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BioAssay: AID 1762

Activator binding kinetics on Ras and Ras-related GTPases, specifically Cdc42_act (activated mutant)

The objective of the HTS associated with this secondary assay was to identify small molecule regulators of Ras and Ras-related GTPases (see Summary Report and PubChem AIDs 757, 758, 759, 760, 761, 764). 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 more ..
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AID: 1762
Data Source: NMMLSC (UNM_GTPase_activator_kinetics_cdc42_act)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-05-14

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: cell division cycle 42 (GTP binding protein, 25kDa) [Homo sapiens]
Description ..   

     More BioActivity Data..
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
757HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac wildtypeScreening depositor-specified cross reference: HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac_w
758HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeScreening depositor-specified cross reference: HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7_
759HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtypeScreening depositor-specified cross reference: HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras_w
760HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2 wildtypeScreening depositor-specified cross reference: HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rab2_
761HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeScreening depositor-specified cross reference: HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42
764HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac activated mutantScreening depositor-specified cross reference: HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Rac_a
1333Multiplex dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 activated mutantConfirmatory depositor-specified cross reference: Dose response for Cdc42_ACT
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasesSummary5 depositor-specified cross reference
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
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
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
720688Assessing protein source dependence on dose response curves of small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeOther same project related to Summary assay
720689Assessing protein source dependence on dose response curves of small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeOther same project related to Summary assay
720699Comparison of test compound incubation dependence on dose response curves of small molecule inhibitors of Ras and Ras-related GTPases specifically Rab7 wildtypeOther same project related to Summary assay
720712Comparison of test compound incubation dependence on dose response curves of small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypeOther 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, Danuta Wlodek, Terry Foutz, Mark Carter, Anna Waller
Target Team Leader for the Center: Larry Sklar (lsklar@salud.unm.edu)

Assay Background and Significance:
The objective of the HTS associated with this secondary assay was to identify small molecule regulators of Ras and Ras-related GTPases (see Summary Report and PubChem AIDs 757, 758, 759, 760, 761, 764). 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 wildtype and activated mutants exhibit measurably distinct affinities for Bodipy-FI-GTP. The assay involves the binding of fluorescent GTP to G protein-GST fusion proteins on GSH beads. A set of six G proteins (Rac1 wt, Rab7 wt, Rac1 activated, Ras wt, Rab2 wt, CDC wt) are arrayed under conditions of divalent molecule depletion.

In the assay described here, real-time binding kinetics between GTP and each of the protein targets were characterized in a multiplex assay (Schwartz, et al 2008). The resulting binding of fluorescent GTP after 3 minutes was utilized in determining Bmax and Kd for each target in the presence of 10 microM small molecule activator compound MLS000088004 versus a DMSO control.
Protocol
Each protein target (4 microM) was bound to glutathione beads overnight at 4 degrees C. Protein on GSH-beads was depleted of nucleotide by incubating with 10 milliM EDTA containing buffer for 20 min at 30 degrees C, washing twice with 0.01% NP-40 containing HPS buffer, then resuspending in the same buffer containing 1 milliM EDTA, 1 milliM DTT and 0.1% BSA. Kinetic assays were performed by incubating 50 microliter of GST-target protein-GSH-bead suspension for 2 min with either DMSO, or 10 microM MLS000088004 and subsequently adding 50 microL of various concentration ice cold BODIPY-GTP. Association of the fluorescent nucleotide was measured using a FacSCAN flow cytometer in the kinetic mode. Data were converted to ASCII format using IDLQuery.
Measured values of bead-bound BODIPY-GTP after 3 minutes of binding are converted to molecular equivalent soluble fluoresceine (MESF) with the aid of calibration beads (Bangs Lab) by the following equation;
kMESF = Slope * MCF + Intercept
where MCF is the Median Channel Fluorescence measurement of bead-bound BODIPY-GTP, kMESF are kiloMESF (1000 * MESF), and Slope and Intercept are the from the linear regression fit of the 5 different levels of calibration beads.
The resulting values of kMESF are graphed versus the different concentrations of BODIPY-GTP in GraphPad Prism and fitted by non-linear regression to one site binding pre the following equation;
kMESF = Bmax * ConcBODIPY-GTP / (Kd + ConcBODIPY-GTP)
where ConcBODIPY-GTP is the concentration of BODIPY-GTP, Bmax is the maximum binding of BODIPY-GTP per bead and Kd is the equilibrium binding constant of BODIPY-GTP to protein on bead.
Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, GTPase, multiplex bead-based screening
Comment
Only one compound was tested in this assay. Potentially additional compounds will be evaluated.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1BMAX_kMESF_DMSOTotal number of bead-bound BODIPY-GTP on bead with Cdc42ACT in presence of 1% DMSOFloat
2KD_microM_DMSOEquilibrium binding constant for BODIPY-GTP to Cdc42ACT in presence of 1% DMSOFloatμM
3BMAX_kMESF_COMPOUNDTotal number of bead-bound BODIPY-GTP on bead with Cdc42ACT in presence of 10 microM testing compoundFloat
4KD_microM_COMPOUND (10μM**)Equilibrium binding constant for BODIPY-GTP to Cdc42ACT in presence of 10 microM testing compoundFloatμM

** Test Concentration.
Additional Information
Grant Number: NIH I RO3 MH081231-01

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