Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy

Li, Yan; Wong, Yiik Diew; Ng, Fui Mee; Liu, Boping; Wong, Yun Xuan; Poh, Zhin Ying; Liu, Shuang; Then, Siew Wen; Lee, Michelle Yueqi; Ng, Hui Qi; Huang, Qiang; Hung, Alvin W.; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H.; Kang, CongBao

doi:10.1074/jbc.m116.737429

Cited by 15 publications

(6 citation statements)

References 42 publications

(84 reference statements)

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“…The averaged chemical shift changes (δCSP) were calculated as described previously. 23 Crystallization and Data Collection. Crystal screening and optimization for PaTrmD have been described elsewhere.…”

Section: ■ Conclusionmentioning

confidence: 99%

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N¹G37) Methyltransferase (TrmD) Inhibitors

et al. 2019

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Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N 1 G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based highthroughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD (PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-L-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAMnoncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.

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Section: ■ Conclusionmentioning

confidence: 99%

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N¹G37) Methyltransferase (TrmD) Inhibitors

et al. 2019

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“…Nevertheless, residue Asp73 of GyrB formed van dan Waals interactions with astaxanthin and novobiocin, respectively (Figure S1c and d ). Regarding topoi IV ParE, Arg1131, Val1153, and Val1136 that formed hydrogen and Pi-alkyl bonds at the active site with astaxanthin (Figures S2c ) were identified as catalytically important residues, though, not in agreement with Gly71, Arg72, Gly73, Ile68, Arg72, Val39, Ile40, Ser43, and Val44 previously reported by Li et al [ 40 ].…”

Section: Discussionmentioning

confidence: 57%

Astaxanthin‐Mediated Bacterial Lethality: Evidence from Oxidative Stress Contribution and Molecular Dynamics Simulation

Aribisala

Nkosi

Idowu

et al. 2021

Oxidative Medicine and Cellular Longevity

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The involvement of cellular oxidative stress in antibacterial therapy has remained a topical issue over the years. In this study, the contribution of oxidative stress to astaxanthin-mediated bacterial lethality was evaluated in silico and in vitro. For the in vitro analysis, the minimum inhibitory concentration (MIC) of astaxanthin was lower than that of novobiocin against Staphylococcus aureus but generally higher than those of the reference antibiotics against other test organisms. The level of superoxide anion of the tested organisms increased significantly following treatment with astaxanthin when compared with DMSO-treated cells. This increase compared favorably with those observed with the reference antibiotics and was consistent with a decrease in the concentration of glutathione (GSH) and corresponding significant increase in ADP/ATP ratio. These observations are suggestive of probable involvement of oxidative stress in antibacterial capability of astaxanthin and in agreement with the results of the in silico evaluations, where the free energy scores of astaxanthins’ complexes with topoisomerase IV ParC and ParE were higher than those of the reference antibiotics. These observations were consistent with the structural stability and compactness of the complexes as astaxanthin was observed to be more stable against topoisomerase IV ParC and ParE than DNA Gyrase A and B. Put together, findings from this study underscored the nature and mechanism of antibacterial action of astaxanthin that could suggest practical approaches in enhancing our current knowledge of antibacterial arsenal and aid in the novel development of alternative natural topo2A inhibitor.

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“…19 F-NMR was acquired as described previously on a Bruker 400-MHz magnet equipped with a BBO probe. 29,30 Titration experiments were carried out on a Bruker 700 MHz equipped with a cryo-probe. The 1 H-15 N-HSQC spectra of 0.5 mM of 15 Nlabeled HD in the absence and presence of different amounts of DNA were collected and compared.…”

Section: Dynamic Analysismentioning

confidence: 99%

Secondary structures, dynamics, and DNA binding of the homeodomain of human SIX1

Loh

et al. 2021

Journal of Peptide Science

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Human sine oculis homeobox homolog (SIX) 1 contains a homeodomain (HD), which is important for binding to DNA. In this study, we carried out structural studies on the HD of human SIX1 using nuclear magnetic resonance (NMR) spectroscopy. Its secondary structures and dynamics in solution were explored. HD is well-structured in solution, and our study shows that it contains three α-helices. Dynamics study indicates that the N-and C-terminal residues of HD are flexible in solution. HD of human SIX1 exhibits molecular interactions with a short double-strand DNA sequence evidenced by the 1 H-15 N-heteronuclear single quantum correlation (HSQC) and 19 F-NMR experiments. Our current study provides structural information for HD of human SIX1. Further studies indicate that this construct can be utilized to study SIX1 and DNA interactions.

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Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy

Abstract: Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the ␣4 and residues close to the loop of ␤1-␣2 and residues in the loop of ␤3-␤4 might improve the inhibitor potency.

Cited by 15 publications

References 42 publications

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N¹G37) Methyltransferase (TrmD) Inhibitors

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N¹G37) Methyltransferase (TrmD) Inhibitors

Astaxanthin‐Mediated Bacterial Lethality: Evidence from Oxidative Stress Contribution and Molecular Dynamics Simulation

Secondary structures, dynamics, and DNA binding of the homeodomain of human SIX1

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Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy

Abstract: Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the ␣4 and residues close to the loop of ␤1-␣2 and residues in the loop of ␤3-␤4 might improve the inhibitor potency.

Cited by 15 publications

References 42 publications

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors

Astaxanthin‐Mediated Bacterial Lethality: Evidence from Oxidative Stress Contribution and Molecular Dynamics Simulation

Secondary structures, dynamics, and DNA binding of the homeodomain of human SIX1

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Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N¹G37) Methyltransferase (TrmD) Inhibitors

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N¹G37) Methyltransferase (TrmD) Inhibitors