interrogation of small gtpase activity
TRANSCRIPT
PowerPoint-PräsentationInterrogation of small GTPase Activity
Screening GAPs and GEFs with the PHERAstar® FSX from BMG
LABTECH and Transcreener® Assays from BellBrook Labs
Introducing the New PHERAstar® FSX
· Most sensitive reader available for fluorescence intensity and polarization
· New TRF laser creating the fastest, most sensitive HTRF® reader
· Simultaneous Dual Emission in all modes
· Two onboard reagent injectors with zero delay reading
· Three integrated barcode readers
Introducing the New PHERAstar® FSX
· Most sensitive reader available for fluorescence intensity and polarization
• Sensitivity Ø FI (top) v < 0.15 pM Fluorescein [384 well] v < 0.5 pM Fluorescein [1536 well]
Ø FI (bottom) v < 1.0 pM Fluorescein [384 well]
Ø FP (at 1 nM Fluorescein) v 0.5 mP SD [384 well] v 1.5 mP SD [1536 well]
Introducing the New PHERAstar® FSX
· Most sensitive reader available for fluorescence intensity and polarization
· New TRF laser creating the fastest, most sensitive HTRF® reader
• Up to 60 flashes per second Ø Increased throughput Ø Increased precision
FSX Transcreener® Certification
FSX Transcreener® Certification
FSX Transcreener® Certification
Interrogation of Modulators of small GTPase Activity: Screening GAPs and GEFs with the PHERAstar® FSX from BMG LABTECH and Transcreener ® GDP Assays from BellBrook Labs
Selective Nucleotide Detection
N
NN
N
NH2
O
OHOH
HH
HH
OPO
OH
O
P
O
O
OH
P
O
HO
OH
ADP vs. ATP GDP vs. GTP AMP vs cAMP or ATP
GMP vs cGMP or GTP GDP vs GTP UDP vs UDP Sugar
Transcreener® assays rely on antibodies that differentiate between nucleotides on the basis of subtle structural differences. This makes detection of the product nucleotide possible even in the presence of excess substrate nucleotide.
HTS Assays
10
Transcreener relies on direct detection of GDP. Binding of tracer to antibody causes a change in fluorescence. There are just two components, and no intermediate steps.
Simplified protocol
The Only Direct detection method available for GDP
GDP Detection All other GDP assays use indirect detection and are more complex. In a series of enzymatic steps, GDP is converted to a detectable product. Each step is subject to inhibition by library compounds.
0 10 20 30 40 50 60 70 80 90 100 0
25 50 75
y = 1.9x + 7.8
[GTP] (mM) [G
Antibody/Tracer optimizations
Highly Selective Antibody Enables Sensitive Detection of GDP in the Presence of Excess GTP
0.0 00
1 0.0
0 10
00 10
00 0
0.0001 0.01 1 100 10000 0
20000
40000
60000
0.5
1.0
GTP Conversion 1 µM 10 µM 100 µM
10% 0.91 0.92 0.90 7.5% 0.88 0.87 0.90 5% 0.68 0.82 0.84 3% 0.60 0.78 0.77 2% 0.05 0.55 0.67
FI
20000
40000
60000 1 mM GTP 10 mM GTP 100 mM GTP
GDP, mM
RF U
0.0 00
1 0.0
[GTP] mM
R at
io (6
65 /6
100
200
300
GDP, mM
m P
% GTP Conversion 1 µM 10 µM 100 µM
10 0.84 0.81 0.83 5 0.72 0.79 0.76
2.5 0.69 0.66 0.73 1 0.27 0.4 0.45
% GTP Conversion 1 µM 10 µM 100 µM
10 0.86 0.91 0.88 7.5 0.85 0.84 0.88 5 0.73 0.83 0.85 3 0.75 0.77 0.75 2 0.86 0.75 0.71
FP FI TR-FRET
%conv
SD
Ave
100%
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2.3586873038
267
Z'
%conv
0.9562066472
100%
0.9482598977
60
0.9366144006
30
0.9283740764
20
0.9246730804
15
0.9107589038
10
0.8961936321
7.5
0.8123614897
5
0.7953367884
3
0.634583602
1.5
0.0598136255
0.75
300.860.850.91
200.860.840.85
100.830.810.84
50.760.790.72
2.50.730.660.69
10.450.40.27
Sheet1
300.860.850.91
200.860.840.85
100.830.810.84
50.760.790.72
2.50.730.660.69
10.450.40.27
Sheet1
100.860.910.88
7.50.850.840.88
50.730.830.85
100
200
300
100
200
300
10000
20000
30000
40000
50000
10000
20000
30000
40000
50000
0.5
1.0
1.5
2.0
5
10
[GDP] uM D
enzyme is inactivated.
The GEF (guanine nucleotide exchange factor) upstream from the site promotes the release of the GDP so GTP can replace and activate the GTPase.
The GTPase remains active until the GTP is hydrolyzed to GDP, this is catalyzed by the GAP (GTPase Activating Protein).
GTPase Activity of Monomeric GTPases
0.1 1 10 100 1000 0
50
100
150
[E] ng/mL
D m
20000
30000
40000
50000
[E] ng/mL
R FU
2
4
6
8
[E] ng/mL
D Ra
tio (6
65 /6
20 )
Measuring Activity of GAP and GEF Proteins Using the Transcreener® GDP Assay
GEFs accelerate steady state GTP hydrolysis rates. By accelerating the rate limiting step of the GTPase catalytic cycle, GEFs enhance the steady state rates of GDP formation by GTPases, which can be detected using the Transcreener® GDP Assay.
10 100 1000 10000
P
GTPases are first titrated to identify the concentration that produced 20% of the maximal signal: 39 nM Cdc42 and 78 nM RhoA.
Titration of GEF protein, Dbs into limiting GTPase to determine optimal Dbs concentration (125 nM).
0.01 1 100 10000 0
40
80
120
[DBS] nM
D m
P Measuring Activity of GEF Proteins Using the Transcreener® GDP Assay
10 100 1000 10000
P
GTPases are first titrated to identify the concentration that produced 20% of the maximal signal: 39 nM Cdc42 and 78 nM RhoA.
Titration of GEF protein, Dbs into limiting GTPase to determine optimal Dbs concentration (125 nM).
0.01 1 100 10000 0
40
80
120
[DBS] nM
D m
P Measuring Activity of GEF Proteins Using the Transcreener® GDP Assay
Measuring GEF Activity of Monomeric GTPases
0 20 40 60 0
40
80
GEF Activity
Time, min
D m
0.05
0.10
0.15
0.20
0.25
Cdc42
40
80
GEF Activity
Time, min
D m
0.05
0.10
0.15
RhoA
0
20
40
60
0.3
0.6
0.9
1.2
The GEF Effect of Dbs Protein
J Biomol Screen. 2015 Jul 20. pii: 1087057115596326. [Epub ahead of print] A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay Reichman M, Schabdach A, Kumar M, Zielinski T, Donover PS, Laury-Kleintop LD, Lowery RG.
0 2000 4000 6000 8000 30000
35000
40000
45000
50000
55000
60000
65000
RhoA
0.1
0.2
0.3
0.4
RhoA+GEF RhoA
RhoA+RhoGAP 14.60 3X RhoA+Dbs 16.00 3X
RhoA+RhoGAP+Dbs 31.28 7X
Time, min
G D
P, mM
0 2000 4000 6000 8000 20000
40000
60000
80000
CDC42+GAP CDC42
0.1
0.2
0.3
0.4
CDC42+GEF CDC42
CDC42+RhoGAP 27.9 4X CDC42+Dbs 37.48 5X
CDC42+RhoGAP+Dbs 92.5 13X
Time, min
G D
P, mM
0 60 120 180 240 0
50
100
150
200
P-Rex1 (ng/ml)
Time, min
Dm P
0.2
0.4
0.6
0.8
PRex(ng/μl) Rac 1 GTPase (nM/min) GEF Effect None 0.53
1.1 3.1 5.8x 2.3 4.1 7.8x 4.5 5.6 10.6x 8.9 7.5 14.1x
Orthogonal Pooled Screening
Each well contains 10 compounds from a 104,000 compound library with many core scaffolds not found elsewhere.
Each compound is present in two wells, amongst a unique combination of 9 other compounds
To be tallied as a hit, the compound must show reactivity in both wells.
P-Rex1/Rac1 Pilot OPS™ Screen 6400 compounds (10 x, N = 2) in 4 pre-dispensed plates
Screen Statistics
Raw Hits (> 3 Std Devs) 21
Dose response 5
20
40
60
80
100 1 3 4 18 19
Cmpds 1 3 4 18 19 [IC50],µM 2.5 2.3 3.2 40.5 1
Cmpds, mM %
In hi
bi tio
n
For more information, visit our Transcreener® GDP Assay webpage or contact us at [email protected]
The Transcreener® GDP Assays are the only HTS method for direct detection of GDP formed by GTPases.
The Transcreener® GDP Assays are available in FP, FI and TR-FRET format.
These assays are robust, reliable, with outstanding signal stability and extensive validation in industrial HTS.
The Transcreener® GDP Assays enable detection of any enzyme that produces GDP and can also be used for detection of GAPs and GEFs.
ADP Protein Kinases (many) Protein substrates Peptide substrates Autophosphorylation
Various ATP-utilizing enzymes Acetyl CoA carboxylase Glutamine synthetase ATP Citrate Lyase Viral Helicase RNA Triphosphatase Hsp 70, 90 RecA
Lipid Kinases Sphingosine kinase Shikimate kinase Pantothenate kinase PI3K – 6 isoforms
Carbohydrate Kinases Ketohexokinase Hexokinase phosphofructokinase
AMP/GMP PDEs - several Ub, SUMO Ligases NAD Synthetase Acyl CoA Synthetase Sialyltransferases (CMP)
GDP Gα proteins Cdc42 H-ras Rho A Arf 1 Fucosyltransferases 1,3
UDP α-1,3 Galactosyltransferase Glucosylceramide Synthase Hepatic UGTs
Examples of Enzymes Validated in Assays
EPIGEN HMTs EZH2 G9a DOT1L MLL4 SET7/9SET8 SUV39H1
PRMTs PRMT1 PRMT3 PRMT4 PRMT8
DNMTs DNMT1 DNMT3
LCGC- Mel Reichman Scott Donover
Interrogation of small GTPase Activity
Introducing the New PHERAstar® FSX
Introducing the New PHERAstar® FSX
Introducing the New PHERAstar® FSX
FSX Transcreener® Certification
FSX Transcreener® Certification
FSX Transcreener® Certification
Slide Number 8
Slide Number 9
Slide Number 10
Slide Number 11
Slide Number 12
Slide Number 13
Slide Number 14
Slide Number 22
Slide Number 23
Slide Number 24
Slide Number 25
LABTECH and Transcreener® Assays from BellBrook Labs
Introducing the New PHERAstar® FSX
· Most sensitive reader available for fluorescence intensity and polarization
· New TRF laser creating the fastest, most sensitive HTRF® reader
· Simultaneous Dual Emission in all modes
· Two onboard reagent injectors with zero delay reading
· Three integrated barcode readers
Introducing the New PHERAstar® FSX
· Most sensitive reader available for fluorescence intensity and polarization
• Sensitivity Ø FI (top) v < 0.15 pM Fluorescein [384 well] v < 0.5 pM Fluorescein [1536 well]
Ø FI (bottom) v < 1.0 pM Fluorescein [384 well]
Ø FP (at 1 nM Fluorescein) v 0.5 mP SD [384 well] v 1.5 mP SD [1536 well]
Introducing the New PHERAstar® FSX
· Most sensitive reader available for fluorescence intensity and polarization
· New TRF laser creating the fastest, most sensitive HTRF® reader
• Up to 60 flashes per second Ø Increased throughput Ø Increased precision
FSX Transcreener® Certification
FSX Transcreener® Certification
FSX Transcreener® Certification
Interrogation of Modulators of small GTPase Activity: Screening GAPs and GEFs with the PHERAstar® FSX from BMG LABTECH and Transcreener ® GDP Assays from BellBrook Labs
Selective Nucleotide Detection
N
NN
N
NH2
O
OHOH
HH
HH
OPO
OH
O
P
O
O
OH
P
O
HO
OH
ADP vs. ATP GDP vs. GTP AMP vs cAMP or ATP
GMP vs cGMP or GTP GDP vs GTP UDP vs UDP Sugar
Transcreener® assays rely on antibodies that differentiate between nucleotides on the basis of subtle structural differences. This makes detection of the product nucleotide possible even in the presence of excess substrate nucleotide.
HTS Assays
10
Transcreener relies on direct detection of GDP. Binding of tracer to antibody causes a change in fluorescence. There are just two components, and no intermediate steps.
Simplified protocol
The Only Direct detection method available for GDP
GDP Detection All other GDP assays use indirect detection and are more complex. In a series of enzymatic steps, GDP is converted to a detectable product. Each step is subject to inhibition by library compounds.
