The Story of Zoliflodacin – A MedChemica Review

 

Figure 1: The Structure of zoliflodacin

 

Introduction

Zoliflodacin (Figure 1, above) is an orally administered DNA gyrase (topoisomerase) inhibitor, under study for the treatment of Gonorrhoea. The Global Antibiotics Research and Development Partnership (GARDP) recently announced that the primary endpoint for its phase 3 clinical trial (noninferiority to ceftriaxone/azithromycin) was met.^1,2 In isolation, this represents a significant milestone in the development of new and effective treatments for Gonorrhoea, a disease that impacts millions. From a slightly higher perspective, the progress of zoliflodacin may lead the way for this compound to be used in trials against other bacterial infections. Furthermore, this may reignite interest in the discovery of new broad-spectrum antibiotics amidst a challenging landscape of antimicrobial-resistant pathogens.

Here, I will summarise the significance and story of this remarkable compound in several parts: Firstly, the problem of antimicrobial resistance will be introduced. Next, the mode of action will be mentioned within the context of currently marketed antibiotics. Finally, a development history timeline of zoliflodacin, including relevant precursor molecules, will be provided.

I .Antibiotic-resistant infections, ever on the rise

Each year, there are millions of infection-related deaths.³ Of these, it is estimated⁴ that 1.27 million are attributable to bacterial antimicrobial resistance (AMR). This represents a significant global health burden and has long been identified as an emerging threat that will require a large amount of effort and investment to overcome. Clinically, AMR manifests itself as a gradual erosion of efficacy of key frontline antibiotics such as penicillin, tetracycline and ciprofloxacin leading to intractable multi-drug resistant “superbugs”.^5,6  The US Centre for Disease Control and Prevention (CDC) describes antibiotic resistance (AR) as “one of the greatest global public health challenges of our time.” Among the pathogens listed in their 2019 report on AR threats⁷was Neisseria gonorrhoeae, a species of bacteria that causes Gonorrhoea. At the time it was estimated that 550,000 antibiotic resistant infections of N. Gonorrhoeae occurred per year and this was classified as urgent.

 

Figure 2: Timeline for efficacy erosion of various frontline antibiotics vs Gonococcal infections (source: https://www.cdc.gov/antimicrobial-resistance/media/pdfs/2019-ar-threats-report-508.pdf)

The plot in Figure 2, published by the CDC,⁷ of % resistant N. Gonorrhoeae infections vs time presents a sobering overview of the changing efficacy of various families of antibiotic drugs over the last 20 years. It does demonstrate the importance of case surveillance, and the almost immediate impact that central recommendations for treatment usage can have on clinically evolved resistance. Despite seeing drops in resistant cases after withdrawing recommendations for treatment usage, resistant cases have been rising steadily to the point that a significant portion of N. Gonorrhoeae cases today are resistant to tetracycline, penicillin and ciprofloxacin: spanning 3 different families of widely used and relied-upon antibiotics.

(For a further discussion of the evolution, emergence, and spread of AR in the context of N. Gonorrhoeae, read the following review by Magnus Unemo and William Shafer: “Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future”)⁸

II. MoA matters

Antibiotics currently in use can be classified by their mode of action (MoA) (Figure 3, below). As an example of how antibiotic resistance can emerge, Efficacy of some well-known β-lactam antibiotics such as penicillin has diminished among certain bacterial strains due to the production of β-lactamase enzymes, that destroy the antibiotic agent. It is through resistance mechanisms such as this that may have led to the emergence of dangerous pathogens such as Methicillin Resistant Staphylococcus Aureus (MRSA).

Figure 3: Classification of antibiotics by mode of action. Source: https://journals.lww.com/joacp/fulltext/2017/33030/action_and_resistance_mechanisms_of_antibiotics__a.4.aspx

A key weapon against antibiotic resistance is the continued development and introduction of new antibiotic medicines, particularly with novel modes of action to avoid cross-resistance. One class of broad-spectrum antibiotics currently in use against serious urinary tract infections (UTIs), when other treatments fail, are the fluoroquinolones, (e.g. ciprofloxacin). The mechanism of action is by inhibition of bacterial DNA topoisomerases.

