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Biology at Work
Transcreener® HTS Enzyme Assay Development
Service
Karen Kleman, PhD
February 7, 2012
Biology at Work
Assays must be robust, reproducible, & automation-friendly
Drug Discovery World Summer 2010 High Throughput Screening 2010: Effective Strategies,
Innovative Technologies, and Use of Better Assays. Published
by HighTech Business Decisions.
Roadblocks in HTS Assay Development
Average HTS biochemical assay development time= 4.1 months
Lack of high quality protein and assay reagents • 44% of those surveyed indicate this to be the greatest hurdle
Capacity, cost, and inefficient communication outside HTS lab
One off assay development is typically required for each enzyme class
• Novel or complex targets can be difficult
Biology at Work
Universal assay platform based on the immunodetection of nucleotides enables simple screening within target families and across target families:
Same protocol, same data analysis
One-stop shopping: one assay platform for many diverse targets
BellBrook Labs has a solution! Fast turnaround, low costs
Methyltransferases
Acetyltransferases
G protein/RGS
Biology at Work
Experienced and knowledgeable scientists will lead project from start to finish.
Flexible service: quick enzyme validation to 1100 compound pilot screen
Transcreener ® assays are available in FP, TR-FRET and FI formats and are validated for HTS.
More than 50 million wells in high throughput screens
Time and Cost-savings: if project progresses to screen, part of the development costs
will be refunded.
Biology at Work
Case Study 1- p97 ATPase
Customer’s Project: Identify chemical probes for p97 ATPase mechanistic studies.
Disease implications:
• Human dementia
• Amyotrophic lateral sclerosis (ALS)
• Parkinson's disease
• Multiple cancers
ATP ADP
P97 Uses ATPase to “pull” polypeptide chain into
the cytosol from the ER where it is then degraded
by the proteosome
Biology at Work
Case Study 1- p97 ATPase
Assay challenges: Initial attempts by the customer to develop a TR-FRET assay resulted in a small assay window. Limited amounts or very dilute enzyme preparations were provided (10 µL).
Customer’s request: develop a TR-FRET (or FP) HTS assay to identify p97 ATPase modulators. Evaluate two enzyme preparations.
Biology at Work
Case Study 1- p97 ATPase- Approach
Exp 1: Antibody/tracer optimizations
for the ADP FP & TR-FRET
Assays
Exp 2: ATP/ADP Standard Curves
Exp 3: Enzyme titrations in both
formats.
Biology at Work
0.001 0.01 0.1 1 10 100 10000
100
200
300
EC85 = 3 g/mL
5 ATP
ADP2 antibody, g/mL
mP
0.01 0.1 1 10 100 10000
5
10
15
20
25
EC85 = 46 nM tracer
ADP HiLyte 647 Tracer, nM
Em
66
5/E
m6
20
Optimization of Transcreener assay reagents for FP and TR-FRET assays
Case Study 1- p97 ATPase
Biology at Work
0.1 1 10 1000
100
200
300
5 M ATP/ADP Standard Curve
% Conversion of 5 M ATP
mP
ADP, µM % Conv ∆mP Z`
5 100 224 0.92
3.5 70 220 0.919231
2.5 50 217 0.921307
1 20 194 0.903611
0.5 10 168 0.871974
0.25 5 109 0.75758
0.125 2.5 37 0.209121
0.1 1 10 1000
5
10
15
20
5 M ATP/ADP Standard Curve
% Conversion of 5 M ATP
Em
66
5/E
m6
20
ADP, µM % Conv Z`
5 100 0.876805
3.5 70 0.871366
2.5 50 0.867278
1 20 0.800815
0.5 10 0.735323
0.25 5 0.606629
0.125 2.5 -0.14226
AT/ADP Standard Curves demonstrate excellent Z’ values <10% ATP substrate conversion
Case Study 1- p97 ATPase
Biology at Work
0.01 0.1 1 10 100 1000
0
25
50
75
100
125
150
175
200
Human+ATP
Human-ATP
Mouse+ATPMouse-ATP
EC80 = 20 ng/mL
mP= 160 units
EC80 = 4 ng/mL
mP= 140 units
[P97], ng/mL
mP
0.001 0.01 0.1 1 10 1000
50
100
150
200
250
300
Human, +ATP
Human, -ATP
Mouse, +ATP
Mouse, -ATP
[P97], ng/mL
mP
0.01 0.1 1 10 100 10000
5
10
15
20Human+ATP
Human-ATP
Mouse+ATP
Mouse-ATP
[P97], ng/mL
Em
66
5/E
m6
20
Case Study 1- p97 ATPase
Excellent screening assay window for p97 ATPase was best achieved with the FP Assay
< 10% ATP was converted to ADP
Biology at Work
• An HTS-ready Transcreener ADP2 FP Assay (>140 mP shift) was developed for p97 ATPase and was the better choice over the TR-FRET assay format for these particular enzyme preparations.
