Crystal structure and stability of gyrase–fluoroquinolone cleaved complexes from Mycobacterium tuberculosis Tim R. Blowera,1, Benjamin H. Williamsonb, Robert J. Kernsb, and James M. Bergera,2 aDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bDivision of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA 52242 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2013. Contributed by James M. Berger, December 22, 2015 (sent for review October 28, 2015; reviewed by Benjamin Bax and Yuk-Ching Tse-Dinh) Mycobacterium tuberculosis (Mtb) infects one-third of the world’s threatens both first-line and second-line use (11). The wide- population and in 2013 accounted for 1.5 million deaths. Fluoro- spread testing of fluoroquinolones against TB has revealed quinolone antibacterials, which target DNA gyrase, are critical considerable variation in efficacy of different drug variants agents used to halt the progression from multidrug-resistant tu- against Mtb. For example, ciprofloxacin is only marginally active, berculosis to extensively resistant disease; however, fluoroquino- and its early use with Mtb was halted in favor of ofloxacin and lone resistance is emerging and new ways to bypass resistance are levofloxacin (7). These two agents are now proving to be less required. To better explain known differences in fluoroquinolone effective than moxifloxacin and gatifloxacin (7, 10); however, the action, the crystal structures of the WT Mtb DNA gyrase cleavage newest two compounds also exhibit some nonideality. For ex- core and a fluoroquinolone-sensitized mutant were determined in ample, gatifloxacin can elicit side effects such as hypo/hypergly- complex with DNA and five fluoroquinolones. The structures, cemia (12), whereas moxifloxacin has potential cardiovascular ranging from 2.4- to 2.6-Å resolution, show that the intrinsically risks (13). Although recent clinical trials that have included low susceptibility of Mtb to fluoroquinolones correlates with a re- fluoroquinolones as part of an alternative drug regimen have duction in contacts to the water shell of an associated magnesium faltered (14), there are prospects for other nonfluoroquinolone ion, which bridges fluoroquinolone–gyrase interactions. Surprisingly, molecules to make an impact on the treatment of MDR- and the structural data revealed few differences in fluoroquinolone– XDR-TB (15, 16). Moreover, a promising recent trial that uses enzyme contacts from drugs that have very different activities against moxifloxacin as part of a three-drug regimen together with pre- Mtb. By contrast, a stability assay using purified components showed tomanid and pyrazinamide reports superior bactericidal activity a clear relationship between ternary complex reversibility and inhibi- against TB and MDR-TB versus current regimens (17). These tory activities reported with cultured cells. Collectively, our data new data make it clear that new quinolone-class agents, which indicate that the stability of fluoroquinolone/DNA interactions is a are also capable of circumventing known resistance mutations, major determinant of fluoroquinolone activity and that moieties might be useful therapeutic agents in the treatment of TB. that have been appended to the C7 position of different quinolone scaffolds do not take advantage of specific contacts that might be Significance made with the enzyme. These concepts point to new approaches for developing quinolone-class compounds that have increased potency Although tuberculosis is a curable disease, its etiological agent, against Mtb and the ability to overcome resistance. Mycobacterium tuberculosis (Mtb), remains a major human pathogen. Control of Mtb is hindered by multidrug-resistant Mycobacterium tuberculosis | antibiotic resistance | fluoroquinolone | strains, which can be currently treated with second-line agents gyrase | complex stability that include fluoroquinolones such as moxifloxacin. Un- fortunately, fluoroquinolone resistance is increasing, making he causative agent of tuberculosis (TB), Mycobacterium tu- improvements to quinolone efficacy clinically important. Fluo- Tberculosis (Mtb), is one of the most important pathogens of roquinolones act by forming complexes that poison Mtb. humans, second only to the HIV in the number of deaths caused To our knowledge, this study describes the first X-ray crystal annually (1). Mtb is estimated to latently infect one-third of the structures of the Mtb gyrase cleavage core complexed with world’s population (2), thereby also creating a huge reservoir for DNA and five fluoroquinolones. These comparative structures future disease. Particularly problematic are cases of multidrug- inform efforts