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 coval ent attachment of myristate to the N-terminal glycine of a small subse t of cellular proteins produced during vegetative growth of Candida al bicans. nmt447D is a mutant NMT allele encoding an enzyme with a Gly(4 47)-->Asp substitution and reduced affinity for myristoyl-CoA. Among i sogenic NMT/NMT, NMT/Delta nmt and nmt Delta/nmt447D strains, only nmt Delta/nmt447D cells require myristate for growth on yeast/peptone/dex trose media (YPD) at 24 or 37 degrees C. When switched from YPD/myrist ate to YPD alone, 60% of the organisms die within 4 h. Chesterfield Pa rkway, Antibodies raised against the C-terminal eight residues of Sacc haromyces cerevisiae Arf1p were used to probe Western blots of total c ellular proteins prepared from these isogenic Candida strains. N-Myris toylation of C. albicans ADP-ribosylation factor (Arf) produced a chan ge in its electrophoretic mobility during SDS-PAGE:the myristoylated s pecies 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 ce lls were incubated at 24 degrees C in YPD/myristate, < 25% cellular Ar f was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myri state, greater than or equal to 50% of total cellular Arf was nonmyris toylated. This finding suggests that greater than or equal to 50% redu ction in Arf N-myristoylation is a biochemical marker of a growth-arre sted cell. A similar conclusion was made after assaying isogenic S. ce revisiae strains containing various combinations of NMT1, nmt1-451D, A RF1, arf1 Delta, ARF2 and arf2 Delta alleles and grown at 24-37 degree s C on YPD or YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevis iae Arf. SC-59383 has an IC50 of 1.45 +/- 0.08 mu M for purified C. al bicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransferase. It had an EC(50) of 51 +/- 17 and 67 +/- 6 mu M, 24 and 48 h after a single administration of the drug to cultures o f C. albicans. The Arf gel mobility shift assay indicated that a singl e dose of 200 mu M produced a greater than or equal to 50% reduction i n Arf IV-myristoylation after 4 h, which is consistent with the fungis tatic, but not fungicidal, activity. The effect on Nmt was specific: a n enantiomer, SC-59840, had no inhibitory effect on purified C. albica ns Nmt (IC50 > 1000 mu M), and 200 mu M of the compound produced no de tectable reduction in Arf: N-myristoylation in vivo. SC-58272, which i s related to SC-59383, was a more potent inhibitor in vitro (IC50 0.05 6 +/- 0.01 mu M), but had no growth inhibitory activity and did not pr oduce 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.