Activator binding kinetics on Ras and Ras-related GTPases, specifically Rab2_wt
The objective of the HTS associated with this secondary assay was to identify small molecule regulators of Ras and Ras-related GTPases (see Summary Report and PubChem AIDs 757, 758, 759, 760, 761, 764). The primary HTS assay was a no-wash fluorescent GTP-binding assay adapted to multiplexed, high-throughput measurements whereby multiple GTPases were simultaneously screened against the MLSCN more ..
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, Danuta Wlodek, Terry Foutz, Mark Carter, Anna Waller
Target Team Leader for the Center: Larry Sklar (firstname.lastname@example.org)
Assay Background and Significance:
The objective of the HTS associated with this secondary assay was to identify small molecule regulators of Ras and Ras-related GTPases (see Summary Report and PubChem AIDs 757, 758, 759, 760, 761, 764). The primary HTS assay was a no-wash fluorescent GTP-binding assay adapted to multiplexed, high-throughput measurements whereby multiple GTPases were simultaneously screened against the MLSCN library. The specificity is based on the observation that individual GTPases including wildtype and activated mutants exhibit measurably distinct affinities for Bodipy-FI-GTP. The assay involves the binding of fluorescent GTP to G protein-GST fusion proteins on GSH beads. A set of six G proteins (Rac1 wt, Rab7 wt, Rac1 activated, Ras wt, Rab2 wt, CDC wt) are arrayed under conditions of divalent molecule depletion.
In the assay described here, real-time binding kinetics between GTP and each of the protein targets were characterized in a multiplex assay (Schwartz, et al 2008). The resulting binding of fluorescent GTP after 3 minutes was utilized in determining Bmax and Kd for each target in the presence of 10 microM small molecule activator compound MLS000088004 versus a DMSO control.
Each protein target (4 microM) was bound to glutathione beads overnight at 4 degrees C. Protein on GSH-beads was depleted of nucleotide by incubating with 10 milliM EDTA containing buffer for 20 min at 30 degrees C, washing twice with 0.01% NP-40 containing HPS buffer, then resuspending in the same buffer containing 1 milliM EDTA, 1 milliM DTT and 0.1% BSA. Kinetic assays were performed by incubating 50 microliter of GST-target protein-GSH-bead suspension for 2 min with either DMSO, or 10 microM MLS000088004 and subsequently adding 50 microL of various concentration ice cold BODIPY-GTP. Association of the fluorescent nucleotide was measured using a FacSCAN flow cytometer in the kinetic mode. Data were converted to ASCII format using IDLQuery.
Measured values of bead-bound BODIPY-GTP after 3 minutes of binding are converted to molecular equivalent soluble fluoresceine (MESF) with the aid of calibration beads (Bangs Lab) by the following equation;
kMESF = Slope * MCF + Intercept
where MCF is the Median Channel Fluorescence measurement of bead-bound BODIPY-GTP, kMESF are kiloMESF (1000 * MESF), and Slope and Intercept are the from the linear regression fit of the 5 different levels of calibration beads.
The resulting values of kMESF are graphed versus the different concentrations of BODIPY-GTP in GraphPad Prism and fitted by non-linear regression to one site binding pre the following equation;
kMESF = Bmax * ConcBODIPY-GTP / (Kd + ConcBODIPY-GTP)
where ConcBODIPY-GTP is the concentration of BODIPY-GTP, Bmax is the maximum binding of BODIPY-GTP per bead and Kd is the equilibrium binding constant of BODIPY-GTP to protein on bead.
Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, GTPase, multiplex bead-based screening .
** Test Concentration.
Data Table (Concise)