HTS-Luminescent assay for inhibitors of ALR by detection of hydrogen peroxide production Measured in Biochemical System Using Plate Reader - 2036-02_Inhibitor_SinglePoint_HTS
Keywords: Mitochondria, biogenesis, protein import, intermembrane space, FAD, sulfhydryl oxidase, ALR, Erv1 ..more
BioActive Compounds: 10857
Keywords: Mitochondria, biogenesis, protein import, intermembrane space, FAD, sulfhydryl oxidase, ALR, Erv1
Screen for inhibitors of purified, recombinant ALR to oxidize the generic substrate DTT. Oxidation of DTT by ALR produces hydrogen peroxide, which was detected using horseradish peroxidase (HRP) and luminol. A solution of ALR and HRP was added to plates containing compounds, followed by the addition of DTT. The plates were incubated for 2 hours prior to the addition of the luminol solution. After a minute incubation, plates were read to detect the flash luminescence signal.
Human ALR is expressed and purified from E. coli. Enzyme preparations were supplied from the assay provider. HRP was purchased from Sigma and DTT and the SuperSignal ELISA Femto Luminol/Enhancer Solution were purchased from Thermo Scientific.
With the exception of the luminol solution, all solutions were prepared with screening buffer (30mM HEPES, 100mM NaCl, 1mM EDTA, pH7.4). All solutions are kept chilled through the duration of the assay.
ALR and HRP were diluted with screening buffer to a final concentration of 0.67uM and 0.027U/ml respectively in black, 384-well, low-volume plates (Greiner 784076), at a volume of 5ul. Inactivated ALR with HRP was used as a positive control, and was added to 32 designated control wells, in the same manner as the active enzyme. 7.5nl of compounds in DMSO were previously transferred to the plates via acoustic transfer (Echo555, LabCyte). 2.5ul of DTT in solution was added to all wells after the ALR solutions, to a final concentration of 133.3uM. The final reaction volume was 7.5ul. Plates were incubated at 37 degrees C for 2 hours in a Liconic STX220 incubator . At the completion of the incubation, 2.5ul luminol solution was added for a minute incubation. Plates were imaged for 25s to detect luminescence using a ViewLux (PerkinElmer).
The assay was initiated by set-up in CBIP (Broad Chemical Biology Informatics Platform) and scheduled with Cellario (HighRes Biosolutions) to be executed on an integrated screening system (HighRes Biosolutions). Plates were moved from different instruments with a Staubli robot. ALR/HRP and DTT solutions were added using a Beckman BioRAPTR FRD Microfluidic Workstation. The luminol solution was added using a Multidrop Combi nL (Thermo).
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -75.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
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Data Table (Concise)