0 10 20 30 40 50 60 70 80 90 100 0
25 50 75
y = 1.9x + 7.8
[GTP] (mM) [G
Antibody/Tracer optimizations
Highly Selective Antibody Enables Sensitive Detection of GDP in the Presence of Excess GTP
0.0 00
1 0.0
0 10
00 10
00 0
0.0001 0.01 1 100 10000 0
20000
40000
60000
0.5
1.0
GTP Conversion 1 µM 10 µM 100 µM
10% 0.91 0.92 0.90 7.5% 0.88 0.87 0.90 5% 0.68 0.82 0.84 3% 0.60 0.78 0.77 2% 0.05 0.55 0.67
FI
20000
40000
60000 1 mM GTP 10 mM GTP 100 mM GTP
GDP, mM
RF U
0.0 00
1 0.0
[GTP] mM
R at
io (6
65 /6
100
200
300
GDP, mM
m P
% GTP Conversion 1 µM 10 µM 100 µM
10 0.84 0.81 0.83 5 0.72 0.79 0.76
2.5 0.69 0.66 0.73 1 0.27 0.4 0.45
% GTP Conversion 1 µM 10 µM 100 µM
10 0.86 0.91 0.88 7.5 0.85 0.84 0.88 5 0.73 0.83 0.85 3 0.75 0.77 0.75 2 0.86 0.75 0.71
FP FI TR-FRET
%conv
SD
Ave
100%
41
42
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43
42
42
42
41
43
42
43
44
43
42
42
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0.9325048082
42
60
53
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49
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51
53
50
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48
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1.3831281496
51
30
72
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1.8097962092
70
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85
82
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85
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2.0602342627
82
15
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1.9318084941
97
10
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2.1157621027
117
7.5
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2.2506037837
134
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2.9142329391
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2.3586873038
267
Z'
%conv
0.9562066472
100%
0.9482598977
60
0.9366144006
30
0.9283740764
20
0.9246730804
15
0.9107589038
10
0.8961936321
7.5
0.8123614897
5
0.7953367884
3
0.634583602
1.5
0.0598136255
0.75
300.860.850.91
200.860.840.85
100.830.810.84
50.760.790.72
2.50.730.660.69
10.450.40.27
Sheet1
300.860.850.91
200.860.840.85
100.830.810.84
50.760.790.72
2.50.730.660.69
10.450.40.27
Sheet1
100.860.910.88
7.50.850.840.88
50.730.830.85
100
200
300
100
200
300
10000
20000
30000
40000
50000
10000
20000
30000
40000
50000
0.5
1.0
1.5
2.0
5
10
[GDP] uM D
enzyme is inactivated.
The GEF (guanine nucleotide exchange factor) upstream from the site promotes the release of the GDP so GTP can replace and activate the GTPase.
The GTPase remains active until the GTP is hydrolyzed to GDP, this is catalyzed by the GAP (GTPase Activating Protein).
GTPase Activity of Monomeric GTPases
0.1 1 10 100 1000 0
50
100
150
[E] ng/mL
D m
20000
30000
40000
50000
[E] ng/mL
R FU
2
4
6
8
[E] ng/mL
D Ra
tio (6
65 /6
20 )
Measuring Activity of GAP and GEF Proteins Using the Transcreener® GDP Assay
GEFs accelerate steady state GTP hydrolysis rates. By accelerating the rate limiting step of the GTPase catalytic cycle, GEFs enhance the steady state rates of GDP formation by GTPases, which can be detected using the Transcreener® GDP Assay.
10 100 1000 10000
P
GTPases are first titrated to identify the concentration that produced 20% of the maximal signal: 39 nM Cdc42 and 78 nM RhoA.
Titration of GEF protein, Dbs into limiting GTPase to determine optimal Dbs concentration (125 nM).
0.01 1 100 10000 0
40
80
120
[DBS] nM
D m
P Measuring Activity of GEF Proteins Using the Transcreener® GDP Assay
10 100 1000 10000
P
GTPases are first titrated to identify the concentration that produced 20% of the maximal signal: 39 nM Cdc42 and 78 nM RhoA.
Titration of GEF protein, Dbs into limiting GTPase to determine optimal Dbs concentration (125 nM).