• Both the mouse and human p97 enzyme preparations can be used for
screening, requiring only picograms of enzyme/well. • < 10% of the ATP was consumed with both p97 assays: ideal for HTS
campaigns.
Case Study 1- P97 ATPase- Solution!
Customer’s request: develop a HTS assay for p97 ATPase and evaluate two enzyme preparations.
Assay development time was ~ 1 week!
Biology at Work
Case Study 2- GTPase (LRRK2)
Customer’s goal: To identify compounds
that affect the GTP-ase activity of the
multifunctional enzyme LRRK2
Disease implications: Mutations in this gene
have been associated with Parkinson’s and
Crohn’s disease
Dominant missense mutations in North
African Arabs, the G2019S mutation can
cause up to 30% of sporadic PD.
In most Western populations, the
commonest known mutation G2019S
underlies between 1 and 5% of cases.
Biology at Work
Assay Challenges: • Low turn-over GTPase activity • LRRK2 has a kinase and a GTPase domain (can these be distinguished) • Dilute commercial enzyme preparation (packaged for kinase); 10 µg
Customer request: Demonstrate LRRK2 activity using the Transcreener GDP FP Assay. Did not observe signal above background.
Case Study 2- LRRK2
Biology at Work
Case Study 2- LRRK2- Approach
Exp 1: Quick evaluation of enzyme activity in the Transcreener GDP Assay. Identify the amount of enzyme required to achieve a >100 mP assay and Z’ value >0.5. Exp 2: Determine if the GTPase activity is specific to the GTP active domain. Exp 3: Assay optimization (temperature, reducing reagent).
Biology at Work
10 M GTP4 hr incubation at 30 C
0.1 1 10 1000
100
200
300
-GTP
+ GTP
LRRK2, nM
mP
1 10 1000
50
100
150
200
1.5h
2h
3h4h
LRRK2, nM
mP
Case Study 2- LRRK2
An assay window > 100 mP units was achieved with 55-75 nM LRRK2 with a 2-3 h incubation
Biology at Work
1 10 100 10000
20
40
60
80
100
120
140
160
Wild Type
Kinase inactive mutant
GTPase mutant
10 M GTP2 hr incubation at 30 C
LRRK2, nM
mP
Case Study 2- LRRK2
GDP production by LRRK2 does not occur at the kinase domain, but is dependent on a functional GTPase domain.
Biology at Work
Case Study 2- LRRK2- Solution!
• LRRK2 GTPase activity can be monitored with the Transcreener GDP FP Assay.
• An assay window of >100 mP units can be achieved with 50-100 nM LRRK2 when incubated at 37°C for 2-3 hours
• GTPase activity is independent of functional kinase domain as
demonstrated with the LRRK2 kinase mutant. • Enzyme requirements may be reduced with further improvements in
reaction buffer and conditions.
Assay development time was < 1 week!