to design new quinolone-class agents and es- resistant (MDR)-TB, which is defined as resistance to two pri- tablish that the low intrinsic susceptibility of Mtb to clinically mary anti-TB drugs, rifampicin and isoniazid. MDR-TB now used fluoroquinolones is due to a paucity of specific gyrase– represents 3.5% of new TB cases; of these, 9% are classified as drug interactions. extensively drug-resistant (XDR)-TB (1), which is defined as MDR-TB with additional resistance to any fluoroquinolone and Author contributions: T.R.B. and J.M.B. designed research; T.R.B. performed research; T.R.B., B.H.W., and R.J.K. contributed new reagents/analytic tools; T.R.B., R.J.K., and J.M.B. ana- one injectable second-line drug (3). TB isolates are also being lyzed data; and T.R.B., R.J.K., and J.M.B. wrote the paper. – recovered that are totally drug-resistant (3 5). Thus, controlling Reviewers: B.B., GlaxoSmithKline; and Y.-C.T.-D., Florida International University. TB, in particular drug-resistant TB, is a major health problem. The authors declare no conflict of interest. Fluoroquinolones are one of the most successful classes of Data deposition: Atomic coordinates and structure factors have been deposited in the drugs against bacterial pathogens, accounting for 24% of the $10 Protein Data Bank, www.pdb.org (PDB ID codes 5BS8, 5BTA, 5BTC, 5BTD, 5BTF, 5BTG, billion antibiotic market (6). Fluoroquinolones are also currently 5BTI, 5BTL, and 5BTN). receiving considerable attention in the treatment of TB, with 1Present address: School of Biological and Biomedical Sciences and Department of Chem- two new C8-methoxy derivatives, moxifloxacin and gatifloxacin, istry, Durham University, Durham DH1 3LE, United Kingdom. currently under evaluation as promising first-line therapeutics 2To whom correspondence should be addressed. Email: [email protected]. – (7 10). These compounds have been used to restrict the develop- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ment of XDR-TB from MDR-TB; however, emerging resistance 1073/pnas.1525047113/-/DCSupplemental. 1706–1713 | PNAS | February 16, 2016 | vol. 113 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1525047113 Downloaded by guest on September 29, 2021 Progress is presently being made toward developing new flu- panel of four different, clinically used fluoroquinolones (cipro- oroquinolone derivatives. For example, a methoxy group at flu- floxacin, levofloxacin, gatifloxacin, and moxifloxacin) and one INAUGURAL ARTICLE oroquinolone position C8 (Fig. 1A) increases activity against new fluoroquinolone derivative (C8-Me-moxifloxacin). X-ray mycobacteria, particularly resistant mutants (18–20). Indeed, crystallography revealed that Mtb gyrase indeed makes an in- moxifloxacin, along with a more active C8-methyl derivative, trinsically low number of interactions with a magnesium ion that retains high inhibitory activity against purified Mtb gyrase even accompanies fluoroquinolone binding, and that the introduction when the enzyme contains commonly acquired fluoroquinolone- of a drug-sensitizing mutation (GyrA A90S) restores interactions resistance substitutions [as described in the accompanying paper seen in nonresistant gyrase homologs. In vitro, we find that C8- by Aldred et al. (21)]. In another example, quinazolinediones Me-moxifloxacin and moxifloxacin are most effective at promoting (diones) have been shown to have an ability to bypass existing cleaved complex formation and inhibiting DNA supercoiling by resistance within mycobacteria and other bacterial species (20, Mtb gyrase, followed by gatifloxacin, and then ciprofloxacin and 22–24). Thus, there still exist opportunities to design more ef- levofloxacin; however, crystal structures of the DNA-binding-and- fective quinolone-class molecules for treatment of TB. cleavage core of Mtb gyrase with both DNA and drug surprisingly DNA gyrase is a heterotetrameric (GyrA2GyrB2) enzyme that failed to reveal any substantial differences in the contacts formed transiently catalyzes dsDNA breaks as it negatively supercoils between the protein and different drugs. Further biochemical in- DNA. Fluoroquinolones prevent the resealing of the ds breaks vestigations using an assay that monitors the stability of preformed that normally follows DNA strand passage (25), generating cleaved complexes largely corroborated the rank order of in- persistent, covalent enzyme–DNA adducts called cleaved com- hibition seen in DNA cleavage experiments and moreover resulted plexes. Cleaved-complex formation, which is reversible, blocks in drug efficacy trends that closely accord with clinical effective- bacterial growth; at elevated fluoroquinolone concentrations, ness.