| Assay Method Information | |
| | Inhibition Assay |
| Description: | Primary screening was performed using PKM2 in the presence of FBP. Compounds were chosen as potential "hits" if the compound demonstrated inhibition of PKM2 activity greater than 50%. Activity was measured by monitoring the concentration of NADH. Pyruvate, produced by the enzymatic activity of pyruvate kinase, is converted into lactate by lactate dehydrogenase, which requires the consumption of NADH (NADH→NAD+). Thus, the activity of PKM2 was indirectly measured by monitoring the consumption of NADH through fluorescence assays. Additionally, the activity of the PKM2 enzyme was directly monitored by measuring the production of ATP, as ATP is produced when phosphoenolpyruvate is converted to pyruvate by PKM2. |
| Affinity data for this assay | |
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