HTS to Identify Inhibitors of Demethylase GASC-1 Measured in Biochemical System Using Plate Reader - 2043-05_Inhibitor_SinglePoint_HTS_Activity
Keywords: GASC-1 (gene amplified in squamous cell carcinoma-1), JMJD2C, KDM4C, histone demethylase, LANCE, HTS ..more
BioActive Compounds: 138
Keywords: GASC-1 (gene amplified in squamous cell carcinoma-1), JMJD2C, KDM4C, histone demethylase, LANCE, HTS
The goal of this project is to identify inhibitors of the histone demethylase GASC-1 (gene amplified in squamous cell carcinoma-1; also known as JMJD2C and KDM4C). GASC-1 has roles in cancer, androgen receptor signaling and the maintenance of pluripotency. GASC-1 directly and specifically removes repressive histone H3 lysine 9 tri-methylation in an iron and 2-oxoglutarate dependent hydroxylation mechanism. A LANCE (Lanthanide Chelate Excite, Perkin Elmer) assay was developed for an HTS. Basically, 10 nM of GASC-1 (N-terminal GST fusion of amino acids 2-372; BPS Bioscience, 50105) is incubated with a histone 3 peptide trimethylated at lysine 9 position in the presence of iron and 2-oxoglutarate for 30 minutes before LANCE reagents are added. The reaction is carried out in 2 uL volume in 1536 well plate. Test or control compounds are present in certain wells depending on experimental design. After an hour incubation of the LANCE reagents, the plates are read by ViewLux (Perkin Elmer) on ex 340/em 618 and em 671. The ratio of emissions 671 over 618 is used to inidcate normalized signal strength.
Expected Outcome: Decrease of signal
1.#Prepare 1 mM of 2,4-Pyridinedicarboxylic acid (2,4-PDCA) in the Base Buffer (50 mM HEPES pH7.5, 0.01% Tween 20). 2,4-PDCA is used as positive control.
2.#Prepare Enzyme Mix of 20 nM of JMJD2C (BPS Bioscience, 50105), 600 nM of HeK9me3 (Anaspec 64360), 200 uM of ascorbic acid in the Assay Buffer (50 mM HEPES pH7.5, 0.01% Tween 20, 0.01% BSA).
3.#Prepare Cofactor Mix of 100 uM of alpha-ketoglutaric acid potassium salt (2OG), 50 uM Ammonium iron(II) sulfate hexahydrate (Fe(II)) in Assay Buffer.
4.#Onto Aurora 1536-well white high base plates, dispense 100 nL/well of 2,4-PDCA with Combi NL (Thermo) according to plate map.
5.#Dispense 1 uL/well of Cofactor Mix and Enzyme Mix respectively with Combi NL (Thermo) to start the reaction.
6.#Incubate at room temperature for 30 minutes.
7.#Dispense 2 uL/well of Lance Mix (4 nM of Eu-Ab (PerkinElmer), 100 nM of Ulight streptA (PerkinElmer),, 2 mM EDTA, 1x lance buffer (PerkinElmer))
8.#Incubate at room temperature for 60 minutes.
9. Read plate on ViewLux (PerkinElmer) on ex 340/em 618 and em 671.
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 (v10.0.2):
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.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Additive)' in Genedata (v10.0.2) 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 25.
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 0.55.
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
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)