Fluorescence Cell-Free Homogeneous Primary HTS to Identify Inhibitors of Histone Deacetylase 3
Assay Overview: HDAC3 is incubated with compounds for 10 minutes before addition of a coumarin-linked tripeptide substrate containing a terminal acetylated lysine, and trypsin. If HDAC3 is not inhibited it will deacetylate the substrate, which renders the peptide a substrate for trypsin. Trypsin can then liberate the coumarin, increasing fluorescence ..more
BioActive Compounds: 483
Keywords: histone deacetylase, HDAC3, epigenetics
Assay Overview: HDAC3 is incubated with compounds for 10 minutes before addition of a coumarin-linked tripeptide substrate containing a terminal acetylated lysine, and trypsin. If HDAC3 is not inhibited it will deacetylate the substrate, which renders the peptide a substrate for trypsin. Trypsin can then liberate the coumarin, increasing fluorescence
Expected Outcome: HDAC3 inhibitors will prevent deacetylation of the peptide substrate, rendering it an unsuitable substrate for trypsin. This will prevent the liberation of the coumarin fluorophore and fluorescence levels will be reduced compared to noninhibited (DMSO) wells.
1) Assay runs were 100 1536 plates each (Aurora 00029844). Assay ready plates (ARPs) with 7.5 nL compound per plate (10 mM stock) were stored vacuum sealed at -20 C until 12 hours before use when they were unsealed and thawed at RT.
2) Positive control suberoylanilide hydroxamic acid (SAHA 1.4 mM stock concentration, 7.5 mM final concentration, synthesized in house) was applied to 112 wells/plate (50 nL/well) using a MultiDrop Combi nL liquid dispenser (Thermo Scientific, model: 838).
3) For each run of 100 plates, 1.5L of HDAC assay buffer was utilized (HAB; 100 mM KCl (EMD cat# 7300, lot# 0827B018), 50 mM HEPES pH 7.4 (GIBCO cat# 15630-114, lot# 697278), 0.05% BSA (CalBiochem cat# 126626, lot# D00085439), and 0.001% Tween-20 (Zymed cat# 00-3005, lot# D00007104)).
4) For 100 plates, 149.25 ul HDAC3 enzyme stock (BPS Biosciences cat# 50003, lot# 91014, 0.67 mg/mL) was diluted into 1 L of HAB. Six microliters of this HDAC3 solution was added to each well (HDAC3 final concentration in each well = 0.78 nM) using a MultiDrop Combi nL liquid dispenser (Thermo Scientific, model: 838).
5) Each plate was then incubated at RT for 10 minutes before addition of 3 uL substrate and trypsin solution addition (S+T) (For 500 mL S+T stock; 141.05 uL 100 mM MAZ1600 substrate (final concentration 9.4 uM), 4.95 mL of 45 uM Trypsin stock (45 uM stock made from lyophilized powder, Worthington cat# 3744 lot# 57E965B) (final concentration in well 150 nM), 494.9 ml of R.T. HAB), using a second MultiDrop Combi nL liquid dispenser (Thermo Scientific, model: 838).
6) After incubation at RT for 45 minutes, plates were spun for 1 minute @ 1000 rpm. Following centrifugation, plates were read using the Viewlux (Perkin Elmer model 1430-002) reader @ 355/460nm.
HTS Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
PC: SAHA, 7.6uM
Active compounds result in decreased 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 NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
For one run, the positive control did not work, so the -100 full inhibition value was set to (0.33)(median of NC), and the compound wells scaled accordingly.
The plate pattern correction algorithm 'Runwise Pattern (Additive)' in Genedata applied to the normalized plate data.
The replicate activity scores were multiplied by -1 to convert Genedata negative percent inhibition values to Pubchem positive percent activity values.
The final PUBCHEM_ACTIVITY_SCORE was set as equal to the mean of the valid replicates.
The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an inhibition threshold of 25% inhibition:
Activity_Outcome = 1 (inactive)
Percent of active replicates is < 50%
Activity_Outcome = 2 (active)
Percent of active replicates = 100% AND the count of active replicates > 1
Activity_Outcome = 3 (inconclusive)
Percent of active replicates = 100% AND the count of replicates = 1
Percent of active replicates is < 100% AND >=50%
** Test Concentration.
Data Table (Concise)