N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Box 8103, St Louis, MO 63110, USA Department of Medicinal and Structural Chemistry, G. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198, USA 3 Author for correspondence: Jeffrey I. Gordon. Tel: + 1 314 362 7243. Fax: + 1 314 362 7058. e-mail: igordon@pharmdec.wustl.edu ABSTRACT 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 Gly 447 Asp substitution and reduced affinity for myristoyl-CoA. Among isogenic NMT/NMT, NMT/ nmt and nmt /nmt447D strains, only nmt /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/nmt , strain, 100% of the Arf is N -myristoylated based on this mobility shift assay. When exponentially growing nmt /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, arf1 , ARF2 and arf2 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 IC 50 of 1.45 + 0.08 µM for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N -myristoyltransf erase. It had an EC 50 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