Biology at Work
Case Study 3- Methyltransferases (DNMTs)
Customer’s goal: To identify DMNT1 inhibitors
Disease implications: Cancer
Hypermethylated tumor suppressor genes
are a direct result of aberrant DNMT
targeting
Epigenome may play a role in tumor cell
resistance to oxidative stress and
highlights a potential role for DNMT1 as a
potential molecular target in cancer
therapy.
Biology at Work
Customer request: - Seeking an orthogonal HTS activity-based assay to complement binding assays. - Compare mouse and human DMNT1 full-length and catalytic domains.
Enzyme Assay Challenges: In Customer’s hands control MT enzyme worked (histone MT), but the assay window for DMNT1 was unsatisfactory. DNA substrate source is difficult to quantitate Enzyme preparations were uncharacterized
Case Study 3- Methyltransferases (DNMT1)
Biology at Work
Case Study 3- Methyltransferases (DNMTs)-Approach
Exp 1 Determine optimum antibody concentrations and perform standard curve with EPIGEN Methyltransferase reagents. Exp 2 Perform DNMT1 titrations in 3 buffers and determine optimum enzyme concentration for HTS. Exp 3 Calculate Z’ values. Exp 4 Assay improvement for catalytic domain.
15 µL enzyme + 2.5 µL stop + 2.5 µL AMP Detection Mix
J Biomol Screen. January 2012 vol. 17 no. 1 59-70 Development and Validation of a Generic Fluorescent Methyltransferase Activity Assay Based on the
Transcreener AMP/GMP Assay. Klink TA, Staeben M, Twesten K, Kopp AL, Kumar M, Schall Dunn R,
Pinchard CA, Kleman-Leyer KM, Klumpp M, Lowery RG.
Biology at Work
3 M SAM/SAH Standard Curveswith 0.5 mU/uL of Poly(dI-dC)
(n=24)
0.001 0.01 0.1 10
50
100
150
200
0.0
0.2
0.4
0.6
0.8
1.0
Z`
SAH ( M)m
P
Case Study 3- Methyltransferases (DNMTs)
Using the optimum AMP/GMP antibody concentration, excellent Z’ values are achieved at < 10% SAM consumption
3 M SAM + 0.5 mU/ L of Poly(dI-dC)
0.01 0.1 1 10 100 10000
100
200
300
Simple Buffer (1)
Simple Buffer + EDTA (2)
Simple Buffer+EDTA+ Triton (3)
EC85= 4.5 ug/mL
AMP2/GMP2 Ab
mP
Z’= 0.5 with 2.5% SAM consumption (LLD) Z’=0.75 with 10% SAM consumption
Biology at Work
Increased DNMT1 activity is observed with the inclusion of EDTA and Triton X-100
Case Study 3- Methyltransferases (DNMT1)
0 25 50 75 100 1250
50
100
150 EC80 = 35 nM
DNMT1, nM
mP
0.1 1 10 100 10000
50
100
150
FL-Buffer 1
Cat-Buffer 1
FL-Buffer 2
Cat-Buffer 2
FL-Buffer 3
Cat-Buffer 3
DNMT1, nM
mP
Biology at Work
EC80 [nM]
Ave mP
∆ mP Std. Dev
Z′ Condition
DNMT1, Full Length
35 117 100 3. 8 0.68 E+ Poly(dI-dC)+ SAM
218 7.5 No SAM
215 8.9 No Poly (dI-dC)
DNMT1, Cat domain
220 203 37 5.3 0.34 E+ Poly(dI-dC)+ SAM
243. 3.4 No SAM
236 3.7 No Poly (dI-dC)
Case Study 3- Methyltransferases (DNMTs)
A sensitive HTS-ready assay has been developed for full length DNMT1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250
50
100
150
200
250
300
Z`=0.7
DNMT1 + SAM +DNA
DNMT1 + SAM
Well Number
mP
Biology at Work
0.0001 0.001 0.01 0.1 1 10 100100
150
200
250
300
Buffer
Buffer + 3 uM SAM
Buffer + 2.7uM SAM+0.3 uM SAH
Poly d (I-C) mU/uL
mP
Case Study 3- Methyltransferases (DNMTs)
0.0001 0.001 0.01 0.1 1 100
20
40
60
80 DNMT1(cat domain) + SAM
Poly(dI-dC), Units/ L
mP
Optimization of the DNA substrate concentration improves DMNT1-catalytic domain activity.
