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BioAssay: AID 2375

Panel results of Cell based ELISA Assessing Activation of Cdc42 and Rac1

Active GTP-bound Rac1 and Cdc42 bind the downstream effector p21 activated kinase (PAK). The extent of PAK binding is thus a routine measure for quantifying activated Rac and Cdc42. Dr. Surviladze with the UNM Center for Molecular Discovery has demonstrated that the assay can be used to score for small molecules that activate or inhibit Rac nucleotide binding in vitro and in vivo. For further more ..
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 Tested Compounds
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 Related BioAssays
 Related BioAssays
AID: 2375
Data Source: NMMLSC (UNM_ELISA_Cdc42_Rac1)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-02-18
Hold-until Date: 2010-08-15

Data Table ( Complete ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasesSummary5 depositor-specified cross reference: GTPase Project Summary report
757HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeScreening same project related to Summary assay
758HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeScreening same project related to Summary assay
759HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeScreening same project related to Summary assay
760HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeScreening same project related to Summary assay
761HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeScreening same project related to Summary assay
764HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantScreening same project related to Summary assay
1333Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
1334Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatory same project related to Summary assay
1335Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
1336Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
1337Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
1339Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantConfirmatory same project related to Summary assay
1340Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeConfirmatory same project related to Summary assay
1341Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
1758Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_wtScreening same project related to Summary assay
1759Activator binding kinetics on Ras and Ras-related GTPases, specifically Ras_wtScreening same project related to Summary assay
1760Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab7_wtScreening same project related to Summary assay
1761Activator binding kinetics on Ras and Ras-related GTPases, specifically Ras_act (activated mutant)Screening same project related to Summary assay
1762Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_act (activated mutant)Screening same project related to Summary assay
1763Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab2_wtScreening same project related to Summary assay
1769Profiling Assay to determine GST-GSH interactions in multiplex bead-based assaysConfirmatory same project related to Summary assay
2009Oxadiazole SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
2019Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype proteinConfirmatory same project related to Summary assay
2020Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
2021Additional SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory same project related to Summary assay
2022Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatory same project related to Summary assay
2027Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatory same project related to Summary assay
2031Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
2033Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
2036Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
2037Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeConfirmatory same project related to Summary assay
2038Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
2039Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatory same project related to Summary assay
2040Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatory same project related to Summary assay
2041Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeConfirmatory same project related to Summary assay
2042Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
2043Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
2045Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
2046Additional SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeConfirmatory same project related to Summary assay
2047Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
2048Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatory same project related to Summary assay
2050Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutantConfirmatory same project related to Summary assay
2051Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 activated mutantConfirmatory same project related to Summary assay
2053Oxadiazole SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeConfirmatory same project related to Summary assay
2055Pyrazoline SAR compounds tested via Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac1 wildtypeConfirmatory same project related to Summary assay
2372Compound effect on equilibrium binding with Cdc42 under varying GTP conditions: nucleotide depletion with Mg bufferOther same project related to Summary assay
2373Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with Mg bufferOther same project related to Summary assay
2374PAK-Effector Pull-down Assay for Quantification of Rac/Cdc42 Activation Using 3T3 CellsOther same project related to Summary assay
2376Dose Response of compounds with constant GTP under the condition of nascent nucleotide depletion and Mg bufferConfirmatory same project related to Summary assay
2378Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with EDTA bufferOther same project related to Summary assay
2393Dose Response of compounds for six proteins with constant GTP under the condition of Mg bufferOther same project related to Summary assay
2418Assessment of Cdc42 inhibitors on Bradykinin activation of 3T3 cellsOther same project related to Summary assay
588369Cellular toxicity assessed by Trypan Blue for compounds active in GTPase screenOther same project related to Summary assay
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 1Confirmatory same project related to Summary assay
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 1Confirmatory same project related to Summary assay
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 1Confirmatory same project related to Summary assay
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 1Confirmatory same project related to Summary assay
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 1Confirmatory same project related to Summary assay
588385SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 1Confirmatory same project related to Summary assay
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 1Confirmatory same project related to Summary assay
588388SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras wildtype, round 1Confirmatory same project related to Summary assay
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 1Confirmatory same project related to Summary assay
588394SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 1Confirmatory same project related to Summary assay
588410Cellular toxicity assessed by Trypan Blue for compounds in active GTPase screen, set2Other same project related to Summary assay
588427Cellular toxicity assessed by Trypan Blue for compounds in active GTPase screen, Set1Other same project related to Summary assay
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 bufferOther same project related to Summary assay
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 bufferOther same project related to Summary assay
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 bufferOther same project related to Summary assay
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 2Confirmatory same project related to Summary assay
588626SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype, round 2Confirmatory same project related to Summary assay
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 2Confirmatory same project related to Summary assay
588630SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically H-Ras wildtype, round 2Confirmatory same project related to Summary assay
588631SAR compounds tested by Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtype, round 2Confirmatory same project related to Summary assay
602137Counter screen to determine effect of SAR compounds for Rab7 project on GST-GSH binding, round 1Other same project related to Summary assay
602145Counter screen to determine effect of SAR compounds for Rab7 project on GST-GSH binding, round 2Other same project related to Summary assay
602146EGFR degradation assay in SCC-12F cellsOther same project related to Summary assay
602148Active GTPase Screen Compounds affects on binding of VLA-4 specific ligandOther same project related to Summary assay
651732Cellular toxicity assessed by Trypan Blue for compounds in active Cdc42 Probe Extension ProjectOther same project related to Summary assay
652107Actin Polymerization inhibition by compounds from Cdc42 Probe Extension ProjectOther same project related to Summary assay
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: Elsa Romero, Anna Waller

