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The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus. Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease. The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by [3H]diisopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by [3H]diisopropyl fluorophosphate. Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site. Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

Covalent attachment of myristic acid (C14:0) to the amino-terminal glycine residue of a variety of eukaryotic cellular and viral proteins can have a profound influence on their biological properties. The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identified as a potential target for antiviral and antifungal therapy. Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide. Perturbations in the binding of its acyl-CoA substrate would therefore be expected to have an important influence on catalysis. We have synthesized 56 analogs of myristic acid (C14:0) to further characterize the acyl-CoA binding site of Saccharomyces cerevisiae NMT. The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S. cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr55gag polyprotein precursor of human immunodeficiency virus I (HIV-I). Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme's binding site is able to accommodate a dipolar protrusion from C4 through C13. This includes the region of the acyl chain occurring near C5-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-150 degrees. The activities of NMT's acyl-CoA substrates decrease with increasing polarity. This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon. This inverse relationship between polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide-containing tetradecanoyl-CoA derivatives. Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr55gag for propagation. 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 microM without associated cellular toxicity.