| Assay Method Information | |
| | HSD17B13 Enzymatic Blocking Assay (50nM) |
| Description: | Compounds of the disclosure were screened for their ability to block HSD17B13 function using an enzymatic blocking assay. HSD17B13 activity was quantified using a NADH Glo luminescence assay (Promega, #Cat. No. G9062) to measure the formation of NADH from the oxidation of LTB4, a known HSD17B13 substrate. Compounds were over a 10-point dose curve using an Echo-555 liquid handling dispenser. HSD17B13 (SEQ ID NO: 1, expressed in E. coli) was added at either a final concentration of 50 nM, depending on the assay run, in a buffer containing 0.2M Tris-HCl pH 7.5 containing 0.01% Triton-X 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 10 μM LTB4 substrate (Cayman chemicals, Cat. No. 20110) and 500 μM NAD, and the reaction mixture was incubated for 1 or 2 hours at room temperature depending on the assay run. DMSO at a concentration of 0.5% was used as a negative control, and a ‘no substrate’ control was used as a positive control (100% inhibition) 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. Generation of NADH 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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