## 3 Mechanism of Action of Benzothiazinones  
An investigation of mechanism of action of BTZ was carried out within the European Union (EU)-funded consortium New Medicines for Tuberculosis (NM4 TB), which was formed in 2006 under the leadership of Prof. [PERSON]. The compound BTZ038 was initially chosen as a lead molecule. Since it contains a chiral center, pure enantiomers BTZ043 (S) and BTZ044 (R) were prepared and subjected to MIC determination. Both forms showed comparable activities in vitro in _M. tuberculosis_. Accordingly, only one of them, BTZ043, was subjected to mechanistic studies [9]. Genetic experiments aimed at identification of the BTZ target were carried out by Prof. [PERSON] and her team at University of Pavia. The two approaches which were chosen for this purpose pointed out to the role of the gene _rv3790_ in conferring resistance against the drug. In the first approach, _M. smegmatis_ was transformed with a cosmid library containing DNA fragments from the same organism. An ortholog of the _rv3790_ gene from _M. smegmatis_ mc\({}^{2}\)155, _MSMEG_6382_, was identified on a cosmid, which resulted in an increase in MICs towards BTZ043. In the second approach, BTZ-resistant strains of _M. smegmatis_, _M. bovis_ BCG and _M. tuberculosis_ were isolated and their resistance was traced to mutations in the same codon in _rv3790_ and its orthologs. In all mutant strains codons corresponding to Cys387 in _rv3790_ gene product were replaced by Ser or Gly, which increased the MICs of the resistant strains up to 10,000 times. Accordingly, the mycobacterial species _M. avium_ and _M. aurum_, in which this position was occupied by Ala or Ser, respectively, were naturally resistant against BTZ, which supported identification of _rv3790_ as a target [9]. Coincidentally, the function of the protein encoded by this gene was described only a short while before the data on the BTZ target became available. In the course of studies focused on the metabolism of arabin in mycobacteria carried out by Prof. [PERSON] with collaborators at Colorado State University in Fort Collins, the _rv3790_ and _rv3791_ gene products were proposed to be involved in a unique epimerisation of decaprenylphosphoryl ribose (DPR) to decaprenylphosphoryl arabinose (DPA) [14]. The latter molecule serves as a sole donor of arabinose residues for synthesis of the essential arabian polymers of the mycobacterial cell wall [15]. It was shown that the conversion takes place via oxidation of the DPR substrate, followed by reduction of the resulting keto-intermediate and thus the two oxidoreductase, _rv3790_ and _rv3791_, from the so-called arabinogalactan biosynthesis cluster [16] were ideal candidates to catalyse these reactions. However, at the time it was not possible to conclude which of the two is responsible for oxidation and which for the reduction step. Examination of the effects of BTZ043 on enzymatic conversion of DPR to DPA proved that the compound inhibited the oxidation step. Given the confirmed roles of Rv3790 and Rv3791 in epimerization of DPR, we named them DprE1 and DprE2, respectively (Figure 2) [9]. An important step towards understanding the mechanism of action of BTZs was isolation of a covalent adduct of BTZ043 and DprE1 by [PERSON] et al. in 2010 [17]. The authors concluded that reduction of the nitro-group to nitroso derivative is critical for formation of the semimercaptal adduct of BTZ043 and DprE1 through the Cys387, which was verified by mass spectrometry. The following study proved that DprE1 itself is responsible for the reductive activation of DprE1. The electrons needed for the reaction are provided by the enzyme's prosthetic group, FAD, which gets reduced during the catalysis of DPR oxidation (Figure 2) [18]. Accordingly, BTZ represents a typical example of a suicide inhibitor. The proposed mechanism of action was confirmed by crystallographic studies in which structures of DprE1 from _M. smegmatis_ were obtained in native and BTZ043-bound forms.