# Mycobacterial Cell Wall: A Source of Successful Targets for Old and New Drugs
## 3 Old TB Drugs
### 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'_-diisopropylenediamine 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; 94; 95]. Its activity against _M. tuberculosis_ in infected mice was four times more potent than streptomycin and protected mice infected with streptomycin- or isoniaizd-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 \(\upmu\)m 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. snegmatis_ 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 arabinolateral 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 arabinolateral 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 arabinosyltransferases responsible for the formation of arabinan in the cell wall [105]. Recently, EMB was postulated to target the glutamate racemase, Murl (Rv1338) [106]. Murl racemizes L-glutamate to D-glutamate, which is required for peptidoglycan biosynthesis. In an enzymatic assay, EMB partially inhibited the Murl racemisation reaction. Docking experiments suggested that EMB could bind to Murl and act as a competitive inhibitor of Murl substrate. Further experiments are required to confirm the role of Murl 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 (_emb CAB_, _Rv3793-3795_) [107; 108] encoding arabinosyltransferase 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 strainswere 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; 113; 114; 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].