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BioAssay: AID 2393

Dose Response of compounds for six proteins with constant GTP under the condition of Mg buffer

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 more ..
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 Tested Compounds
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Inconclusive(32)
 
 
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AID: 2393
Data Source: NMMLSC (UNM_Mg_DRCompounds_MultiPlex _Panel)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-02-23
Hold-until Date: 2010-08-19

Data Table ( Complete ):           All
Targets
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasessummarySummary for GTPase
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.

Dose Response Assay Background and Significance:

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.

Ras and Ras-related GTPase functions are tightly regulated, and dysregulation is causal in a wide variety of human diseases. Ras mutations resulting in impaired GTP hydrolysis and plasma membrane hyperactivation are linked to many human cancers [Farnsworth et al. 1991; Sukumar et al. 1983; Taparowsky et al. 1982; Boylan et al. 1990; Hruban et al. 2004; Abrams et al. 1996]. Point mutations in the Rab and Rho GTPases are also causal in diverse human diseases affecting pigmentation, immune, and neurologic functions [Houlden et al. 2004; Verhoeven et al 2003; Williams et al. 2000; Bahaderan et al. 2003; and preliminary findings]. Rab and Rho mutants identified in human disease act as dominant negatives either due to a failure to bind GTP or due to inappropriate coupling of the active proteins with downstream effectors. To date, inhibition of Ras and Ras-related proteins has relied largely on altering membrane recruitment with various drugs affecting prenylation [Morgillo F and Lee HY, 2006; Russell RG, 2006; Park, et al. 2002]. Generally, Ras proteins must be farnesylated for proper membrane localization, while Rab and Rho proteins are geranylated. Such strategies lack specificity and are problematic because each of these prenylation machineries is required for the proper function of many Ras superfamily members. Rational drug design has only recently been applied to identify the first two small molecule inhibitors of Rho GTPase family members [Gao, et al. 2004; Nassar et al. 2006]. Therefore, broadly testing the Ras-related GTPases as targets for small molecule inhibitors and activators is expected to identify new classes of compounds that may be useful in the treatment of human disease, as well as in unraveling the molecular details of how Ras-related GTPases function.

The primary HTS screen for compounds effecting the binding of fluorscent GTP to various Ras-related GTPases yielded a number of interesting compounds. In this assay a number of compounds and their effect on different target proteins were assessed in a dose response manner under different conditions than the primary screen. For this assay, the binding buffer has 1 milliM MgCl2 buffer (the primary screen were carried out in 1 milliM EDTA buffer).
Panel Information
Multiplex Dose Response of Test compounds - Six different target proteins binding with fluorescent GTP under the condition of varying test compound concentrations
    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1Rac1ACT32GTPase activating protein 1 activated mutant
2Rac1WT329GTPase activating protein 1 wildtype
3Cdc42ACT1616Cell Division Cycle 42 activated mutant
4Cdc42WT1715Cell Division Cycle 42 wildtype
5RhoACT32Rho GTPase activating protein 1 activated mutant
6RhoWT131Rho GTPase activating protein 1 wildtype

§ Panel component ID.
Protocol
Individual protein targets (4 microM) are bound to glutathione beads overnight at 4 degrees C. Binding assays are performed by incubating 50 microL of GST-target protein-GSH-bead suspension for 2 min with 1 milliM MgCl2 and either DMSO or test compound (6 point 10-fold dilution series 100 microM to 0.02 microM) and subsequently adding 50 microL of a fixed concentration (1.5 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.

Calculations:

The binding measurements for each protein target were normalized to the amount of binding in DMSO alone:
%Response = 100*(MCF/MCFwDMSO)
where MCF is the binding of fluorescent GTP at different concentrations of test compound and MCFwDMSO is the binding of fluorescent GTP in the presense of DMSO alone.

The different %Response values were fit to 4-parameter sigmoidal dose-response curve with variable slope:
%Response= Bottom + (Top - Bottom)/(1 +10^((LogEC50-X)*HillSlope))
where Bottom and Top are estimates of minimum and maximum of %Response over the concentration range of test compounds, LogEC50 is the log of the Effective Concentration of test compound yielding 50% change in the %Response, and HillSlope is variable slope of the dose response curve.