DNA Topoisomerase inhibitors to the rescue?

DNA topoisomerases are a family of enzymes that induce changes in the coiled state of DNA, allowing it to be properly metabolised and transcribed for replication and other processes to occur as normal. Disturbance of these targets by chemical inhibition thus turns out to be cytotoxic, with intriguing applications of such compounds as anticancers and antibiotics. Topoisomerases can be classified as type I or type II by which type of DNA (single stranded- or double stranded-, respectively) they recognise, and further categorised by their dependence on ATP and Mg²⁺ to carry out their role.

Figure 4: Classification of DNA topoisomerases, source: https://onlinelibrary.wiley.com/doi/10.1002/bies.202000286

One of the most famous examples is DNA gyrase, which is found in both Prokaryotes and Eukaryotes. The key structural differences between human and bacterial gyrase makes it a good target for antibacterial chemotherapy.

While the DNA gyrase inhibitor ciprofloxacin remains an important frontline treatment for many conditions, resistance began to emerge in the late 1990s⁹ due to frequent use against minor infections¹⁰ and instances of drug inefficacy are now common. In fact, outbreaks of a particular bacterium: C. Difficile have even been attributed to fluoroquinolone (mis)use.^11,12 The revelation of a post-antibiotic era encouraged a flurry of discovery efforts at the time, in an attempt to find agents that were still effective against these resistant strains.

III. Spiro-Pyrimidine-Triones (SPTs): A new class of topoisomerase inhibitors

Figure 5: Structure of PNU-286607, hit antibacterial compound from the Pharmacia Research Compound Collection

In the 2000s, research into novel antibacterial compounds was in full swing. Researchers at the (then known as) Pharmacia-Upjohn corporation in Kalamazoo, Michigan published some of their work in this area in 2008.¹³ The authors noted that although target-based approaches for hit finding were state-of-the-art, and promising, due to the publication of numerous bacterial genomes, they largely fail to produce clinical antibiotic candidates due to a lack of whole-cell activity (WCA). Thus, they opted for a phenotypic screening approach based on finding compounds with WCA, in order to mitigate this expected attrition. The reward for this effort came in the form of a remarkable hit compound, PNU-286607 (Figure 5), which came from the Pharmacia Research Compound Collection.

Figure 6: Example of the Tert-Amino effect, and its use in the synthesis of PNU-286607, source: [https://pubs.acs.org/doi/pdf/10.1021/ja808014h]

Figure 8: DNA Decatenation assays carried out with PNU-286607 for (A) DNA gyrase and (B) purified bacterial topoisomerase IV, identifying these proteins as the probable antibacterial targets. NB – the axes may have been mis-labelled on plot A, as the stated IC50 for DNA gyrase in the text is 9 μM, while this doesn’t seem to be correct when you look at the data. Source: [https://doi.org/10.1128/aac.00247-08]

PNU-286607 was found to have decent activity against numerous multi-drug resistant (MDR) bacteria, including strains of MDR S. Aureus. Comparable, in fact, to contemporary marketed antibiotics. The likely mode of action was investigated by reverse genomics studies, and comparison of activity with compounds of known MoA. The results pointed to a similar MoA to ciprofloxacin (but with important subtle differences) namely: the inhibition of bacterial DNA topoisomerase (DNA gyrase). This was concluded by testing PNU-286607 in a functional DNA decatenation assay (Figure 8) suggesting IC₅₀ values of 9 μM (DNA gyrase, E. Coli) and 30 μM (Topoisomerase IV, E. Coli).

The value of this hit compound was further demonstrated by the fact that, without chemical modification, it displayed: high oral bioavailability and in-vivo efficacy in a lethal systemic infection mouse model of MRSA and no activity against purified human topo II or in a eukaryotic cell proliferation assay, demonstrating a promising safety margin.

The race towards a clinical SPT candidate antibiotic

Figure 9: Evolution of nitrobenzene group from PNU-286607 in patent filings between 2000-2010

The discovery of PNU-286607 attracted significant interest, but its properties were not suitable for progression into the clinic at the time. A number of patents were filed, but a key breakthrough in this work came from the discovery that the nitrobenzene moiety could be exchanged with heterocycles (Figure 9 above),^16–18 opening the door to a more promising drug-like property space.

