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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Structural aspects of Mycobacterium tuberculosis DNA gyrase targeted by novel bacterial topoisomerase inhibitors Maja Kokot , Martina Hrast Rambaher , Lipeng Feng , Lesley A Mitchenall , David M. Lawson , Anthony Maxwell , Tanya Parish , Nikola Minovski , Marko Anderluh Acs Medicinal Chemistry Letters DOI: 10.1021/acsmedchemlett.4c00447 Diamond Proposal Number(s): [25108] Open Access Show Abstract ▼ Abstract: In this Letter, we present a small series of novel bacterial topoisomerase inhibitors (NTBIs) that exhibit both potent inhibition of Mycobacterium tuberculosis DNA gyrase and potent antimycobacterial activity. The disclosed crystal structure of M. tuberculosis DNA gyrase in complex with DNA and compound 5 from this NBTI series reveals the binding mode of an NBTI in the GyrA binding pocket and confirms the presence and importance of halogen bonding for the excellent on-target potency. In addition, we have shown that compound 5 is a promising M. tuberculosis DNA gyrase inhibitor, with an IC50 for M. tuberculosis gyrase of 0.096 μM, and it has potent activity against M. tuberculosis, with an IC50 of 0.165 μM.	Nov 2024
I04-Macromolecular Crystallography	Exploring the interaction of N-(benzothiazol-2-yl)pyrrolamide DNA gyrase inhibitors with the GyrB ATP-binding site lipophilic floor: A medicinal chemistry and QTAIM study Nace Zidar , Andrej Emanuel Cotman , Wessel Sinnige , Ondrej Benek , Michaela Barančokova , Anamarija Zega , Lucija Peterlin Mašič , Tihomir Tomašič , Janez Ilaš , Sara R. Henderson , Julia E. A. Mundy , Anthony Maxwell , Clare E. M. Stevenson , David M. Lawson , Geert Jan Sterk , Rodrigo Tosso , Lucas Gutierrez , Ricardo D. Enriz , Danijel Kikelj Bioorganic & Medicinal Chemistry 43 DOI: 10.1016/j.bmc.2024.117798 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.	Jun 2024
I03-Macromolecular Crystallography	Exploring the 5-substituted 2-aminobenzothiazole-based DNA Gyrase B inhibitors active against ESKAPE pathogens Maša Sterle , Martina Durcik , Clare E. M. Stevenson , Sara Henderson , Petra Eva Szili , Marton Czikkely , David M. Lawson , Anthony Maxwell , Dominique Cahard , Danijel Kikelj , Nace Zidar , Csaba Pal , Lucija Peterlin Mašič , Janez Ilaš , Tihomir Tomašič , Andrej Emanuel Cotman , Anamarija Zega Acs Omega DOI: 10.1021/acsomega.3c01930 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 μg/mL for most Gram-positive strains and 4–16 μg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.	Jun 2023
I04-Macromolecular Crystallography	New dual inhibitors of bacterial topoisomerases with broad-spectrum antibacterial activity and in vivo efficacy against vancomycin-intermediate Staphylococcus aureus Martina Durcik , Andrej Emanuel Cotman , Žan Toplak , Štefan Možina , Žiga Skok , Petra Eva Szili , Márton Czikkely , Elvin Maharramov , Thu Hien Vu , Maria Vittoria Piras , Nace Zidar , Janez Ilaš , Anamarija Zega , Jurij Trontelj , Luis A. Pardo , Diarmaid Hughes , Douglas Huseby , Tália Berruga-Fernández , Sha Cao , Ivailo Simoff , Richard Svensson , Sergiy V. Korol , Zhe Jin , Francisca Vicente , Maria C. Ramos , Julia E. A. Mundy , Anthony Maxwell , Clare E. M. Stevenson , David M. Lawson , Björn Glinghammar , Eva Sjöström , Martin Bohlin , Joanna Oreskär , Sofie Alvér , Guido V. Janssen , Geert Jan Sterk , Danijel Kikelj , Csaba Pal , Tihomir Tomašič , Lucija Peterlin Mašič Journal Of Medicinal Chemistry 1703 DOI: 10.1021/acs.jmedchem.2c01905 Diamond Proposal Number(s): [25108] Open Access Show Abstract ▼ Abstract: A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125–0.25 μg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1–4 μg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.	Mar 2023
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Discovery and hit-to-lead optimization of benzothiazole scaffold-based DNA gyrase inhibitors with potent activity against Acinetobacter baumannii and Pseudomonas aeruginosa Andrej Emanuel Cotman , Martina Durcik , Davide Benedetto Tiz , Federica Fulgheri , Daniela Secci , Maša Sterle , Štefan Možina , Žiga Skok , Nace Zidar , Anamarija Zega , Janez Ilaš , Lucija Peterlin Mašič , Tihomir Tomašič , Diarmaid Hughes , Douglas L. Huseby , Sha Cao , Linnéa Garoff , Talía Berruga Fernández , Paraskevi Giachou , Lisa Crone , Ivailo Simoff , Richard Svensson , Bryndis Birnir , Sergiy V. Korol , Zhe Jin , Francisca Vicente , Maria C. Ramos , Mercedes De La Cruz , Björn Glinghammar , Lena Lenhammar , Sara R. Henderson , Julia E. A. Mundy , Anthony Maxwell , Claren E. M. Stevenson , David M. Lawson , Guido V. Janssen , Geert Jan Sterk , Danijel Kikelj Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.2c01597 Diamond Proposal Number(s): [18565, 25108] Open Access Show Abstract ▼ Abstract: We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.	Jan 2023
