**2. The First Discovery of DprE1 and MmpL3 as Drug Target**

Dr. Vadim Makarov and collaborators, including our Laboratory, published in 2009 the first report in which DprE1 was described as the target of 1,3-benzothiazin-4-ones (BTZ), a new class of agents endowed with antimycobacterial activity [30]. To find the BTZ target, two genetic approaches were performed in parallel using both a *Mycobacterium smegmatis* cosmid library for resistance to BTZ, and the selection and characterization of *M. smegmatis*, *Mycobacterium bovis* BCG and *M. tuberculosis* spontaneous resistant mutants. Both methods revealed that *dprE1* gene was responsible for BTZ resistance. It is noteworthy that all the mutants carried mutations in the same codon of *dprE1*, leading to the replacement of Cys387 with a Ser or Gly residue [30]. DprE1 was known to be involved in the arabinogalactan biosynthesis, a key precursor required for the synthesis of the cell-wall [14,31], and it was demonstrated that its inhibition abolishes the formation of decaprenylphosphoryl arabinose (DPA), thus provoking cell lysis and bacterial death [30]. Later, new antimycobacterial compounds targeting DprE1 were identified by independent whole cell-based screens, like dinitrobenzamides (DNB) and bromoquinoxaline (VI-9376) [32,33], and afterward, numerous DprE1 inhibitors have come out since that groundbreaking report in 2009.

In this context, it has been demonstrated that DprE1 is essential for mycobacterial cell growth and survival [34,35]. Moreover, using *M. tuberculosis* conditional knockdown mutants, it has been shown that depletion of the DprE1 mRNA and protein level leads to a rapid cell death in vitro and ex vivo, suggesting that some enzymatic reactions are more sensitive to inhibition [35], providing the proof-of-principle of why it is so successful hitting DprE1 enzyme.

MmpL3 was firstly identified in our laboratory as the cellular target of BM212, the hit compound of a pyrrole-derivative class of antimycobacterial agents by screening the *M. smegmatis* cosmid library for BM212 resistance, as well as the selection and characterization of spontaneous resistant mutants [36]. MmpL3 is a transporter belonging to the mycobacterial membrane protein large (MmpL) family, specific for Actinobacteria. Since MmpL3 protein belongs to the resistance-nodulation-cell division (RND) permease superfamily, involved mainly in drug efflux, there was concern that the BM212 resistance could have been due to an enhanced efflux of the drug. Using efflux pump inhibitors, the authors provided evidence that mutations in *mmpL3* gene were instead responsible for resistance to BM212 per se and did not mediate resistance to BM212 via drug efflux [36]. Moreover, *M. smegmatis* spheroplasts assay showed that BM212 inhibits the MmpL3 transport activity, corroborating the evidence that MmpL3 was the BM212 cellular target [15].

Immediately after this first publication, MmpL3 was demonstrated to be the target of SQ109, an antitubercular in clinical trials, for which the mechanism of action was still unclear, and adamantyl urea AU1235, a compound displaying potent bactericidal activity against Mtb [37,38]. Genetic and biochemical studies indicated a clear effect of inhibiting MmpL3 on the translocation of trehalose monomycolate (TMM) in Mtb, thus abolishing the trehalose dimycolate (TDM) formation and mycolic acid transfer onto arabinogalactan [37,38]. Subsequently, several papers have reported the discovery of new MmpL3 inhibitors.

MmpL3 is essential for both mycobacterial growth and intracellular survival [16,39]. Strikingly, its broad promiscuity can be explained not only for its essentiality for tubercle bacilli, but also because the proton motive force needed to TMM transport across the membrane is dissipated by different MmpL3 inhibitors [16], a possible indirect mechanism.

The importance of these promiscuous targets can be exemplified by the numerous papers that report novel compound(s) able to exert antimycobacterial activity targeting these proteins, but the presence of drug candidates in the antitubercular pipeline better highlight the relevance of promiscuous targets. Indeed, among the new drug candidates, Macozinone (MCZ), BTZ-043 and TBA-7371 (or AZ 7371), present in Phase I or II, inhibit DprE1, while SQ109, hitting MmpL3, has completed Phase IIb study. Moreover, the Phase IIa of telacebec (Q203) has given positive results: telacebec has a high specificity for the cytochrome bc1 complex of Mtb, particularly for QcrB, another promiscuous target.
