*4.2. 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 (adamantyl urea), C215 (benzimidazole), NITD-349 (indolecarboxamides), THP P (tetrathydropyrazolo pyrimidine), Spiro (*N*-benzyl-6- ,7- -dihydrospiro[piperidine-4,4- -thieno[3,2-c]pyrans]), PIPD1 (piperidinol), E11 (acetamide) and HC2091 (carboxamide) [36–38,51,93–97].


**Table 2.** Main MmpL3 inhibitors showing antitubercular activity.

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]. 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 [16,103,104]. In order to solve this controversy and to shed more light on the mechanism of action of these inhibitors, Li 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 ΔpH and ΔΨ (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]. Analysis of the transmembrane region has shown the existence of two pairs of hydrophilic residues (D256-Y646 and Y257-D645) on TM4 and TM 10 which links these two helices by forming hydrogen bonds. These residues are similar to the Asp-Asp-Lys triad found in AcrB and the Asp-Asp-Thr triad in SecDF, which are known to be involved in the proton relay pathway [106,107]. Consistently with this, three out of four of these residues have been shown to be essential for MmpL3 activity [81]. Furthermore, this catalytic tetrad is conserved in most MmpL transporters, with the exception of MmpL7, so it is likely that all MmpL transporters use the same mechanism to drive its substrate translocation [81].

The crystal structure of MmpL3 bound to SQ109, Au1235 and ICA38 has shown that these MmpL3 inhibitors bind to the same pocket in the center of the transmembrane region of the protein (Figure 3A), inducing conformational changes which in turn disrupt the interaction of the two Asp-Tyr pairs involved in proton translocation (Figure 3B,C). Furthermore, molecular docking assays with six other compounds (BM212, NITD-349, GSK2200150A, C215, PIPD1 and HC2091) showed that they can fit the same binding pocket within MmpL3. Thus, MmpL3 inhibitors seem to have an identical mechanism of action, binding to the same pocket and blocking the proton translocation relay pathway that is essential for substrate transport. This is also supported by the fact that the majority of resistance mutations found in MmpL3 lie in the TM region, close to the binding pocket. Zhang et al. purified 12 different mutant versions of MmpL3 and showed that most of the mutations affected the binding of the inhibitors to the pocket by disrupting hydrophobic/electrostatic interactions, inducing conformational changes or causing steric hindrance which leads to a decrease or abolition of the binding. On the other hand, there is still no clear evidence as to how mutations located further for the binding pocket induce resistance. Although MmpL3 is the only essential MmpL protein in *M. tuberculosis*, several other MmpL proteins have been shown to be required for virulence and/or drug efflux [108,109]. The fact that all inhibitors act by disrupting the proton relay pathway, a conserved feature of all MmpL transporters, opens the avenue for the development of molecules that target not only MmpL3 but all the MmpL family of transporters and thus affect virulence and resistance to other drugs.

**Figure 3.** (**A**) Crystal structure of Mtb MmpL3 and detail of the inhibitors binding site (SQ109, PDB: 6AJG with carbon atoms in green; AU1235, PDB: 6AJH with carbon atoms in magenta; ICA38 PDB: 6AJJ with carbon atoms in cyan [25]). (**B**) Mechanism of transport of MmpL3: The flow of protons through charged residues in the transmembrane region of MmpL3 drives conformational changes that lead to the transport of TMM from the membrane to the periplasmic space. (**C**) MmpL3 inhibitors bind to a pocket in the transmembrane region of MmpL3, disrupting the electrostatic interactions between D256 and Y257 with Y646 and D645, respectively. This blocks the proton relay pathway and ultimately TMM translocation.

In summary, these findings will lead to a new era in MmpL3 inhibitor drug discovery since structure-guided molecules can now be designed with better anti-tubercular efficacy and pharmacokinetic/pharmacodynamic (PK/PD) properties.

#### **5. Future Perspectives for DprE1 and MmpL3 Inhibitors in Clinical Therapy**

Promiscuous targets could be an obstacle in the discovery of novel antitubercular drug targets, being recurrently found in HTS [110], nevertheless, they have been proven really useful for the development of novel drug candidates. This is well demonstrated by the fact that five compounds currently in clinical trials inhibit these targets, with DprE1 inhibitors TBA-7379, BTZ043 and Macozinone in Phase I, while the QcrB inhibitor Telacebec, the MmpL3 inhibitor SQ109 and again the Macozinone are in Phase II [26–29].

In particular, Macozinone has completed a dose-escalation phase I study in healthy male volunteers and a multiple ascending dose study in Russia, which demonstrated the good safety profile of the compound. Moreover, in 2018 it completed a Phase IIa Early Bactericidal Activity (EBA) study in Russia and Belarus in drug sensitive-TB (DS-TB) patients, confirming the safety and efficacy of the drug. In parallel, a Phase I clinical study has been initiated in Switzerland, by the nonprofit Innovative Medicines for Tuberculosis (iM4TB) foundation [24].

The MmpL3 inhibitor SQ109 has completed a Phase 2b study in Russia which demonstrated its efficacy in MDR-TB patients, with a good tolerability profile [111]. Furthermore, SQ109 was demonstrated to enhance the activity of isoniazid, rifampicin and bedaquiline and shortened clearance of TB in a mice model. The drug now has also completed three Phase I studies in the USA and two Phase II studies in Africa in DS-TB patients.

These studies thus demonstrate the great potential of these targets for the development of a new drug to reach the objective to eradicate TB.

**Author Contributions:** Conceptualization, G.R. and L.R.C.; writing, G.D., J.M.B. and L.R.C., review and editing, M.R.P., E.D.R., G.R. and L.R.C.; visualization, J.M.B. and L.R.C.; project administration, G.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Dept. of Biology and Biotechnology "L. Spallanzani", University of Pavia (G.D., M.R.P., E.D.R., G.R. and L.R.C.).

**Conflicts of Interest:** The authors declare no conflict of interest.
