Interrogation of small GTPase Activity. Screening GAPs and GEFs with the PHERAstar FSX from BMG LABTECH and Transcreener Assays from BellBrook Labs

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1 Interrogation of small GTPase Activity Screening GAPs and GEFs with the PHERAstar FSX from BMG LABTECH and Transcreener Assays from BellBrook Labs

2 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 All microplate formats to 3456 well AlphaScreen/AlphasLISA laser, now with AlphaPlex TM technology

3 Introducing the New PHERAstar FSX Most sensitive reader available for fluorescence intensity and polarization Sensitivity FI (top) v <.15 pm Fluorescein [384 well] v <.5 pm Fluorescein [1536 well] FI (bottom) v < 1. pm Fluorescein [384 well] FP (at 1 nm Fluorescein) v.5 mp SD [384 well] v 1.5 mp SD [1536 well]

4 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 6 flashes per second Increased throughput Increased precision

5 FSX Transcreener Certification

6 FSX Transcreener Certification

7 FSX Transcreener Certification

8 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

9 HTS Assays Selective Nucleotide Detection Transcreener assays rely on antibodies that differentiate between nucleotides on the basis of subtle structural differences. This makes NH 2 detection of the product nucleotide possible even in the presence of excess substrate nucleotide. N N O O O N N HO P O P O P O O OH OH OH H H H OH H OH ADP vs. ATP GDP vs. GTP AMP vs camp or ATP GMP vs cgmp or GTP GDP vs GTP UDP vs UDP Sugar

10 The Only Direct detection method available for GDP 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 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. 1

11 Antibody/Tracer optimizations [GDP HiLyte647 Tracer] (nm) y = 1.9x [GTP] (mm)

12 Highly Selective Antibody Enables Sensitive Detection of GDP in the Presence of Excess GTP mp GTP GDP FP FP RFU GTP GDP FI FI Ratio 665/ GTP GDP TR-FRET TR-FRET mm Competitor Nucleotide, mm Nucleotide, mm Selectivity for GDP over GTP is 138 fold

13 Great Sensitivity and Robustness mp mm GTP 1 mm GTP 1 mm GTP FI FP FI TR-FRET GDP, mm RFU mm GTP 1 mm GTP 1 mm GTP GDP, mm Ratio (665/615) [GTP] mm 1 mm GTP 1 mm GTP 1 mm GTP 1 1 GTP Conversion 1 µm 1 µm 1 µm 1% % % % % % GTP Conversion 1 µm 1 µm 1 µm % GTP Conversion 1 µm 1 µm 1 µm

14 Outstanding Reagent and Signal Stability mp Hour 24 Hours 2. FP FI FI TR-FRET FP 5 1 Hour 4 1 Hour Hour 24 Hours 3 1. RFU 2 1 D Ratio (665/62) [GDP] mm [GDP] mm [GDP], mm mp Hour 24 Hours FP RFU Hour 24 Hours FI TR-FRET FP FI TR-FRET D Ratio (665/62) Hours 1 Hour [GDP] mm [GDP] mm [GDP] um

15 GTPases When GDP is attached to the GTPase, the 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).

16 GTPase Activity of Monomeric GTPases DmP cdc42 Arf RAC1 RhoA RFU cdc42 Arf RAC1 RhoA [E] ng/ml [E] ng/ml DRatio (665/62) cdc42 Arf RAC1 RhoA [E] ng/ml

17 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.

