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BioAssay: AID 1772

Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases

Assay Implementation: Zurab Surviladze, Lin Hong, Danuta Wlodek, Terry Foutz, Mark Carter, Anna Waller ..more
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 Tested Compounds
 Tested Compounds
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Probe(5)
 
 
Active(5)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Probe(5)
 
 
Active(5)
 
 
AID: 1772
Data Source: NMMLSC (UNM_GTPase_Multiplex_Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-05-19
Modify Date: 2012-10-10

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 5    Active: 5
Related Experiments
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AIDNameTypeComment
757HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeScreeningdepositor-specified cross reference: Primary HTS for RacWT
758HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeScreeningdepositor-specified cross reference: Primary HTS for Rab7WT
759HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeScreeningdepositor-specified cross reference: Primary HTS for RasWT
760HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeScreeningdepositor-specified cross reference: Primary HTS for Rab2WT
761HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeScreeningdepositor-specified cross reference: Primary HTS for Cdc42WT
764HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantScreeningdepositor-specified cross reference: Primary HTS for RacACT
1333Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatorydepositor-specified cross reference: Dose response for Cdc42ACT
1334Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatorydepositor-specified cross reference: Dose response for Cdc42W
1335Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatorydepositor-specified cross reference: Dose response for RasWT
1336Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatorydepositor-specified cross reference: Dose response for Rab7WT
1337Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatorydepositor-specified cross reference: Dose response for Rab2WT
1339Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantConfirmatorydepositor-specified cross reference: Dose response for RacAC
1340Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeConfirmatorydepositor-specified cross reference: Dose response for RacWT
1341Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatorydepositor-specified cross reference: Dose response for RasACT
1758Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_wtScreeningdepositor-specified cross reference: Activator binding kinetics for Cdc42_wt
1759Activator binding kinetics on Ras and Ras-related GTPases, specifically Ras_wtScreeningdepositor-specified cross reference: Activator binding kinetics for Ras_wt
1760Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab7_wtScreeningdepositor-specified cross reference: Activator binding kinetics for Rab7_wt
1761Activator binding kinetics on Ras and Ras-related GTPases, specifically Ras_act (activated mutant)Screeningdepositor-specified cross reference: Activator binding kinetics for Ras_act
1762Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_act (activated mutant)Screeningdepositor-specified cross reference: Activator binding kinetics for Cdc42_act
1763Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab2_wtScreeningdepositor-specified cross reference: Activator binding kinetics for Rab2_wt
1769Profiling Assay to determine GST-GSH interactions in multiplex bead-based assaysConfirmatorydepositor-specified cross reference: Profiling Assay to determine GST-GSH interactions in multiplex bead-based assays, counter screen for
2009Oxadiazole SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested by Multiplex dose response to identify specific small molecule inhib
2019Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype proteinConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2020Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2021Additional SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatorydepositor-specified cross reference: Additional SAR compounds tested by Multiplex dose response to identify specific small molecule inhib
2022Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatorydepositor-specified cross reference: Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2027Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2031Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2033Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2036Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatorydepositor-specified cross reference
2037Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2038Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatorydepositor-specified cross reference: Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2039Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatorydepositor-specified cross reference: Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2040Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatorydepositor-specified cross reference: Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2041Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2042Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2043Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatorydepositor-specified cross reference: Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2045Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2046Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatorydepositor-specified cross reference: Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2047Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2048Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2050Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2051Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2053Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatorydepositor-specified cross reference: Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2055Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatorydepositor-specified cross reference: Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhi
