RT Journal Article SR Electronic(1) A1 Lodge, Jennifer K. A1 Jackson-Machelski, Emily A1 Devadas, Balekudru A1 Zupec, Mark E. A1 Getman, Daniel P. A1 Kishore, Nandini A1 Freeman, Sandra K. A1 McWherter, Charles A. A1 Sikorski, James A. A1 Gordon, Jeffrey I.YR 1997 T1 N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase JF Microbiology, VO 143 IS 2 SP 357 OP 366 DO https://doi.org/10.1099/00221287-143-2-357 PB Microbiology Society, SN 1465-2080, AB 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/dnmt and nmtd/nmt447D strains, only nmtd/nmt447D 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 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/nmtd, strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmtd/nmt447D 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 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, arf1d, ARF2 and arf2d alleles and grown at 24-37 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 M for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransf erase. It had an EC50 of 51 + 17 and 67 + 6 M, 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 M 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 > 1000 M), and 200 M 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 + 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., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-143-2-357