title: Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm).
abstract: The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium.
authors.affiliation: Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia.
authors.forename: Veronika
authors.initials: V
authors.lastname: Škedelj
authors.forename: Andrej
authors.initials: A
authors.lastname: Perdih
authors.forename: Matjaž
authors.initials: M
authors.lastname: Brvar
authors.forename: Ana
authors.initials: A
authors.lastname: Kroflič
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubbée
authors.forename: Victoria
authors.initials: V
authors.lastname: Savage
authors.forename: Alex J
authors.initials: AJ
authors.lastname: O'Neill
authors.forename: Tom
authors.initials: T
authors.lastname: Solmajer
authors.forename: Marija
authors.initials: M
authors.lastname: Bešter-Rogač
authors.forename: Didier
authors.initials: D
authors.lastname: Blanot
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.forename: Sophie
authors.initials: S
authors.lastname: Magnet
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.forename: Jure
authors.initials: J
authors.lastname: Stojan
authors.forename: Anamarija
authors.initials: A
authors.lastname: Zega
mesh_terms: D026603:D-Aspartic Acid / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D016984:Enterococcus faecium / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D026603:D-Aspartic Acid; D008025:Ligases
keywords: ATP; ATP binding site; Antibacterial agents; Drug design; Enterococcus faecium; ITC; Isothermal titration calorimetry; Kinetic measurements; MIC; PDB; Pharmacophore modeling; Protein databank; RA; SARs; adenosine-5′-triphosphate; d-Aspartate ligase; isothermal titration calorimetry; minimal inhibitory concentration; residual activity; structure–activity relationships