Advances in structure-based drug design of novel bacterial topoisomerase inhibitors

Abstract

Future Medicinal ChemistryVol. 2, No. 11 EditorialFree AccessAdvances in structure-based drug design of novel bacterial topoisomerase inhibitorsKatherine Widdowson & Alan HennessyKatherine Widdowson† Author for correspondenceAntibacterial Discovery Performance Unit, Infectious Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA. Search for more papers by this authorEmail the corresponding author at katherine.l.widdowson@gsk.com & Alan HennessyAntibacterial Discovery Performance Unit, Infectious Diseases CEDD, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UKSearch for more papers by this authorPublished Online:11 Nov 2010https://doi.org/10.4155/fmc.10.250AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: antibacterial mode of actionbacterial topoisomerase inhibitorsnovel bacterial topoisomerase inhibitorsquinolone antibacterialstreatment of resistant bacterial infectionx-ray structure-based drug designThe quinolone class of antibacterial agents, exemplified by ciprofloxacin, represents one of the most important classes of antibacterial agents [1]. Although the targets of these antibacterial agents, bacterial type II topoisomerases (BTs) have been investigated for the past 40 years [2–5], x-ray crystal structures of the quinolone–BT binding mode have only appeared very recently [6–8]. In addition, x-ray structures of a novel class of BT inhibitors that are structurally distinct from the quinolones have recently been published [9]. The novel bacterial topoisomerase inhibitors (NBTIs) and quinolones occupy different binding pockets, providing a structural basis for their lack of cross-resistance to quinolone-resistant strains. The knowledge gained by these recent crystal structures generates unprecedented opportunities for structural optimization and design of new topoisomerase inhibitors.What was previously known about NBTIs?We have been working in the NBTI field for over a decade, our first patent application appearing in 1999 [101]. In 2007, two communications describing a series of nonquinolone pyrazole and tetrahydroindazole inhibitors of type II topoisomerases were published by a group at Johnson and Johnson [10,11]. Although topoisomerase enzymatic activity and antibacterial potencies were given, there was only a brief insight into the mode of action of these inhibitors. The evidence to suggest a novel mode of action for these inhibitors was the absence of DNA cleavage complexes (in contrast to quinolones) and also similar antibacterial potencies against both quinolone-susceptible and -resistant strains of Staphylococcus aureus. Novel binding to the topoisomerase enzymes is not surprising given the structural differences between quinolones and these NBTIs.In 2008, a Novexel group published a detailed description of the mode of action of their lead compound NXL-101 [12]. Again, a nonquinolone mode of action was cited due to mutational findings, which show resistance caused by different topoisomerase enzyme mutations to those observed in quinolone-resistant strains. The key mutations conferring quinolone resistance are alterations to Ser-84 of S. aureus GyrA (equivalent to Ser-83 in Escherichia coli GyrA), whereas key mutations conferring NXL-101 resistance are alterations to S. aureus GyrA Asp-83. The relative potencies of NXL-101 and representative quinolones (ciprofloxaxin and moxifloxacin) in functional assessments of the topoisomerase II (gyrase) and IV enzymes were also discussed, which again highlighted the differences between quinolones and NBTIs.In 2009, this Novexel group reviewed the clinical progress of NBTIs, especially Viquidicin (NXL-101), which was discontinued due to QT prolongation in Phase I clinical trials [13]. The paper asserts that this is an extremely active patent field with activity from GlaxoSmithKline, Pfizer, Astra-Zeneca, Aventis, Morphochem and Toyama.While models and crystal structures of the gyrase enzyme were known, it was clear that little was truly known about the precise binding mode of the NBTIs and, therefore, progress on medicinal chemistry relied on classical structure–activity relationship (SAR) techniques such as pharmacophore modeling and optimization of ‘Lipinski-like’ properties.For GlaxoSmithKline, this changed when a crystal structure of a S. aureus DNA gyrase–DNA–NBTI complex was solved, using a multidisciplinary in-house team [9]. The crystal structures of quinolones and related quinazolinediones inhibitors bound to topoisomerase IV–DNA complexes have now also been published elswehere [6–8].General comments on the S. aureus DNA gyrase–DNA–NBTI complexPrior to solving the crystal structure, it had already been discovered that NBTI GSK299423 had the previously discussed key differences of quinolones: no dsDNA cleavage complexes and lack of cross-resistance to quinolone-resistant bacterial strains (caused by both topoisomerase II [gyrase] and IV mutations). A broad spectrum of activity against multidrug-resistant bacteria was also noted for GSK299423.The 2.1Å crystal structure of a complex of an NBTI with DNA and S. aureus gyrase also clarified a number of points regarding the mode of action of NBTIs.Replacement of the DNA-cleaving catalytic S. aureus GyrA Tyr-123 residues with phenylalanine allowed a precleavage complex to be isolated. We believe that the uncleaved DNA is stretched upon