1887

Abstract

Myristoyl-CoA:protein -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 is a mutant allele encoding an enzyme with a Gly ? Asp substitution and reduced affinity for myristoyl-CoA. Among isogenic and strains, only cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 . When switched from YPD/myristate to YPD alone, 60% of the organisms die within 4 h. Antibodies raised against the C-terminal eight residues of Arf1p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains. -Myristoylation of 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 , strain, 100% of the Arf is -myristoylated based on this mobility shift assay. When exponentially growing cells were incubated at 24 in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, = 50% of total cellular Arf was nonmyristoylated. This finding suggests that = 50% reduction in Arf -myristoylation is a biochemical marker of a growth-arrested cell. A similar conclusion was made after assaying isogenic strains containing various combinations of and alleles and grown at 24-37 on YPD or YPD/myristate. Peptidomimetic inhibitors of Nmt were synthesized based on the N-terminal sequence of an Arf. SC-59383 has an IC of 1.45 + 0.08 M for purified Nmt and is 560-fold selective for the fungal compared to human -myristoyltransf erase. It had an EC of 51 + 17 and 67 + 6 M, 24 and 48 h after a single administration of the drug to cultures of The Arf gel mobility shift assay indicated that a single dose of 200 M produced a < 50% reduction in Arf -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 Nmt (IC > 1000 M), and 200 M of the compound produced no detectable reduction in Arf -myristoylation SC-58272, which is related to SC-59383, was a more potent inhibitor (IC 0.056 + 001 M), 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.

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1997-02-01
2021-10-20
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