Characterization and optimization of in vitro assay conditions for (1,3)β-Glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening

Abstract

(1,3)β-D-Glucan synthase (E.C.2.4.1.34. UDP-glucose: 1,3-β-D-glucan 3-β-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-β-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (l,3)β-glucan synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 μM guanosine 5'-O-(3-thiotriphosphate) (GTPγS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10 mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 μM GTPγS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1,3)β-glucan synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen > 1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1,3)β-glucan synthase inhibitors.