*3.1. Why DprE1 Is a Promiscuous Target*

DprE1 (Decaprenylphosphoryl-β-d-ribofuranose 2-oxidoreductase, EC 1.1.98.3), is involved along with DprE2 (Decaprenylphosphoryl-2-keto-β-d-erythro-pentose reductase, EC 1.1.1.333), in the two-step epimerization of decaprenylphosphoribose (DPR) to decaprenylphosphoarabinose (DPA), a key arabinosyl donor essential for the biosynthesis of cell-wall arabinan polymers [40].

As a flavoprotein, after the first half-reaction in which DPR is oxidized into decaprenylphosphorylerythropentose (DPX), DprE1 needs to reoxidize the reduced flavin to begin a new catalytic cycle, thus, to complete this half-reaction the enzyme must use an electron acceptor. Initially, DprE1 was considered an oxidase, which uses molecular oxygen as an electron acceptor [41]. However, the enzyme was found to utilize more efficiently organic compounds, including bacterial membrane-embedded quinones, such as menaquinone, which was postulated as a physiological electron acceptor [41]. For this reason, the enzyme should be more precisely defined as an oxidoreductase.

In this context, DprE1 was found to have a very broad specificity for the electron acceptor, being able to use several compounds belonging to very different chemical classes. Indeed, the enzyme can reduce not only quinones, but also indophenols such as 2,6-dichlorophenolindophenol, or the phenoxazine moiety of resazurin, both compounds used in DprE1 activity assays [41,42], as well as different nitroaromatic moieties that characterize several covalent inhibitors of this enzyme [30,32,33,43,44].

To date, more than 15 chemical classes of compounds inhibiting DprE1 have been reported. These classes encompass both covalent and noncovalent inhibitors, and although the covalent ones are the most successful, several noncovalent inhibitors with significant antitubercular activity have actually been described [45,46].

The success of DprE1 covalent inhibitors in antitubercular drug discovery partly resides in this peculiar reactivity of the enzyme with the nitroaromatic moiety. Indeed, the most potent class of these inhibitors, the benzothiazinones, has been demonstrated to be suicide prodrugs that need activation to covalently bind DprE1 [47]. This activation consists of the reduction of the nitro group to nitroso, which rapidly forms a semimercaptal covalent adduct with a cysteine residue, located in a pocket just in front of the flavin cofactor. In this way, since the electron acceptor site is occupied, the enzyme is unable to reoxidize the flavin cofactor, resulting in being irreversibly inhibited [41,48] (Figure 1).

**Figure 1.** Mechanism of activation of the BTZ043 suicide inhibitor of DprE1.

This mechanism of action has been demonstrated for benzothiazinones and benzothiazinone analogs [41,43,49,50]. However, other different classes of nitro-compounds have been identified as potent DprE1 inhibitors, including dinitrobenzamides [32], nitroquinoxalines [33] and nitroimidazoles [51], that conceivably share the same mechanism of action of benzothiazinones [52].

Currently, 33 DprE1 structures from *M. tuberculosis* or *M. smegmatis* have been deposited in the Protein Data Bank, as apoenzyme [41,53] or in complex with both covalent [41,43,44,49,54] and noncovalent inhibitors [43,50,55–58]. The enzyme is characterized by the two-domain topology of the vanillyl-alcohol oxidase family of oxidoreductases, which includes a flavin adenine dinucleotide (FAD)-binding domain and the substrate-binding domain [41]. The isoalloxazine ring of FAD is located at the interface of the substrate-binding domain and the cofactor-binding domain, where are located in two disordered loops, which have been supposed to be involved in interactions with the DPR substrate, or with other proteins involved in the DPA biosynthesis [42].

In all structures of DprE1 in complex with inhibitors, all molecules showed binding sites that are significantly overlapped (Figure 2). This binding pocket, which conceivably constitutes the binding site of the physiological electron acceptor of DprE1, shows a very broad specificity, thus explaining the promiscuity of DprE1 for inhibitors.

**Figure 2.** Crystal structure of *Mycobacterium tuberculosis* (Mtb) DprE1 and detail of the inhibitor binding sites. The superimposition of the structures of DprE1, cocrystallized with both covalent and noncovalent inhibitors (PBTZ-169, PDB: 4NCR [49] with carbon atoms in grey; Ty38c, PDB: 4P8K [58] with carbon atoms in cyan; TCA1, PDB: 4KWS [55] with carbon atoms in magenta; CT325, PDB: 4FF6 [43] with carbon atoms in violet), shows how the compounds have significantly overlapping binding sites, located in front of the isoalloxazine ring of the FAD cofactor (with carbon atoms in green). The black arrow points to the Cys387 side chain.

A broad specificity for inhibitors is a necessary but not sufficient condition for a target, to be considered promiscuous. Indeed, as promiscuous targets usually emerge during phenotypic screening, this implies that the inhibitors identified have antitubercular activity, so they can reach the cellular compartment in which the target resides. It is noteworthy that among the obstacles for the antitubercular drug discovery process, there is the difficulty of the compounds to cross the cell wall and the cellular membranes. Moreover, the presence of efflux pumps that can extrude the compounds outside the cells, and potential cytosolic inactivation processes of drugs lead to a lack of activity. In this context, it is noteworthy that one of the most successful pathways for antitubercular discovery is the cell wall compartment [59].

Recently, it was demonstrated that DPA biosynthesis partly occurs also outside the cytosolic membrane, and in particular, DprE1 was localized into the periplasm [60]. How DprE1 can reach the periplasmic compartment is still not clear, despite this protein, as well as its partner DprE2, do not possess any export signal for the translocation across the cytosolic membrane [60]. Anyhow, this localization allows drugs against DprE1 not only to easily reach the target, but also to escape several resistance mechanisms such as efflux pump or potential cytoplasmic inactivating enzymes.
