Profiling Assay to determine GST-GSH interactions in multiplex bead-based assays
The objective of the HTS associated with this counterscreen was to identify small molecule regulators of Ras and Ras-related GTPases (see Summary Report and PubChem AIDs 1333, 1334, 1335, 1336, 1337, 1339, 1340, 1341). 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 more ..
BioActive Compounds: 186
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
Profiling Assay Background and Significance:
The objective of the HTS associated with this counterscreen was to identify small molecule regulators of Ras and Ras-related GTPases (see Summary Report and PubChem AIDs 1333, 1334, 1335, 1336, 1337, 1339, 1340, 1341). 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 wt and activated forms exhibit measurably distinct affinities for Bodipy-FI-GTP vs GTP. The assay involves the binding of fluorescent GTP to G protein-GST fusion proteins on GSH beads. A set of six G proteins (Rac 1 wt, Rab7 wt, Rac 1 activated, Ras wt, Rab 2 wt., CDC wt) are arrayed under conditions of divalent molecule depletion.
In this counterscreen assay, compounds were evaluated for the ability to interfere with the binding of the GST fusion proteins to the GSH beads (thus producing potential false positive results). Dose response experiments were constructed in which compounds at 10 mM concentration in DMSO were serially diluted 1:3.16 eight times for a total of nine different test compound concentrations. Final compound dilutions in DMSO ranged from 1 microM to 10 mM. These dilutions were then further diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM. GSH beads were incubated overnight with 50 nM GST-green fluorescent protein similar to conditions in the in the screening assay.
Sample acquisition and preliminary analysis was conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates. The stream of particles is excited at 488 nm and 635 nM, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis of the time-resolved acquisition data file uses IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to segregate data for each target and the fluorescence scavenger in the multiplex. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with GFP, and the green median fluorescence intensity (MFI) per bead population (well) is calculated.
In dose response GST-GSH experiments, the assay was performed without compound and with nine different concentrations of compound, from 10 nanoM to 100 microM, to produce a series of 9 data points. IDLeQuery calculates the median channel fluorescence (MCF) for each of these ligand concentrations, generating competition curves.
Ligand competition curves were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.
In order to be considered active and get a score > 0, the compounds have to pass the following criteria:
- -8 < LOGEC50 < -4 (the computed EC50 value should be in the interval of tested concentrations)
- 0.5 < |HILLSLOPE| < 2 (the absolute value of HILLSLOPE should be higher than 0.5 and lower than 2)
- [TOP - STD_TOP] > [BOTTOM + STD_BOTTOM] (the amplitude of the biological signal should be statistically significant)
- |LOGEC50| > STD_LOGEC50 (the standard error of LOGEC50 should be lower than the absolute value of LOGEC50)
- |HILLSLOPE| > STD_HILLSLOPE (idem for the HILLSLOPE)
- [TOP - BOTTOM] scavenger < 0.5*[TOP - BOTTOM] target (the inherent fluorescence of the test compound should be lower than 50% of the biological signal)
- [TOP - BOTTOM]/TOP for GST-GFP < 0.5*[TOP - BOTTOM]/TOP for the target (the interference of the compound with the GST/GSH interaction should be lower than 50% of the biological signal)
The PUBCHEM_ACTIVITY_SCORE was based on the following equation;
PUBCHEM_ACTIVITY_SCORE = 1000 * (-4 - LOGEC50)/4 * [(TOP-STD_TOP)-(BOTTOM+STD_BOTTOM)]/TOP * (1 - ||HILLSLOPE|-1|) * (1-STD_LOGEC50/|LOGEC50|)
In this assay active compounds have the activity score higher or equal than 1, and for inactive compounds the activity score is 0.
Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, Rac 1 wt, Rab7 wt, Rac 1 activated, Ras wt, Rab 2 wt., CDC wt, GST-GSH counterscreen.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)