# Mycobacterial Cell Wall: A Source of Successful Targets for Old and New Drugs
## 4 New Generation of Cell Wall Inhibitors
### Target: Mycolic Acids
#### 4.1.2 Pretomanid (PA-824)  
_Discovery._ Another challenge in TB drug discovery is to discover new molecules that will eliminate dormant or persistent _M. tuberculosis_, a subpopulation of bacteria that are genetically drug-susceptible but'resistant' (or tolerant) to drug treatment. One of the most used methods to study persistent _M. tuberculosis_ is the Wayne model where _M. tuberculosis_ is subjected to gradual oxygen depletion to allow for a slow entry into anaerbiosis. To find potential inhibitors of dormant _M. tuberculosis_, nitroimidazoles were tested, as they inhibit bacterial anaerobes. Metronidazole, an antibacterial and antiprotozoan drug with activity against Gram-positive and Gram-negative anaerbes [192] showed little, if any, activity against _M. tuberculosis_ in mice [193]. However, a bicyclic nitroimidazole (CGI 17341) was reported to have potent in vitro and in vivo antimycobacterial activity. CGI 17341 showed no cross-resistance with other TB drugs but was highly mutagenic [194]. A series of more than 300 analogs was then synthesized to solve the CGI 17341 mutagenicity issue [195]. This led to the discovery of the nitroimidazole PA-824 now called pretomarid (PMD). _Activity._ PMD is an oral drug, poorly soluble in water (0.012 g/L). PMD is active against replicating and non-replicating (anaerobic), drug-susceptible and drug-resistant (MIC ranging from 0.08 to 0.7 \(\upmu\)m) _M. tuberculosis_ strains. In a mouse model of _M. tuberculosis_ infection, PMD given orally for 10 days performed as well as INH in lowering organ bacterial burdens. Increasing the concentration of PMD (from 25 mg/kg to 100 mg/kg) led to a significant reduction in organ burden compared to INH [195]. Similar results were obtained in _M. tuberculosis_-infected guinea pigs treated with PMD. The toxic threshold in mice was 1 g/kg for a single dose of PMD and 0.5 g/kg for a daily dose given for 28 days [195]. In a drug combination experiment, the lung burden in mice infected with the drug-susceptible _M. tuberculosis_ H37 Rv strain was 2.5 log10 lower in mice receiving PMD/BDQ/PZA than in mice treated with standard regimen (INH/RIF/EMB/PZA) after one month of treatment. None of the mice treated for 2 months with PMD/BDQ/PZA relapsed (mice were kept for an additional three months at the end of the treatment to assess relapse). The addition of moxifloxacin (MXF) to this combination did not improve the outcome during the first month of treatment but did decrease the number of mice relapsing after 1.5 month treatment [196]. In a subsequent experiment,_M. tuberculosis_-infected mice were treated with BDQ/MXF/PZA +/- PMD [197]. The addition of PMD to the BDQ/MXF/PZA treatment decreased the lung burden an extra 1 log\({}_{10}\) after one month of treatment compared to BDQ/MXF/PZA alone. In both groups, mice were relapse free after two months of treatment [197]. In addition, treatment of _M. tuberculosis_-infected mice with BDQ/linezolid (LZD) +/- PMD revealed that the addition of PMD had a significant impact on the lung burden. PMD in combination with BDQ/LZD resulted in a 7.2 log\({}_{10}\) CFU _M. tuberculosis_ killing in the lungs and relapse-free mice after two and three months of treatment, respectively. In contrast, the BDQ/LZD treatment resulted in a 5.2 log reduction in CFU in the lungs, but more than 90% of the mice relapsed even after 4 months of treatment [197]. _Mode of action._ Like many other TB drugs, PMD is a prodrug. PMD is activated by a deazaflavin (F420)-dependent nitroreductase (Ddn, Rv3547) to generate a des nitro metabolite releasing nitric oxide (NO) [198]. The presence of the des nitro metabolite and rate of NO release were linked specifically to the anaerobic killing activity of PMD. PMD treatment of _M. tuberculosis_ also causes a decrease in ketomycolates production and an accumulation of hydroxyymcolates leading to the hypothesis that PMD inhibits an enzyme or affects a cofactor responsible for the oxidation of hydroxyymcolic acids to ketomycolic acids [195]. A transcriptional analysis of _M.