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/ delta nmt and nmt delta/nmt447D strains, only nmt delta/nmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 degrees C. When switched from YPD/myristate to YPD alone, 60% of the organisms die with 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 delta strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmt delta/nmt447D cells were incubated at 24 degrees C in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, > or = 50% of total cellular Arf was nonmyristoylated. This finding suggests that > or = 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 delta, ARF2 and arf2 delta alleles and grown at 24-37 degrees C on YPD of YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevisiae Aft. SC-59383 has an IC50 of 1.45 +/- 0.08 microM 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 microM, 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 microM 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 > 1,000 microM), and 200 microM 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 microM), 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.
Lodge
Jennifer K
JK
Department of Molecular Biology and Pharmacology Washington University School of Medicine Box 8103, St Louis, MO 63110 USA.
Jackson-Machelski
Emily
E
Department of Molecular Biology and Pharmacology Washington University School of Medicine Box 8103, St Louis, MO 63110 USA.
Devadas
Balekudru
B
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
Zupec
Mark E
ME
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
Getman
Daniel P
DP
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
Kishore
Nandini
N
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
Freeman
Sandra K
SK
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
McWherter
Charles A
CA
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
Sikorski
James A
JA
Department of Medicinal and Structural ChemistryG. D. Searle & Co., 700 Chesterfield Parkway, St Louis, MO 63198USA.
Gordon
Jeffrey I
JI
Department of Molecular Biology and Pharmacology Washington University School of Medicine Box 8103, St Louis, MO 63110 USA.
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005069:Evaluation Studies as Topic; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D018716:Molecular Mimicry; D009842:Oligopeptides / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*
D000935:Antifungal Agents; D007093:Imidazoles; D009842:Oligopeptides; C104792:SC 58272; C104793:SC 59383; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors