Development of an AlphaLisa assay for Screening Histone Methyltransferase Inhibitors (NB-SC107)

A top pharmaceutical company based in US approached us to develop an AlphaLisa assay for the histone methyltransferase G9a/ EHMT2. The experimental design and results are as follows:

Assay Buffer:

Used for the enzyme reaction and diluting G9a enzyme, biotinylated histone H3 (1-21) peptide, SAM and inhibitors.

Formulation: 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 0.01% Tween-20, 0.005% BSA.

HMT Buffer 1:

Used to dilute the acceptor and donor beads.

Assay Protocol:

Dilute G9a enzyme, biotinylated histone H3 (1-21) peptide, SAM and inhibitors in assay buffer just before use.

Add the following to 384-well white plate wells:

5 μL assay buffer or inhibitor (2X)

2.5 μL of enzyme (4X)

2.5 μL of biotinylated Histone H3 (1-21) peptide/ SAM mix (4X)

Cover the plate with a sealer and incubate at RT.

Prepare H3K9me2 acceptor beads in 1X Epigenetics Buffer 1. Add 5 μL Acceptor beads to each well. Cover the plate with a sealer and incubate for 60 min at RT in dark.

Prepare streptavidin donor beads in 1X Epigenetics Buffer 1. Add 10 μL Donor beads in subdued light. Cover the plate with a sealer and incubate for 30 min at RT in dark.

Read signal in Alpha enabled readers. Novatein Biosciences used BMG Pherastar.

Effect of time on G9a enzyme activity:

G9a was incubated with 100 nM biotinylated Histone-H3 (1-21) peptide and 100 µM S-adenosyl methionine (SAM) for various time points. Acceptor and donor beads were used at 5 μg/ml and 20 μg/ml. As the counts obtained with 5 μg/ml were good, we used both the beads at 5 μg/ml final concentration in the assay.

Time (min)	Alpha Counts (blank subtracted)
180	2755792	2870244	2572242
120	2630541	2718970	2885052
60	1685737	1353978	1692881
30	905154	854907	967214
15	172824	80978	204934
5	14535	6422	17062
0	0	0	0

For all the subsequent experiments, enzyme assay incubation was carried out for 1 hour.

 

 

Effect of G9a concentration on enzyme activity:

G9a was incubated with biotinylated Histone-H3 (1-21) peptide and S-adenosyl methionine (SAM) for 1 hour. Acceptor and donor beads were used at 5 μg/ml. Beads alone was used as blank.

G9a (nM)	Alpha counts (blank subtracted)
0.05	5985	8417	3401
0.1	83733	98553	129029
0.5	2049663	1785278	2162409
1	2999625	3370296	3142182
3.6	4724996	4659807	4927650