title: New high-throughput fluorimetric assay for discovering inhibitors of UDP-N-acetylmuramyl-L-alanine: D-glutamate (MurD) ligase.
abstract: A novel assay for monitoring the activity of the bacterial enzyme UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5'-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC50 value of 105 microM, was discovered.
authors.affiliation: Drug Discovery, Lek Pharmaceuticals d.d., Ljubljana, Slovenia. katja.kristan@sandoz.com
authors.forename: Katja
authors.initials: K
authors.lastname: Kristan
authors.forename: Miha
authors.initials: M
authors.lastname: Kotnik
authors.forename: Marko
authors.initials: M
authors.lastname: Oblak
authors.forename: Uros
authors.initials: U
authors.lastname: Urleb
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005470:Fluorometry / Q000379:methods*; D018698:Glutamic Acid / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C076606:UDP-N-acetylmuramylalanine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase