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| | nhibition Assay against Klebsiella pneumoniae LpxC |
| Description: | LpxC inhibition assays were performed using liquid chromatography with tandem mass spectrometry. Assays were performed, in duplicate, in opaque, 96-well microplates in a total assay volume of 50 μL. The incubation mixture contained: LpxC (0.2 nM Kpn), 0.8 μM UDP-3-O [(R)-3-hydroxymyristoyl]-N-acetyl-glucosamine, 40 mM Bis-Tris/HCl buffer (pH 5.9), 5 mM sodium phosphate buffer (NaH2PO4/Na2HPO4, pH 7.0), 1 mM DTT, 0.1% (w/v) fatty-acid free BSA, 10% DMSO (v/v, with or without compound). The reactions were incubated at 22° C. for 60 minutes (with mild shaking), then terminated by the addition of 25 μL 0.25 N HCl. Samples were analyzed using a LC-MS system to measure native LpxC substrate and reaction product. IC50 analysis was done using GeneData Screener and a four parameter variable slope normalized to controls. Test compounds were prepared as 8-point dose-response curves (factor dilution 2) in triplicate, starting at 1 μM final concentration. Each assay plate included 6 wells used for the Z′ factor calculation, 3 as a positive control for the assay and 3 as a negative control. The robustness was calculated as the median Z′ factor for 5 plates. Chir-90, a well-known inhibitor of LpxC activity was used as inhibitor control standard. |
| Affinity data for this assay | |
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