0.01 1 100 10000 0
40
80
120
[DBS] nM
D m
P Measuring Activity of GEF Proteins Using the Transcreener® GDP Assay
Measuring GEF Activity of Monomeric GTPases
0 20 40 60 0
40
80
GEF Activity
Time, min
D m
0.05
0.10
0.15
0.20
0.25
Cdc42
40
80
GEF Activity
Time, min
D m
0.05
0.10
0.15
RhoA
0
20
40
60
0.3
0.6
0.9
1.2
The GEF Effect of Dbs Protein
J Biomol Screen. 2015 Jul 20. pii: 1087057115596326. [Epub ahead of print] A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay Reichman M, Schabdach A, Kumar M, Zielinski T, Donover PS, Laury-Kleintop LD, Lowery RG.
0 2000 4000 6000 8000 30000
35000
40000
45000
50000
55000
60000
65000
RhoA
0.1
0.2
0.3
0.4
RhoA+GEF RhoA
RhoA+RhoGAP 14.60 3X RhoA+Dbs 16.00 3X
RhoA+RhoGAP+Dbs 31.28 7X
Time, min
G D
P, mM
0 2000 4000 6000 8000 20000
40000
60000
80000
CDC42+GAP CDC42
0.1
0.2
0.3
0.4
CDC42+GEF CDC42
CDC42+RhoGAP 27.9 4X CDC42+Dbs 37.48 5X
CDC42+RhoGAP+Dbs 92.5 13X
Time, min
G D
P, mM
0 60 120 180 240 0
50
100
150
200
P-Rex1 (ng/ml)
Time, min
Dm P
0.2
0.4
0.6
0.8
PRex(ng/μl) Rac 1 GTPase (nM/min) GEF Effect None 0.53
1.1 3.1 5.8x 2.3 4.1 7.8x 4.5 5.6 10.6x 8.9 7.5 14.1x
Orthogonal Pooled Screening
Each well contains 10 compounds from a 104,000 compound library with many core scaffolds not found elsewhere.
Each compound is present in two wells, amongst a unique combination of 9 other compounds
To be tallied as a hit, the compound must show reactivity in both wells.
P-Rex1/Rac1 Pilot OPS™ Screen 6400 compounds (10 x, N = 2) in 4 pre-dispensed plates
Screen Statistics
Raw Hits (> 3 Std Devs) 21
Dose response 5
20
40
60
80
100 1 3 4 18 19
Cmpds 1 3 4 18 19 [IC50],µM 2.5 2.3 3.2 40.5 1
Cmpds, mM %
In hi
bi tio
n
For more information, visit our Transcreener® GDP Assay webpage or contact us at [email protected]
The Transcreener® GDP Assays are the only HTS method for direct detection of GDP formed by GTPases.
The Transcreener® GDP Assays are available in FP, FI and TR-FRET format.
These assays are robust, reliable, with outstanding signal stability and extensive validation in industrial HTS.
The Transcreener® GDP Assays enable detection of any enzyme that produces GDP and can also be used for detection of GAPs and GEFs.
ADP Protein Kinases (many) Protein substrates Peptide substrates Autophosphorylation
Various ATP-utilizing enzymes Acetyl CoA carboxylase Glutamine synthetase ATP Citrate Lyase Viral Helicase RNA Triphosphatase Hsp 70, 90 RecA
Lipid Kinases Sphingosine kinase Shikimate kinase Pantothenate kinase PI3K – 6 isoforms
Carbohydrate Kinases Ketohexokinase Hexokinase phosphofructokinase
AMP/GMP PDEs - several Ub, SUMO Ligases NAD Synthetase Acyl CoA Synthetase Sialyltransferases (CMP)
GDP Gα proteins Cdc42 H-ras Rho A Arf 1 Fucosyltransferases 1,3
UDP α-1,3 Galactosyltransferase Glucosylceramide Synthase Hepatic UGTs
Examples of Enzymes Validated in Assays
EPIGEN HMTs EZH2 G9a DOT1L MLL4 SET7/9SET8 SUV39H1
PRMTs PRMT1 PRMT3 PRMT4 PRMT8
DNMTs DNMT1 DNMT3
LCGC- Mel Reichman Scott Donover
Interrogation of small GTPase Activity
Introducing the New PHERAstar® FSX
Introducing the New PHERAstar® FSX
Introducing the New PHERAstar® FSX
FSX Transcreener® Certification
FSX Transcreener® Certification
FSX Transcreener® Certification
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