complex formation with the gyrase enzyme, but that GSK299423 then arrests the complex prior to the double-strand cleavage. This is a key mechanistic difference from the quinolones as they stabilize the postcleavage DNA–gyrase complex. Crystallographic data show that for a similar DNA–topoisomerase IV–quinolone construct, a quinolone molecule intercalates at each of two DNA-binding sites at the site of DNA cleavage, close to Acinetobacter baumannii ParC Ser-84 (equivalent to Ser-83 in E. coli GyrA), thus explaining the differences in resistance profiles for quinolones versus NBTIs [8].Although an unprecedented view of the catalytic cycle of the DNA gyrase enzyme is observed, as medicinal chemists, we focused on the key binding regions of our NBTI ligand.DNA-binding regionThe cyano-quinoline (LHS) unit of GSK299423 intercalates between the two central base pairs of a stretched 20-base pair DNA duplex, midway between the two gyrase active sites on the twofold axis of symmetry.An important feature for medicinal chemistry has been that the LHS, linker, central unit and oxathiolo-pyridine (RHS) can generally be independently changed and key subunits can therefore be evaluated systematically. To date, over 100 examples of LHS, linker and central units, and over 550 RHS units, have been assessed.With knowledge from the crystal structure, we examined the LHS–DNA binding interactions. These LHS–DNA interactions have been difficult to measure but we have used computational tools such as ab initio calculations to prioritize new series, especially where arduous syntheses are involved. Detailed analysis showed the accuracy of this system in predicting active and inactive LHS units for a variety of systems.The twofold symmetry of the binding site led us to scaffolds with alternative LHS linking points, which in turn further drove the SAR of the series.The search to find additional interactions in the DNA-binding region continues, in order to improve the potency of our novel NBTIs. A variety of LHS systems exists in the NBTI patent literature and more will no doubt appear in the future.Gyrase protein-binding regionA noncatalytic pocket appears upon complex formation allowing the RHS unit of GSK299423 to enter and bind via a series of van der Waals interactions with the Ala-68, Gly-72, Met-75 and Met-121 residues.This pocket is hydrophobic and its size appears to offer limited scope for a large variety of structural change. An additional interaction involves the methylene group flanked by oxygen and sulfur atoms, which appear to form an unusual hydrogen bond with the carbonyl oxygen of Ala-68 (distance of 3.2Å) [14]. The observation of this interaction helped clarify some of the SAR observed for this unit. Apart from the RHS unit, aryl-azinone units have also been described as being alternative RHS units of potent NBTIs.These key residues are largely conserved across Gram-positive and Gram-negative bacteria (both gyrase and Topo IV enzymes), thereby offering us a chance to design dual-targeting, broad-spectrum antibacterials. One difference is the change of Met-121 in S. aureus to Ile or Leu in many Gram-negative species. This modest alteration may offer some opportunities to develop more potent Gram-negative agents, exploiting the slightly larger pocket found in Gram-negative bacteria.The size of the pocket does place some restriction on the size of the unit that can bind within, and the patent literature on NBTIs appears largely focused on small bicyclic ring systems.Just outside this pocket, the basic nitrogen of GSK299423 forms an interaction with the other Asp-83 residue. We and the Novexel group have shown that mutation of Asp-83, or the nearby in space Met-121 residue, reduces both target and antibacterial potency of NBTIs and so this finding is in excellent correlation with previous data.This crystal structure provides strong evidence for retaining this interaction. Replacing this amine with a view to reducing hERG liability may have seemed favorable, but we had more success in keeping this key feature and instead lowering the overall logD of our compounds. Indeed, in the NBTI patent literature this basic amine is rarely absent, indicating challenges in finding replacements.Given that no major additional interactions are present it would appear that the central unit’s key role is to ensure the correct positioning of the LHS and RHS units. Using the vectors from the LHS and RHS units it is possible to predict the optimal linker geometry. In our experience an eight-atom link between the LHS and RHS is generally optimal. Many variants have appeared already and the central link provides an opportunity to modulate physicochemical properties without compromising target potency.ConclusionEmerging knowledge of the crystal structures of NBTI and quinolone ligands bound into topoisomerase–DNA complexes has given medicinal chemists an unprecedented opportunity to design novel antibacterials, with a view to overcoming clinical resistance, especially to marketed quinolones.The future may bring yet more advances in our knowledge of the mechanism of these toposisomerase enzymes and allow de novo design of the next generation of NBTIs. The complex nature of the catalytic cycle permits several points of intervention, in addition to those relevant to the quinolones and NBTIs, which may offer yet more novel antibacterial lead starting points.There can now be little