| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | Leucyl-tRNA Synthetasea. Single Point Percentage InhibitionThe activity of the pathogenic aaRSs was monitored by measuring AMP production using the commercial kit AMP-Glo (Promega, Madison, USA).Ligand stock solutions were prepared in 100% DMSO at 10 mM concentration. An assay buffer consisting of 100 mM Tris HCl pH 7.6, 40 mM MgCl, 20 mM KCl and 150 mM NaCl was prepared in dH2O. An enzyme solution containing 72.95 μM LeuRS was prepared in assay buffer to provide 20 nM final assay concentration and 50 nM reservoir concentration for a 1:2.5 dilution in the assay. A substrate solution was also prepared in assay buffer with 50 mM L-leucine, 10 mM ATP and 100 mg/mL tRNA. A final assay concentration of 500 μM L-leucine, 16.7 μM ATP and 0.5 mg/mL tRNA and reservoir concentration of 833.33 μM L-leucine, 27.83 μM ATP and 0.83 mg/mL tRNA was used for a 1:1.66 dilution in assay.Enzyme percentage inhibition values were recorded in triplicate at a single point concentration of 100 μM. If the tested compound inhibited the aminoacylation reaction above 25% in at least two replicates, an IC50 was performed with the same enzymatic assay. |
| Affinity data for this assay | |
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