N-myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA: protein N-myristoyltransferase.
Myristoyl-CoA: protein N-myristoyltransferase (Nmt) catalyses the covalent attachment of myristate to the N-terminal glycine of a small subset of cellular proteins produced during vegetative growth of Candida albicans. nmt447D is a mutant NMT allele encoding an enzyme with a Gly447-->ASP substitution and reduced affinity for myristoyl-CoA. Among isogenic NMT/NMT, NMT/ delta nmt and nmt delta/nmt447D strains, only nmt delta/nmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 degrees C. When switched from YPD/myristate to YPD alone, 60% of the organisms die with 4 h. Antibodies raised against the C-terminal eight residues of Saccharomyces cerevisiae Arf1p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains. N-Myristoylation of C. albicans ADP-ribosylation factor (Arf) produced a change in its electrophoretic mobility during SDS-PAGE: the myristoylated species migrated more rapidly than the nonmyristoylated species. In an NMT/nmt delta strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmt delta/nmt447D cells were incubated at 24 degrees C in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, > or = 50% of total cellular Arf was nonmyristoylated. This finding suggests that > or = 50% reduction in Arf N-myristoylation is a biochemical marker of a growth-arrested cell. A similar conclusion was made after assaying isogenic S. cerevisiae strains containing various combinations of NMT1, nmt1-451D, ARF1, arf1 delta, ARF2 and arf2 delta alleles and grown at 24-37 degrees C on YPD of YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevisiae Aft. SC-59383 has an IC50 of 1.45 +/- 0.08 microM for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransferase. It had an EC50 of 51 +/- 17 and 67 +/- 6 microM, 24 and 48 h after a single administration of the drug to cultures of C. albicans. The Arf gel mobility shift assay indicated that a single dose of 200 microM produced a < 50% reduction in Arf N-myristoylation after 4 h, which is consistent with the fungistatic, but not fungicidal, activity. The effect on Nmt was specific: an enantiomer, SC-59840, had no inhibitory effect on purified C. albicans Nmt (IC50 > 1,000 microM), and 200 microM of the compound produced no detectable reduction in Arf N-myristoylation in vivo. SC-58272, which is related to SC-59383, was a more potent inhibitor in vitro (IC50 0.056 +/- 0.01 microM), but had no growth inhibitory activity and did not produce any detectable reduction in Arf N-myristoylation. These findings highlight the utility of the Arf protein gel mobility shift assay for demonstrating the mechanism-based antifungal activity of SC-59383, a selective inhibitor of C. albicans Nmt.
Lodge, JK; Jackson-Machelski, E; Devadas, B; Zupec, ME; Getman, DP; Kishore, N; Freeman, SK; McWherter, CA; Sikorski, JA; Gordon, JI
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