Thiocarlide: a promising antituberculosis drug and a novel tool for study of fatty acid and mycolic acid biosynthesis in Mycobacterium species

Abstract

This dissertation describes various lessons learned from a study of the effects of thiocarlide (THC) on mycobacteria: the antimycobacterial activity, the mode of action, the gene encoding the enzymatic target, and the characteristics of the target enzyme of THC. Some fundamental aspects of fatty acid and mycolic acid synthesis were unraveled through the use of THC as a study tool. Thiocarlide (THC), a thiourea, has considerable antimycobacterial activity against various species of mycobacteria with a minimal inhibitory concentration (MIC) of 0.5-2.5 μg/ml on 7H11 agar. When used in a range of 1-10 μg/ml, THC showed effectiveness against various clinical isolates of M. tuberculosis which all have differences in drug resistance phenotypes and IS6110-based genotypes. In a murine macrophage model, THC exhibited bactericidal activity against viable intracellular M. tuberculosis in a dose-dependent manner (0.05-2.5 μg/ml). No acute toxicity was observed against primary macrophage cell cultures as demonstrated by diminution of redox activity in an Alamar Blue assay. A homologous series of thiourea derivatives were synthesized and in an agar proportion assay most derivatives were as effective or more effective than THC. With the purpose of identification of drug targets, the mode of action of THC was determined. Through the use of [1,2-14C] acetate whole-cell labeling and analysis by thin layer chromatography, THC was shown to inhibit the synthesis of short-chain fatty acids and all types of mycolic acids in M. tuberculosis, M. bovis BCG and M. aurum A+. Gas chromatographic analysis revealed that THC specifically inhibited oleic (C18:1 Δ9) and hence tuberculostearic acid (brC19:0) synthesis compensated by a partial increase of stearic acid and other saturated fatty acids (C20:0, C22:0, C24:0, and C26:0). The decreased synthesis of oleic acid concomitant with the increase of stearic acid suggests that THC acts by inhibiting Δ9 desaturase. The inhibition of the synthesis of tuberculostearic acid is a direct consequence of the inhibition of oleic acid synthesis, indicating that oleic acid is in turn metabolized to tuberculostearic acid. The selective reduction of oleic acid was dose-dependent and THC completely inhibited oleic acid synthesis at 3 μg/ml. The supplementation of 7H11 agar medium with oleic acid in the form of oleic acid-albumin-dextrose-catalase (OADC) reversed the bactericidal effect of THC, supporting the definitive effect of THC on oleic acid synthesis. Inhibition of oleic acid synthesis was not observed with isoniazid (INH) and ethionamide (ETH), which are inhibitors of mycolic acid biosynthesis, suggesting that the mode of action of THC is unique. Attempts were made to identify the M. tuberculosis gene that encodes the Δ9 desaturase by the strategy of target overexpression. The M. tuberculosis desA genes designated desA1, desA2 and desA3 were cloned and expressed in M. bovis BCG. The overexpression of the desA3 gene from a constitutive promoter and the use of [1,2-14C]acetate cell labeling revealed an obvious increase of oleic acid synthesis. Imminunoblotting of whole-cells and subcellular fractions of recombinant M. bovis BCG expressing His-tagged DesA3 indicates that the product of the desA3 gene is a membrane-associated protein. By [1,2-14C]acetate whole-cell labeling, it was shown that the Δ9 desaturase activity of the desA3 gene product was completely inhibited by sterculic acid, a known Δ9 desaturase inhibitor. Unlike THC, sterculic acid did not inhibit mycolic acid synthesis suggesting that oleic and mycolic acid syntheses are not linked and that THC has a second target in the mycolic acid synthesis pathways. An in vitro assay for Δ9 desaturation was developed to demonstrate the enzymatic function of the mycobacterial Δ9 desaturase. A cell lysate fraction of M. bovis BCG exhibited the ability to catalyze the desaturation of stearoyl-CoA to the corresponding Δ9 monounsaturated derivative, oleic acid, indicating the presence of the Δ9 desaturase in this strain. The in vitro assay also demonstrated that DesA3 is indeed the Δ9 desaturase. In summary, THC shows promise as an effective antituberculosis drug whose mechanism is unique, and the Δ9 desaturase has therapeutic value as a novel target for a new generation of antituberculosis drugs.