25 24 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Macromolecular Crystallography Group Beamline I04 Newantibiotics with a surprisingmode of action Related publication: Kolarič, A., Germe, T., Hrast, M., Stevenson, C. E. M., Lawson, D. M., Burton, N. P., Vörös, J., Maxwell, A., Minovski, N., & Anderluh, M. Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds. Nature Communications , 12 , 150 (2021). DOI :10.1038/s41467-020-20405-8 Publication keywords: DNA gyrase; Antibiotics; NBTIs; Halogen bonds R elatively fewnew antibiotics were developed in the late 20 th Century, and overuse has led to the development of significant bacterial resistance. With some bacterial infections becoming untreatable, antibiotic discovery is now a priority. An international collaboration, involving two academic groups in Slovenia and an academic group and a small company in Norwich, designed a series of new molecules with antibiotic potential. These new antibiotics, Novel Bacterial Topoisomerase Inhibitors (NBTIs), kill bacteria and act against a well-validated target, DNA gyrase. However, to enable the design of further molecules, the teamneeded to know the molecular structure of the newmolecules bound to gyrase. They used state-of-the-art macromolecular crystallography beamline I04 at Diamond Light Source to investigate how these work at the molecular level. The team evaluated the relevant potency of several NBTIs against gyrase and showed that they stabilised intermediates between the enzyme and the DNA, with a single DNA strand cleaved, which is important as it leads to bacterial killing. In addition, they solved the crystal structure of gyrase bound to one of the new inhibitors, revealing the existence of bifurcated halogen bonds between the enzyme and the inhibitor molecule - an unprecedented observation in a biological system. This new information informs the intelligent design of new molecules based on the new biochemical and structural information. The ultimate aim is to develop new antibiotics with desirable properties for use in clinical medicine. Novel Bacterial Topoisomerase Inhibitors (NBTIs) are a promising class of new antibacterial agents that are active against the bacterial target, DNA gyrase. By forming a complex with the enzyme and DNA, they stabilise single-strand DNA cleavage breaks, preventing the enzyme from functioning, which leads to cell death, in a manner related to the well-known quinolone antibiotics 1,2 . Since this type of antibacterial has now been known for many years, one would think that there would be little left to be discovered. Yet, the exact mechanism of NBTI-single-strand stabilisation has remained only hypothetical. The present study started as a classical medicinal chemistry project with the in-silico design of NBTIs with innovative fragments binding to DNA gyrase, the so-called “right-hand side (RHS)” of the starting compounds. In particular, careful examination of the binding site revealed that the RHS part of the molecule could form halogen/hydrogen bonds with the backbone carbonyl oxygen of at least one of the two amino acid residues (Alanines) in gyrase. NBTIs emerging from these in-silico protocols were synthesised, their biological activity was evaluated and the exact binding mode was clarified by crystallography 3-5 . To our delight, NBTI compounds with a p-halo-substituted phenyl RHS fragment showed remarkable inhibition of Staphylococcus aureus DNA gyrase: IC 50 =35 nM for chloro, IC 50 =7 nM for bromo, and IC 50 =11 nM for iodo derivatives. This gave us an indication that halogen binding is likely to be behind the measured potency. Surprisingly, such excellent enzyme inhibitory potency was due to the formation of a symmetrical bifurcated halogen bond between the halogen atom and a backbone carbonyl oxygen of Ala68 from both gyrase A (GyrA) subunits (PDB ID: 6Z1A) in the heterotetrameric enzyme. It was surprising to see a symmetric bifurcated halogen bond in a biological system. Obtaining the crystal structure was not trivial, in fact, we found that bromo and iodo derivatives stabilised the ternary complex so strongly and quickly that no crystals would form. The crystal was obtained in the end, but with a chloro derivative. Serendipitously, this is the first time that such a so- called symmetrical bifurcated halogen bond has been identified in a relevant biological systemwhere a ligand interacts with its macromolecular target. The crystal structure had more to offer and allowed a genuine breakthrough in understanding the mechanism of action of NBTIs. Namely, since gyrase is a symmetrical enzyme, all previously reported crystal structures housed NBTIs in two orientations rotated by 180° within the same crystal. Our crystal structure is the first in which an NBTI binds in a single conformation (without static disorder), which allowed us to elucidate the mechanism of how an NBTI stabilises the gyrase-DNA complex with only one DNA strand being nicked. Indeed, the intercalation of the asymmetric NBTI causes a shift of the DNA backbone away from the cleavage site on only one side. Consequently, the scissile phosphate can get close to the catalytic metal on one side only, which is why the NBTI stabilises single-strand cleavage. Our discovery of the mechanism of action as well as the identification of the symmetric bifurcated halogen bonds are the starting point for further structural modifications and optimisation of NBTIs. More importantly, it expands the repertoire of the medicinal chemist’s interaction toolbox to design molecules of the future. References: 1. Bush, N. G. et al. Quinolones: mechanism, lethality and their contributions to antibiotic resistance. Molecules , 25 , 5662 (2020). DOI: 10.3390/molecules25235662 2. Kolarič, A. et al . Two decades of successful SAR-grounded stories of the novel bacterial topoisomerase inhibitors (NBTIs). Journal of Medicinal Chemistry , 63 , 5664–5674 (2020). DOI: 10.1021/acs.jmedchem.9b01738 3. Kolaric, A. et al . Structure-based design of novel combinatorially generated NBTIs as potential DNA gyrase inhibitors against various Staphylococcus aureus mutant strains. Molecular BioSystems , 13 , 1406–1420 (2017). DOI: 10.1039/C7MB00168A 4. Kolarič, et al . Cyclohexyl amide-based novel bacterial topoisomerase inhibitors with prospective GyrA-binding fragments. Future Medicinal Chemistry , 11 , 935–945 (2019). DOI: 10.4155/fmc-2018-0472 5. Kolarič, et al . Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds. Nature Communications , 12 , 150 (2021). DOI: 10.1038/s41467-020-20405-8 Funding acknowledgement: The financial support of this work from the Slovenian Research Agency (Grants P1-0017 and P1-0208) is gratefully acknowledged. Work in A.M.’s laboratory is supported by the Biotechnology and Biosciences Research Council (BBSRC; UK) Institute Strategic Programme Grant BB/P012523/1, and theWellcome Trust (Investigator Award 110072/Z/15/Z); J.V.’s work was also supported by an FTMA award from BBSRC (BB/S507921/1). Diamond Light Source is acknowledged for access to beamline I04 under proposal MX18565. Corresponding authors: Prof. Anthony Maxwell, John Innes Centre,
[email protected] Prof. David Lawson, John Innes Centre,
[email protected] Figure 1: The crystal structure of p-chloro NBTI bound to a S. aureus gyrase-DNA complex (PDB ID: 6Z1A); (a) Overview showing gyrase in ribbons with the two halves of the complex in pale cyan and pale brown, respectively. A molecular surface for the DNA is shown in green and the inhibitor is shown as magenta van der Waals spheres; (b) Close up of the inhibitor binding site showing intercalation with the DNA at the top and the bifurcated halogen bond at the bottom that the NBTI makes with the backbone carbonyl oxygens of the two symmetry-related Ala68 residues of GyrA. Also shown as a transparent magenta surface is omit electron density for the inhibitor calculated at 2.3 Å resolution and contoured at 1.6 σ.