Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis

Abstract

The thiourea isoxyl (thiocarlide; 4,4-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 g/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a ⌬9-stearoyl desaturase as the drug target. Development of a cell-free assay for ⌬9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of ⌬9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased ⌬9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound ⌬9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.The prevalence of tuberculosis, particularly in concert with human immunodeficiency virus infection and AIDS, has been well documented (1). An equally serious public health problem is increasing multi-drug-resistant tuberculosis (2). At present only a few alternative chemotherapeutic regimens are available, resulting in poor therapeutic outcomes and high mortality rates among multi-drug-resistant tuberculosis patients (3). There is an urgent need to develop new effective antituberculosis drugs with bactericidal mechanisms different from those of the presently available agents.It is prudent to re-examine drugs that were formerly deemed effective against tuberculosis. Isoxyl (ISO) 1 (thiocarlide) (Fig. 1) is a thiourea derivative that was successfully used in the 1960s to treat tuberculosis (4 -7). Recently, ISO was shown to have considerable antimycobacterial activity in vitro and to be effective against various clinical isolates of multidrug-resistant strains of Mycobacterium tuberculosis in the range of 1-10 g/ml (8). An early note reported that ISO, like isoniazid (INH) and ethionamide (ETH), strongly inhibits the synthesis of mycolic acids (9), a result since confirmed with the demonstration that all types of mycolic acids are affected (8). In addition it was noted that ISO also inhibited shorter chain fatty acid synthesis (8 -11), suggesting inhibitory effects dif...

Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions

“…This combination of phenotypes is reminiscent of isoxyl, a drug used in the 1960s for the treatment of TB. Isoxyl (1,3-bis[4-(3-methylbutoxy)phenyl]thiourea) inhibits the biosynthesis of mycolic acids (43), short-chain fatty acids (26), oleic acid, and subsequently tuberculostearic acid (27). Isoxyl targets the ⌬ 9 -desaturase DesA3, resulting in the inhibition of oleic acid biosynthesis (27), but the targets involved in mycolic and fatty acid biosyntheses are still unknown.…”

“…Isoxyl (1,3-bis[4-(3-methylbutoxy)phenyl]thiourea) inhibits the biosynthesis of mycolic acids (43), short-chain fatty acids (26), oleic acid, and subsequently tuberculostearic acid (27). Isoxyl targets the ⌬ 9 -desaturase DesA3, resulting in the inhibition of oleic acid biosynthesis (27), but the targets involved in mycolic and fatty acid biosyntheses are still unknown. One of the advantages of antibacterials having multiple targets is the reduced development of drug resistance (32).…”

The use of microarray analysis to determine the gene expression profiles of Mycobacterium tuberculosis in response to anti-bacterial compounds

“…Interestingly, it has also recently been shown to target a delta-9 desaturase in mycobacteria 19 . Genes coding for enzymes involved in FAS-II were upregulated after exposure to both drugs: fabD (coding for a malonyl-CoA::acyl carrier protein (ACP) transferase), acpM (an acyl carrier protein), kasA and kasB (both -ketoacyl ACP synthases).…”

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

“…DesA3 shares the conserved eight His motif found in membrane acyl-lipid and acyl-CoA desaturases and in the related bacterial membrane enzymes alkane hydroxylase and xylene monooxygenase (5,(26)(27)(28). By this assessment, DesA3 is likely to be an integral membrane diiron enzyme.…”

“…One product of this reaction, oleic acid, is an essential constituent of mycobacterial membrane phospholipids and triglycerides (5,(23)(24)(25). Thus DesA3 is suggested to be a target of isoxyl (also known as thiocarlide), an anti-tuberculosis drug that inhibits the synthesis of oleic and mycolic acids (5).DesA3 shares the conserved eight His motif found in membrane acyl-lipid and acyl-CoA desaturases and in the related bacterial membrane enzymes alkane hydroxylase and xylene monooxygenase (5,(26)(27)(28). By this assessment, DesA3 is likely to be an integral membrane diiron enzyme.…”