Fungal IPC synthase assay

Details Fungal IPC synthase assay Fungal IPC synthase assay

1997-10-06

2000-02-08

Achutamurthy, Ponnathapu

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

435 29, 435 32, 435 35, 435 15, 435194, C12Q 100, C12Q 102

Patent

active

060226846

ABSTRACT:
The presently-disclosed IPC synthase-inhibitor assays comprise the steps of: (1) expression of the IPC1 gene in a cell; (2) introducing labeled starting substrates for ceramide conversion as well as potential inhibitor(s) of such conversion to the expressed gene product in an environment which allows time and conditions for conversion, and (3) identifying those potential inhibitors which actually inhibit conversion. The present invention also provides methods to determine the ability of a test compound to inhibit fungal growth, comprising the steps of (1) presenting active inositolphosophotidylceramide synthase in a manner such that synthesis of inositolphosphotidylceramide can occur; (2) introducing ceramide and phosphotylinositol, said ceramide or phosphatidylinositol carrying label for identification; (3) subjecting said active inositolphosophotidylceramide synthase, ceramide and phosphotylinositol to ordinary conditions necessary for ceramide conversion to phosphoinositol ceramide; and (4) identifying those test compounds which inhibit ceramide conversion to phosphoinositol ceramide.

REFERENCES:
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Heidler et al. The AUR1 gene in Saccharomyces cerevisiae encodes dominant resistance to the antifungal agent aureobasidin A (LY295337). Antimicrob Agents Chemother. 1995;39(12):2765-9, Dec. 1, 1995.
Hashida-Okado et al. AUR1, a novel gene conferring aureobasidin resistance on Saccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells. Mol Gen Genet. 1996;251(2):236-44, May 23, 1996.
Nagiec et al Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene. J Biol Chem. 1997;272(15):9809-17, Apr. 1, 1997.
Becker et al. Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae. J. Bacteriol. 1980;142(3):747-54, Jun. 1, 1980.
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