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BioAssay: AID 2373

Compound effect on equilibrium binding with Cdc42 under varying GTP conditions with Mg 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
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 2373
Data Source: NMMLSC (UNM_Mg_GTPEqBinding_Cdc42)
Depositor Category: NIH Molecular Libraries Probe Production 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 GTPasessummaryGTPase summary
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 Mg buffer.
Protocol
The protein target (GST-Cdc42, 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 MgCl2 and either DMSO or 10 microM compound and subsequently adding 50 microL of varying concentration (range 0-100 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_1.56nanoMAmount of 1.56 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
2BoundGTP_Compound_3.125nanoMAmount of 3.125 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
3BoundGTP_Compound_6.25nanoMAmount of 6.25 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
4BoundGTP_Compound_12.5nanoMAmount of 12.5 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
5BoundGTP_Compound_25nanoMAmount of 25 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
6BoundGTP_Compound_50nanoMAmount of 50 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
7BoundGTP_Compound_100nanoMAmount of 100 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of 10 microM test compoundFloat
8FitBmax_CompoundCalculated maximum number of binding sites for GTP on Cdc42 bound beads in presense of 10 microM test compoundFloat
9FitKd_Compound_nanoMCalculated affinity of GTP to Cdc42 in presense of 10 microM test compoundFloatnM
10BoundGTP_DMSO_1.56nanoMAmount of 1.56 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
11BoundGTP_DMSO_3.125nanoMAmount of 3.125 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
12BoundGTP_DMSO_6.25nanoMAmount of 6.25 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
13BoundGTP_DMSO_12.5nanoMAmount of 12.5 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
14BoundGTP_DMSO_25nanoMAmount of 25 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
15BoundGTP_DMSO_50nanoMAmount of 50 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
16BoundGTP_DMSO_100nanoMAmount of 100 nanoM fluorescent GTP bound to Cdc42 bound beads in presense of DMSOFloat
17FitBmax_DMSOCalculated maximum number of binding sites for GTP on Cdc42 bound beads in presense of DMSOFloat
18FitKd_DMSO_nanoMCalculated affinity of GTP to Cdc42 in presense of DMSOFloatnM
Additional Information
Grant Number: NIH I RO3 MH081231-01

Data Table (Concise)
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