## 4 Mmpl3 Transporter: The Other Hot TB Target of the Moment
### MmpL3: The Achilles Heel of Mycobacterium Tuberculosis  
In recent years, a diversity of scaffolds have been reported to inhibit MmpL3 (Table 2): SQ109 (diamine), DA5 (SQ109 related compound), BM212 (diarylpyrrole), Au1235 (adamantly1 urea), C215 (benzimidazole), NITD-349 (indolecarboxamides), THP P (tetrathydropyrazole pyrimidine), Spiro (\(N\)-benzyl-6',7'-dihydropyipro[piperidine-4,4'-thieno[3,2-c]pyrans]), PIPD1 (piperidinol), E11 (acetamide) and HC2091 (carboxamide) [36; 37; 38; 38; 51; 93; 94; 95; 96; 97]. Among these compounds, SQ109 is the most advanced in the TB drug pipeline and has completed Phase I clinical trials in the US and Phase II clinical trials in Africa. SQ109 was identified originally from a combinatorial library screening based on the 1,2-ethylenediamine structure of ethambutol. However, other structural dissimilarities between SQ109 and ethambutol, the significant activity of SQ109 against drug-resistant strains of _M. tuberculosis_, including those that were ethambutol-resistant, suggested that SQ109 had a different target than its parent molecule [98]. SQ109 showed bactericidal activity against _M. tuberculosis_ in vitro, including MDR and XDR-TB. Furthermore, it had activity against intracellular bacilli as well as an extremely low spontaneous resistance rate [37]. In addition to that, it showed synergistic activity with isoniazid and rifampicin and additive effects with streptomycin in vitro [99]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Chemical Class** & **Compound** & **Structure** & _M. tuberculosis_ **MIC** & **Ref. ** \\ \hline \multirow{3}{*}{Darylpyroles} & \multirow{3}{*}{BM212} & \multirow{3}{*}{3.6 \(\mu\)M} & \multirow{3}{*}{[36]} \\  & & & \\  & & & \\  & DA5 & & 9.97 \(\mu\)M & [37] \\  & & SQ  
SQ109 also increases the activity of the new TB drug bedaquiline in vitro [100]. Replacement of ethambutol by SQ109 in the standard regimen in mice led to a 1.5 n log decrease in colony-forming units (CFUs) [101]. Furthermore, SQ109 was safe and well-tolerated in Phase I and Phase IIa clinical trials [102]. MmpL3 seems to be a promiscuous target since a plethora of compounds with dissimilar scaffolds have been shown to affect its activity and every new library screening seems to lead to novel scaffolds targeting the same enzyme. However, the reason for this extreme susceptibility is not clear. One possibility is that the compounds, due to their high hydrophobicity, get vastly enriched in the membrane where they can bind readily to MmpL3. This, in addition to the extreme vulnerability of MmpL3, would explain the variety of compounds targeting MmpL3. There has been some controversy as to the direct or indirect inhibition of these compounds against MmpL3, it has been suggested that some compounds may act indirectly on MmpL3 by disrupting the pH gradient and/or membrane potential which will disrupt the proton relay needed by MmpL3 to pump out TMM. Indeed, while SQ109 and BM212 display a broad spectrum of activity and are active against non-replicating Mtb bacilli, some others specifically target mycobacteria and do not kill nonreplicating bacilli [103; 104; 16]. In order to solve this controversy and to shed more light on the mechanism of action of these inhibitors, [PERSON] et al. have recently developed assays both in vitro and in whole cells to identify direct inhibitors of MmpL3. Their use of fluorescent competition assays and surface plasmon resonance experiments with MmpL3 purified protein has shown that SQ109, BM212, Au1235, NITD304, NITD349 and THPP1 all bind to MmpL3. The authors also showed that only SQ109 and BM212 dissipated both \(\Delta\)pH and \(\Delta\Psi\) (although the latter only at high concentrations). These assays could be a great tool for assessing the specificity of new and previously identified drugs that are believed to act against MmpL3 [105]. Recently, the crystal structure of _M. smegmatis_ MmpL3 alone and in complex with four TB drug candidates has been solved [25].