# Structure-Based Drug Design for Tuberculosis: Challenges Still Ahead
## 3 DNA Gyrase: A Validated Drug Target
**(D)** Generic chemical structure of the novel bacterial topoisomerase inhibitor (NBTI) series of compounds. Analog 1 is a classical NBTI, whereas analogs 2 and 3 combine the three favorable features for _M. tuberculosis_ DNA gyrase inhibitor (MGI). and coworkers [89]. Although the ATP binding sites have been less successfully exploited as antibacterial targets, with the exception of the natural products, such as coumarins [90; 91], it is worth noting that a candidate drug emerged from these studies. SPR720 (Table 3) is an orally bioavailable prodrug of SPR719, an aminobenzimidazole inhibitor of both gyrase and topoisomerase IV. Indeed, this novel class of antimicrobials targets the ATPase subunits of gyrase and topoisomerase IV, and it was optimized using SSBD and structure-activity relationship (SAR) studies of potency against both Gram-positive and some Gram-negative bacterial species [92; 93]. Crystal structures of novobiocin (Table 3) bound to _Escherichia coli_ GyrB subunit served as a starting point for ligand optimization [94], while further optimization of the metabolic profile led to the identification of SPR720 (formerly VXc-486) [95], whose suitability to be a drug candidate for the treatment of tuberculosis and non-tuberculosis mycobacterial infections was thoroughly evaluated [96; 97]. The compound completed Phase I clinical trials in 2019 (study ID NCT03796910, sponsored by Spero Therapeutics, LLC), aimed at evaluating the safety, tolerability, and pharmacokinetics (PK) profile in healthy volunteers. Moreover, an in vivo combination of SPR720 with rifampicin and pyrazinamide (two first-line drugs used in TB treatment) showed comparable efficacy to the combination of three drugs, including moxifloxacin, rifampicin, and pyrazinamide [98]. Work on the DNA-binding site has also been productive, as GlaxoSmithKline (GSK) created a new class of type IIA topoisomerase inhibitors, called novel bacterial topoisomerase inhibitors (NBTIs) (Table 3) [99]. The crystallographic structures of the DNA-bound _Staphylococcus aureus_ DNA gyrase with either NBTI or FQ, all obtained at high resolution, revealed that the binding sites for each drug are different [100]. Indeed, the NBTI 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, remaining close to the active sites and FQs binding sites (Figure 2B). As NBTIs display relatively poor activity against the _M. tuberculosis_ DNA gyrase, the antitubercular profiles of 3000 compounds, representative of the chemical diversity of this family, were evaluated by high-throughput phenotypic screenings in vitro and in vivo [101]. MIC determination on _M. tuberculosis_ H37 Rv showed a high hit rate (68% of compounds with MIC values lower than 10 uM), with the most potent derivatives matching or even improving the MIC values for currently used TB drugs, including last-generation FQs. Overall, 29% of the compounds had MICs of \(<\) 1 uM, and 18% had MICs of \(<\) 0.1 uM. By using structural data provided by the work of [PERSON] et al., the general structures of these TB-active compounds were divided into three different regions, each of them interacting with one of the three topologically important target-gyrase interacting regions [100]. While a left-hand side (LHS) is responsible for key contacts with the gyrase DNA substrate, a right-hand side (RHS) is embedded into the enzyme, potentially contributing to the protein target selectivity (Figure 2D). Last, a central linker unit (CU) establishes key interactions with the gyrase and offers the opportunity to modulate key physicochemical properties (Figure 2D).