Figure 10: Synthesis of the fused benzisoxazole ring system of (-)-1 from 2,3,4-trifluorobenzoic acid

Researchers at AstraZeneca identified this series as a significant opportunity to gain a development candidate in this field, and set about making and testing analogues of PNU-287707 and other patented inhibitors.^19,20They were able to adequately differentiate their work by discovering that the nitrobenzene could be replaced by a fused benzisoxazole, which was prepared by a double SN_Ar synthetic route from 2,3,4-trifluorobenzoic acid (Figure 10).

With a solid route towards benzisoxazoles in hand, a number of analogues were prepared and screened against a DNA supercoiling assay²¹ and whole-cell-activity assays against numerous bacterial strains to obtain minimum inhibitory concentration (MIC) values.

Plasma protein binding (PPB) is a key property in drug discovery, a candidate needs to exhibit a high enough free drug concentration in the blood (measured in-vitro as “fraction unbound”, fu) to be able to perform the desired pharmacological effect. On the other hand, a lower fu will have the effect of protecting the drug compound from metabolism and clearance by having it retained as a reservoir of protein-bound complex. Very low %fu values were observed for most of the prepared analogues in this work, which is expected for acidic, moderate lipophilicity compounds.²²

Figure 11:  Plot demonstrating in-vivo efficacy against MSSA in immunocompetent and neutropenic mice. CFU = colony forming unit. Source: https://pubs.acs.org/doi/10.1021/jm501174m

An option for the antibacterial assays employed was to add 50% human serum into the assay medium, this gave an early indication of the total drug levels required to cause the desired antibacterial effect. Large increases in MIC values were seen when repeating the antibacterial assay in the presence of 50% serum, apart from their picked compound (-)-1  which showed only a 3x drop-off upon serum incorporation.

(-)-1 was shown to dose-dependently inhibit the uptake of labelled bases/cofactors, such as ³H-thymidine, ³H-uridine and ¹⁴C-N-acetyl-glucosamine. This aligned with the previously published results for PNU-286607, and suggested the two compounds had a common MoA of interfering with DNA, RNA and cell wall biosynthesis. Measurement of in-vivo efficacy against mouse models of MRSA (Figure 11.)  and a NOAEL of 500 mg kg^-1 day^-1, leading to a good therapeutic window, gave the researchers encouragement that the SPT series showed great promise (assuming that the next generation inhibitor showed better antibacterial activity and spectrum vs streptococci while maintaining favourable PK and tolerability).

 

The discovery of zoliflodacin / ETX-0914

Figure 12: Key data showing variation in antibacterial activity, PK and preclinical safety properties upon variation of benzisoxazole 3-position. Source: https://www.nature.com/articles/srep11827

Like clockwork, such a next generation TopoIV inhibitor: zoliflodacin (ETX0914) was patented and disclosed soon after,^23–26 once it had entered Phase I clinical trials as a potential treatment for gonococcal infections including the notorious N. Gonorrhoea. Reporting on the continuation of discovery efforts from (-)-1 towards zoliflodacin,²⁶ the authors noted which areas of the SPT scaffold were amenable to variation and which were essential (figure 12). By varying the benzisoxazole 3-position, changes to important in-vitro and in-vivo measured properties could be made while maintaining and/or gaining antibacterial activity across the desired spectrum. ETX-0914 displayed very high bioavailability in dog and, importantly, no measurable toxicity signals of concern seen for some fluoroquinolone antibiotics including genotoxicity (measured by mouse micronucleus aberration assay, MMA) and bone marrow suppression (measured by suppression of erythroid and myeloid cell lines).

The authors suggest that planar aromatic or carboxamide substitutions at the benzisoxazole 3-position lead to more problematic safety profiles than those that were more sp3-rich, suggesting that the latter were less susceptible to binding significant off-targets.

