*3.2. Ethambutol—Target: Arabinogalactan*/*LAM*

*Discovery.* Ethambutol ((+)-2,2- -(ethylenediimino)di-1-butanol, EMB) was discovered at the Lederle Laboratories division of the American Cyanamid Company in New York (USA) in 1961. During a random screening of synthetic compounds in mice, *N***,***N*- -diisopropylethylenediamine was found to protect mice from an *M. tuberculosis* infection. Following an intensive campaign of structure–activity relationship (SAR), ethambutol, a di-hydroxylated derivative of *N***,***N*- -diisopropylethylenediamine, was synthesized [93–95]. Its activity against *M. tuberculosis* in infected mice was four times more potent than streptomycin and protected mice infected with streptomycin- or isoniazid-resistant *M. tuberculosis* strains.

*Activity and toxicity.* EMB is water soluble (solubility 7.58 g/L) and easily taken up by *M. tuberculosis* [96]. EMB is bacteriostatic, with a MIC ranging from 5 to 34 μm against *M. tuberculosis*. EMB is a first-line TB drug, given for the first two months of TB treatment alongside INH, RIF and PZA. EMB adverse effects include liver and ocular toxicity (decreased vision, color blindness) although these effects are reversible once EMB treatment is stopped [97].

*Mode of action.* The target of EMB has not been definitively determined. It was initially thought that EMB inhibited the synthesis of metabolites needed for *M. tuberculosis* replication [96] or hampered RNA synthesis [98]. A set of studies in *M. smegmatis* determined that EMB treatment resulted in the inhibition of mycolic acid transfer to the cell wall [99]; the cellular accumulation of TMM, TDM and free mycolic acids [100]; the inhibition of D-arabinose incorporation into the arabinogalactan complex and arabinomannan [101]; and the accumulation of DPA [102], the arabinose donor in the synthesis of arabinan [103]. While the synthesis of arabinan for the arabinogalactan complex was completely inhibited by EMB, EMB only partially inhibited the synthesis of the arabinan of LAM [104], leading to the conclusion that EMB inhibited different arabinosyl transferases responsible for the formation of arabinan in the cell wall [105].

Recently, EMB was postulated to target the glutamate racemase, MurI (Rv1338) [106]. MurI racemizes L-glutamate to D-glutamate, which is required for peptidoglycan biosynthesis. In an enzymatic assay, EMB partially inhibited the MurI racemisation reaction. Docking experiments suggested that EMB could bind to MurI and act as a competitive inhibitor of MurI substrate. Further experiments are required to confirm the role of MurI in *M. tuberculosis* inhibition by EMB.

*Resistance.* Characterization of EMB-resistant *M. tuberculosis* clinical isolates revealed a connection between EMB resistance and a cluster of genes (*embCAB***,** *Rv3793***–***3795*) [107,108] encoding arabinosyltransferases involved in the biosynthesis of the mycobacterial cell wall arabinan [109]. Initially, the most common mutations found in EMB-resistant but not in EMB-susceptible clinical isolates were at position 306 of *embB* (M306I, M306V and M306L) [107]. When the *embB* mutations M306L, M306V and M306I were introduced into wild-type *M. tuberculosis* strains, the resulting strains

were found to be EMB-resistant, suggesting that the *embB* mutations were a molecular marker for EMB resistance [110,111]. However, *embB* mutations were found in EMB-susceptible clinical isolates, leading to questioning the role of *embB* in EMB resistance [112–115]. Additionally, EMB-monoresistant clinical isolates did not harbor mutation in *embB*, suggesting a different target for EMB [113]. EMB-susceptible strains carrying *embB* mutations were resistant to other antituberculosis drugs pointing to the *emb* locus as a marker for MDR or XDR *M. tuberculosis* strains [110,112,115].

Safi and colleagues have postulated that the high-level EMB resistance found in clinical isolates was a result of an accumulation of various mutations, each individually causing a low-level resistance to EMB but together provided a high degree of resistance [110]. The authors demonstrated that, in vitro, a stepwise introduction of specific mutations in *embB***,** *Rv3806c* (*ubiA*) and *embC* in *M. tuberculosis* led to a strain 8-fold more resistant to EMB than wild-type *M. tuberculosis*. *ubiA* encodes a DPPR synthase involved in DPA biosynthesis. Mutations in *ubiA* increase intracellular DPA levels, which might bind to Emb, leading to EMB resistance [116]. However, the role of *ubiA* in EMB resistance is questionable since mutations in *ubiA* are found in both EMB-susceptible and EMB-resistant *M. tuberculosis* clinical isolates [117].

*Area of investigation.* SAR on ethambutol was performed to improve EMB antimycobacterial activity while reducing its toxicity. The outcome was SQ109, a compound now in phase 2 clinical trial, and with a very different mechanism of action than EMB (see below).
