Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin

Abstract

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a ‘pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase

“…These structural data showed that compound 1 bound to the protein at a site remote from the DNA-cleavage site and devoid of any direct DNA contacts. As far as we know, neither of these features has been previously reported in topoisomerase II poisons (15,19,25,26). The binding pocket lies at the interface between the GyrB topoisomerase-primase (TOPRIM) domain (27) and GyrA winged helix domain (WHD) and opens as a groove toward the outer side of the enzyme (Fig.…”

“…In parallel with the biochemical and genetic studies, we explored cocrystallization of the inhibitor with DNA gyrase. We have found that the S. aureus DNA gyrase crystallography platform can be used to gain structural information on compounds that target both Grampositive and Gram-negative organisms (15,19,24). Using this crystallographic platform, we obtained a 1.98-Å structure of compound 1 complexed to an S. aureus DNA gyrase core fusion [lacking the gyrase A (GyrA) C-terminal domain, the gyrase B (GyrB) Greek-key domain, and the GyrB N-terminal domain] and DNA ( Fig.…”

“…The structure contained two complexes in the asymmetric, each with two molecules of compound 1 bound. The central four base pairs of DNA, between the two DNA-cleavage sites, were largely disordered, as in a binary complex, with the same doubly nicked DNA duplex (19). The complex with compound 1 was in a very similar cell to a previously determined structure of S. aureus DNA gyrase with ciprofloxacin and DNA (PDB ID code 2XCT; cell P2 1 89.0 123.2 170.4, beta = 90.25°compared with compound 1 cell, P2 1 89.4 120.7 168.9, beta = 90.0°), suggesting that compound 1 and ciprofloxacin can stabilize similar conformations of the enzyme.…”

“…Proteins were purified and complexes were prepared and crystallized and structures were determined using methods as previously described (19,24). The X-ray data collection (35) and refinement statistics for the two thiophene structures described in this paper are shown in SI Appendix, Table S1.…”

“…This NBTIbinding site is different from the FQ-binding sites, and gepotidacin retains minimum inhibitory concentration (MIC) activity against FQresistant strains (16). The spiropyrimidinetriones, exemplified by ETX0914, also in phase II trials, bind the topoisomerase complex at a site that overlaps with that of FQs, but remains effective against FQ-resistant strains (18,19). In contrast to the FQ, triazaacenaphthylene, and spiropyrimidinetrione classes, which all bind to the DNA gate, the aminocoumarins, represented by novobiocin, target the gyrase ATPase domain.…”