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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 structure of HemD (PA5259). Secondary-structural elements are colour-coded in yellow (β-strands) and red (α-helices). The N- and C-termini are shown as blue and red spheres, respectively.
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fig1: Schematic view of the structure of HemD (PA5259). Secondary-structural elements are colour-coded in yellow (β-strands) and red (α-helices). The N- and C-termini are shown as blue and red spheres, respectively.

Mentions: P. aeruginosa HemD is a monomeric enzyme comprising two globular α/β-domains linked by a pair of antiparallel β-strands (Fig. 1 ▶ and Supplementary Fig. S1a). The cavity at the domain interface is sufficiently large to provide space for binding of the linear tetrapyrrole substrate hydroxymethylbilane. The overall fold is identical to that of other known uroporphyrinogen III synthases, but the orientation of the domains is quite different to some of these enzymes (PDB entries 1jr2, 3re1, 3d8n, 3d8r and 1wcx; Mathews et al., 2001 ▶; Schubert et al., 2008 ▶; Peng et al., 2011 ▶; E. Mizohata, T. Matsuura, K. Murayama, H. Sakai, T. Terada, M. Shirouzu, S. Kuramitsu & S. Yokoyama, unpublished work), as reflected by the significant variation in r.m.s.d. values from 1.9 to 5.2 Å upon superposition of the crystal structures. The conformation of PA5259 in the crystals corresponds to the ligand-free closed state of the enzyme (Schubert et al., 2008 ▶). The low level of conservation of active-site residues when compared with the human enzyme (Mathews et al., 2001 ▶) suggests that the development of selective inhibitors of PA5259 might be feasible.


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 structure of HemD (PA5259). Secondary-structural elements are colour-coded in yellow (β-strands) and red (α-helices). The N- and C-termini are shown as blue and red spheres, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Schematic view of the structure of HemD (PA5259). Secondary-structural elements are colour-coded in yellow (β-strands) and red (α-helices). The N- and C-termini are shown as blue and red spheres, respectively.
Mentions: P. aeruginosa HemD is a monomeric enzyme comprising two globular α/β-domains linked by a pair of antiparallel β-strands (Fig. 1 ▶ and Supplementary Fig. S1a). The cavity at the domain interface is sufficiently large to provide space for binding of the linear tetrapyrrole substrate hydroxymethylbilane. The overall fold is identical to that of other known uroporphyrinogen III synthases, but the orientation of the domains is quite different to some of these enzymes (PDB entries 1jr2, 3re1, 3d8n, 3d8r and 1wcx; Mathews et al., 2001 ▶; Schubert et al., 2008 ▶; Peng et al., 2011 ▶; E. Mizohata, T. Matsuura, K. Murayama, H. Sakai, T. Terada, M. Shirouzu, S. Kuramitsu & S. Yokoyama, unpublished work), as reflected by the significant variation in r.m.s.d. values from 1.9 to 5.2 Å upon superposition of the crystal structures. The conformation of PA5259 in the crystals corresponds to the ligand-free closed state of the enzyme (Schubert et al., 2008 ▶). The low level of conservation of active-site residues when compared with the human enzyme (Mathews et al., 2001 ▶) suggests that the development of selective inhibitors of PA5259 might be feasible.

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