| Assay Method Information | |
| | Human GOAT Enzymatic Assay |
| Description: | Prepare test compounds in DMSO to make up a 0.2 mM stock solution. Serially dilute the stock solution in DMSO for ten concentrations with final compound concentrations ranging from 10 μM to 0.5 nM in a 96-well round-bottom plate. Prepare enzyme and substrate solutions in assay buffer (0.02% TWEEN™-20 in 50 mM Tris, pH 7.5 containing 250 mM sucrose, 1 mg/mL BSA and 10 mM EDTA). Add diluted compound (1 μL) to each well of row A to N of a corresponding low protein binding 384 well plate. Add human GOAT substrate mix (10 μL), consisting of human desacyl-ghrelin-biotin (CPC Scientific Inc., 6.0 μM final), octanoyl-CoA (Sigma, 60 μM final) and an AG specific antibody (WO 2006/091381) (1.0 μg/mL final), to the compounds. Add GOAT-His/sf9 enzyme preparation, that has been prepared in assay buffer (9 μL), to each well of the plate containing substrate and test compounds resulting in a final concentration of 0.01 μg/mL to initiate the reaction. Incubate the mixture for 1 h at RT on a gently rotating oscillator. Add 4 M guanidine hydrochloride (20 μL) to all wells, mix, and incubate for 3 h to stop the reaction.Prepare ELISA plates (STREPTAVIDIN SPECTRAPLATE 384, Perkin Elmer) by blocking with 2% Heat-Inactivated FBS in PBS (40 μL) (Invitrogen) blocking buffer for 3 h. Aspirate the blocking buffer from ELISA plate and add blocking buffer (23 μL) to columns 1-24, rows A-N. Reserve rows O and P for the acylghrelin standard curve. Add the reaction mixture (2 μL) to the ELISA plates. Prepare a 10 point standard curve (biotin-labeled octanoyl-ghrelin) by serial 2× dilution in blocking buffer containing 0.2M Guanidine hydrochloride starting at 2.5 μM. Incubate the reaction mixture or biotin-labeled AG standard in the ELISA plate overnight at 4° C. The following day, wash the plate 3× with wash buffer (0.1% TWEEN-20/PBS, 100 μL per well in each wash cycle). Add AG specific antibody (WO 2006/091381) (25 μL of 0.5 μg/mL in blocking buffer) to each well and incubate at RT for 1 h. Wash the plate 3× with the wash buffer, similarly to the previous step. Add Protein G-HRP (25 μL) (Southern Biotech) diluted 3,000× in blocking buffer and incubate 1 h at RT. Wash the late 3× with wash buffer, as in the previous steps. Add TMB reagent (25 μL) (Kirkegaard & Perry Laboratories, Inc.) to each well and let develop for 20 min and stop with 1 M phosphoric acid (25 μL per well). Read plates at 450 nm using an ENVISION Multilabel plate reader. AG levels are calculated versus a fitted standard curve and percent inhibition calculated. The 10-point inhibition curve is plotted and fitted with the four-parameter logistic equation to obtain IC50 values using ACTIVITYBASE (ver. 7.3.2.1).Following a protocol essentially as described above, all of the compounds of the Examples herein were tested and exhibited an IC50 for the in vitro cell free human GOAT enzymatic assay of lower than 1 μM. |
| Affinity data for this assay | |
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