| Assay Method Information | |
| | Human ACLY (hACLY) RF/MS Activity Assay |
| Description: | Table 2: Compounds of the disclosure were evaluated for their efficacy in inhibiting hACLY using rapid fire mass spectrometry (RF/MS). The final reaction volume for each compound was 20 μl and consisted of buffer (50 mM Hepes pH 8.0, 10 mM MgCl2, 0.003% BSA, 0.01% Brij35, 50 mM NaCl, 4 mM DTT) and 1 nM hACLY, EV12992, PP6692) using Greiner, 384 well small volume, deep well plates (Cat #784201). A two-fold dilution series with a top concentration of 10 μM was used to record a concentration response curve. Both the substrate (CoA) and product (Acetyl-CoA) were quantified, and given a ratio. The ratio was normalized using both a negative (0% inhibition) and positive (100% inhibition) control to determine the % inhibition. The final DMSO-concentration was 1% (v/v). Compounds were pre-incubated for 30 min with the buffered enzyme solution at RT (20° C.), and substrate solution was added (final concentrations: 15 μM Coenzyme A, 50 μM ATP and 50 μM citrate) to initiate the enzyme reaction. The enzyme reaction was incubated for additional 30 min at RT. The reaction was quenched upon addition of 40 μl of 5% Formic acid in H2O and centrifuged (4350 rpm at 20° C. for 10 min). |
| Affinity data for this assay | |
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