*3.3. Ethionamide*/*Prothionamide—Target: Mycolic Acids*

*Discovery.* In 1954, Thomas Gardner and colleagues published the synthesis of an INH analog, where a thioamide group replaced the acyl hydrazide group in INH [118]. This thioisonicotinamide compound was active in a mouse model of *M. tuberculosis* infection but was found to be less potent than INH. This publication was noticed by a French team who found that the thioamide derivative was more active than streptomycin and more importantly, potent against INH-, streptomycin- and PAS-resistant *M. tuberculosis* strains. They synthesized a series of α-alkyl derivatives of the thioisonicotinamide and found that adding an ethyl or propyl group at the α position generated two compounds with activity in vitro and in mice greater than streptomycin but not as potent as INH [119]. They had synthesized ethionamide (ETH) and prothionamide (PTH).

*Activity and toxicity.* ETH is a bactericidal drug, with a MIC ranging from 6 to 15 μm against drug-susceptible *M. tuberculosis.* ETH and PTH are poorly soluble in water (0.84 and 0.28 g/L, respectively). ETH and PTH are in the group C (other core second-line agents) of second-line TB drugs used interchangeably for the treatment of MDR and XDR TB cases. ETH and PTH are oral drugs with some severe adverse effects (hepatoxicity, gastrointestinal disorders, neurotoxicity) [120].

*Mode of action.* Winder and colleagues were the first to demonstrate that ETH inhibited mycolic acid biosynthesis [121]. They noticed that ETH affected *Mycobacterium bovis* BCG similarly to INH although they observed no cross resistance between INH and ETH leading to the conclusion that the mode of action of INH and ETH were not identical [121]. Actually, cross resistance between INH and ETH had been observed in TB patients a few years earlier. Several studies noted that TB patients treated with INH developed resistance to both INH and ETH although the patients had never received ETH [122–124]. This suggested that ETH and INH shared a common mechanism of action. Winder and colleagues had postulated that INH and ETH "might differ in the means by which they reach the sensitive site". That was prescient since INH and ETH were eventually revealed as prodrugs activated by different enzymes. While INH is activated by a catalase peroxidase, ETH is activated by the NADPH-specific flavin adenine dinucleotide-containing Baeyer–Villiger monooxygenase EthA (Rv3854c also called EtaA) [125,126]. In vitro, activation of ETH by EthA leads first to the formation of ethionamide S-oxide, which is further metabolized by EthA to form either 2-ethyl-4-amidopyridine [126] or (2-ethyl-pyridin-4-yl) methanol [125] via radical intermediate(s). Once activated, the mechanism of action of ETH is very similar to INH. The activated form of ETH

reacts with NAD<sup>+</sup> to form an ETH\*-NAD adduct [127]. The structures of this adduct as well as the PTH\*-NAD adduct were determined by X-ray crystallography, revealing an ethyl-isonicotinoyl or a propyl-isonicotinoyl covalently attached to the nicotinamide portion of reduced NAD<sup>+</sup> and bound to InhA. Inhibition of InhA by these adducts results in mycolic acid biosynthesis inhibition.

Wang and colleagues postulated that the activated form of ETH was an iminoyl radical but expressed doubt that this activation reaction was caused by EthA alone and hypothesized that additional enzymes might be involved [127]. Another study also concluded that another mechanism of activation might exist for ETH when demonstrating that deletion of *ethA* and its regulator *ethR* (*Rv3855*) in *M. tuberculosis* caused only a modest increase (3-fold) in ETH resistance [128]. EthA has two close homologs: the monooxygenases Rv3083 (MymA) and Rv0565c [125]. Grant and colleagues determined that MymA was indeed an activator of ETH by showing that loss of function of MymA or overexpression of *mymA* conferred resistance or hypersusceptibility to ETH, respectively [129]. In this study, the authors also noted that transposon mutants in *Rv0565c* as well as in two other genes encoding Baeyer–Villiger monooxygenases (*Rv1393c* and *Rv3049c*) did not lead to ETH resistance. However, a recent study determined that overexpression or deletion of *Rv0565c* led to ETH hypersusceptibility or low resistance, respectively, in *M. tuberculosis*, leading the authors to conclude that Rv0565c was an additional activator of ETH in *M. tuberculosis* [130]. In both studies on *mymA* and *Rv0565c*, no biochemical evidence was provided to show that MymA or Rv0565c actually activates ETH and what would be the resulting activated molecule(s).

