Method of screening for compounds that inhibit the enzymatic activity of GWT1 gene product
The present invention enables screening for compounds that inhibit the transport of GPI-anchored proteins to fungal cell walls, using a simple assay for transacylation to GlcN-PI using membrane fraction expressing GWT1 protein. New antifungal agents can be created that inhibit the synthesis of fungal cell walls and also inhibit adhesion to host cells by inhibiting the transport of GPI-anchored proteins to fungal cell walls.
1. A method of screening for a sample that comprises a compound that inhibits the activity of the GWT1 protein, wherein the compound decreases GlcN-(acyl)PI, wherein the method comprises the steps of: (1) contacting a test sample with an overexpressed protein encoded by the GWT1 gene of the following (a) or (b); (a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:2; (b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; (2) adding glucosaminyl-acylphosphatidylinositol (GlcN-(acyl)PI) precursor to the mixture of the test sample and the protein; (3) detecting GlcN-(acyl)PI; and (4) selecting the test sample that comprises a compound that inhibits the activity of the GWT1 protein, wherein the compound decreases GlcN-(acyl)PI.
2. The method of claim 1 , wherein the step of detecting the acylated glycosylphosphatidylinositol (GPI) is thin-layer chromatography.
3. The method of claim 2 , wherein the method further comprises a step 4, of determining whether the selected test sample inhibits the process of transporting a glycosylphosphatidylinositol-anchored (GPI-anchored) protein to a fungal cell wall, whether the test sample inhibits the expression of a GPI-anchored protein on a fungal cell surface, or whether the test sample inhibits the proliferation of a fungi.
4. The method of claim 1 , wherein the method further comprises a step 5, of determining whether the selected test sample inhibits the process of transporting a GPI-anchored protein to a fungal cell wall, whether the test sample inhibits the expression of a GPI-anchored protein on a fungal cell surface, or whether the test sample inhibits the proliferation of a fungi.