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 mu l 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 C-14-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 mu M 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.