| Assay Method Information | |
| | Kinetic Assay |
| Description: | Table 1: A kinetic assay to monitor FECH enzymatic activity was adapted from a published protocol (Burden et a., Biochim Biophys Acta 1435, 191-197 (1999)). Recombinant human FECH activity was established by monitoring the increase of Ni2+ mesoporphyrin IX reaction product at 550 nm over time (FIG. 1 ).20,000 compounds were analyzed in the initial screen (FIG. 2 ). Because each plate had both negative (uninhibited FECH activity) and positive (NMPP) controls, and there was considerable inter-plate variability, a hit was established as reduction >3 standard deviations from the average activity of the negative control (100% activity) for each plate. Using this metric, 664 (3.3%) of the compounds tested in the initial screen were designated hits. All these compounds were selected for secondary screening.Secondary screening with these hits was run so that the assay was set up identically to the initial screen. However, analysis for hits from this secondary screen differed from the initial screen. Hits were defined from the secondary screen by their percent inhibition of FECH activity (the average of the activity from negative controls from two plates used in the secondary screen) (FIG. 3 ). The 93 compounds (0.5%) that inhibited FECH activity by 50% or more in this secondary screen were subjected to further scrutiny. Two were removed after pan-assay interference compound assessment. Intriguingly, the 91 remaining ‘hit’ compounds from the secondary screen (0.5% of the initial screen) included 62 triazolopyrimidinones. |
| Affinity data for this assay | |
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