Target_SCORE is based on the comparison of the estimated EC50 to the least amount of acceptable EC50, 20 microM, for compounds that had a magnitude of change greater than 15%. Thus Target_SCORE = 100*(1 - (EC50inMicroM/20) where EC50inMicroM is the calculated estimate of EC50 in microM. Active compounds have Target_SCORE greater than 0. The desired outcome was to find compounds with specific Cdc42 activity, however many compounds also affected other protein targets, namely Rac1. Thus the overall PUBCHEM_SCORE was left at 0 and compounds were designated 'inconclusive' due to this mixture of activity.
Comment
Abbreviations: microM for micromolar, milliM for millimolar, nanoM for nanomolar, milliL for milliliter
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Rac1ACT_ScoreScore for Rac1 activated mutant1Integer
2Rac1ACT_OutcomeOutcome for Rac1 activated mutant1Outcome
3Rac1ACT_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit1FloatμM
4Rac1ACT_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate1FloatμM
5Rac1ACT_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate1FloatμM
6Rac1ACT_BOTTOMResponse value at the bottom plateau1Float%
7Rac1ACT_TOPResponse value at the top plateau1Float%
8Rac1ACT_HILLSLOPEHill slope estimate for the fitted dose response curve1Float
9Rac1ACT_PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata difference1Float
10Rac1ACT_RSQRCorrelation coefficient for the fitted dose response curve1Float
11Rac1ACT_N_POINTSNumber of data points for each dose response curve1Integer
12Rac1ACT_RESPONSE_100_MICROM (100μM**)Median channel fluorescence measured with 100.00 micromolar concentration of test compound1Float%
13Rac1ACT_RESPONSE_33.33_MICROM (33.33μM**)Median channel fluorescence measured with 33.33 micromolar concentration of test compound1Float%
14Rac1ACT_RESPONSE_11.11_MICROM (11.11μM**)Median channel fluorescence measured with 11.11 micromolar concentration of test compound1Float%
15Rac1ACT_RESPONSE_3.7_MICROM (3.7μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compound1Float%
16Rac1ACT_RESPONSE_1.23_MICROM (1.23μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compound1Float%
17Rac1ACT_RESPONSE_0.41_MICROM (0.41μM**)Median channel fluorescence measured with 0.41 micromolar concentration of test compound1Float%
18Rac1ACT_RESPONSE_0.14_MICROM (0.14μM**)Median channel fluorescence measured with 0.14 micromolar concentration of test compound1Float%
19Rac1ACT_RESPONSE_0.05_MICROM (0.05μM**)Median channel fluorescence measured with 0.05 micromolar concentration of test compound1Float%
20Rac1ACT_RESPONSE_0.02_MICROM (0.02μM**)Median channel fluorescence measured with 0.02 micromolar concentration of test compound1Float%
21Rac1WT_ScoreScore for Rac1 wildtype2Integer
22Rac1WT_OutcomeOutcome for Rac1 wildtype2Outcome
23Rac1WT_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit2FloatμM
24Rac1WT_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate2FloatμM
25Rac1WT_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate2FloatμM
26Rac1WT_BOTTOMResponse value at the bottom plateau2Float%
27Rac1WT_TOPResponse value at the top plateau2Float%
28Rac1WT_HILLSLOPEHill slope estimate for the fitted dose response curve2Float
29Rac1WT_PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata difference2Float
30Rac1WT_RSQRCorrelation coefficient for the fitted dose response curve2Float
31Rac1WT_N_POINTSNumber of data points for each dose response curve2Integer
32Rac1WT_RESPONSE_100_MICROM (100μM**)Median channel fluorescence measured with 100.00 micromolar concentration of test compound2Float%
33Rac1WT_RESPONSE_33.33_MICROM (33.33μM**)Median channel fluorescence measured with 33.33 micromolar concentration of test compound2Float%
34Rac1WT_RESPONSE_11.11_MICROM (11.11μM**)Median channel fluorescence measured with 11.11 micromolar concentration of test compound2Float%
35Rac1WT_RESPONSE_3.7_MICROM (3.7μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compound2Float%
36Rac1WT_RESPONSE_1.23_MICROM (1.23μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compound2Float%
37Rac1WT_RESPONSE_0.41_MICROM (0.41μM**)Median channel fluorescence measured with 0.41 micromolar concentration of test compound2Float%
