JMJD2A counterscreen for GASC1 inhibitors Measured in Biochemical System Using Plate Reader - 2043-02_Inhibitor_Dose_CherryPick_Activity
The goal of this project is to identify inhibitors of the histone demethylase GASC-1 (gene amplified in squamous cell carcinoma-1). 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. Probes that inhibit more ..
BioActive Compounds: 43
Keywords: GASC-1 (gene amplified in squamous cell carcinoma-1), JMJD2A, histone demethylase, DELFIA
Broad Institute MLPCN GASC-1 Project
Project ID: 2043
Stefan Kubicek, Broad Institute, firstname.lastname@example.org, Cambridge, MA.
Christina Scherer, Broad Institute, email@example.com, Cambridge, MA.
The goal of this project is to identify inhibitors of the histone demethylase GASC-1 (gene amplified in squamous cell carcinoma-1). 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. Probes that inhibit GASC-1 activity in a DELFIA primary assay will be tested for specificity in counter screens to determine if their mechanism of activity is specific to GASC-1 alone, to all JMJ demethylases, to JMJ demethylases and non-JMJ demethylases, or to hydroxylases in general. Following classification into one of the previous selectivity classes, lead compounds from each group will be tested in a cell-based secondary assay for GASC-1 inhibitory activity. Finally, probe candidates will be tested in a cell-based counter screen to determine if their cell-based activity is specific to GASC-1.
Compounds were assayed for inhibitory activity against the JMJD2A histone demethylase using a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). Compounds that cause a reduction in JMJD2A demethylase activity in addition to the primary JMJD2C assay are not Jumonji demethylase isoform specific but may still be specific for this subclass of histone demethylases.
Assays were conducted in 384-well neutravidin coated DELFIA plates (Perkin Elmer, CC11-H10), using recombinant JMJD2A (GST-JMJD2C 1-420). Compounds were pinned (25 nL, 2.35 uM final concentration) into 20 uL JMJD2A enzyme mix immediately before addition of 20 uL of 2X trimethyl jmjA peptide/cofactor mix. This mixture was allowed to incubate at room temperature for 1 h, while JMJD2A could carry out the demethylation reaction and the peptide substrate could attach to the plate surface. Following incubation, plates were washed three times with 100 uL wash buffer. Following washing and aspiration, 50 uL of antibody mix was added to each well and allowed to incubate for 1 h at room temperature. The antibody mix contained both a monoclonal antibody specific to the dimethyl peptide (anti-2x-di-H3K9 IgG (Abcam, ab32521)) and a secondary polyclonal europium labeled antibody specific to the primary (Eu-anti-rabbit (PerkinElmer, AD0106)). Plates were washed three times as before and 50 uL of DELFIA enhancement solution (Perkin Elmer, 4001-0010) was then added and europium emission was measured after 60 min (ex. 360 nm, em. 620 nm).
1. The positive control compound pyridine-2,4-dicarboxylic acid (Sigma, P63395) was added to each of the pre-assigned poscon wells of the 384-well neutravidin coated assay plate (Perkin Elmer, CC11-H10) using a Combi nL liquid dispenser (Thermo Scientific) at an off-line workstation.
2. Assay plates were then loaded into a room temperature Liconic incubator (STX 2201C) on an enclosed automated screening system (HiRes Biosolutions). Each run was initiated and scheduled with the Cellario software (HiRes Biosolutions) and runs were tracked in CBIP (Broad Chemical Biology Informatics Platform). Staubli arms moved plates from different instruments on the robotic system.
3. Assay plates were filled with 20 uL/well of GASC-1 enzyme mix (150 ng GST-JMJD2A (1-420) in 40 mM TRIS pH 8.5, 8 mM glutathione) using a Multidrop Combi liquid dispenser. Enzyme mix was kept on ice and warmed to room temperature using a 6 ft Combi tube draped in a room temperature water bath.
4. Assay plates were then pinned with 25 nL 3.75 mM compound (final concentration = 2.35 uM) each with a 25 nL pin head using a MicroPin pin tool (HiRes Biosolutions).
5. Plates with compound then had 20 uL/well 2x jmjA peptide/cofactors (100 uM 2-oxogluterate, 10 uM iron sulfate, 0.5 uM peptide bio-H3(1-20)-cys-biotin) added using a second Multidrop Combi liquid dispenser and were incubated at room temperature 60 minutes.
6. Plates were then washed 3x with 100 uL/well wash buffer (50 mM TRIS pH 7.5, 150 mM sodium chloride, 0.05 % Tween 20) using a BioTek plate washer.
7. Plates then had 50 uL/well antibody mix (anti-2x-di-H3K9 IgG (ab32521 lot 400724) (1:5000) and Eu-anti-rabbit (Wallac/PerkinElmer AD0106 batch 392-158-A) (1:1000) in 50 uL FI-buffer (50 mM TRIS pH 7.5, 150 mM sodium chloride, 0.05 % Tween 40, 25 mM diethylenetriaminepentaacetic acid, 0.2 % bovine serium albumin, 0.05 % bovine gamma globulins)) added using a thrid MultiDrop Combi and were incubated at room temperature for 1 h.
8. Plates were then washed again in the same manner as in step 6.
9. Plates then had 50 uL/well enhancement solution (Wallac/PerkinElmer 4001-0010) added using a fourth MultiDrop Combi and were incubated at room temperature for 60 min.
10. Europium emission was then measure using a Envision plate reader (Perkin Elmer, Excitation: 360 nm, Emission 620 nm, integration 400 ms).
11. Plates were then discarded.
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.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
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.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)