Development of Small Molecule Probes of the Histone Methyltransferase, NSD2 Measured in Biochemical System Using Plate Reader - 7053-01_Inhibitor_SinglePoint_HTS_Activity_Set2
H3K36, histone methyltransferase, cancer, multiple myeloma, NSD2 small molecule inhibitors, histone-modifying, t(4;14) chromosomal translocation, AlphaScreen ..more
BioActive Compounds: 1662
H3K36, histone methyltransferase, cancer, multiple myeloma, NSD2 small molecule inhibitors, histone-modifying, t(4;14) chromosomal translocation, AlphaScreen
The NSD2 methyltransferase reaction is designed to monitor the lysine methylation of a 16-amino acid biotinylated peptide (Ac-RLARRGGVKRISGLI[K-Ahx-Biot]amide) (H4 peptide). NSD2 transfers a methyl group from S-adenosylmethionine (SAM) to the lysine residue of the H4 peptide. The enzyme reaction is allowed to proceed for 120 mins. The assay uses an AlphaScreen technology as the detection mode. The degree of lysine methylation is determined in the detection step when the Rabbit polyclonal anti-histone H3 (monomethyl K36) antibody reacts with the peptide methylated lysine group. Subsequent binding of the streptavidin coated Donor bead to the biotinylated peptide and Protein A coated Acceptor bead binding to the anti-histone H3 (monomethyl K36) antibody results in Donor/Acceptor proximity. Excitation at 680 nm enables Donor bead singlet oxygen transfer to the Acceptor bead that emmits light at 520-620 nm.
Active compounds inhibiting NSD2 are expected to give diminished methylation at H3K36 relative to controls and to decrease the measured alpha screen intensity. Further orthogonal assays will determine which compounds are authentic inhibitors and which may be artifacts.
This assay is a 2-step procedure. In the first step the NSD2 enzyme, a methyl transferase, transfers a methyl group from S-adenosylmethionine (SAM) to the lysine residue of a 16-amino acid biotinylated peptide (Ac-RLARRGGVKRISGLI[K-Ahx-Biot]amide) (H4 peptide). The assay buffer is 50 mM Tris pH 8.5, 5 mM MgCl2, 0.01% Tween 20, and 1 mM DTT.
1.)10 nL of a 10 mM stock compound solution was added to Perkin Elmer shallow 384-well AlphaPlates (#CUSG01602) resulting in a final screening compound concentration of 12.5 uM in a 8 uL reaction.
2.)The NSD2 stock enzyme solution (376.5 uM) is diluted in the above buffer to a concentration of 60 nM and 4 uL is added to the enzyme reaction.
3.)The peptide and SAM substrates are mixed together resulting in a solution containing 600 nM peptide and 5 uM SAM. 8 uL is added to the enzyme reaction and incubate at room temperature for 120 mins.
4.)The final reagent concentrations in the enzyme reaction are: 30 nM NSD2, 300 nM peptide, 2.5 uM SAM and 12.5 uM compound.
Detection Step: The detection step buffer is 50 mM Tris pH7.5, 1 M NaCl, 0.1% Tween 20, 1 mM DTT.
1.) A detection mixture is prepared using the detection step bufffer containing 20 ug/mL of both Acceptor and Donor beads (5 mg/mL stock solution). Dilute the stock anti-histone H3 (mono methyl K36) antibody (100 ug/mL) in the acceptor bead solution to 50 ng/mL. Keep both bead solutions, stock and diluted, in the dark.
2.)Add 8 uL of the bead/antibody mixture to the reaction.
5.)The final concentrations of the detection step in 16 uL are 10 ug/mL both acceptor and donor beads and 25 ng/mL antibody.
6.) Incubate 90 mins at room temperature.
7.)Read on the Envision.
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 (Multiplicative)' 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 50.
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 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)