2372Compound effect on equilibrium binding with Cdc42 under varying GTP conditions: nucleotide depletion with Mg bufferOtherdepositor-specified cross reference: Compound effect on equilibrium binding with Cdc42 under varying GTP conditions: nucleotide depletion
2373Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with Mg bufferOtherdepositor-specified cross reference: Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with Mg buffer other
2374PAK-Effector Pull-down Assay for Quantification of Rac/Cdc42 Activation Using 3T3 CellsOtherdepositor-specified cross reference: PAK-Effector Pull-down Assay for Quantification of Rac/Cdc42 Activation Using 3T3 Cells other
2375Panel results of Cell based ELISA Assessing Activation of Cdc42 and Rac1Otherdepositor-specified cross reference: Panel results of Cell based ELISA Assessing Activation of Cdc42 and Rac1 other
2376Dose Response of compounds with constant GTP under the condition of nascent nucleotide depletion and Mg bufferConfirmatorydepositor-specified cross reference: Dose Response of compounds with constant GTP under the condition of nascent nucleotide depletion and
2378Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with EDTA bufferOtherdepositor-specified cross reference: Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with EDTA buffer othe
2393Dose Response of compounds for six proteins with constant GTP under the condition of Mg bufferOtherdepositor-specified cross reference: Dose Response of compounds for six proteins with constant GTP under the condition of Mg buffer other
2418Assessment of Cdc42 inhibitors on Bradykinin activation of 3T3 cellsOtherdepositor-specified cross reference: Assessment of Cdc42 inhibitors on Bradykinin activation of 3T3 cells other
588369Cellular toxicity assessed by Trypan Blue for compounds active in GTPase screenOtherdepositor-specified cross reference
588373SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutant, round 1Confirmatorydepositor-specified cross reference
588377SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 1Confirmatorydepositor-specified cross reference
588381SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras activated mutant, round 1Confirmatorydepositor-specified cross reference
588383SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtype, round 1Confirmatorydepositor-specified cross reference
588384SAR compounds for Rab7 project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutant, round 1Confirmatorydepositor-specified cross reference
588385SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 1Confirmatorydepositor-specified cross reference
588387SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras activated mutant, round 1Confirmatorydepositor-specified cross reference
588388SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras wildtype, round 1Confirmatorydepositor-specified cross reference
588393SAR compounds for Cdc42 probe extension project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 1Confirmatorydepositor-specified cross reference
588394SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 1Confirmatorydepositor-specified cross reference
588410Cellular toxicity assessed by Trypan Blue for compounds in active GTPase screen, set2Otherdepositor-specified cross reference
588427Cellular toxicity assessed by Trypan Blue for compounds in active GTPase screen, Set1Otherdepositor-specified cross reference
588477Dose Response of round 1 SAR compounds for Cdc42 probe extension project tested by multiplex of eight GTPase proteins under the condition of Mg Buffer, non-chelator bufferOtherdepositor-specified cross reference
588479Dose Response of round 1 SAR compounds for GTPase inhibitor project tested by multiplex of eight GTPase proteins under the condition of Mg Buffer, non-chelator bufferOtherdepositor-specified cross reference
588622Dose Response of round 2 SAR compounds for GTPase inhibitor project tested by multiplex of eight GTPase proteins under the condition of Mg Buffer, non-chelator bufferOtherdepositor-specified cross reference
588624SAR compounds for Rab7 project tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutant, round 2Confirmatorydepositor-specified cross reference
588626SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 2Confirmatorydepositor-specified cross reference
588628SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras activated mutant, round 2Confirmatorydepositor-specified cross reference
588630SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras wildtype, round 2Confirmatorydepositor-specified cross reference
588631SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 2Confirmatorydepositor-specified cross reference
602137Counter screen to determine effect of SAR compounds for Rab7 project on GST-GSH binding, round 1Otherdepositor-specified cross reference
602145Counter screen to determine effect of SAR compounds for Rab7 project on GST-GSH binding, round 2Otherdepositor-specified cross reference
602146EGFR degradation assay in SCC-12F cellsOtherdepositor-specified cross reference
602148Active GTPase Screen Compounds affects on binding of VLA-4 specific ligandOtherdepositor-specified cross reference
651732Cellular toxicity assessed by Trypan Blue for compounds in active Cdc42 Probe Extension ProjectOtherdepositor-specified cross reference
652107Actin Polymerization inhibition by compounds from Cdc42 Probe Extension ProjectOtherdepositor-specified cross reference
720688On Hold
720689On Hold
720699On Hold
720712On Hold
743330On Hold
Description:
University of New Mexico Assay Overview:
Assay Support: NIH I RO3 MH081231-01
HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Development: Zurab Surviladze, Ph.D.
Assay Implementation: Zurab Surviladze, Lin Hong, Danuta Wlodek, Terry Foutz, Mark Carter, Anna Waller