Figure 13: Dependence of DNA topology on concentration of Mg²⁺ ions for topoisomerase IV in the presence of ciprofloxacin (left) and zoliflodacin (right). Source: https://www.nature.com/articles/srep11827

As with the studies of previous iterations of potential SPT antibiotics, differentiation of MoA  from fluoroquinolone antibiotics was demonstrated by mechanistic studies and efficacy against fluoroquinolone-resistant bacterial isolates, including demonstration of a lack of cross resistance. Binding and inhibition of zoliflodacin to topoisomerases of S. Aureus were found to be independent of the concentration of Mg²⁺ (Figure 13), in contrast to ciprofloxacin, which is thought to form a chelation complex with a Mg ion.²⁷ This result suggests a different mode of binding of zoliflodacin and ciprofloxacin to TopoIV / DNA gyrase.

The generation of zoliflodacin-resistant strains of S. Aureus was carried out, where the authors add that the incidences of drug resistance were much lower than that observed in similar studies done with fluoroquinolones. While zoliflodacin-resistant strains were found, they were also found to be fully susceptible to ciprofloxacin and novobiocin, further differentiating their MoA. Resistance mutations were found only in the gyrB domain of DNA gyrase, near to the binding site for ciprofloxacin but distant from ciprofloxacin resistance determinants.

Summary – Zoliflodacin:  a promising representative of a new class of antibiotic compounds

In conclusion, antimicrobial resistance may present a defining medical challenge of our time. Clinically observed bacterial infections that are resistant to key antibiotic drugs on the market today, are now a significant percentage of total infections and this percentage is set to rise. Work started in the late 1990s on identifying novel antibiotic compounds with distinct modes of action has culminated in the introduction of zoliflodacin, a clinical antibiotic candidate drug with hopes of treating gonococcal infections that are resistant to fluoroquinolones such as ciprofloxacin.

Zoliflodacin has a colourful development history, beginning from an extraordinary high-throughput screening hit compound (that displayed in-vivo efficacy vs MRSA). Contributions to the development of this compound series came from numerous organisations, including Pharmacia & Upjohn, Werner-Lambert, Pfizer, Entasis, AstraZeneca and more. The complex polycyclic chemical structure presented a synthetic challenge in scaling up, and lead to the revival and utilisation of some old but highly effective rearrangement processes. Final lead optimisation lead to zoliflodacin, which entered clinical trials soon after.

Trials have since demonstrated that zoliflodacin shows promise for treating gonococcal infections, but the broader goal of becoming a broad-spectrum antibiotic to counter fluoroquinolone-resistant infections has yet to be ruled out. This work has also stimulated further studies on the SAR and antibiotic spectrum of the SPT inhibitor family, leading to some promising progress towards addressing antibiotic-resistant strains of Mycobacterium tuberculosis and multidrug resistant Staphylococcus aureus.^28–31

While AMR seems like a daunting challenge ahead, serendipity in science has a track record of finding treatments to bacterial infection (perhaps I should write about penicillin next).   The discovery of PNU-286607 followed by the hard development work of numerous institutions and hundreds of people giving us zoliflodacin, offers hope for a future of new broad spectrum antibiotic drugs.

Dr David Cousins – Senior Medicinal Chemist, MedChemica. Ltd.

References:

(1)        Zoliflodacin in Uncomplicated Gonorrhoea; NCT03959527; Global Antibiotics Research and Development Partnership. https://www.clinicaltrials.gov/study/NCT03959527 (accessed 2024-11-04).

(2)        Positive Results Announced in Largest Pivotal Phase 3 Trial of a First-in-Class Oral Antibiotic to Treat Uncomplicated Gonorrhoea. https://gardp.org/positive-results-announced-in-largest-pivotal-phase-3-trial-of-a-first-in-class-oral-antibiotic-to-treat-uncomplicated-gonorrhoea/ (accessed 2024-11-04).

