Myristoyl-CoA: protein N-myristoyltransf erase (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 G l F 7 4 Asp substitution and reduced affinity for myristoyl-CoA. Among isogenic NMTINMT, NMTlhnmt and nmthlnmt447D strains, only nmthlnmt447D 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 A r f l p 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 NMrhmtA strain, 100% of the Arf is Nmyristoylated based on this mobility shift assay. When exponentially growing nmtMnmt447D cells were incubated at 24 "C in YPDfmyristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, 2 50 YO of total cellular Arf was nonmyristoylated. This finding suggests that 2 5 0 % reduction in Arf N-myristoylation is a biochemical marker of a growtharrested cell. A similar conclusion was made after assaying isogenic 5. cerevisiae strains containing various combinations of NMT7, nmt1=451D, ARF1, arflA, ARF2 and ad2A alleles and grown at 24-37 "C on YPD or YPD/myristate.Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the Nterminal sequence of an 5. cerevisiae Arf. SC-59383 has an I C, , of 145 k0.08 pM for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransferase. It had an EC, , of 51 f 17 and 67+6 pM, 24 and 48 h after a single administration of the drug to cultures of C. albicans. The Atf gel mobility shift assay indicated that a single dose of 200 pM 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 (I C, > 1000 pM), and 200 pM 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 (IC,, 0056 &0*01 pM), but had no growth inhibitory activity and did not produce any detectable reduction in Arf J. K. LODGE a n d OTHERS 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.

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these ...

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr555 ¶9) is necessary for its proteolytic processing and viral assembly. We have shown recently that sever al analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4' H9 cells without accompanying cellular toxicity.To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production.

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MyristoylCoA:protein N-myristoyltransferase (NMT) covalently attaches the 14-carbon saturated fatty acid myristate, via an amide bond, to the N-terminal glycine residues of a variety of cellular proteins. Genetic studies have shown that NMT is essential for the viability of the principal fungal pathogens which cause systemic infection in immunosuppressed humans and hence is a target for development of fungicidal drugs. We have generated a class of potent peptidomimetic inhibitors of the NMT from one such fungal pathogen, Candida albicans. The N-terminal tetrapeptide from a substrate analog inhibitor, ALYASKL-NH2, was replaced with an omega-aminoalkanoyl moiety having an optimal 11-carbon chain for inhibition (11-aminoundecanoyl-SKL-NH2, 3a, IC50 = 1.2 +/- 0.14 microM). A series of replacements for the C-terminal Leu established that residues containing a lipophilic side chain were most effective, with cyclohexylalanine having the greatest potency (3g, IC50 = 0.36 +/- 0.06 microM). Removal of the carboxamide moiety led to a metabolically stable dipeptide inhibitor containing an N-(cyclohexylethyl)lysinamide (17e, IC50 = 0.11 +/- 0.03 microM). Partial rigidification of the flexible aminoundecanoyl chain produced the dipeptide p-(omega-aminohexyl)phenacetyl-L-seryl-L-lysyl-N-(cyclohexyleth yl)amide (26b, IC50 = 0.11 +/- 0.04 microM). Subsequent incorporation of an alpha-methyl substituent into 26b provided the dipeptide analog [2-[p-(omega-aminohexyl)phenyl]propionyl]-L-seryl-L-lysyl-N-(cyclohex ylethyl)amide, a very potent inhibitor (48, IC50 = 0.043 +/- 0.006 microM), which retained the three essential elements required for recognition by the acyl transferase's peptide binding site.