doubt as to the need for novel mechanism antibacterials to combat the ever-growing threat of resistant bacterial infection and, given the torturous process from hit compound to marketed antibacterial, the time to act is now.The development of novel quinolones and NBTI antibacterials is now at a pivotal point, history may judge us poorly if we fail.AcknowledgementsWe thank all GlaskoSmithKline scientists, past and present, who have worked on this program, for their efforts. In particular we wish to acknowledge the two program leaders Neil Pearson and Mick Gwynn.Financial & competing interests disclosureAlan Hennessy was funded by the Wellcome Trust Seeding Drug Discovery Initiative and contract HDTRA1–07–9–0002 with the US Department of Defence Joint Science and Technology Office for Chemical and Biological Defence and the Defence Threat Reduction Agency’s Transformational Medical Technologies. Katherine Widdowson is an employee of GlaxoSmithKline. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the US Department of Defence or the US Government. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. 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Springer, NY, USA (1991).Google Scholar101 GlasxoSmithKline Beecham PLC: WO9937635 (1999).Google ScholarFiguresReferencesRelatedDetailsCited ByMolecular docking and molecular dynamics simulation identify a novel Radicicol derivative that predicts exclusive binding to Plasmodium falciparum Topoisomerase VIB2 March 2021 | Journal of Biomolecular Structure and Dynamics, Vol. 40, No. 15Elucidation of an essential function of the unique charged domain of Plasmodium topoisomerase III24 December 2020 | Biochemical Journal, Vol. 477, No. 241,3-Dioxane-Linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition1 May 2019 | ACS Infectious Diseases, Vol. 5, No. 7Novel bacterial topoisomerase inhibitors: challenges and perspectives in reducing hERG toxicityAnja Kolarič & Nikola Minovski14 September 2018 | Future Medicinal Chemistry, Vol. 10, No. 19Four Ways to Skin a Cat: Inhibition of Bacterial Topoisomerases Leading to the Clinic26 May 2017Structure-based design of novel combinatorially generated NBTIs as potential DNA gyrase inhibitors against various Staphylococcus aureus mutant strains1 January 2017 | Molecular BioSystems, Vol. 13, No. 7Quinazolinone azolyl ethanols: potential lead antimicrobial agents with dual action modes targeting methicillin-resistant Staphylococcus aureus DNAXin-Mei Peng, Li-Ping Peng, Shuo Li, Srinivasa Rao Avula, Vijaya Kumar Kannekanti, Shao-Lin Zhang, Kin Yip Tam & Cheng-He Zhou26 September 2016 | Future Medicinal Chemistry, Vol. 8, No. 16Recent advances in the rational design and optimization of antibacterial agents1 January 2016 | MedChemComm, Vol. 7, No. 9Topoisomerase II from Human Malaria ParasitesJournal of Biological Chemistry, Vol. 290, No. 33Structure activity relationship of substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-4)Bioorganic & Medicinal Chemistry Letters, Vol. 25, No. 11Novel DNA Gyrase Inhibiting Spiropyrimidinetriones with a Benzisoxazole Scaffold: SAR and in Vivo Characterization17 October 2014 | Journal of Medicinal Chemistry, Vol. 57, No. 21Extending the N-linked aminopiperidine class to the mycobacterial gyrase domain: Pharmacophore mapping from known antibacterial leadsEuropean Journal of Medicinal Chemistry, Vol. 85Confronting the challenges of discovery of novel antibacterial agentsBioorganic & Medicinal Chemistry Letters, Vol. 24, No. 16Food-Borne Topoisomerase InhibitorsN- versus O-alkylation: Utilizing NMR methods to establish reliable primary structure determinations for drug discoveryBioorganic & Medicinal Chemistry Letters, Vol. 23, No. 16De novo design of novel DNA–gyrase inhibitors based on 2D molecular fingerprintsBioorganic & Medicinal Chemistry Letters, Vol. 23, No. 14Novel quinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IVBioorganic & Medicinal Chemistry Letters, Vol. 23, No. 10Multitarget ligands in antibacterial research: progress and opportunities19 December 2012 | Expert Opinion on Drug Discovery, Vol. 8, No. 2Exploiting bacterial DNA gyrase as a drug target: current state and perspectives9 September 2011 | Applied Microbiology and Biotechnology, Vol. 92, No. 3 Vol. 2, No. 11 Follow us on social media for the latest updates Metrics History Published online 11 November 2010 Published in print November 2010 Information© Future Science LtdKeywordsantibacterial mode of actionbacterial topoisomerase inhibitorsnovel bacterial topoisomerase inhibitorsquinolone antibacterialstreatment of resistant bacterial infectionx-ray structure-based drug designAcknowledgementsWe thank all GlaskoSmithKline scientists, past and present, who have worked on this program, for their efforts. In particular we wish to acknowledge the two program leaders Neil Pearson and Mick Gwynn.Financial & competing interests disclosureAlan Hennessy was funded by the Wellcome Trust Seeding Drug Discovery Initiative and contract HDTRA1–07–9–0002 with the US Department of Defence Joint Science and Technology Office for Chemical and Biological Defence and the Defence Threat Reduction Agency’s Transformational Medical Technologies. Katherine Widdowson is an employee of GlaxoSmithKline. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the US Department of Defence or the US Government. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.PDF download