(3)        Ikuta, K. S.; et al. Global Mortality Associated with 33 Bacterial Pathogens in 2019: A Systematic Analysis for the Global Burden of Disease Study 2019. The Lancet 2022, 400 (10369), 2221–2248. https://doi.org/10.1016/S0140-6736(22)02185-7

(4)        Murray, C. J. L.; et al. Global Burden of Bacterial Antimicrobial Resistance in 2019: A Systematic Analysis. The Lancet 2022, 399 (10325), 629–655. https://doi.org/10.1016/S0140-6736(21)02724-0

(5)        Algammal, A.; Hetta, H. F.; Mabrok, M.; Behzadi, P. Editorial: Emerging Multidrug-Resistant Bacterial Pathogens “Superbugs”: A Rising Public Health Threat. Front. Microbiol. 2023, 14, 1135614. https://doi.org/10.3389/fmicb.2023.1135614

(6)        Painuli, S.; Semwal, P.; Sharma, R.; Akash, S. Superbugs or Multidrug Resistant Microbes: A New Threat to the Society. Health Science Reports 2023, 6 (8), e1480. https://doi.org/10.1002/hsr2.1480

(7)        ANTIBIOTIC RESISTANCE THREATS IN THE UNITED STATES 2019. https://www.cdc.gov/antimicrobial-resistance/media/pdfs/2019-ar-threats-report-508.pdf (accessed 2024-11-04).

(8)        Unemo, M.; Shafer, W. M. Antimicrobial Resistance in Neisseria Gonorrhoeae in the 21st Century: Past, Evolution, and Future. Clin Microbiol Rev 2014, 27 (3), 587–613. https://doi.org/10.1128/CMR.00010-14

(9)        Vatopoulos, A. C.; Kalapothaki; Network, G.; for the Surveillance of Antimicrobial Resistance; Legakis, N. J. Bacterial Resistance to Ciprofloxacin in Greece: Results from the National Electronic Surveillance System. Emerg. Infect. Dis. 1999, 5 (3), 471–476. https://doi.org/10.3201/eid0503.990325

(10)     Wu, H. M.; Harcourt, B. H.; Hatcher, C. P.; Wei, S. C.; Novak, R. T.; Wang, X.; Juni, B. A.; Glennen, A.; Boxrud, D. J.; Rainbow, J.; Schmink, S.; Mair, R. D.; Theodore, M. J.; Sander, M. A.; Miller, T. K.; Kruger, K.; Cohn, A. C.; Clark, T. A.; Messonnier, N. E.; Mayer, L. W.; Lynfield, R. Emergence of Ciprofloxacin-Resistant Neisseria Meningitidis in North America. N Engl J Med 2009, 360 (9), 886–892. https://doi.org/10.1056/NEJMoa0806414

(11)     Gaynes, R.; Rimland, D.; Killum, E.; Ken Lowery, H.; Johnson Ii, T. M.; Killgore, G.; Tenover, F. C. Outbreak of Clostridium Difficile Infection in a Long‐Term Care Facility: Association with Gatifloxacin Use. CLIN INFECT DIS 2004, 38 (5), 640–645. https://doi.org/10.1086/381551

(12)     Gerding, D. N. Clindamycin, Cephalosporins, Fluoroquinolones, and Clostridium Difficile –Associated Diarrhea: This Is an Antimicrobial Resistance Problem. CLIN INFECT DIS 2004, 38 (5), 646–648. https://doi.org/10.1086/382084

(13)     Miller, A. A.; Bundy, G. L.; Mott, J. E.; Skepner, J. E.; Boyle, T. P.; Harris, D. W.; Hromockyj, A. E.; Marotti, K. R.; Zurenko, G. E.; Munzner, J. B.; Sweeney, M. T.; Bammert, G. F.; Hamel, J. C.; Ford, C. W.; Zhong, W.-Z.; Graber, D. R.; Martin, G. E.; Han, F.; Dolak, L. A.; Seest, E. P.; Ruble, J. C.; Kamilar, G. M.; Palmer, J. R.; Banitt, L. S.; Hurd, A. R.; Barbachyn, M. R. Discovery and Characterization of QPT-1, the Progenitor of a New Class of Bacterial Topoisomerase Inhibitors. Antimicrob Agents Chemother 2008, 52 (8), 2806–2812. https://doi.org/10.1128/AAC.00247-08

(14)     Ruble, J. C.; Hurd, A. R.; Johnson, T. A.; Sherry, D. A.; Barbachyn, M. R.; Toogood, P. L.; Bundy, G. L.; Graber, D. R.; Kamilar, G. M. Synthesis of (−)-PNU-286607 by Asymmetric Cyclization of Alkylidene Barbiturates. J. Am. Chem. Soc. 2009, 131 (11), 3991–3997. https://doi.org/10.1021/ja808014h

