol, except that ergosterol has a methyl group and two double bonds not present in cholesterol. In yeast, ergosterol affects membrane fluidity and permeability and plays an essential role in the yeast cell cycle. Yeast cells can take up cholesterol and decrease their requirement for ergosterol to very low levels, but cholesterol alone cannot completely substitute for ergosterol (Gaber, R. F., et al.,
Mol. Cell. Biol
. 9: 3447-3456 (1989)).
Though sterol biosynthesis in different organisms have many common steps, the biosynthesis of ergosterol involves steps distinct from cholesterol biosynthesis. These occur after ring formation from squalene, in the transformation of lanosterol to other sterols. One such step is sterol methylation at position 24 in the ergosterol biosynthetic pathway. The enzyme S-adenosylmethionine:&Dgr;24-sterol-C-methyl transferase is responsible for the alkylation. The ERG6 gene encoding this enzyme has been cloned and found to be not essential for growth, but erg6 mutants lacked normal membrane function (Gaber, et al., cited above, and McCammon, M. T., et al.,
J. Bact
. 157: 475-483 (1984)).
Another enzyme unique to ergosterol biosynthesis is sterol &Dgr;22 desaturase, which catalyzes the desaturation of ergosta-5,7-dien-3-beta-ol, forming a double bond in ergosterol between carbons at positions 22 and 23 that are not present in cholesterol. This appears to be catalyzed by a cytochrome P450 (Hata, S., et al.,
Biochem. Biophys. Res. Com
. 116: 162-166 (1983)).
The term “cytochrome P450” is a trivial name for a class of cytochromes that includes a number of heme proteins that have a characteristic absorption maximum at 450 nm when combined with Co in the reduced state (‘P’ denotes pigment; hence, the name). These cytochromes occur in many animal tissues, plants and microorganisms and catalyze the monooxygenation of a vast variety of hydrophobic substances including lipophilic endogenous compounds and xenobiotics, serving as oxygenating catalysts in the presence of one or more electron-transfer proteins or redox enzymes. These cytochromes act in concert with a reductase, a flavoprotein that serves as an electron-transfer enzyme from NADPH to cytochrome P450.
A second cytochrome P450 implicated in the ergosterol biosynthetic pathway has a counterpart in cholesterol biosynthesis. This is lanosterol 14-&agr;-demethylase, which in yeast catalyzes the oxidative removal of a methyl group at carbon position 14 of lanosterol during ergosterol biosynthesis, and the mammalian orthologue catalyzes the identical reaction in cholesterol biosynthesis (Turi, T. G., and Loper, J. C.,
J. Biol. Chem
. 276: 2046-2056 (1992)). Though in higher organisms the diversity of oxidative reactions and broad substrate specificity of the cytochrome P450s are due primarily to the presence of multiple P450s encoded by a gene superfamily that may contain more than 100 genes (Kalb, V. F. et al.,
DNA
6: 529537 (1987)), yeast has been suggested as a model for cytochrome-P450 systems in all eukaryotes (Kalb, V. F., et al., Gene 45: 237-245 (1986)). Active enzyme from a mammalian cytochrome P450 clone has been expressed in
Saccharomyces cerevisiae
(Oeda, K., et al.,
DNA
4: 203-210 (1985)).
Cytochrome P450 reductases from a variety of sources have been isolated and compared, and the baker's yeast (
Saccharomyces cerevisiae
) cytochrome P450 reductase gene has been cloned and characterized (Yabusaki, Y., et al.,
J. Biochem
. 103: 1004-1010 (1988)). Studies of yeast mutants lacking the cytochrome P450 reductase gene showed that they were viable (Sutter, T. R., and Loper, J. C.,
Blochem. Biophys. Res. Com
. 160: 1257-1266 (1989)). However, the cytochrome P450 reductase yeast mutants were 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-&agr;-demethylase mentioned above, indicating that the mutants were partially defective. No evidence that these mutants had a second cytochrome P450 reductase gene could be found, so apparently an alternate pathway provides for the functions of this reductase in
S. cerevisiae.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a screening test for the identification of agents exhibiting potential fungicidal activity for a wide variety of agricultural, medical, and veterinary uses, and for the identification of potential antihypercholesterolemic agents.
It is a further and more specific object of this invention to identify agents that inhibit cytochrome P450 reductase or other enzymes or electron-transfer proteins associated with cytochrome P450 reductase in the ergosterol biosynthetic pathway.
These and other objects are accomplished by the present invention, which provides a method for the identification of agents which inhibit cytochrome P450 reductase involved in ergosterol biosynthesis. The method is a screening test whereby test samples are simultaneously incubated in a culture of a yeast strain supersensitive to inhibitors of cytochrome P450 reductase enzymes or electron-transfer proteins and a second yeast strain exhibiting less sensitivity to these compounds. Agents that are positive in the test inhibit growth more in the supersensitive strain than in the less sensitive strain.
In the practice of this invention's method for screening for the presence or absence of inhibition of cytochrome P450 reductase or cytochrome P450 reductase related enzymes or electron-transfer proteins (cytochrome P450s) by a test sample, the test sample is added to a culture or culture area having a
Saccharomyces cerevisiae
strain having a disrupted cytochrome P450 reductase gene. At the same time, the test sample is added to a control culture or culture area having a
Saccharomyces cerevisiae
strain exhibiting enhanced membrane permeability. The cultures are incubated with the test sample for such time under such conditions sufficient to observe yeast cell growth, ordinarily monitored in corresponding cultures or culture areas of the strains containing no test sample. The extent of inhibition of growth in the culture or culture area containing the cytochrome P450 reductase mutant is then compared with the extent of inhibition in the culture or culture area containing the strain having enhanced membrane permeability. The presence of inhibition of-cytochrome P450 reductase or cytochrome P450 reductase related enzymes or electron-transfer proteins is determined by observation of whether culture growth inhibition in the reductase mutant exceeds that in the permeable mutant.
In a preferred screening test, a
Saccharomyces cerevisiae
strain having a disrupted cytochrome P450 reductase gene is grown in culture in the presence of test samples. At the same time, a
S. cerevisiae
strain having an erg6 mutation is grown in culture with the same test samples. Potentially active agents are identified by the observation of enhanced inhibition of the cytochrome P450 reductase disrupted strain over the erg6 strain. A positive control is employed to assist in the identification of potential agents. In these embodiments, a known inhibitor of cytochrome P450 is used, such as, for example, the lanosterol 14-&agr;-demethylase inhibitors ketoconazole, miconazole, dinaconazole or econazole. Controls are added to both cultures or culture areas, and relative inhibition is compared with that observed with the test samples.
In a particularly preferred embodiment, the cytochrome P450 reductase disrupted strain and the erg 6 yeast strain are grown in a solidified media in a plate or dish, so that test samples and positive controls can be observed visually and simultaneously as regions of the same culture. Actives produce a much larger zone (~≧8 mm) around test samples grown in a lawn of
Hsien-Fen Kuh Lai Margaret
Kirsch Donald Richard
BASF - Aktiengesellschaft
Finnegan, Henderson, Farabow, Garrett & Dunner.
Murthy Ponnathapu
Walicka Malgorzata A.
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