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Assay Method Information

Assay Name:  Scintillation Proximity Assay (SPA) for Detection of SMYD2 Enzymatic Inhibition
Description:  SMYD2 inhibitory activities of the compounds described in the present invention were quantified using a scintillation proximity assay (SPA) which measures methylation by the enzyme of the synthetic, biotinylated peptide Btn-Ahx GSRAHSSHLKSKKGQSTSRH (SEQ ID NO: 3) Amid x TFA (Biosyntan) derived from p53 and referred to from here on as p53 Peptide. The SMYD2 full length enzyme was produced in-house by expression (with an N-terminal 6×His tag) in E. coli and purification by affinity chromatography on a Ni-NTA Sepharose column followed by a size exclusion chromatography step on a Superdex 200 16/60 column (GE Healthcare).In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One), from which 50 nl of compound solutions were transferred into a white low volume test microtiter plate from the same supplier. Subsequently, 2.5 μl SMYD2 in aqueous assay buffer [50 mM Tris/HCl pH 9.0 (AppliChem), 1 mM dithiothreitol (DTT, Sigma), 0.01% (w/v) bovine serum albumine (BSA, Sigma), 0.0022% (v/v) Pluronic (Sigma)] were added to the compounds in the test plate to a final enzyme concentration of typically 3 nM (this parameter was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay). The samples were then incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the methylation reaction, which was initiated by the addition of 2.5 μl 2-fold concentrated solution (in assay buffer) of titrated S-Adenosyl-L-Methionine (3H-SAM, Perkin Elmer, final concentration: 60 nM) and p53 Peptide substrate (final concentration: 1.0 μM). The resulting mixture (5 μl final volume) was shortly centrifuged (2 min., 1500 rpm) and incubated at 22° C. during 30 min. Thereupon the reaction was stopped by adding 3 μl of Streptavidin PS SPA imaging beads (Perkin Elmer, final concentration of 3.12 μg/μl) and ¿cold¿ SAM (AK Scientific, 25 μM final concentration) for non-specific binding reduction. Plates containing the stopped reaction were sealed with transparent adhesive foil (Perkin Elmer), centrifuged (2 min., 1500 rpm), and further incubated for¿at least¿1 h at RT (or overnight at 4° C.) in order to allow the SPA signals to develop. Subsequently, the amount of product was evaluated by measuring the energy transfer from the β-particles emitted by the 3H-labeled substrate to the Europium scintillator co-polymerized in the polystyrene matrix of the PS imaging beads, using the standard settings for this purpose of a Viewlux (Perkin-Elmer) CCD plate imaging device (emission filter 613/55 (IFP). The resulting scintillation counts were taken as indicator for the amount of methylated peptide per well. The data were normalised using two sets of control wells (typically 16 each) for high- (=enzyme reaction with DMSO instead of test compound=0%=Minimum inhibition) and low- (=all assay components without enzyme=100%=Maximum inhibition) SMYD2 activity. IC50 values were calculated by fitting the normalized inhibition data to a 4-parameter logistic equation using the ¿Screener¿ analysis software from Genedata.
Affinity data for this assay
 

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Last update November 1, 2007
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