Burkholderia pseudomallei D-alanine-D-alanine ligase

detailed characterisation and assessment of a potential antibiotic drug target

Laura Diaz Saez, Leah Torrie, Stuart McElroy, David Gray, Bill Hunter (Lead / Corresponding author)

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Abstract

Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We selected the essential enzyme D-alanine-D-alanine ligase (BpDdl), required for the ATP assisted biosynthesis of a peptidoglycan precursor, and have characterised the enzyme. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+D-Ala-D-Ala), and comparisons with orthologous enzymes suggest a ligand-induced conformational change occurs that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow up and then in combination with a computational analysis of the active site we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches
Original languageEnglish
Number of pages16
JournalFEBS Journal
Early online date1 Jul 2019
DOIs
Publication statusE-pub ahead of print - 1 Jul 2019

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Burkholderia pseudomallei
Adenosine Monophosphate
Anti-Bacterial Agents
Ligands
Enzymes
Pharmaceutical Preparations
Peptidoglycan
Biosynthesis
Pathogens
Ligases
Small Molecule Libraries
Recombinant Proteins
Assays
Screening
Drug Discovery
Adenosine Triphosphate
Libraries
Catalytic Domain
D-alanylalanine synthetase
Infection

Keywords

  • antibacterial
  • enzyme assay
  • enzyme inhibition
  • peptidoglycan
  • target validation

Cite this

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title = "Burkholderia pseudomallei D-alanine-D-alanine ligase: detailed characterisation and assessment of a potential antibiotic drug target",
abstract = "Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We selected the essential enzyme D-alanine-D-alanine ligase (BpDdl), required for the ATP assisted biosynthesis of a peptidoglycan precursor, and have characterised the enzyme. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+D-Ala-D-Ala), and comparisons with orthologous enzymes suggest a ligand-induced conformational change occurs that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow up and then in combination with a computational analysis of the active site we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches",
keywords = "antibacterial, enzyme assay, enzyme inhibition, peptidoglycan, target validation",
author = "{Diaz Saez}, Laura and Leah Torrie and Stuart McElroy and David Gray and Bill Hunter",
note = "Funding: European Synchrotron Radiation Facility; Diamond Light Source; The Wellcome Trust (GrantNumber(s): 082596, 094090); Defense Science and Technology Laboratory.",
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AU - Diaz Saez, Laura

AU - Torrie, Leah

AU - McElroy, Stuart

AU - Gray, David

AU - Hunter, Bill

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N2 - Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We selected the essential enzyme D-alanine-D-alanine ligase (BpDdl), required for the ATP assisted biosynthesis of a peptidoglycan precursor, and have characterised the enzyme. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+D-Ala-D-Ala), and comparisons with orthologous enzymes suggest a ligand-induced conformational change occurs that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow up and then in combination with a computational analysis of the active site we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches

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