Compound effect on equilibrium binding with Cdc42 under varying GTP conditions: nucleotide depletion 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 ..
Sequence: cell division cycle 42 (GTP binding protein, 25kDa) [Homo sapiens]
More BioActivity Data..
BioActive Compound: 1
Depositor Specified Assays
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 nascent nucleotide depletion with Mg buffer.
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-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 0-1000 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).
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.
Abbreviations: microM for micromolar, milliM for millimolar, nanoM for nanomolar, milliL for milliliter
Data Table (Concise)