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BioAssay: AID 2378

Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with EDTA buffer

Assessement of equilibrium binding of guanine nucleotide to the guanine nucleotide binding protein can be utilized to evaluate if a inhibitory compound is acting at the binding site of guanine nucleotide or at an alternative, allosteric site. A direct competitive inhibitor does not hinder the binding of the guanine nucleotide from reaching the maximum level of binding at high concentrations of more ..
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
 Tested Substances
 Tested Substances
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Active(1)
 
 
AID: 2378
Data Source: NMMLSC (UNM_EDTA_GTPEqBinding_Cdc42)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-02-18
Hold-until Date: 2010-08-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
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 summary assay
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
2375Panel results of Cell based ELISA Assessing Activation of Cdc42 and Rac1Other 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
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: Zurab Surviladze, Ph.D., Anna Waller, Ph.D.

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:

Assessement of equilibrium binding of guanine nucleotide to the guanine nucleotide binding protein can be utilized to evaluate if a inhibitory compound is acting at the binding site of guanine nucleotide or at an alternative, allosteric site. A direct competitive inhibitor does not hinder the binding of the guanine nucleotide from reaching the maximum level of binding at high concentrations of guanine nucleotide. However, the binding of guanine nucleotide in the presense of a non-competitive inhibitor could potentially not reach the same maximum as without the inhibitory compound. In other words, the outcome of non-competitive inhibitors would be that the maximum binding sites available have been lowered. This assay were carried out to assess the mechanism of action for this compound in the condition of EDTA buffer.
Protocol
The protein target (4 microM) is bound to glutathione beads overnight at 4 degrees C. Binding assays are performed by incubating 50 microL of GST-CDC42-GSH-bead suspension for 2 min with 1 milliM EDTA and either DMSO or 10 microM compound and subsequently adding 50 microL of varying concentration (range 0-125 nanoM) ice cold BODIPY-GTP. Association of the fluorescent nucleotide is measured using a FacSCAN flow cytometer. The flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nanometer (FL1)are analyzed by IDLQuery software to determine the median fluorescence per bead population. Non-specific binding of the BODIPY-GTP were assessed in the presence of excess non-fluorescent GTP (10 microM).

Calculations:
The specific binding of fluorescent GTP (SpecMCF) were calculated from the median values measured at different fluorescent GTP concentrations in the presense of blocking non-fluorescent GTP (RawMCFwNFGTP) and DMSO (RawMCFwDMSO):
SpecMCF = RawMCFwDMSO - RawMCFwNFGTP

These specific binding values were then fit to an One site binding (hyperbola) equation:
BoundGTP = Bmax * [GTP]/(Kd + [GTP])
where BoundGTP is the bound fluorescent GTP, [GTP] is the concentration of fluorescent GTP in nanoM, Bmax is the calculated value of maximum binding sites for GTP, and Kd is the observed affinity of GTP to the guanine nucleotide binding protein under those conditions.

PUBCHEM_SCORE is based on the comparison of the different extimates of Bmax in the presense of DMSO or 10 microM inhibitory compound. Thus PUBCHEM_SCORE = 100 *(BmaxDMSO-BmaxCmpd)/BmaxDMSO where BmaxDMSO is the Bmax calculated in the presense of DMSO and BmaxCmpd is the Bmax calculated in presense of 10 microM inhibitory compound. Active compounds have PUBCHEM_SCORE greater than 50.
Comment
Abbreviations: microM for micromolar, milliM for millimolar, nanoM for nanomolar, milliL for milliliter
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1BoundGTP_Compound_3nanoMAmount of 3 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compoundFloat
2BoundGTP_Compound_6nanoMAmount of 6 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compoundFloat
3BoundGTP_Compound_12.5nanoMAmount of 12.5 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compoundFloat
4BoundGTP_Compound_25nanoMAmount of 25 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compoundFloat
5BoundGTP_Compound_50nanoMAmount of 50 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compoundFloat
6BoundGTP_Compound_100nanoMAmount of 100 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compoundFloat
7FitBmax_CompoundCalculated estimate of maximum GTP binding sites on Cdc42 covered beads in presense of 10 microM test compoundFloat
8FitKd_Compound_nanoMCalculated affinity of GTP to Cdc42 in the presense of 10 microM test compoundFloat
9BoundGTP_DMSO_3nanoMAmount of 3 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSOFloat
10BoundGTP_DMSO_6nanoMAmount of 6 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSOFloat
11BoundGTP_DMSO_12.5nanoMAmount of 12.5 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSOFloat
12BoundGTP_DMSO_25nanoMAmount of 25 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSOFloat
13BoundGTP_DMSO_50nanoMAmount of 50 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSOFloat
14BoundGTP_DMSO_100nanoMAmount of 100 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSOFloat
15FitBmax_DMSOCalculated estimate of maximum GTP binding sites on Cdc42 covered beads in presense of DMSOFloat
16FitKd_DMSO_nanoMCalculated affinity of GTP to Cdc42 in the presense of DMSOFloat
Additional Information
Grant Number: NIH I RO3 MH081231-01

Data Table (Concise)
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