I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108]
Open Access
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.
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Nov 2024
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[18565]
Open Access
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.
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Jun 2024
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I03-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[18565]
Open Access
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.
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Jun 2023
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[25108]
Open Access
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.
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Mar 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[18565, 25108]
Open Access
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.
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Jan 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1856]
Open Access
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.
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Mar 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
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.
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Jan 2021
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[18565]
Open Access
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.
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Oct 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Diamond Proposal Number(s):
[18565]
Open Access
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.
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Jul 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475, 13467]
Open Access
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.
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Jan 2019
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