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic Lipeng Feng , Julia E. A. Mundy , Clare E. M. Stevenson , Lesley A. Mitchenall , David M. Lawson , Kaixia Mi , Anthony Maxwell Proceedings Of The National Academy Of Sciences 118 DOI: 10.1073/pnas.2016705118 Diamond Proposal Number(s): [1856] Open Access Show Abstract ▼ Abstract: DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA–MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.	Mar 2021
I04-Macromolecular Crystallography	Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds Anja Kolarič , Thomas Germe , Martina Hrast , Clare E. M. Stevenson , David M. Lawson , Nicolas P. Burton , Judit Voros , Anthony Maxwell , Nikola Minovski , Marko Anderluh Nature Communications 12 DOI: 10.1038/s41467-020-20405-8 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase–DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.	Jan 2021
I04-Macromolecular Crystallography	Exploring the chemical space of benzothiazole-based DNA gyrase B inhibitors Žiga Skok , Michaela Barančoková , Ondřej Benek , Cristina Durante Cruz , Päivi Tammela , Tihomir Tomašič , Nace Zidar , Lucija Peterlin Mašič , Anamarija Zega , Clare E. M. Stevenson , Julia E. A. Mundy , David M. Lawson , Anthony Maxwell , Danijel Kikelj , Janez Ilaš Acs Medicinal Chemistry Letters DOI: 10.1021/acsmedchemlett.0c00416 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: We designed and synthesized a series of inhibitors of the bacterial enzymes DNA gyrase and DNA topoisomerase IV, based on our recently published benzothiazole-based inhibitor bearing an oxalyl moiety. To improve the antibacterial activity and retain potent enzymatic activity, we systematically explored the chemical space. Several strategies of modification were followed: varying substituents on the pyrrole carboxamide moiety, alteration of the central scaffold, including variation of substitution position and, most importantly, modification of the oxalyl moiety. Compounds with acidic, basic, and neutral properties were synthesized. To understand the mechanism of action and binding mode, we have obtained a crystal structure of compound 16a, bearing a primary amino group, in complex with the N-terminal domain of E. coli gyrase B (24 kDa) (PDB: 6YD9). Compound 15a, with a low molecular weight of 383 Da, potent inhibitory activity on E. coli gyrase (IC50 = 9.5 nM), potent antibacterial activity on E. faecalis (MIC = 3.13 μM), and efflux impaired E. coli strain (MIC = 0.78 μM), is an important contribution for the development of novel gyrase and topoisomerase IV inhibitors in Gram-negative bacteria.	Oct 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase Sara R. Henderson , Clare E. M. Stevenson , Brandon Malone , Yelyzaveta Zholnerovych , Lesley A Mitchenall , Mark Pichowicz , David H Mcgarry , Ian R Cooper , Cedric Charrier , Anne-Marie Salisbury , David M. Lawson , Anthony Maxwell Journal Of Antimicrobial Chemotherapy 22 DOI: 10.1093/jac/dkaa286 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Objectives: To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. Methods: Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. Results: Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. Conclusions: Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.	Jul 2020
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Architecture of microcin B17 synthetase: an octameric protein complex converting a ribosomally synthesized peptide into a DNA gyrase poison Dmitry Ghilarov , Clare E. M. Stevenson , Dmitrii Y. Travin , Julia Piskunova , Marina Serebryakova , Anthony Maxwell , David M. Lawson , Konstantin Severinov Molecular Cell DOI: 10.1016/j.molcel.2018.11.032 Diamond Proposal Number(s): [9475, 13467] Open Access Show Abstract ▼ Abstract: The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.	Jan 2019

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