Counterscreen for JMJD2E Inhibitors: qHTS Assay for Inhibitors of Formaldehyde Dehydrogenase (FDH)
This assay is a counterscreen for a related assay for inhibitors of JMJD2E. See AID 2147 for details. The JMJD2E assay utilized an FDH-coupled protocol. ..more
BioActive Compounds: 16
Depositor Specified Assays
This assay is a counterscreen for a related assay for inhibitors of JMJD2E. See AID 2147 for details. The JMJD2E assay utilized an FDH-coupled protocol.
To screen for false positive results in the primary assay due to inhibition of the formaldehyde dehydrogenase coupled enzyme, an FDH-only assay was run using FDH as the enzyme (0.000025 U/uL final concentration) and 10 uM formaldehyde and 0.25 mM NAD+ as the substrate, and following the same fluidic protocol as described in AID 2147 for the coupled HTS assay.
Actives in this assay are likely false positives of the primary HTS.
Assay is initiated by dispensing 3 uL of enzyme solution (4/3x final concentration) into a 1,536-well assay plate using a nanoliter solenoid-based dispenser (Beckman-Coulter, Fullerton, CA). After pintool transfer of compound solution, the assay plate is incubated at room temperature for 10 minutes, followed by a 1 uL addition of 4x substrate solution to initiate the reaction (final assay volume of 4 uL). The plate is then manually transferred into the ViewLux reader for kinetic measurements.
Keywords: FDH, JMJD2, JMJD2E, Human 2-Oxoglutarate Oxygenase, qHTS
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)