Download Development and Validation of a Generic

Development and Validation of a Generic Fluorescence Methyltransferase Activity Assay
Using the Transcreener® AMP2/GMP2 Assay
Tony A. Klink, Matt Staeben, Rebecca Josvai, Meera Kumar, Karen Kleman-Leyer, and Robert G. Lowery
BellBrook Labs, LLC, Madison, WI, USA
150
100
1 µM
5 µM
10 µM
50 µM
50
0
0
5
10
15
The Transcreener® EPIGEN Methyltransferase Assay Principle.
SAH
produced by the target methyltransferase is converted to AMP by coupling enzymes which allows homogenous
fluorescent detection using the Transcreener ® AMP2/GMP2 Assay.
B)
100
125
∆ mP
∆ mP
100
150
∆ mP
125
50
100
0
0
5
10
15
20
25
0
% Conversion
15 minutes
30 minutes
1 hour
2 hours
5
10
15
20
25
% Conversion
5 hours
8 hours
48 hours
0 Hour
2.5 Hours
4.5 Hours
6.5 Hours
SAM/SAH standard curves
(10 µM initial SAM) were used to examine A) equilibration and stability of signal after addition to coupling
enzyme reactions and B) stability of pre-combined detection reagents prior to addition to the plate. Both
studies were performed at room temperature.
0.35
0.70
0.80
0.78
0.86
0.85
0.85
0.85
0.87
0.87
0.89
0.84
0.88
0.87
0.89
0.93
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0.91
0.91
75
50
50
Set7/Set9
25
25
G9a
SUV39H1
0
8.5 Hours
14 Hours
19 Hours
Figure 3. Signal and Reagent Stability for >16 hours.
50 µM
0.06
0.53
0.72
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25
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10 µM
-0.49
0.04
0.43
0.59
0.73
0.72
0.82
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0.86
0.83
0.85
A) Standard curves
mimic MT reactions (formation of SAH from SAM) show large assay window (>100 mP signal) at less than 20%
SAM conversion over the full range of initial SAM concentrations tested. B) Table with Z’ values at percent SAM
conversion showing the lower limit of detection (yellow) and initial values that are ≥0.5 (green).
150
50
25
5 µM
-1.7
-1.3
-0.22
0.11
0.49
0.50
0.70
0.72
0.79
0.76
0.80
Figure 2. Initial Velocity Detection from 1 µM to 50 µM SAM.
175
75
20
1 µM
% SAM Conversion
Figure 1.
125
100
% SAM
Conversion
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1.0
2.0
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5.0
7.5
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0
1000
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0
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[G9a] ng/mL
5000
[Enzyme] ng/mL
Figure 4. Universal HMT Detection.
Enzyme titrations demonstrate dose responses for three HMTs,
Set7/Set9, G9a, and SUV39H1 using Histone H3 peptide (1-25) at the SAM Km (6 µM, 2 µM, and 12 µM, respectively).
HMT reactions were run for 60 minutes followed by addition of quench and detection reagents including the SAM-SAH
®
2
coupling enzyme mixture and the Transcreener AMP /GMP2 monoclonal antibody and tracer.
without acceptor substrate
1 µM Histone H3 (full length)
10 µM Histone H3 Peptide 1-25
100 µM Histone H3 Peptide 1-25
Figure 5. Accommodation of Different Acceptor Substrates.
G9a histone
methyltransferase reactions were performed with native protein (1 µM) or peptide substrates (10 µM or 100 µM)
at 1 µM SAM. Reactions were processed as described in Figure 4.
Conclusions
6
3
SAH, pmol
4
2
• Excellent Z’ values and >100 mP signal are achieved using [SAM] from 1 µM to 50 µM
under initial velocity conditions.
3
• Greater than 16 hour deck and signal stability ensures compatibility with automated HTS
infrastructure.
2
sinefungin
chaetocin
1
1
0
• The Transcreener® EPIGEN Methyltransferase Assay combines coupling enzymes with the
well characterized Transcreener® AMP2/GMP2 FP reagents to enable universal
homogenous methyltransferase enzyme detection.
4
234 ng/mL
117 ng/mL
59 ng/mL
29 ng/mL
14.6 ng/mL
5
SAH, pmol
∆ mP
A)
B)
A)
∆ mP
Overview
Methylation is a ubiquitous covalent modification used to control the function of
diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins,
nucleic acids and lipids. Histone methyltransferases (HMTs) are currently of high
interest as drug targets because of their role in epigenetic regulation, however
most HMT assay methods are either not amenable to an HTS environment or are
applicable to a limited number of enzymes.
We developed a generic
methyltransferase assay method using fluorescent immunodetection of AMP, which
is formed from the MT reaction product S-adenosylhomocysteine in a dual enzyme
coupling step. The assay format shows >100 mP signal with Z’>0.5 at initial rate
conditions for 1 µM to 50 µM SAM. The suitability for HTS is demonstrated using
384-well plates with >16 hour deck (prior to plate addition) and signal (stability after
plate addition) stability at room temperature. The activity of three HMTs (G9a,
Set7/Set9, SUV39H1) were followed with using histone H3 peptides while G9a
activity was assessed using both peptide and full length histone H3. In addition,
inhibitor potencies were determined with G9a HMT. By combining a novel
enzymatic coupling step with the well characterized Transcreener® AMP2/GMP2
Assay, we have developed a robust HTS assay for HMTs which should be broadly
applicable to other types of methyltransferases as well.
• The universal nature of the assay is demonstrated using three HMTs with either peptide or
full-length protein substrates in a single assay.
• Inhibitor potency measurements were demonstrated using G9a HMT and two known HMT
inhibitors, sinefungin and chaetocin.
0
10
20
30
40
Time (minutes)
50
Figure 6. G9a Histone Methyltransferase Initial Rates.
60
Initial velocity conditions were
determined using saturating histone H3 peptide (1-25) of 50 µM with [SAM] at the Km = 2 µM. Quantity of SAH
was determined using a 2 µM SAM/SAH standard curve as shown in Figure 2.
0
0.1
1
10
100
1000
[Inhibitor] µM
Figure 7. Pharmacology.
Dose response curves were generated for G9a methyltransferase sensitivity
with known inhibitors (chaetocin and sinefungin). The IC50 values for sinefungin and chaetocin were 4.7 and 770 µM,
respectively. The G9a enzyme reactions progressed to 25% SAM conversion with a Z’ >0.5.
Acknowledgements
Funding for this work was provided by the National Institute of General Medical Sciences grant
#R44GM073290
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