*3.2. DprE1 Inhibitors, Which Are the Current Status and Future Perspectives*

Currently, at least 11 different scaffolds have been reported as effective DprE1 inhibitors, either covalent and noncovalent, showing different efficacy in vitro, ex vivo and in vivo [46]. It is noteworthy that the majority of these compounds have been identified through phenotypic screening, then subjected to optimization processes. The most representative compounds are reported in Table 1.


**Table 1.** Main covalent and not covalent DprE1 inhibitors showing antitubercular activity.



The first DprE1 inhibitors identified is the BTZ043, belonging to the class of the benzothiazinones [30]. Moreover, nearly in the same period other classes of compounds, such as dinitrobenzamides, nitroquinoxaline and triazoles, have been described as covalent inhibitors [32,33,51].

As above reported, all these compounds possess a nitro group, that is converted to nitroso through the oxidation of the FADH2 cofactor of DprE1, allowing the formation of a covalent bond with a cysteine residue. However, an alternative and similarly probable mechanism of formation of the nitroso group has been proposed. In this model, the thiol group of the cysteine is able to start the redox reaction which converts the nitro group into nitroso similarly to the von Richter reaction [65].

Despite BTZ043 was extremely potent against *M. tuberculosis* with an MIC of 1 ng/mL (2.3 nM) [30], its great efficacy was not translated into a mouse model, probably due to its relatively high lipophilicity, and low pharmacokinetic properties. Several attempts were then done to improve this class of compounds, and in particular, a structure activity relationship (SAR) study of piperazine benzothiazinone derivatives afforded the PBTZ-169 [49]. This compound, bearing a cyclohexylmethyl substituted piperazine showed improved in vitro and in vivo potency, and resulted in being less susceptible to the attack of nitroreductases [66]. PBTZ-169 is currently in a clinical trial under the name macozinone (MCZ) [29].

Although the nitro group is essential for the activity of the benzothiazinones [30,66], although potentially mutagenic, several attempts to substitute it, for instance by a pyrrole ring, or by an azide group were performed [67,68]. However, all these attempts led to noncovalent inhibitors, with much-reduced efficacy.

Also, noncovalent inhibitors, although less potent in vitro, can show in-vivo efficacy. For instance, the first discovered noncovalent inhibitor was the benzothiazole TCA1. This compound showed a relatively high MIC against *M. tuberculosis* of 0.5 μM, nevertheless, it proved to be effective in mice, both in acute and chronic infection models [55]. A series of pyrimidine conjugate of the benzothiazole give a further improvement of this compound, with the best derivatives showing MIC of 0.08–0.09 μM [61]. More recently a new class of inhibiting DprE1 benzothiazoles, benzothiazole-N-oxides, have been identified through a whole cells screening [44], but despite their promising bactericidal activity they showed toxicity issues.

The high vulnerability of DprE1 as an antitubercular drug target was confirmed by the number of screening campaigns that identified effective noncovalent inhibitors, including thiadiazoles [42], carboxy-quinoxalines [58], aminoquinolones [62], pyrazolopyridones [63], and azaindoles [69,70].

The latter are the most promising drug candidates among the noncovalent DprE1 inhibitors, the best inhibitor showing potent antitubercular activity in vitro and in in vivo chronic infection models, low toxicity and no antagonistic activity with other TB drugs [64]. Currently, the azaindole TBA-7371 has started phase I clinical trials [27], further confirming the great potential of DprE1 inhibitors in anti-tubercular drug discovery.

## *3.3. DprE2: A Promising Target for Future Drug Discovery*

As above reported, the conversion of DPR to DPA is a two steps reaction, in which the product of DprE1 decaprenyl-phospho-2- -keto-d-arabinose must be reduced to finalize the epimerization of the substrate. This second step is catalyzed by the enzyme DprE2 (decaprenylphosphoryl-2-keto-β-d-erythro-pentose reductase) [14]. As DprE1, also DprE2 is essential for mycobacterial growth [35]. Moreover, the two enzymes have been supposed to strongly interact, and sometimes they were considered two subunits of a single enzyme named decaprenylphosphoryl-β-d-ribofuranose 2-epimerase. Indeed, DprE1 and DprE2 interaction in *Corynebacterium glutamicum*, strongly related to mycobacteria, have been demonstrated by a bacterial two-hybrid system [71]. An in silico study using homology modeling, protein threading and molecular dynamics proposed a model in which DprE1 and DprE2 form a complex [72], but currently, no structural and functional data on DprE2 have been reported yet. Anyhow, to fulfill their reactions it is conceivable that the two enzymes must work in concert, thus, reasonably a strong interaction occurs. In this context, the elucidation of the structural and functional behavior of the full decaprenylphosphoryl-β-d-ribofuranose 2-epimerase complex should be a useful tool for the development of further antitubercular inhibitors [73].
