Neil Osheroff from the Vanderbilt University School of Medicine is working to overcome drug resistance and revitalise the use of established targets for antibacterial agents, as we discover here
Resistance to antibacterial drugs represents one of the greatest public health challenges of our time and is a threat to modern medicine. The Centers for Disease Control and Prevention estimate that in the United States alone, there are >2.8 million cases of resistant bacterial infections annually that result in ~36,000 deaths. Worldwide, it is estimated that >700,000 people die each year from resistant microbial infections and that number could rise to ~10,000,000 by the year 2050.
Despite extensive drug discovery, there are a limited number of targets for antibacterial agents. Two of the most important are the type II topoisomerases, gyrase and topoisomerase IV. These bacterial enzymes are the targets of fluoroquinolones (such as ciprofloxacin), which are among the most efficacious and broad-spectrum oral antibacterials currently in clinical use. Fluoroquinolones are used as front-line treatments for a wide variety of bacterial infections in humans, including urinary tract infections, sexually transmitted diseases, skin and tissue infections, chronic bronchitis, pneumonia, and pelvic infections. Fluoroquinolones are also the first-line of prophylactic treatment for bioterrorist agents, such as anthrax, and they are commonly used to treat tuberculosis.
Unfortunately, fluoroquinolone usage is being threatened by an increasing prevalence of resistance, which extends to every country and every bacterial infection treated by this drug class. The most common and clinically relevant form of resistance is target-mediated, which is caused by specific mutations in gyrase and topoisomerase IV.
The vast majority of bacteria encode two closely related type II topoisomerases, gyrase and topoisomerase IV, which are responsible for altering the three-dimensional structure of DNA (deoxyribonucleic acid). DNA, which is the genetic material in humans and bacteria, is essentially a very long double-stranded “rope” that is made up of two interwound strands. This double helix is highly compacted in human nuclei and bacterial cells. As a result, DNA often gets tangled or knotted during cellular processes. DNA tangles and knots are extremely harmful to cells because they do not allow newly synthesised chromosomes to segregate properly or the two strands of DNA to separate, respectively. In addition, as protein complexes move through the double helix to synthesise new DNA (replication) or express genes (transcription), the genetic material ahead of these complexes becomes overwound. This overwinding makes it difficult to separate the two strands of the genetic material and inhibits these critical cellular processes.
Gyrase and topoisomerase IV work in concert to alleviate DNA overwinding ahead of replication and transcription complexes and to remove tangles and knots from DNA. These enzymes act by generating transient breaks in both strands of the double helix, passing another segment of DNA through the DNA gate, and resealing the original break.
Gyrase and topoisomerase IV are very powerful enzymes because their actions make DNA invisible to itself. However, their mechanism of action also makes them extremely dangerous enzymes, because if they fail to rejoin the DNA that they have cleaved, they have the potential to fragment the genome. Thus, levels of DNA cleavage by gyrase and topoisomerase IV are precariously balanced. If enzyme activity falls below threshold levels, cells die because of the loss of essential enzyme functions.
Bacterial type II topoisomerases: Life and death
Conversely, if cleavage levels are too high, the accumulation of DNA strand breaks leads to mutagenesis and cell death. The Osheroff Laboratory has a long-standing interest in how drugs interact with topoisomerases and how they function. We have demonstrated that fluoroquinolones interact with gyrase and topoisomerase IV through a chelated metal ion that is coordinated through water molecules that interact with the amino acids that are mutated in resistant bacteria. Fluoroquinolones kill bacteria by inserting themselves into the DNA bonds that have been cleaved by gyrase and topoisomerase IV, thereby increasing the levels of double-stranded DNA breaks that are generated by these enzymes.
Novel topoisomerase-targeted antibacterials
If new classes of antibacterials can be identified that target gyrase and topoisomerase IV but interact differently than fluoroquinolones, they have the potential to overcome drug resistance. Thus, the Osheroff laboratory has been characterising interactions between two novel classes of antibacterial compounds with gyrase and topoisomerase IV. Members of these two classes, gepotidacin and zoliflodacin, respectively, are currently in phase III clinical trials and appear to overcome fluoroquinolone resistance. If successful, they will become the first new classes of antibacterials approved for human use since 1980.
Gepotidacin is a “novel bacterial topoisomerase inhibitor” (NBTI). Compared to fluoroquinolones, NBTIs are distinct in three major respects. First, structural studies demonstrate that only a single NBTI molecule interacts with the DNA in the active site of gyrase. It binds between the two scissile bonds and elongates the DNA in the active site of the enzyme. This is in contrast to the two fluoroquinolones (one at each cut scissile bond) that interact with cleaved DNA in the active site. Second, whereas fluoroquinolones stabilise double-stranded DNA breaks generated by gyrase or topoisomerase IV, NBTIs induce only single-stranded DNA breaks. Third, it appears that NBTIs may kill some bacterial by increasing levels of gyrase- or topoisomerase IV-mediated DNA cleavage, while in others, they act by robbing cells of the essential functions of these enzymes.
Zoliflodacin is a spiropyrimidinetrione (SPT). Although these compounds interact with different residues than fluoroquinolones, they induce enzyme-mediated double-stranded DNA breaks.
Hopefully, these and other new drug classes will allow gyrase and topoisomerase IV to remain important antibacterial targets for decades to come.
K.J. Aldred, T. Blower, J.M. Berger, R.J. Kerns, and N. Osheroff (2016) Proc. Natl. Acad. Sci. USA 113, E839–E846. “Fluoroquinolone Interactions with Mycobacterium tuberculosis Gyrase: Enhancing Drug Activity Against Wild-Type and Resistant Gyrase.”
R.E. Ashley, R.H. Lindsey, Jr., S.A. McPherson, C.L. Turnbough, Jr., R.J. Kerns, and N. Osheroff (2017) Biochemistry 56, 4191-4200. “Gyrase-Quinolone Interactions and the Basis of Drug Resistance.”
E.G. Gibson, R.E. Ashley, R.J. Kerns, and N. Osheroff (2018) in Antimicrobial Resistance in the Twenty-first Century (B. Fong, D. Shlaes, and K. Drlica, eds.), pp. 507-529. Springer, New York. “Fluoroquinolone Interactions with Bacterial Type II Topoisomerases and Target-mediated Drug Resistance.”
E.G. Gibson, B. Bax, P.F. Chan, and N. Osheroff (2019) ACS Infect. Dis. 5, 570-581. “Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase.”
E.G. Gibson, A.A. Oviatt, M. Cacho, K.C. Neuman, P.F. Chan, and Neil Osheroff (2019) Biochemistry 58, 4447-4455, “Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial that Targets Bacterial Type II Topoisomerases.”
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