(15)     Verboom, W.; Reinhoudt, D. N.; Visser, R.; Harkema, S. “Tert-Amino Effect” in Heterocyclic Synthesis. Formation of N Heterocycles by Ring-Closure Reactions of Substituted 2-Vinyl-N,N-Dialkylanilines. J. Org. Chem. 1984, 49 (2), 269–276. https://doi.org/10.1021/jo00176a011

(16)     Barbachyn, M. R.; Dobrowolski, P. J.; Hurd, A. R.; Mcnamara, D. J.; Palmer, J. R.; Romero, A. G.; Ruble, J. C.; Sherry, D. A.; Thomasco, L. M.; Toogood, P. L.; Bundy, G. L.; Martin, G. E.; Romero, D. L. Tricyclic Tetrahydroquinoline Antibacterial Agents. WO2004031195A1, April 15, 2004. https://patents.google.com/patent/WO2004031195A1/en?oq=WO2004031195 (accessed 2024-11-04).

(17)     Barbachyn, M. R.; Dobrowolski, P. J.; Hagen, S. E.; Heimbach, T. H.; Hurd, A. R.; Johnson, T. A.; Mcnamara, D. J.; Ruble, J. C.; Sherry, D. A.; Thomasco, L. M.; Toogood, P. L. Antibacterial Agents. WO2006120563A2, November 16, 2006. https://patents.google.com/patent/WO2006120563A2/en?oq=WO2006120563 (accessed 2024-11-04).

(18)     Johnson, T. A.; Sherry, D. A.; Mcnamara, D. J.; Toogood, P. L. 8-Pyrazinyl-s-Spiropyrimidinetrione-Oxazinoquinoline Derivatives as Antibacterial Agents. WO2007072151A1, June 28, 2007. https://patents.google.com/patent/WO2007072151A1/en?oq=WO2007072151 (accessed 2024-11-04).

(19)     Basarab, G. S.; Brassil, P.; Doig, P.; Galullo, V.; Haimes, H. B.; Kern, G.; Kutschke, A.; McNulty, J.; Schuck, V. J. A.; Stone, G.; Gowravaram, M. Novel DNA Gyrase Inhibiting Spiropyrimidinetriones with a Benzisoxazole Scaffold: SAR and in Vivo Characterization. J. Med. Chem. 2014, 57 (21), 9078–9095. https://doi.org/10.1021/jm501174m.

(20)     Barvian, K.; Basarab, G. S.; Gowravaram, M. R.; Hauck, S. I.; Zhou, F. Fused, Spirocyclic Heteroaromatic Compounds for the Treatment of Bacterial Infections. US8658641B2, February 25, 2014. https://patents.google.com/patent/US8658641B2/en?oq=US8658641 (accessed 2024-11-04)

(21)     Shapiro, A.; Jahic, H.; Prasad, S.; Ehmann, D.; Thresher, J.; Gao, N.; Hajec, L. A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay. SLAS Discovery 2010, 15 (9), 1088–1098. https://doi.org/10.1177/1087057110378624

(22)     Smith, D. A.; Di, L.; Kerns, E. H. The Effect of Plasma Protein Binding on in Vivo Efficacy: Misconceptions in Drug Discovery. Nat Rev Drug Discov 2010, 9 (12), 929–939. https://doi.org/10.1038/nrd3287

(23)     Basarab, G. S.; Gowravaram, M. R.; Hauck, S. I.; Zhou, F. Compounds and Methods for Treating Bacterial Infections. US20140206677A1, July 24, 2014. https://patents.google.com/patent/US20140206677A1/en?oq=US20140206677 (accessed 2024-11-04).

