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New High-Throughput Fluorimetric Assay for Discovering Inhibitors of UDP-N-Acetylmuramyl-l-Alanine: d-Glutamate (MurD) Ligase

Drug Discoverys, Lek Pharmaceuticals d.d., Ljubljana, Slovenia, katja.kristan{at}sandoz.com

Miha Kotnik

Drug Discoverys, Lek Pharmaceuticals d.d., Ljubljana, Slovenia

Marko Oblak

Sandoz Development Center Slovenia, Bioanalytics, Lek Pharmaceuticals d.d., Ljubljana, Slovenia

Uro Urleb

Drug Discoverys, Lek Pharmaceuticals d.d., Ljubljana, Slovenia

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^TM assay kit from DiscoveRx. The novel assay has been validated by obtaining K_M,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 µM, was discovered. (Journal of Biomolecular Screening 2009:412-418)

Key Words: Mur ligases • MurD inhibitors • high-throughput screening • fluorimetric assay • cell wall biosynthesis

Journal of Biomolecular Screening, Vol. 14, No. 4, 412-418 (2009)
DOI: 10.1177/1087057109332597


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