Chemistry: University of Kansas Specialized Chemistry Center
KU Specialized Chemistry Center PI: Jeff Aube, Ph.D.
KU SCC Project Manager: Jennifer E. Golden. Ph.D.
KU SCC Chemists on this project: Chad Schroeder, M.S., Denise Simpson, Ph.D., Julica Noeth, B.S.

Assay Background and Significance:

Active GTP-bound Rac1 and Cdc42 bind the downstream effector p21 activated kinase (PAK). The extent of PAK binding is thus a routine measure for quantifying activated Rac and Cdc42. Dr. Surviladze with the UNM Center for Molecular Discovery has demonstrated that the assay can be used to score for small molecules that activate or inhibit Rac nucleotide binding in vitro and in vivo. For further evaluation and dose dependence assays of compounds found to be active in cell based morphological assays we will use an ELISA based version of the assay developed by Cytoskeleton, Inc. (GLISA, Denver, CO).
Panel Information
ELISA for activated Cdc42 and Rac1 - Cell based ELISA assessing for active Cdc42 and Rac1
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1Cdc4234Cell Division Cycle 42 GTP binding protein
2Rac132GTPase-activating protein 1

§ Panel component ID.
Protocol
In the ELISA format PAK coated on microtiter wells was reconstituted in cold water immediately prior to the assay. 3T3 cells cultured in serum free medium and treated +/- epidermal growth factor (10 ng/ml) and sample molecules. No growth factor lysate served as baseline negative control, + growth factor lysate served as measure of maximal response. Assays conducted on different days were normalized based on maximal response. Cell lysates were prepared, snap frozen and equal amounts of cellular protein tested for Cdc42 or Rac1 activity. Dose range was measured in duplicate in multiple trials with sample molecule concentration of 1 microM and 10 microM. Readout was measured by colorimetric assay. Assay was run with a negative control (no cell lyaste, buffer with all other assay components) and a positive control (1 ng GTP-bound GTPase, with all other assay components) to allow calculation of ng active GTPase in each lysate sample.

Calculations:
The measured raw absorbance data were normalized between the different experiments as follows:
a) Subtract GLISA buffer blank from all Abs. values
b) Calculate nanograms active GTPase based on standard run in each experiment that corresponds to 1 nanogram GTP-Rac or GTP-Cdc42
c) Calculate average value for plus growth factor samples for each experiment and normalize all nanogram GTPase values to this value.
PUBCHEM_SCORE is based on the magnitude the response signal was depressed due to the presense of 10 microM sample compound. Thus PUBCHEM_SCORE = 100 * (1 - normalized nanogram GTPase). Active compounds have PUBCHEM_SCORE greater than 15.
Comment
Abbreviations: microM for micromolar, ng for nanogram, ml for milliliter
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: 3T3
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Cdc42_OutcomeOutcome for Cdc421Outcome
2Cdc42_ScorePubchem Score for Cdc42 based on measured value at 10 microM test compound1Integer
3AveCdc42_1microM (1μM**)Average normalized measurement of active Cdc42 in the presence of 1 microM test compound1Float
4StdCdc42_1microM (1μM**)Standard deviation of normalized measurement of active Cdc42 in the presence of 1 microM test compound1Float
5AveCdc42_10microM (10μM**)Average normalized measurement of active Cdc42 in the presence of 10 microM test compound1Float
6StdCdc42_10microM (10μM**)Standard deviation of normalized measurement of active Cdc42 in the presence of 10 microM test compound1Float
7Rac1_OutcomeOutcome for Rac12Outcome
8Rac1_ScorePubchem Score for Rac1 based on measured value at 10 microM test compound2Integer
9AveRac1_1microM (1μM**)Average normalized measurement of active Rac1 in the presence of 1 microM test compound2Float
10StdRac1_1microM (1μM**)Standard deviation of normalized measurement of active Rac1 in the presence of 1 microM test compound2Float
11AveRac1_10microM (10μM**)Average normalized measurement of active Rac1 in the presence of 10 microM test compound2Float
12StdRac1_10microM (10μM**)Standard deviation of normalized measurement of active Rac1 in the presence of 10 microM test compound2Float

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: NIH I RO3 MH081231-01

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