| Assay Method Information | |
| | HSD17B4 Enzymatic Blocking Assay |
| Description: | To screen compounds of the disclosure for their ability to block HSD17B4 function, an enzymatic blocking assay was performed. For the assay, HSD17B4 activity was measured by detecting NADH formation from oxidation of Estradiol using a NADH Glo luminescence assay (Promega, #Cat. No. G9062). Compounds of the disclosure were evaluated in a dose titration assay where the compounds were titrated at 10-point dose curve using an Echo-555 liquid handling dispenser. HSD17B4 (SEQ ID NO: 4, expressed in mammalian cells) was added at a final concentration of 2.5 ng/μL in a buffer containing 50 mM Tris-HCl, pH 7.5, Pluronic F-127 0.05%, Tween20 0.01%, BSA 0.01%, into a low volume white 384-well assay plate. Compounds were incubated with the enzyme for 15 min at 20° C. The assay reaction was then initiated by addition of 23.46 μM Estradiol substrate (Sigma, catalog #E2758-250MG, CAS: 50-28-2) and 1500 μM NAD co-factor, and the reaction mixture was incubated for 45 min at 20° C. DMSO at a concentration of 0.5% was used as a negative control and a ‘no substrate’ control was used a positive control in the assay. Detection reagent was then added as per manufacturer's instructions, and the plate was subsequently incubated in the dark for 1 hour at room temperature, NADH generated was detected in the luminescence mode on the Envision Perkin Elmer plate reader. |
| Affinity data for this assay | |
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