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 ..
Related BioAssays
Related BioAssays
Target
BioActive Compound: 1
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 1772 | Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases | summary | GTPase 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.
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.
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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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | BoundGTP_Compound_3nanoM | Amount of 3 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 2 | BoundGTP_Compound_6nanoM | Amount of 6 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 3 | BoundGTP_Compound_12.5nanoM | Amount of 12.5 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 4 | BoundGTP_Compound_25nanoM | Amount of 25 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 5 | BoundGTP_Compound_50nanoM | Amount of 50 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 6 | BoundGTP_Compound_100nanoM | Amount of 100 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 7 | FitBmax_Compound | Calculated estimate of maximum GTP binding sites on Cdc42 covered beads in presense of 10 microM test compound | | Float | ||
| 8 | FitKd_Compound_nanoM | Calculated affinity of GTP to Cdc42 in the presense of 10 microM test compound | | Float | ||
| 9 | BoundGTP_DMSO_3nanoM | Amount of 3 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSO | | Float | ||
| 10 | BoundGTP_DMSO_6nanoM | Amount of 6 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSO | | Float | ||
| 11 | BoundGTP_DMSO_12.5nanoM | Amount of 12.5 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSO | | Float | ||
| 12 | BoundGTP_DMSO_25nanoM | Amount of 25 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSO | | Float | ||
| 13 | BoundGTP_DMSO_50nanoM | Amount of 50 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSO | | Float | ||
| 14 | BoundGTP_DMSO_100nanoM | Amount of 100 nanoM fluorescent GTP bound to Cdc42 covered beads in presense of DMSO | | Float | ||
| 15 | FitBmax_DMSO | Calculated estimate of maximum GTP binding sites on Cdc42 covered beads in presense of DMSO | | Float | ||
| 16 | FitKd_DMSO_nanoM | Calculated affinity of GTP to Cdc42 in the presense of DMSO | | Float |
Additional Information
Grant Number: NIH I RO3 MH081231-01
Data Table (Concise)
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