Assay Background and Significance:

This report summarizes the series of assays used to identify small molecule regulators of Ras and Ras-related GTPases. Ras and related small molecular weight GTPases function in the regulation of signaling and cell growth, and collectively serve to control cell proliferation, differentiation and apoptosis [Tekai et al. 2001; Wennerberg et al. 2005]. The Ras-related GTPases are divided into four subfamilies with the Rab proteins regulating membrane transport, Rho proteins (including Rac and Cdc 42) regulating cytoskeletal rearrangements and responses to signaling, Arf/Sar proteins regulating membrane and microtubule dynamics as well as protein transport, and Ran proteins controlling nucleocytoplasmic transport. This project focuses on representative Ras, Rho, and Rab family members to validate the approach for the identification of new chemical compounds with novel therapeutic potential in cell signaling and growth control.

The primary HTS assay was a no-wash fluorescent GTP-binding assay adapted to multiplexed, high-throughput measurements whereby multiple GTPases were simultaneously screened against the MLSCN library. The specificity is based on the observation that individual GTPases including wt and activated forms exhibit measurably distinct affinities for Bodipy-FI-GTP vs GTP. The assay involves the binding of fluorescent GTP to G protein-GST fusion proteins on GSH beads. A set of six G proteins (Rac 1 wt, Rab7 wt, Rac 1 activated, Ras wt, Rab 2 wt., CDC wt) are arrayed under conditions of divalent molecule depletion.

Result of this screening campaign has yielded five compounds as probe compounds:
(SID/CID/Probe#)
57578335 / 888706 / ML099
57578337 / 7345532 / ML098
57578338 / 2160985 / ML097
57578341 / 2950007 / ML141
85747738 / 1067700 / ML282

ML097, ML098, and ML099 are pan-activators for Ras-related GTPases.
ML141 is a potent and selective inhibitor of Cdc42 GTPase.
ML282 is a pan-inhibitor with high efficacy toward Rab7 GTPase.
Protocol
Bead sets are coated with individual GST-small G proteins, blocked with Buffer (0.01% NP-40; 30mM HEPES pH 7.5; 100mM KCl; 20mM NaCl; 1mM EDTA; 0.1% BSA and 1mM DTT), incubated overnight at 4 degrees C, and finally washed in buffer. The different bead sets, acquired from Duke Scientific, have similar size (~ 4 microm diameter) however they are distinguished by varied magnitude of emission at 665 +/-10 nm with excitation at 635 nm.

The assay is conducted in 384-well microplates in a total well volume of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 200nM Bodipy-FL-GTP in buffer containing BSA and DTT for a final concentration of GTP of 100nM). Positive Controls, which contain bead mixture and fluorescent GTP but no test compound, are located in columns 1 and 2 on plate. Negative Controls containing bead mixture with fluorescent GTP and 0.5 mM unlabeled GTP, are collected from a separate test tube. Plates are placed on rotators and incubated for 40-45 minutes at 4 degrees C.

For dose response confirmatory assays, test compounds were cherry-picked from compound storage plates at 10 milliM in DMSO, then serially diluted 1:3 eight times for a total of nine different test compound concentrations in DMSO. Final compound dilutions in DMSO ranged from 1 microM to 10mM. These dilutions were then diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM.

Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter volume sampling from 384-well microtiter plates [Kuckuck, et al., 2001; Ramirez, et al., 2003]. The stream of particles is excited at 488 nm and 635 nm, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis of the time-resolved acquisition data file uses IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to segregate data for each target and the fluorescence scavenger in the multiplex. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the green median fluorescence intensity (MFI) per bead population (well) is calculated.

