A high throughput in vitro assay for fungal (1,3)beta-glucan synthase inhibitors.
An in vitro assay for (1,3)beta-glucan synthase activity from the filamentous ascomycete Neurospora crassa, suitable for use as a high throughput screen for enzyme inhibitors is described. Samples were added to 25 microliters reaction mixtures in 96-well V-bottom microtiter plates and plates incubated at 22 degrees C. Reactions were terminated by the addition of TCA and the contents transferred to a Milliblot D apparatus containing Inotech 201-A glass fiber filters. Filters were washed to remove unincorporated substrate and the amount of 14C-labeled (1,3)beta-glucan formed by each reaction was quantitated using a Phosphorlmager. As little as 50 ng of an inhibitor with a Ki of 4 microM was detected by this assay. This assay is rapid and cost effective, permitting its use as a screen to detect compounds that inhibit (1,3)beta-glucan synthase activity.
Taft
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Mycotox, Inc., Denver, CO 80262.
Enderlin
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Selitrennikoff
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CP
D000935:Antifungal Agents / Q000032:analysis; D005374:Filtration; D005898:Glass; D005936:Glucans / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008565:Membrane Proteins; D009492:Neurospora crassa / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D047071:beta-Glucans
D000935:Antifungal Agents; D005936:Glucans; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; D047071:beta-Glucans; C033363:beta-1,3-glucan; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D014532:Uridine Diphosphate Glucose