4.1.1. SQ109

*Discovery.* A collaboration between a pharmaceutical laboratory (Sequella) and an academic (Laboratory of Host Defenses NIAID/NIH) laboratory led to the synthesis and screening of a chemical library composed of 63,238 molecules based on the ethylenediamine core of EMB [177]. EMB was targeted for its good antimycobacterial activity in TB patients and lack of previous SAR studies. The goal was to create a more potent yet less toxic EMB analog. The screening selected 170 compounds that had an MIC of less than 6 mg/L against *M. tuberculosis* (discarding inactive or compounds that could not cross the cell wall) and had an inhibitory effect on the cell wall (determined by monitoring upregulation of the *iniBAC* operon promoter, a phenotype observed in cell wall inhibitors [178]). Based on the molecular structures of these 170 compounds, a new chemical library composed of 30,000 molecules was synthesized and retested against *M. tuberculosis* (in vitro and in macrophages). Eleven compounds passed those screens and were then assessed in mice [179]. The compound with the best antimycobacterial activity, pharmacological and toxicity data was SQ109.

*Activity.* SQ109 is a lipophilic, oral drug with poor water solubility (1.7 mg/L). SQ109 is bactericidal against drug-susceptible, MDR and XDR *M. tuberculosis* strains, with a MIC ranging from 0.4 to 3 μm. In vitro, SQ109 synergizes with the first-line TB drugs INH and RIF as well as with second-line TB drugs such as cycloserine, moxifloxacin, amikacin and bedaquiline (BDQ) [180]. In *M. tuberculosis*-infected mice, treatment with SQ109 for 28 days resulted in a dose-dependent reduction in lung and spleen burdens [181]. SQ109 was not as effective as INH in eliminating *M. tuberculosis* with organ burdens between 1 and 2.5 log10 higher in the SQ109 treatment compared to INH treatment [181]. However, SQ109 peak concentration in the lung and spleen was higher than SQ109 MIC and 45-fold higher than in plasma [181].

*Mode of action.* Surprisingly, this double SAR based on EMB resulted in a drug, SQ109, with a different mode of action than EMB. EMB-resistant *M. tuberculosis* strains were fully susceptible to SQ109 [179]. Transcriptional response of SQ109-treated *M. tuberculosis,* while consistent with other cell wall inhibitors, did not match EMB's response [182]. Lipid analysis of SQ109-treated *M. tuberculosis* revealed an inhibition of mycolic acid attachment to the arabinogalactan, a depletion of TDM and an accumulation of TMM [183]. To decipher the mechanism behind this result, isolation of spontaneous SQ109-resistant mutants was attempted without success (the mutation rate for SQ109 is exceptionally low (10<sup>−</sup>11)) [183]. Instead, an analog of SQ109 was used to isolate spontaneous resistant mutants. These

mutants were co-resistant to SQ109 and carried a mutation in the mycolic acid flippase encoded by *mmpl3* (*Rv0206c*), involved in the translocation of TMM across the plasma membrane [184]. Analyses carried out in *M. smegmatis* revealed that SQ109 binds to Mmpl3 at the proton transportation site inhibiting the proton motive force for substrate translocation [185].

*Clinical trial.* In a 14 day study where SQ109 was given alone or in combination with RIF, SQ109 was shown to be safe in TB patients, with only gastrointestinal issues being disclosed [186]. The SQ109 plasma concentration was lower than the MIC for the drug and, in contrast to the in vitro results, the combination of SQ109 and RIF led to a decrease in SQ109 availability [186]. In mice, SQ109 had been shown to be metabolized by the cytochrome P450 isoenzymes CYP2D6 and CYP2C19 [181]. Since RIF induces the expression of CYP2C enzymes in humans [187], RIF might lower the effective dose of SQ109. On the other hand, the presence of SQ109 had no effect on the plasma concentration of RIF. The result of this 14 day trial was that SQ109 alone had no bactericidal effect, and there was no synergy or additive effect with the combination RIF and SQ109 [186]. In a phase 2 trial (NCT01785186) performed in newly diagnosed pulmonary TB patients, treatment with SQ109 in combination with RIF/INH/PZA failed to improve culture conversion compared to RIF/INH/PZA [188]. A phase 2b clinical trial in Russia on MDR TB patients revealed that SQ109, combined with standard drug therapy, was safe, well tolerated and effective (80% of sputum negative patients after 24 weeks of treatment with SQ109 compared to 61% without SQ109) [189].

*Area of investigation.* With the success of SQ109 and the essentiality of *mmpl3* in *M. tuberculosis*, novel inhibitors of Mmpl3 have been actively sought. Inhibition of Mmpl3 is not specific to the chemical structure of SQ109 and numerous chemical scaffolds are being identified as Mmpl3 inhibitors [185,190,191].
