FAMEs and MAMEs were then extracted, and equal counts (50,000 cpm) were applied to 2-D, 10% AgNO3-impregnated TLC plates. successive cycles of fatty acid elongation. This is in contrast with the type I fatty acid synthase (FAS-I) found in mammals, which is characterized by a multifunctional enzyme bearing all the necessary enzymatic activities for fatty acid elongation (20). Since FAS-II is absent from humans, this pathway has recently received considerable attention as a good target for the development of new and selective inhibitors (5, 26). The third step of the elongation cycle is carried out by the -hydroxyacyl-acyl carrier protein (ACP) dehydratase (FabZ), which catalyzes the dehydration of -hydroxyacyl-ACP to FabZ and represent the first FabZ inhibitors known to date (19). They have also been shown to inhibit the intraerythrocytic growth of BCG growth. (A) Structures of NAS-91 and NAS-21. (B) Antimycobacterial effect of NAS-91 against BCG. The susceptibility of BCG strains to NAS-91 was determined on Middlebrook Daidzein 7H11 solid medium containing OADC enrichment Egfr with increasing inhibitor concentrations (g/ml). Serial 10-fold dilutions (indicated on the plates) of actively growing culture were plated and incubated at 37C for 10 to 14 days. The MIC, defined as the minimum concentration required to inhibit 99% of the growth, was estimated to be around 10 to 25 g/ml. Mycobacteria are Daidzein unusual in that they possess both FAS-I and FAS-II (3, 10, 21), and numerous antitubercular inhibitors have been shown to inhibit mycolic acids by targeting the FAS-II enzymes (10, 23). Thiolactomycin inhibits the -ketoacyl ACP synthases KasA and KasB (11), whereas isoniazid (INH) and ethionamide inhibit the enoyl-ACP reductase InhA (1, 23); KasA/KasB and InhA are enzymes that catalyze the first and last steps of the repetitive FAS-II cycle, respectively. Although no orthologue genes of have yet been identified in mycobacterial genomes, two recent studies have reported Rv0636 as the gene encoding the FAS-II -hydroxyacyl-ACP dehydratase in (4, 17). In this study, we evaluated the antimycobacterial potential of NAS-91 and NAS-21, which were synthesized as described earlier (19). The activity of these molecules was first assessed against Daidzein BCG 1173P2 on Middlebrook 7H11 agar plates supplemented with oleic acid, albumin, dextrose, and catalase (OADC) enrichment with increasing inhibitor concentrations. Serial 10-fold dilutions of actively growing cultures were plated and incubated at 37C for 10 to 14 days. The MIC was defined as the minimum concentration required to inhibit 99% of the growth. As shown in Fig. ?Fig.1B,1B, NAS-91 exhibited potent antimycobacterial activity, with an MIC of 10 to 25 g/ml. NAS-21 also inhibited BCG growth, although less efficiently than NAS-91, with an MIC of 50 g/ml (data not shown). We next determined the activity of NAS-91 against H37Rv using the agar proportion method. The culture was grown in Middlebrook 7H9 medium at 37C with shaking until the optical density at 600 nm reached 1.0. Serial dilutions of the logarithmically growing culture were made, and an aliquot of the diluted culture expected to give 1,000 CFU on Middlebrook 7H11 agar plates supplemented with OADC was used for plating on both control plates and drug-containing plates and incubated at 37C. Colonies were counted after 15 to 20 days. NAS-91 appeared to be a far better inhibitor than NAS-21, exhibiting 99% growth inhibition at 10 g/ml. Conversely, NAS-91 did not show any inhibition activity against even at high concentrations (up to 100 g/ml) (data not shown). The similar growth inhibitory effects observed in BCG and prompted us to investigate the mechanism of action of NAS-91 in mycobacteria. Since this inhibitor has been shown to target FabZ (19), we examined whether this compound would also inhibit mycolic acids, which are known to be the end products of FAS-II in mycobacteria. Mid-log-phase cultures of BCG (4 ml) were treated with various drug concentrations, followed by further incubation at 37C for 8 h. At this point, 1 Ci/ml of [2-14C]acetate (56 mCi/mmol; Amersham Biosciences) was added to the cultures, followed by further incubation at 37C for 16 h. The 14C-labeled cells were harvested by centrifugation, washed once with phosphate-buffered saline, and subjected to Daidzein alkaline hydrolysis using 15% aqueous tetrabutylammonium hydroxide at 100C overnight, followed by the addition of 4 ml of CH2Cl2, 300 l of CH3I, and 2 ml of water. The entire reaction was then mixed for 1 h. The upper aqueous phase was discarded, and the lower organic phase was washed twice with water and evaporated to dryness. Fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were redissolved in diethyl ether,.