38Rac1WT_RESPONSE_0.14_MICROM (0.14μM**)Median channel fluorescence measured with 0.14 micromolar concentration of test compound2Float%
39Rac1WT_RESPONSE_0.05_MICROM (0.05μM**)Median channel fluorescence measured with 0.05 micromolar concentration of test compound2Float%
40Rac1WT_RESPONSE_0.02_MICROM (0.02μM**)Median channel fluorescence measured with 0.02 micromolar concentration of test compound2Float%
41Cdc42ACT_ScoreScore for Cdc42 activated mutant3Integer
42Cdc42ACT_OutcomeOutcome for Rac1 activated mutant3Outcome
43Cdc42ACT_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit3FloatμM
44Cdc42ACT_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate3FloatμM
45Cdc42ACT_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate3FloatμM
46Cdc42ACT_BOTTOMResponse value at the bottom plateau3Float%
47Cdc42ACT_TOPResponse value at the top plateau3Float%
48Cdc42ACT_HILLSLOPEHill slope estimate for the fitted dose response curve3Float
49Cdc42ACT_PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata difference3Float
50Cdc42ACT_RSQRCorrelation coefficient for the fitted dose response curve3Float
51Cdc42ACT_N_POINTSNumber of data points for each dose response curve3Integer
52Cdc42ACT_RESPONSE_100_MICROM (100μM**)Median channel fluorescence measured with 100.00 micromolar concentration of test compound3Float%
53Cdc42ACT_RESPONSE_33.33_MICROM (33.33μM**)Median channel fluorescence measured with 33.33 micromolar concentration of test compound3Float%
54Cdc42ACT_RESPONSE_11.11_MICROM (11.11μM**)Median channel fluorescence measured with 11.11 micromolar concentration of test compound3Float%
55Cdc42ACT_RESPONSE_3.7_MICROM (3.7μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compound3Float%
56Cdc42ACT_RESPONSE_1.23_MICROM (1.23μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compound3Float%
57Cdc42ACT_RESPONSE_0.41_MICROM (0.41μM**)Median channel fluorescence measured with 0.41 micromolar concentration of test compound3Float%
58Cdc42ACT_RESPONSE_0.14_MICROM (0.14μM**)Median channel fluorescence measured with 0.14 micromolar concentration of test compound3Float%
59Cdc42ACT_RESPONSE_0.05_MICROM (0.05μM**)Median channel fluorescence measured with 0.05 micromolar concentration of test compound3Float%
60Cdc42ACT_RESPONSE_0.02_MICROM (0.02μM**)Median channel fluorescence measured with 0.02 micromolar concentration of test compound3Float%
61Cdc42WT_ScoreScore for Cdc42 wildtype4Integer
62Cdc42WT_OutcomeOutcome for Cdc42 wildtype4Outcome
63Cdc42WT_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit4FloatμM
64Cdc42WT_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate4FloatμM
65Cdc42WT_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate4FloatμM
66Cdc42WT_BOTTOMResponse value at the bottom plateau4Float%
67Cdc42WT_TOPResponse value at the top plateau4Float%
68Cdc42WT_HILLSLOPEHill slope estimate for the fitted dose response curve4Float
69Cdc42WT_PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata difference4Float
70Cdc42WT_RSQRCorrelation coefficient for the fitted dose response curve4Float
71Cdc42WT_N_POINTSNumber of data points for each dose response curve4Integer
72Cdc42WT_RESPONSE_100_MICROM (100μM**)Median channel fluorescence measured with 100.00 micromolar concentration of test compound4Float%
73Cdc42WT_RESPONSE_33.33_MICROM (33.33μM**)Median channel fluorescence measured with 33.33 micromolar concentration of test compound4Float%
74Cdc42WT_RESPONSE_11.11_MICROM (11.11μM**)Median channel fluorescence measured with 11.11 micromolar concentration of test compound4Float%
75Cdc42WT_RESPONSE_3.7_MICROM (3.7μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compound4Float%
76Cdc42WT_RESPONSE_1.23_MICROM (1.23μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compound4Float%
77Cdc42WT_RESPONSE_0.41_MICROM (0.41μM**)Median channel fluorescence measured with 0.41 micromolar concentration of test compound4Float%
78Cdc42WT_RESPONSE_0.14_MICROM (0.14μM**)Median channel fluorescence measured with 0.14 micromolar concentration of test compound4Float%
79Cdc42WT_RESPONSE_0.05_MICROM (0.05μM**)Median channel fluorescence measured with 0.05 micromolar concentration of test compound4Float%
