| Assay Method Information | |
| | HSD10B1 Enzymatic Blocking Assay |
| Description: | To screen compounds of the disclosure for their ability to block HSD17B10 function, an enzymatic blocking assay was performed. For the assay, HSD17B10 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. HSD17B10 (Seq. ID NO: 5, expressed in mammalian cells) was added at a final concentration of 31.25 ng/well in a buffer containing 0.2M Tris-HCl, pH 7.5, into a low volume white 384-well assay plate. Compounds were incubated with the enzyme for 30 minutes at room temperature. The assay reaction was then initiated by addition of 25 μM Estradiol substrate (Sigma, catalog #E2758-250MG, CAS: 50-28-2) and 500 μM NAD co-factor, and the reaction mixture was incubated for 3 hours at room temperature. 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. The IC50 values were determined using Dotmatics analysis software. |
| Affinity data for this assay | |
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