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BioAssay: AID 2376

Dose Response of compounds with constant GTP under the condition of nascent nucleotide depletion and Mg buffer

UNM Cheminformatics: Cristian Bologa, Ph.D., Fabiola Miscioscia, Ph.D., Ramona Curpan, Ph.D., Oleg Ursu, Ph.D. ..more
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AID: 2376
Data Source: NMMLSC (UNM_NucleotideDepletion_Mg_DRCompounds_Cdc42)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-02-18
Hold-until Date: 2010-08-16

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasessummarySummary report for GTPase target project
761HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtypescreeningHTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Cdc42 wildtype
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)
UNM Cheminformatics: Cristian Bologa, Ph.D., Fabiola Miscioscia, Ph.D., Ramona Curpan, Ph.D., Oleg Ursu, 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 the compound found to exhibit most specific response on Cdc42 protein was assessed in a dose response manner with different conditions than the primary screen. For this assay, the nascent nucleotides were depleted from the protein prior to the measurement of binding of fluorescent GTP in the presense of test compound in a 1 milliM MgCl2 buffer (the primary screen were carried out in 1 milliM EDTA buffer).
Protocol
The protein target (GST-Cdc42, 4 microM) is bound to glutathione beads overnight at 4 degrees C. Protein on GSH-beads is depleted of nascent nucleotide by incubating with 10 milliM EDTA-containing buffer for 20 min at 30 degrees C, washing twice with 0.01% detergent, NP-40, containing HPS buffer, then resuspending in the same buffer containing 1 milliM MgCl2, 1 milliM DTT and 0.1% BSA. Binding assays are performed by incubating 50 microL of GST-target protein-GSH-bead suspension for 2 min with either DMSO, or test compound (6 point 10-fold dilution series 100 nanoM to 10 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. 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 test compound concentrations in the presense of blocking, non-fluorescent GTP (RawMCFwNFGTP) and DMSO (RawMCFwDMSO):
SpecMCF = RawMCFwDMSO - RawMCFwNFGTP

These specific binding values were normalize to the amount of binding in DMSO alone sample:
%Response = 100*(SpecMCF/SpecMCFwDMSO)
where SpecMCF is the specific binding of fluorescent GTP at different concentrations of test compound and SpecMCFwDMSO is the specific 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.

PUBCHEM_SCORE is based on the comparison of the estimated EC50 to the least amount of acceptable EC50, 10 microM. Thus PUBCHEM_SCORE = (10-EC50inMicroM)/10 where EC50inMicroM is the calculated estimate of EC50 in microM. Active compounds have PUBCHEM_SCORE greater than 50.
Comment
Abbreviations: microM for micromolar, milliM for millimolar, nanoM for nanomolar, milliL for millilite
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_QUALIFIERQualifier for the value of EC50String
2EC50_MICROM*Effective concentration of half maximal event count as estimated by curve fitFloatμM
3EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
4EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
5BOTTOMResponse value at the bottom plateauFloat%
6TOPResponse value at the top plateauFloat%
7HILLSLOPEHill slope estimate for the fitted dose response curveFloat
8STD_BOTTOMStandard error for the response value at the bottom plateauFloat%
9STD_TOPStandard error for the response value at the top plateauFloat%
10STD_HILLSLOPEStandard error for the HILL SLOPEFloat
11PERCENT_SPANSpan of dose response fitted Top minus Bottom or rawdata differenceFloat
12RSQRCorrelation coefficient for the fitted dose response curveFloat
13N_POINTSNumber of data points for each dose response curveInteger
14RESPONSE_0.12_MICROM (0.12uM) (0.12μM**)Median channel fluorescence measured with 0.12 micromolar concentration of test compoundFloat%
15RESPONSE_0.37_MICROM (0.37uM) (0.37μM**)Median channel fluorescence measured with 0.37 micromolar concentration of test compoundFloat%
16RESPONSE_1.1_MICROM (1.1uM) (1.1μM**)Median channel fluorescence measured with 1.1 micromolar concentration of test compoundFloat%
17RESPONSE_3.3_MICROM (3.3uM) (3.3μM**)Median channel fluorescence measured with 3.70 micromolar concentration of test compoundFloat%
18RESPONSE_10_MICROM (10uM) (10μM**)Median channel fluorescence measured with 1.23 micromolar concentration of test compoundFloat%

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

Data Table (Concise)
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