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Interrogating Gtpase Effector Proteins: Screening Gaps and Gefs with the Transcreener® GDP Assay

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Interrogating Gtpase Effector Proteins: Screening Gaps and Gefs with the Transcreener® GDP Assay Interrogating GTPase Effector Proteins: Screening GAPs and GEFs with the Transcreener® GDP Assay Meera Kumar BellBrook Labs Selective Nucleotide Detection The Transcreener® assays rely on antibodies that are able to 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. NH2 N N N O O O N HO P O P O P O O OH OH OH H H H H OH OH ADP vs. ATP AMP vs cAMP or ATP GDP vs. GTP GMP vs cGMP or GTP GDP vs GTP UDP vs UDP Sugar Transcreener® GDP Assay: 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. 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. Simplified protocol 3 Indirect Detection of GDP is Convoluted: These assays can lead to many false positives GTPase™ Glo Assay ADP Hunter™ Assay Antibody/Tracer optimizations 200 175 y = 1.9x + 7.8 150 125 100 75 50 25 [GDP HiLyte647 Tracer] (nM) 0 0 10 20 30 40 50 60 70 80 90 100 [GTP] (mM) Highly Selective Antibody Enables Sensitive Detection of GDP in the Presence of Excess GTP FI FP FI TRTR-FRET-FRET 300 FP 80000 1.5 250 GTP GTP GDP GDP 200 GTP 60000 GDP 1.0 mP 150 40000 RFU 100 0.5 20000 Ratio 665/617 50 0 0 1 0.0 0.1 10 0.01 100 0.0001 0.01 1 100 10000 0.001 1000 0.0001 0.01 1 100 10000 0.0001 10000 mM Competitor Nucleotide, mM Nucleotide, mM Selectivity for GDP over GTP is 138 fold Great Sensitivity and Robustness FI FP TRTR-FRET-FRET FP 60000 FI 300 6 1 mM GTP 100 mM GTP 10 mM GTP m 100 mM GTP 10 M GTP 40000 200 4 1 mM GTP RFU mP m 100 1 M GTP 20000 2 10 mM GTP Ratio (665/615) 100 mM GTP 0 0 0 0.0001 0.01 1 100 0.0001 0.01 1 100 1 GDP, mM GDP, mM 0.01 100 0.0001 10000 [GTP] mM GTP % GTP Conversion 1 µM 10 µM 100 µM % GTP Conversion 1 µM 10 µM 100 µM Conversion 1 µM 10 µM 100 µM 10 0.84 0.81 0.83 10% 0.91 0.92 0.90 10 0.86 0.91 0.88 5 0.72 0.79 0.76 7.5% 0.88 0.87 0.90 7.5 0.85 0.84 0.88 2.5 0.69 0.66 0.73 5% 0.68 0.82 0.84 5 0.73 0.83 0.85 1 0.27 0.4 0.45 3% 0.60 0.78 0.77 3 0.75 0.77 0.75 2% 0.05 0.55 0.67 2 0.86 0.75 0.71 Robust and Reliable Signal Stability 2.0 TR-FRET FI FI TR-FRET 300 FPFP 50000 1 Hour 1.5 40000 1 Hour 24 Hour 200 1 Hour 24 Hours 30000 24 Hours 1.0 mP RFU 20000 100 Ratio (665/620) 0.5 10000 D 0 0 0.0 0.01 0.1 1 10 0.01 0.1 1 10 0.001 0.01 0.1 1 10 100 [GDP] mM [GDP] mM [GDP], mM FP FI TRTR-FRET-FRET 300 FP 15 50000 FI 24 Hours 1 Hour 40000 1 Hour 200 1 Hour 24 Hours 10 24 Hours 30000 mP RFU 20000 100 5 Ratio (665/620) 10000 D 0 0 0 0.01 0.1 1 10 0.01 0.1 1 10 0.001 0.01 0.1 1 10 100 [GDP] mM [GDP] uM [GDP] mM 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). GTPase Activity of Monomeric GTPases 200 60000 cdc42 cdc42 Arf 50000 Arf 150 RAC1 RAC1 RhoA RhoA 40000 100 mP RFU D 30000 50 20000 0 10000 0.1 1 10 100 1000 0.1 1 10 100 1000 [E] ng/mL [E] ng/mL 