80Cdc42WT_RESPONSE_0.02_MICROM (0.02μM**)Median channel fluorescence measured with 0.02 micromolar concentration of test compound4Float%
81RhoACT_ScoreScore for Rho activated mutant5Integer
82RhoACT_OutcomeOutcome for Rho activated mutant5Outcome
83RhoACT_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit5FloatμM
84RhoACT_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate5FloatμM
85RhoACT_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate5FloatμM
86RhoACT_BOTTOMResponse value at the bottom plateau5Float%
87RhoACT_TOPResponse value at the top plateau5Float%
88RhoACT_HILLSLOPEHill slope estimate for the fitted dose response curve5Float
89RhoACT_PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata difference5Float
90RhoACT_RSQRCorrelation coefficient for the fitted dose response curve5Float
91RhoACT_N_POINTSNumber of data points for each dose response curve5Integer
92RhoACT_RESPONSE_100_MICROM (100μM**)Median channel fluorescence measured with 100.00 micromolar concentration of test compound5Float%
93RhoACT_RESPONSE_33.33_MICROM (33.33μM**)Median channel fluorescence measured with 33.33 micromolar concentration of test compound5Float%
94RhoACT_RESPONSE_11.11_MICROM (11.11μM**)Median channel fluorescence measured with 11.11 micromolar concentration of test compound5Float%
95RhoACT_RESPONSE_3.7_MICROM (3.7μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compound5Float%
96RhoACT_RESPONSE_1.23_MICROM (1.23μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compound5Float%
97RhoACT_RESPONSE_0.41_MICROM (0.41μM**)Median channel fluorescence measured with 0.41 micromolar concentration of test compound5Float%
98RhoACT_RESPONSE_0.14_MICROM (0.14μM**)Median channel fluorescence measured with 0.14 micromolar concentration of test compound5Float%
99RhoACT_RESPONSE_0.05_MICROM (0.05μM**)Median channel fluorescence measured with 0.05 micromolar concentration of test compound5Float%
100RhoACT_RESPONSE_0.02_MICROM (0.02μM**)Median channel fluorescence measured with 0.02 micromolar concentration of test compound5Float%
101RhoWT_ScoreScore for Rho wildtype6Integer
102RhoWT_OutcomeOutcome for Rho wildtype6Outcome
103RhoWT_EC50_MICROMEffective concentration of half maximal event count as estimated by curve fit6FloatμM
104RhoWT_EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimate6FloatμM
105RhoWT_EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimate6FloatμM
106RhoWT_BOTTOMResponse value at the bottom plateau6Float%
107RhoWT_TOPResponse value at the top plateau6Float%
108RhoWT_HILLSLOPEHill slope estimate for the fitted dose response curve6Float
109RhoWT_PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata difference6Float
110RhoWT_RSQRCorrelation coefficient for the fitted dose response curve6Float
111RhoWT_N_POINTSNumber of data points for each dose response curve6Integer
112RhoWT_RESPONSE_100_MICROM (100μM**)Median channel fluorescence measured with 100.00 micromolar concentration of test compound6Float%
113RhoWT_RESPONSE_33.33_MICROM (33.33μM**)Median channel fluorescence measured with 33.33 micromolar concentration of test compound6Float%
114RhoWT_RESPONSE_11.11_MICROM (11.11μM**)Median channel fluorescence measured with 11.11 micromolar concentration of test compound6Float%
115RhoWT_RESPONSE_3.7_MICROM (3.7μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compound6Float%
116RhoWT_RESPONSE_1.23_MICROM (1.23μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compound6Float%
117RhoWT_RESPONSE_0.41_MICROM (0.41μM**)Median channel fluorescence measured with 0.41 micromolar concentration of test compound6Float%
118RhoWT_RESPONSE_0.14_MICROM (0.14μM**)Median channel fluorescence measured with 0.14 micromolar concentration of test compound6Float%
119RhoWT_RESPONSE_0.05_MICROM (0.05μM**)Median channel fluorescence measured with 0.05 micromolar concentration of test compound6Float%
120RhoWT_RESPONSE_0.02_MICROM (0.02μM**)Median channel fluorescence measured with 0.02 micromolar concentration of test compound6Float%

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

Classification
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