# Mycobacterial Cell Wall: A Source of Successful Targets for Old and New Drugs
## 4 New Generation of Cell Wall Inhibitors  
### Target: Mycolic Acids  
#### 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 \(\upmu\)m. In vitro, SQ109 synergizes with the first-line TB drugs INH and RIF as well as with second-line TB drugs such as cyclosporine, mozifloxacin, 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 log\({}_{10}\) 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 arabin galactan, 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 (\(10^{-11}\))) [183]. Instead, an analog of SQ109 was used to isolate spontaneous resistant mutants. Thesemutants 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.