10 cdc42 8 Arf RAC1 6 RhoA 4 Ratio (665/620) Ratio 2 D 0 0.01 0.1 1 10 100 1000 [E] ng/mL Comparison of the Three Detection Formats 50 50 r² 0.9984 40 40 TR-FRET EC50, 30 30 20 20 FI, EC50 10 10 0 0 0 20 40 60 FP, EC50 CDC42 Arf Rac RhoA FI EC50 (ng/uL) 2.438 49.58 5.258 7.219 FP EC50 (ng/uL) 4.005 41.57 5.278 8.587 TR-FRET EC50 (ng/uL) 8.421 45.16 13.5 7.37 Measuring Activity of 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. Measuring Activity of GEF Proteins Using the Transcreener® GDP Assay 200 cdc42 150 RhoA GTPases are first titrated to identify the concentration that produced 20% of the maximal 100 mP D signal: 39 nM Cdc42 and 78 nM RhoA. 50 0 10 100 1000 10000 [E] nM 120 CDC42 80 RhoA Titration of GEF protein, Dbs into limiting GTPase mP No GTPase to determine optimal Dbs concentration (125 nM). D 40 0 0.01 1 100 10000 [DBS] nM Measuring GEF Activity of Monomeric GTPases 120 80 CDC42 120 RhoA RhoB + Dbs CDC42 + Dbs RhoA+ Dbs RhoB Dbs 60 80 Dbs Dbs 80 mP mP GEF Activity mP 40 D D D GEF Activity 40 40 20 0 0 0 0 20 40 60 0 20 40 60 0 60 120 180 Time, min Time, min Time, min 0.15 0.25 1.2 Cdc42 + Dbs RhoA + Dbs 0.20 RhoB + Dbs M M 0.10 0.9 m RhoB m 0.15 M m 0.6 0.10 0.05 RhoA Cdc42 [GDP] [GDP] 0.05 [GDP] 0.3 0.00 0.00 0 20 40 60 0.0 0 20 40 60 0 60 120 180 Time, min Time, min Time, min 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. Measuring GEF Activity of PRex 200 P-Rex1 (ng/ml) 0.8 8.9 150 4.5 0.6 M) 2.3 m mP 100 1.1 0.4 D None 50 GDP ( 0.2 0 0.0 0 60 120 180 240 0 30 60 90 Time, min Time, min P-Rex1 (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 (N=2) in 4 pre-dispensed plates ■ Start reaction with addition of substrates ■ Read plate after 1 hr at RT ■ Data sent to LCGC for deconvolution 100 1 Screen Statistics 80 3 4 Z’-From DMSO Controls 0.8 60 18 19 Z-factor of Entire Screen 0.6 40 % Inhibition Hits > 3 Std Devs 21 20 0 0.1 1 10 100 Cmpds, mM Cmpds 1 3 4 18 19 [IC50],µM 2.5 2.3 3.2 40.5 1 Measuring Activity of GAP Proteins Using the Transcreener® GDP Assay GAP Transcreener Assays for ARFGAP Modulation 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. Time course: ARFGAP1 J Biomol Screen. 2011 Aug;16(7):717-23. Epub 2011 May 18 Screen of LOPAC Library Pilot screen of the LOPAC1280 library. (A) Scatter plot of fluorescence polarization changes after incubation of ARF1 and ARFGAP1 with individual compounds of the LOPAC1280 collection. (B) The correlation coefficient for two parallel screens of the LO PAC 1280 library was 0.98. J Biomol Screen. 2011 Aug;16(7):717-23. Epub 2011 May 18 Hypothesis: Mutation(s) can alter the rate-limiting step of Gα but still allow for RGS protein GAP activity The Problem A Possible Solution * * * * Goal: Increase koff (GDP) /kcat (GTPase) from 0.03 to ≥ 5 GAP Activity of RGS4 A 175 Gai2(R179M,A327S) + RGS4 150 125 A. Dashed line is in the absence and solid 100 line is in the presence of 2.0 ng/μL mP RGS4.
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