Summary and Sequence of Assays Performed

HTS_Rac_wt AID # 757
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >20% change in activity from baseline
# compounds evaluated: 194,635
# active compounds: 521

HTS_Rab_7_wt AID # 758
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >20% change in activity from baseline
# compounds evaluated: 194,635
# active compounds: 107

HTS_Ras_wt AID # 759
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >20% change in activity from baseline
# compounds evaluated: 194,635
# active compounds: 267

HTS_Rab_2_wt AID # 760
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >20% change in activity from baseline
# compounds evaluated: 194,635
# active compounds: 365

HTS_Cdc42_wt AID # 761
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >20% change in activity from baseline
# compounds evaluated: 194,635
# active compounds: 398

HTS_Rac_act_mutant AID # 764
Method: multiplex flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >20% change in activity from baseline
# compounds evaluated: 194,635
# active compounds: 219

Dose_Response_Cdc42_act_mutant AID # 1333
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 37

Dose_Response_Cdc42_wt AID # 1334
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 21

Dose_Response_Ras_wt AID # 1335
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 20

Dose_Response_Rab7_wt AID # 1336
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 0

Dose_Response_Rab2_wt AID # 1337
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 65

Dose_Response_Rac_act_mutant AID # 1339
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 55

Dose_Response_Rac_wt AID # 1340
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 112

Dose_Response_Ras_act_mutant AID # 1341
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal, and interference of test compound with GST/GST interaction; see AID for details
# compounds evaluated: 634
# active compounds: 38

Activator_binding_kinetics_Cdc42_wt AID # 1758
Method: multiplex fluorescent-GTP equilibrium binding curve in the presence and absense of activating compound
Test concentration: 10 microM
Activity criteria: Activity score=100 based on comparison of EC50 calculated from dose response in the presence versus in absense of activating compound; see AID for details
# compounds evaluated: 1
# active compounds: 1

Activator_binding_kinetics_Ras_wt AID # 1759
Method: multiplex fluorescent-GTP equilibrium binding curve in the presence and absense of activating compound
Test concentration: 10 microM
Activity criteria: Activity score=100 based on comparison of EC50 calculated from dose response in the presence versus in absense of activating compound; see AID for details
# compounds evaluated: 1
# active compounds: 1

Activator_binding_kinetics_Rab7_wt AID # 1760
Method: multiplex fluorescent-GTP equilibrium binding curve in the presence and absense of activating compound
Test concentration: 10 microM
Activity criteria: Activity score=100 based on comparison of EC50 calculated from dose response in the presence versus in absense of activating compound; see AID for details
# compounds evaluated: 1
# active compounds: 1

Activator_binding_kinetics_Ras_act_mutant AID # 1761
Method: multiplex fluorescent-GTP equilibrium binding curve in the presence and absense of activating compound
Test concentration: 10 microM
Activity criteria: Activity score=100 based on comparison of EC50 calculated from dose response in the presence versus in absense of activating compound; see AID for details
# compounds evaluated: 1
# active compounds: 1

Activator_binding_kinetics_Cdc42_act_mutant AID # 1762
Method: multiplex fluorescent-GTP equilibrium binding curve in the presence and absense of activating compound
Test concentration: 10 microM
Activity criteria: Activity score=100 based on comparison of EC50 calculated from dose response in the presence versus in absense of activating compound; see AID for details
# compounds evaluated: 1
# active compounds: 1

Activator_binding_kinetics_Rab2_wt AID # 1763
Method: multiplex fluorescent-GTP equilibrium binding curve in the presence and absense of activating compound
Test concentration: 10 microM
Activity criteria: Activity score=100 based on comparison of EC50 calculated from dose response in the presence versus in absense of activating compound; see AID for details
# compounds evaluated: 1
# active compounds: 1