*Resistance.* Mutations in ETH-resistant laboratory and clinical *M. tuberculosis* strains are found in the activators of ETH (*ethA***,** *mymA***,** *Rv0565c*), the negative regulator of *ethA* (*ethR*) and ETH target (*inhA* gene and promoter region) [129–131]. Mutations in the *inhA* promoter region are more frequent than mutations in the activator(s) of ETH [131]. Mutations in *inhA* or in its promoter region are found in up to 68% of ETH-resistant clinical isolates while mutations in ETH activator *ethA* are usually found in no more than 55% of ETH-resistant clinical isolates, and some of these *ethA* mutations can also be found in ETH-susceptible strains [131]. The mutations in *ethA* cover 91% of the gene (from 2 to 1341 bp; *ethA* has 1470 bp) [71]. Mutations in *ethR* have been identified in ETH-resistant clinical strains carrying the c-15t mutation in the *inhA* promoter region [131]. Recently, cyclic dimeric guanosine monophosphate, a bacterial second messenger, was shown to boost the binding of EthR to *ethA* promoter, causing a decrease in *ethA* transcription levels and ETH resistance [132].

Other genes have been implicated in ETH resistance. *M. tuberculosis* strains deleted for *mshA*, a gene encoding the glycosyltransferase involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol, are eight times more resistant to ETH than their parental strains [133]. Mycothiol is thought to play a role in ETH resistance by increasing the rate of ETH activation by EthA [133]. In *M. smegmatis* and *M. bovis* BCG, mutants co-resistant to INH and ETH were isolated carrying mutations in *ndh*, a gene encoding an NADH dehydrogenase whose function is to oxidize NADH into NAD+. In this case, *ndh* mutants were shown to accumulate NADH. Excess NADH could act as a competitive inhibitor for the binding of the ETH-NAD to InhA, triggering ETH resistance [134].

*Area of investigation.* Regulation of *ethR* expression plays a role in ETH resistance and susceptibility. While overexpression of *ethR* was shown to cause ETH resistance, inhibition of *ethR* triggers higher levels of *ethA* expression and increases ETH susceptibility [135]. Baulard and colleagues have taken advantage of this point and generated EthR inhibitors to boost ETH activity. Screening of chemical libraries and SAR on potential EthR inhibitors led to a first series of EthR inhibitors, which by themselves had no activity on *M. tuberculosis*, yet, when combined with ETH, significantly "boosted" ETH activity in vitro and in *M. tuberculosis*-infected mice [136]. The most active of this first generation of boosters, the oxadiazole BDM41906, decreased the MIC for ETH to the nM range [137]; however, BDM41906 had no "boosting" activity in *M. tuberculosis* strains carrying *ethA* mutations [138]. The second generation of ETH booster, the spiroisoxazoline SMARt (Small Molecule Aborting Resistance)-420, does not inhibit EthR but instead triggers a different activation mechanism for ETH. This new activation system uses the oxidoreductase Rv0077c as the activator,

which is negatively regulated by Rv0078. SMARt-420 binds to Rv0078, releasing the activity of Rv0077c. SMARt-420 increases the susceptibility of *M. tuberculosis* to ETH 40-fold. In addition, ETH-resistant *M. tuberculosis* strains carrying an *ethA* mutation regain ETH susceptibility when SMARt-420 is present, both in vitro and in mice [138]. SMARt-420 can, therefore, be used in the context of ETH resistance due to *ethA* mutations but not due to *inhA* mutations.
