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The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Moynie L, Schnell R, McMahon SA, Sandalova T, Boulkerou WA, Schmidberger JW, Alphey M, Cukier C, Duthie F, Kopec J, Liu H, Jacewicz A, Hunter WN, Naismith JH, Schneider G - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

Bottom Line: A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce.The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here.The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, Scotland.

ABSTRACT
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

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Schematic view of the dimer (a) and dodecamer (b) of the probable oxidoreductase PA1648. The catalytic and nucleotide-binding domains are labelled alongside the two consensus motifs and the NADP+-binding site.
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fig8: Schematic view of the dimer (a) and dodecamer (b) of the probable oxidoreductase PA1648. The catalytic and nucleotide-binding domains are labelled alongside the two consensus motifs and the NADP+-binding site.

Mentions: The PA1648 monomer is comprised of a catalytic domain and a nucleotide-binding domain (Fig. 8 ▶ and Supplementary Fig. S1h). The catalytic domain is formed by residues 1–129 and 299–334, which includes α-helices 1, 2 and 11 and β-strands 1–8, 15 and 16. The strands form five-stranded and three-stranded antiparallel twisted sheets and a two-stranded parallel β-sheet. The nucleotide-binding domain comprises residues 130–298 (α-helices 3–­10 and β-strands 9–14) and adopts the characteristic Rossmann fold. The cofactor-binding site is located in a cleft between the catalytic and nucleotide-binding domains. A DALI search of the PDB with the PA1648 dimer revealed structural similarity to numerous members of the medium-chain reductase superfamily. The conservation of sequence is most pronounced in the nucleotide-binding domain and includes the GXXS (residues 246–249) and glycine-rich GXXGXXXG (residues 158–165) motifs, which interact with the adenine and nicotinamide moieties of the NADP+, as observed in the homologue 2j3h (Youn et al., 2006 ▶). This particular homologue shares a sequence identity of 41% with PA1648 and aligns 326 Cα atoms with an r.m.s.d. of 1.4 Å. The catalytic domain showed relatively little sequence identity (5%) to the structural homologues and lacks the polyproline helix observed in some other members of the family.


The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Moynie L, Schnell R, McMahon SA, Sandalova T, Boulkerou WA, Schmidberger JW, Alphey M, Cukier C, Duthie F, Kopec J, Liu H, Jacewicz A, Hunter WN, Naismith JH, Schneider G - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

Schematic view of the dimer (a) and dodecamer (b) of the probable oxidoreductase PA1648. The catalytic and nucleotide-binding domains are labelled alongside the two consensus motifs and the NADP+-binding site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539698&req=5

fig8: Schematic view of the dimer (a) and dodecamer (b) of the probable oxidoreductase PA1648. The catalytic and nucleotide-binding domains are labelled alongside the two consensus motifs and the NADP+-binding site.
Mentions: The PA1648 monomer is comprised of a catalytic domain and a nucleotide-binding domain (Fig. 8 ▶ and Supplementary Fig. S1h). The catalytic domain is formed by residues 1–129 and 299–334, which includes α-helices 1, 2 and 11 and β-strands 1–8, 15 and 16. The strands form five-stranded and three-stranded antiparallel twisted sheets and a two-stranded parallel β-sheet. The nucleotide-binding domain comprises residues 130–298 (α-helices 3–­10 and β-strands 9–14) and adopts the characteristic Rossmann fold. The cofactor-binding site is located in a cleft between the catalytic and nucleotide-binding domains. A DALI search of the PDB with the PA1648 dimer revealed structural similarity to numerous members of the medium-chain reductase superfamily. The conservation of sequence is most pronounced in the nucleotide-binding domain and includes the GXXS (residues 246–249) and glycine-rich GXXGXXXG (residues 158–165) motifs, which interact with the adenine and nicotinamide moieties of the NADP+, as observed in the homologue 2j3h (Youn et al., 2006 ▶). This particular homologue shares a sequence identity of 41% with PA1648 and aligns 326 Cα atoms with an r.m.s.d. of 1.4 Å. The catalytic domain showed relatively little sequence identity (5%) to the structural homologues and lacks the polyproline helix observed in some other members of the family.

Bottom Line: A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce.The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here.The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, Scotland.

ABSTRACT
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

Show MeSH
Related in: MedlinePlus