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/Δnmt and nmtΔ/nmt447D strains, only nmtΔ/nmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37°C. 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 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/nmtΔ strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmtΔ/nmt447D cells were incubated at 24°C 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 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, arf1Δ, ARF2 and arf2Δ alleles and grown at 24-37°C on YPD or YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevisiae Arf. SC-59383 has an IC₅₀ of 1.45 ± 0.08 μM 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 μM, 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 μM 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. alkicans Nmt (IC₅₀ > 1000 μM), and 200 μM 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 μM), but had no growth inhibitory activity and did not produce any detectable reduction in Arf N-myristolation. 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.