| Assay Method Information | |
| | 3β-HSD1 Inhibition Assay |
| Description: | DHEA and NAD+ were enzymatically converted to androstenedione and NADH, respectively. Final NADH concentrations were spectroscopically quantified using a commercially available Amplite™ Colorimetric NADH Assay Kit which utilizes an NADH probe with an absorbance wavelength of 460 nm. Enzymatic assays with a final volume of 25 μL were ran in a 384-well, Greiner Bio-One UV-STAR assay plate in 50 mM TRIS pH 8.0 assay buffer. Compound IC50 values were experimentally determined in duplicate using a 10-point concentration response curve (CRC) with a top concentration of 50 μM and a bottom concentration of 25 pM. Compound stocks at 10 mM in DMSO were serial diluted 5-fold in DMSO to generate an ECHO compatible source plate. 125 nL of DMSO, Trilostane, or serial diluted compounds were then acoustically transferred into an empty UV-STAR assay plate to give a final assay concentration of 0.5% v/v DMSO. The stamp volume and dilution scheme can be adjusted to accommodate compound potency. The assay can tolerate up to a 5% v/v DMSO final assay concentration. 15 μL of 1 μg purified 3β-HSD1 and 50 μM DHEA (final assay volume concentration) was dispensed per well into the stamped plate, centrifuged for 1 minute at 1000 RPM, covered, and allowed to incubate for 15 minutes at room temp. Enzymatic reactions were initiated upon the addition of 10 μL of NAD+ diluted in assay buffer to give a final assay volume concentration of 2 mM. The assay plate was centrifuged for 1 minute at 1000 RPM, covered, and incubated at room temperature for 1 hour. 25 μL Amplite™ Colorimetric NADH Assay Kit detection solution (prepared as recommended) was added to all reaction wells, the plate centrifuged for 1 minute at 1000 RPM, covered, and incubated for 15 minutes. Final absorbance values (λ=460 nm) are spectroscopically determined on BioTek Cytation5 or Synergy4 plate readers. The background signal was determined by averaging 32 fully inhibited reactions (Trilostane treated) and subtracted across the plate. From the background subtracted values, the non-inhibited enzyme signal (DMSO treated) was averaged from 32 control reactions. |
| Affinity data for this assay | |
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