# Structure-Based Drug Design for Tuberculosis: Challenges Still Ahead
## 3 DNA Gyrase: A Validated Drug Target  
Topoisomerases are enzymes that participate in the overwinding or underwinding of DNA. They are commonly split into two classes, depending on the number of DNA strands cut during a single catalysis cycle: class I topoisomerases, which include eukaryotic topoisomerase I, prokaryotic topoisomerase IIIa, or reverse gyrase among others, and class II topoisomerases, whose members include (but are not restricted to) topoisomerase IV and DNA gyrase [76]. In _M. tuberculosis_, DNA gyrase is a heterotetrameric nanomachine, consisting of two GyrB and two GyrA subunits, and is essential for DNA replication, transcription, and repair in living cells [77]. _M. tuberculosis_ is an exception in the prokaryotic world because of the presence of only one type I and one type II topoisomerase, whereas most other eubacteria have two enzymes of each type. In the canonical situation, DNA gyrase and topoisomerase IV, both type IIA topoisomerases, have distinct specific activities: DNA gyrase removes positive supercoils that accumulate ahead of  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Family** & **Name** & **Structure** & **IC\({}_{50}\) (\(\mu\)M)** & **Reference** \\ \hline Tetrahydrobenzothiophenes & AX20017 & 0.39 & [57] \\  & & 5.5 & [75] \\  & & & \\  &  
replication forks [78], whereas topoisomerase IV decatenates replication intermediates [79]. Thus, in _M. tuberculosis_, DNA gyrase being the unique topoisomerase IIA, it must be able to carry out both activities in vivo [80]. Furthermore, with no other topoisomerase IIA in _M. tuberculosis_, the DNA gyrase is the unique target of antibiotics from the fluoroquinolones (FQ) family (quinolones with higher cellular penetration efficiency), which bind and stabilize the DNA-DNA gyrase complex. Stabilization of this ternary complex leads to the cytotoxic accumulation of double-strand cleaved DNA fragments within the cell, inducing bacterial death [81]. Crystallographic structures of the isolated domain and the cleavage core, either in its _apo_ form or co-crystallized with several FQ, brought crucial information to elucidate the mode of binding of these drugs [82; 83; 84; 85; 86; 87]. The ratio FQ/DNA-DNA gyrase in the ternary complex is two FQ molecules per complex, with one molecule binding in each cleavage site, wedging between the two ends of the cleaved DNA strands on both halves of the complex, in a drug-binding pocket whose walls are formed by DNA base stacking (Figure 2A) [82]. The emergence of strains with multidrug resistance (MDR) phenotype, or extensively-drug resistance (XDR) phenotype in which drug resistance includes this family of antibiotics, has made improving the activity of this family of drugs by using structural data to be of utmost importance. The main goals of these studies are to increase specific drug/enzyme interactions, but also to find new molecules that inhibit this validated target without cross-resistance with FQ [88]. A significant amount of effort has thus been invested in discovering new _M. tuberculosis_ gyrase inhibitors, mostly centered on the ATP- and DNA-binding sites, and particularly using in silico methods based on the emerging gyrase structural information, as extensively reviewed by [PERSON]  
Figure 2: Inhibitors of DNA gyrase. **(A)** Top view of the X-ray structure of _M. tuberculosis_ gyrase cleavage core in complex with DNA and moxifloxacin. Protein is shown in cartoon representation with transparent surfaces, with GyrB in blue and GyrA in pink. DNA is shown in orange, with moxifloxacin as red spheres (pdb: 5 BS8). **(B)** Top view of the model of a complex of GSK000 (green spheres) with _M. tuberculosis_ gyrase cleavage core and uncleaved DNA (in orange), based on the crystal structure of GSK299423 with _Staphylococcus aureus_ gyrase (pdb: 2 XCR). The same representation, as in panel A, was used for the protein. **(C)** Ball-and-stick representation of the ligands colored by element.