Group1 of compounds in SAR series for all 8 protein targets
AIDs OnHold: 2021, 2022, 2038, 2039, 2040, 2043, 2046
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal

Group2 of compounds in SAR series for all 8 protein targets
AIDs OnHold: 2009, 2027, 2031, 2033, 2037, 2042, 2051, 2053
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal

Group3 of compounds in SAR series for all 8 targets
AIDs OnHold: 2019, 2020, 2041, 2045, 2047, 2048, 2050, 2055
Method: multiplex dose response flow cytometry bead-based fluorescent ligand binding competition assay in
Test concentration: Nine point concentration curve from 10 nanoM to 100 microM
Activity criteria: Activity score > 0 based on evaluation of EC50, absolute value of Hill slope, amplitude of biological signal

AID greater than 2009 are associated with various SAR compound series and their effects on different GTPase.
Comment
Abbreviations: mM for millimolar, nm for nanometer
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 5
MLP Probe ML# for probe 1: ML099
PubChem Substance ID (SID) for probe 1: 57578335
PubChem Compound ID (CID) for probe 1: 888706
Probe type for probe 1: Activator
IC50/EC50 (nM) for probe 1: 59
Target for probe 1: cdc42 activated mutant (gi: 190938)
Anti-target for probe 1: glutathione S-transferase
Fold selectivity for probe 1: >1000
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK47359/ (ID: 2360326)
Grant number for probe 1: MH081231-01
MLP Probe ML# for probe 2: ML141
PubChem Substance ID (SID) for probe 2: 57578341
PubChem Compound ID (CID) for probe 2: 2950007
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 2600
Target for probe 2: Cdc42 wt (gi: 190938)
Disease relevance for probe 2: Research Tool
Anti-target for probe 2: glutathione S-transferase
Fold selectivity for probe 2: >50
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK51965/ (ID: 2412042)
Grant number for probe 2: MH081231-01
MLP Probe ML# for probe 3: ML282
PubChem Substance ID (SID) for probe 3: 85747738
PubChem Compound ID (CID) for probe 3: 1067700
Probe type for probe 3: Inhibitor
Disease relevance for probe 3: cancer, immunodeficiencies, and neurological disorders
NCBI Book chapter link for probe 3: http://www.ncbi.nlm.nih.gov/books/NBK143553/ (ID: 3036330)
Grant number for probe 3: MH081231-01
MLP Probe ML# for probe 4: ML097
PubChem Substance ID (SID) for probe 4: 57578338
PubChem Compound ID (CID) for probe 4: 2160985
Probe type for probe 4: Activator
IC50/EC50 (nM) for probe 4: 50
Target for probe 4: cdc42 activated mutant (gi: 190938)
Anti-target for probe 4: glutathione S-transferase
Fold selectivity for probe 4: >1000
MLP Probe ML# for probe 5: ML098
PubChem Substance ID (SID) for probe 5: 57578337
PubChem Compound ID (CID) for probe 5: 7345532
Probe type for probe 5: Activator
IC50/EC50 (nM) for probe 5: 355
Target for probe 5: cdc42 activated mutant (gi: 190938)
Anti-target for probe 5: glutathione S-transferase
Fold selectivity for probe 5: >200
PubMed Publication ID (PMID) for probe 1: 20008126
PubMed Publication ID (PMID) for probe 3: 22486388
PubMed Publication ID (PMID) for probe 4: 20008126
PubMed Publication ID (PMID) for probe 5: 20008126
NCBI Book chapter title for probe 1: Three small molecule pan activator families of Ras-related GTPases
NCBI Book chapter title for probe 2: A Potent and Selective Inhibitor of Cdc42 GTPase
NCBI Book chapter title for probe 3: A small molecule pan-inhibitor of Ras-superfamily GTPases with high efficacy towards Rab7
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML#ML probe numberString
2EC50EC50 reported in probe report for specific protein target for the probeFloatμM
Additional Information
Grant Number: NIH I RO3 MH081231-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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