(24)     Jacobsson, S.; Golparian, D.; Alm, R. A.; Huband, M.; Mueller, J.; Jensen, J. S.; Ohnishi, M.; Unemo, M. High In Vitro Activity of the Novel Spiropyrimidinetrione AZD0914, a DNA Gyrase Inhibitor, against Multidrug-Resistant Neisseria Gonorrhoeae Isolates Suggests a New Effective Option for Oral Treatment of Gonorrhea. Antimicrob Agents Chemother 2014, 58 (9), 5585–5588. https://doi.org/10.1128/AAC.03090-14

(25)     Kohlhoff, S. A.; Huband, M. D.; Hammerschlag, M. R. In Vitro Activity of AZD0914, a Novel DNA Gyrase Inhibitor, against Chlamydia Trachomatis and Chlamydia Pneumoniae. Antimicrob Agents Chemother2014, 58 (12), 7595–7596. https://doi.org/10.1128/AAC.03920-14

(26)     Basarab, G. S.; Kern, G. H.; McNulty, J.; Mueller, J. P.; Lawrence, K.; Vishwanathan, K.; Alm, R. A.; Barvian, K.; Doig, P.; Galullo, V.; Gardner, H.; Gowravaram, M.; Huband, M.; Kimzey, A.; Morningstar, M.; Kutschke, A.; Lahiri, S. D.; Perros, M.; Singh, R.; Schuck, V. J. A.; Tommasi, R.; Walkup, G.; Newman, J. V. Responding to the Challenge of Untreatable Gonorrhea: ETX0914, a First-in-Class Agent with a Distinct Mechanism-of-Action against Bacterial Type II Topoisomerases. Sci Rep 2015, 5 (1), 11827. https://doi.org/10.1038/srep11827

(27)     Aldred, K. J.; McPherson, S. A.; Turnbough, C. L.; Kerns, R. J.; Osheroff, N. Topoisomerase IV-Quinolone Interactions Are Mediated through a Water-Metal Ion Bridge: Mechanistic Basis of Quinolone Resistance. Nucleic Acids Research 2013, 41 (8), 4628–4639. https://doi.org/10.1093/nar/gkt124

(28)     Basarab, G. S.; Doig, P.; Eyermann, C. J.; Galullo, V.; Kern, G.; Kimzey, A.; Kutschke, A.; Morningstar, M.; Schuck, V.; Vishwanathan, K.; Zhou, F.; Gowravaram, M.; Hauck, S. Antibacterial Spiropyrimidinetriones with N-Linked Azole Substituents on a Benzisoxazole Scaffold Targeting DNA Gyrase. J. Med. Chem. 2020, 63(20), 11882–11901. https://doi.org/10.1021/acs.jmedchem.0c01100

(29)     Basarab, G. S.; Ghorpade, S.; Gibhard, L.; Mueller, R.; Njoroge, M.; Peton, N.; Govender, P.; Massoudi, L. M.; Robertson, G. T.; Lenaerts, A. J.; Boshoff, H. I.; Joerss, D.; Parish, T.; Durand-Reville, T. F.; Perros, M.; Singh, V.; Chibale, K. Spiropyrimidinetriones: A Class of DNA Gyrase Inhibitors with Activity against Mycobacterium Tuberculosis and without Cross-Resistance to Fluoroquinolones. Antimicrob Agents Chemother 2022, 66 (4), e02192-21. https://doi.org/10.1128/aac.02192-21

(30)     Govender, P.; Müller, R.; Singh, K.; Reddy, V.; Eyermann, C. J.; Fienberg, S.; Ghorpade, S. R.; Koekemoer, L.; Myrick, A.; Schnappinger, D.; Engelhart, C.; Meshanni, J.; Byl, J. A. W.; Osheroff, N.; Singh, V.; Chibale, K.; Basarab, G. S. Spiropyrimidinetrione DNA Gyrase Inhibitors with Potent and Selective Antituberculosis Activity. J. Med. Chem. 2022, 65 (9), 6903–6925. https://doi.org/10.1021/acs.jmedchem.2c00266

(31)     Byl, J. A. W.; Mueller, R.; Bax, B.; Basarab, G. S.; Chibale, K.; Osheroff, N. A Series of Spiropyrimidinetriones That Enhances DNA Cleavage Mediated by Mycobacterium Tuberculosis Gyrase. ACS Infect. Dis. 2023, 9 (3), 706–715. https://doi.org/10.1021/acsinfecdis.3c00012