Biology at Work
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
DNMT1 catyltic domain, nM
mP
Case Study 3- Methyltransferases (DNMT1)
An HTS-ready assay for the catalytic domain is achievable with further assay optimization
Biology at Work
Case Study 3- DNMT- Solution!
Assay development time was 1 week!
• An HTS-ready DMNT1 assay was established with the Transcreener EPIGEN Methyltransferase Assay.
• An assay window of >100 mP units and Z’= 0.7 can be achieved with 35 nM DMNT1 when incubated at room temperature for 1 hour using 3 µM SAM + 0.05 units of Poly(dI-dC).
• Inclusion of EDTA and Triton X-100 in the enzyme reaction and removal of
MgCl2 (data not shown) enhanced the DMNT1 activity. • The assay window for DMNT1(catalytic domain) was improved by
optimizing the Poly(dI-dC) DNA substrate concentration making it amendable for HTS.
Biology at Work
BellBrook Labs Booth # 128
Available services include:
1) Quick enzyme activity test: We will determine the Transcreener reagent concentrations, establish a standard curve
and perform an enzyme titration using user-defined reaction conditions.
Specs: Z’ value > 0.6 at <30% substrate consumption
2) Assay optimization: We will determine the optimal enzyme concentration, buffer composition, and reaction time for
maximal signal under initial velocity conditions.
Specs: Z’ value > 0.6 at <30% substrate consumption
3) IC50 determination: We will perform a 12 point dose response experiment, in triplicate, and determine the IC50 with
inhibitors of your choice.
4) Pilot Screen: We will perform a pilot screen of 1120 small molecule drugs.
Specs: Z value of at least 0.5.
Biology at Work
Protein Kinases Protein
Peptide substrates
Autophosphorylation
(Including Mycobacterial Shikimite kinase &
Pantothenate kinase ; antimicrobial)
Various ATP-utilizing enzymes Acetyl CoA carboxylase
Glutamine synthetase
ATP Citrate Lyase
Viral Helicase
Trypanosoma RNA Triphosphatase
TbCet1
Hsp70
Hsp90
RecA
Lipid Kinases Sphingosine kinase
PI3Ka
PI3Kb
PI3Kd
PI3Kg
PIP4KIIb
PIP4KIIg
Carbohydrate Kinases Ketohexokinase
Hexokinase
phosphofructokinase AMP PDEs
Ub, SUMO Ligases
NAD Synthetase
Acyl CoA Synthetase
Sialyltransferases (CMP)
Methyltransferases
GDP Galpha protein
Cdc42
H-ras
Fucosyltransferase
UDP α-1,3 Galactosyltransferase
Glucosylceramide
Synthase
Hepatic UGTs
PAP SULTs
Enzyme Targets
Biology at Work
Acknowledgements
Bob Lowery
Meera Kumar
Tom Zielinski
Andy Kopp
Transcreener assay development was
supported by the following NIH SBIR
grants:
2 R44 GM59542-02A1
5 R44 GM059542-03
1 R43 GM069258-1
1 R43 CA110535-01A1
2 R44 GM069258-02A1
5 R44 GM069258-03
1 R43 GM073290-01A1
1 R44 CA110535-02
1 R43 NS059082-01
5 R44 CA110535-03
2 R44 GM073290-02
2 R44 GM073290-02
2 R44 NS059082-02
5 R44 GM073290-03
Biology at Work
J Biomol Screen. January 2012 vol. 17 no. 1 59-70
Development and Validation of a Generic Fluorescent Methyltransferase Activity Assay Based on the Transcreener AMP/GMP Assay.
