Characterization and optimization of in vitro assay conditions for (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening.
(1,3)Beta-D-glucan synthase (E.C.2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-beta-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1,3)beta-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 microM guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 microM GTPgammaS. 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)beta-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)beta-glucan synthase inhibitors.
Wood
R L
RL
MycoTox, Inc., Denver, Colorado 80262, USA.
Miller
T K
TK
Wright
A
A
McCarthy
P
P
Taft
C S
CS
Pomponi
S
S
Selitrennikoff
C P
CP
D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology*; D002021:Buffers; D002176:Candida albicans / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins; D015203:Reproducibility of Results; D029702:Schizosaccharomyces pombe Proteins
D000935:Antifungal Agents; D002021:Buffers; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase