*3.5. Thiacetazone—Target: Mycolic Acids*

*Discovery*. Gerhard Domagk was awarded the Nobel Prize in 1939 for the discovery of prontosil, a prodrug that releases *p*-aminobenzenesulfonamide, the first sulfonamide drug active against *Staphylococcus* and *Streptococcus* infections, but not against *M. tuberculosis* infections [163]. While prontosil was quickly replaced by penicillin to fight antibacterial infections, Domagk and his team continued working on the synthesis of related compounds, the thiosemicarbazides, and discovered that one in particular, thiacetazone (TAC, also called Tibione, Tb I, Conteben), had impressive activity against *M. tuberculosis* in guinea pigs [164]. In the late 1940s, TAC was tested in Germany on patients with various forms of TB and found to have promising activity although severe adverse effects were recorded [37,165].

*Activity and toxicity.* TAC is an oral, inexpensive, effective and bacteriostatic drug, with a MIC ranging from 0.3 to 5 μm against drug-susceptible *M. tuberculosis.* TAC is poorly soluble in water (0.09 g/L). TAC is part of the group D3 (add-on agents) of second-line TB drugs. TAC has been given in combination with INH in resource-poor countries [166,167]. Its utilization as a TB drug was mostly discontinued because of its high toxicity (skin disorder, gastrointestinal symptoms, conjunctivitis, vertigo, liver and kidney damage) especially in HIV-positive TB patients [168]. TAC is, therefore, not recommended in HIV-positive TB patients.

*Mode of action.* TAC is a prodrug that is activated by the monooxygenase EthA [127,169]. Once activated, TAC inhibits the dehydratase HadAB of the FAS-II system by forming a disulfide bound with a cysteine (Cys61) residue of HadA [170,171]. During the elongation of fatty acyl-ACPs by FAS-II, HadA binds the acyl-ACP while HadB performs the dehydratase reaction. When covalently bound to the Cys61 residue of HadA, TAC obstructs the acyl-ACP channel preventing the binding of the fatty acyl-ACP [172]. The covalent bond between TAC and HadA Cys61 blocks the activity of HadAB leading to inhibition of mycolic acid biosynthesis [173].

*Resistance.*The mode of action of TAC was discovered through analysis of TAC resistance. Since TAC treatment of *M. tuberculosis*results in inhibition of mycolic acids, Belardinelli and Morbidoni overexpressed every gene involved in the FAS-II system. Only overexpression of the dehydratase operon *hadABC*or its dimer *hadBC*resulted in highly TAC-resistant *M. tuberculosis*strains (MIC > 0.2 mm) [170]. Curiously, the level of TAC resistance was much lower when the dimer *hadAB* was overexpressed in *M. tuberculosis* (MIC 10 μm). This was puzzling since HadAB is the target of TAC. Moreover, sequencing of spontaneous *M. tuberculosis* TAC-resistant mutants revealed mutations in HadA (Cys61Ser, Cys61Gly) but also in HadC (Val85Phe, Thr123Ala, Lys157Arg, Ala151Val) [170,174]. Grzegorzewicz and colleagues set up to unmask the role of *hadC* in TAC resistance [175]. The authors found that mutations in *hadC* protected *M. tuberculosis* from TAC and compensated for HadAB inhibition by TAC. The authors proposed that *hadC* mutations prevented TAC from reaching the active site of HadAB [175].

TAC resistance is also mediated by mutations in *mmaA4* (*Rv0642c*), a gene encoding a methoxy mycolic acid synthase required for the synthesis of keto- and methoxy-mycolic acids [170,176]. The methyltransferases involved in the modifications of the meromycolic acids such as MmaA4 interact with the proteins of the FAS-II system including the dehydratase heterodimers HadAB and HadBC [31]. Mutations in *mmaA4* might, therefore, modify the conformation of HadAB preventing the binding of TAC to HadA and causing TAC resistance [175].

*Area of investigation.*In the late 1940s, the discovery of the antiTB activity of the thiosemicarbazide TAC propelled pharmaceutical companies into an extensive search for TAC analogs with antiTB properties. This led to the discovery of the most important antimycobacterial drug INH (see above). This family of compounds might still reveal interesting molecules with pharmaceutical properties. Recently, new TAC analogs were synthesized with promising activities against *M. tuberculosis* [174].