Klink TA, Staeben M, Twesten K, Kopp AL, Kumar M, Schall Dunn R, Pinchard CA, Kleman-Leyer KM, Klumpp M, Lowery RG.
J Biomol Screen. 2011 Aug;16(7):717-23. Epub 2011 May 18
High-throughput fluorescence polarization assay for the enzymatic activity of GTPase-activating protein of ADP-ribosylation factor (ARFGAP).
Sun W, Vanhooke JL, Sondek J, Zhang Q.
Science. 2011 Jul 22;333(6041):453-6
De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila.
Neunuebel MR, Chen Y, Gaspar AH, Backlund PS Jr, Yergey A, Machner MP.
Mol Cell. 2011 Mar 4;41(5):567-78.
Structure of lipid kinase p110ß/p85ß elucidates an unusual SH2-domain-mediated inhibitory mechanism.
Zhang X, Vadas O, Perisic O, Anderson KE, Clark J, Hawkins PT, Stephens LR, Williams RL.
Molecular and Biochemical Parasitology 2011 Jan: 175(1):21-29
Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds
Crowther GJ, Napuli AJ, Gilligan JH, Gagaring K, Borboa R, Francek C, Chen Z, Dagostino EF, Stockmyer JB, Wang Y, Rodenbough PP, Castaneda LJ,
Leibly DJ, Bhandari J, Gelb MH, Brinker A, Engels IH, Taylor J, Chatterjee AK, Fantauzzi P, Glynne RJ, Van Voorhis WC, Kuhen KL.
Assay Drug Dev Technol. 2010 Jun;8(3):344-55
Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes
Staeben M, Kleman-Leyer KM, Kopp AL, Westermeyer TA, Lowery RG.
J Biomol Screen. 2010
Mar;15(3):279-86. Epub 2010 Feb 10. Rowlands M, McAndrew C, Prodromou C, Pearl L, Kalusa A, Jones K, Workman P,Aherne W. Detection of the
ATPase activity of the molecular chaperones Hsp90 and Hsp72 using the TranscreenerTM ADP assay kit..
J Biomol Screen 2009;
Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity
Thomas Zielinski, Adam J. Kimple, Stephanie Q. Hutsell, Mark D. Koeff, David P. Siderovski, Dr. , and Robert G. Lowery
J Biomol Screen 2009; 14(7): 838-844
High-Throughput, Cell-Free, Liposome-Based Approach for Assessing In Vitro Activity of Lipid Kinases
Douglas J. Demian, Susan L. Clugston, Meta M. Foster, Lucia Rameh, Deborah Sarkes, Sharon A. Townson, Lily Yang, Melvin Zhang, Maura E. Charlton
J Biomol Screen 2009; 14(6): 679-689
Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists
Michael Habig, Anke Blechschmidt, Sigmar Dressler, Barbara Hess, Viral Patel, Andreas Billich, Christian Ostermeier, David Beer, Martin Klumpp
Comb Chem High Throughput Screen 2009; 12(3):258-68
Development and Validation of a High-density Fluorescence Polarization-based Assay for the Trypanosoma RNA Triphosphatase TbCet1
Antczak C, Shum D, Radu C, Seshan VE, Djaballah H.
Assay Drug Dev Technol 2009; 7(1):56-67
Characterization and Optimization of a Red-Shifted Fluorescence Polarization ADP Detection Assay
Karen M. Kleman-Leyer, Tony A. Klink, Andrew L. Kopp, Thane A. Westermeyer, Mark D. Koeff, Brad R. Larson, Tracy J. Worzella, Cori A. Pinchard,
Sebastianus A.T. van de Kar, Guido J.R. Zaman, Jorrit J. Hornberg, Robert G. Lowery
Transcreener® Product References