18 Measuring Activity of GEF Proteins Using the Transcreener GDP Assay DmP cdc42 RhoA GTPases are first titrated to identify the concentration that produced 2% of the maximal signal: 39 nm Cdc42 and 78 nm RhoA [E] nm 12 DmP 8 CDC42 RhoA No GTPase Titration of GEF protein, Dbs into limiting GTPase to determine optimal Dbs concentration (125 nm) [DBS] nm

19 Measuring Activity of GEF Proteins Using the Transcreener GDP Assay DmP cdc42 RhoA GTPases are first titrated to identify the concentration that produced 2% of the maximal signal: 39 nm Cdc42 and 78 nm RhoA [E] nm 12 DmP 8 CDC42 RhoA No GTPase Titration of GEF protein, Dbs into limiting GTPase to determine optimal Dbs concentration (125 nm) [DBS] nm

20 Measuring GEF Activity of Monomeric GTPases DmP CDC42 CDC42 + Dbs Dbs GEF Activity DmP RhoA RhoA+ Dbs Dbs GEF Activity DmP RhoB RhoB + Dbs Dbs GEF Activity Time, min Time, min Time, min [GDP] mm Cdc42 + Dbs Time, min Cdc42 [GDP] mm RhoA + Dbs RhoA Time, min [GDP] mm RhoB + Dbs RhoB Time, min

21 The GEF Effect of Dbs Protein J Biomol Screen. 215 Jul 2. pii: [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.

22 Measuring combined GAP + GEF Activity of Monomeric GTPases 65 RFU Time, sec.4 RhoA RhoA+GAP RhoA+GEF RhoA+GAP+GEF GDP, mm RhoA RhoA+GEF RhoA+GAP RhoA+GAP+GEF Time, min µmoles GDP/min Enhancement RhoA 3.25 RhoA+RhoGAP X RhoA+Dbs 16. 3X RhoA+RhoGAP+Dbs X

23 Measuring combined GAP + GEF Activity of Monomeric GTPases 8 RFU 6 4 CDC42 CDC42+GAP CDC42+GEF CDC42+GAP+GEF Time, sec.4 GDP, mm Time, min CDC42 CDC42+GEF CDC42+GAP CDC42+GAP+GEF µmoles GDP/min Enhancement CDC42.7 CDC42+RhoGAP X CDC42+Dbs X CDC42+RhoGAP+Dbs X

24 Measuring GEF Activity of PRex DmP P-Rex1 (ng/ml) None GDP (mm) Time, min Time, min PRex(ng/μl) Rac 1 GTPase (nm/min) GEF Effect None x x x x

25 Orthogonal Pooled Screening Each well contains 1 compounds from a 14, 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.

26 P-Rex1/Rac1 Pilot OPS Screen 64 compounds (1 x, N = 2) in 4 pre-dispensed plates % Inhibition Screen Statistics Z -From DMSO Controls.8 Z-factor of Entire Screen.6 Raw Hits (> 3 Std Devs) Cmpds, mm Cmpds [IC 5 ],µm Dose response 5

27 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. For more information, visit our Transcreener GDP Assay webpage or contact us at

28 Associated Products Transcreener GDP FP Assay 39-1K Transcreener GDP FI Assay 314-1K Transcreener GDP TR-FRET Assay 321-1K

29 Examples of Enzymes Validated in ADP Protein Kinases (many) Protein substrates Peptide substrates Autophosphorylation Carbohydrate Kinases Ketohexokinase Hexokinase phosphofructokinase Lipid Kinases Sphingosine kinase Shikimate kinase Pantothenate kinase PI3K 6 isoforms Various ATP-utilizing enzymes Acetyl CoA carboxylase Glutamine synthetase ATP Citrate Lyase Viral Helicase RNA Triphosphatase Hsp 7, 9 RecA GDP Gα proteins Cdc42 H-ras Rho A Arf 1 Fucosyltransferases 1,3 UDP α-1,3 Galactosyltransferase Glucosylceramide Synthase Hepatic UGTs AMP/GMP PDEs - several Ub, SUMO Ligases NAD Synthetase Acyl CoA Synthetase Sialyltransferases (CMP) Assays EPIGEN HMTs EZH2 G9a DOT1L MLL4 SET7/9SET8 SUV39H1 PRMTs PRMT1 PRMT3 PRMT4 PRMT8 DNMTs DNMT1 DNMT3

30 Thank you Special Thanks to: BellBrook Labs- Tom Zielinski Bob Lowery LCGC- Mel Reichman Scott Donover