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BioAssay: AID 2295

Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao for SAR compounds

Regulators of G protein signaling (RGS) proteins are a diverse set of intracellular proteins that modulate G protein-coupled receptor (GPCR) signaling [Neitzel and Hepler, 2006]. Their diversity is a result of their localized tissue distribution as well as their preferential regulation of a particular subunit of G protein (Galpha) [Zhong and Neubig, 2001; Neubig and Siderovski, 2002]. Following more ..
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 Tested Compounds
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 Tested Substances
AID: 2295
Data Source: NMMLSC (UNM_RGS4_DoseResponse_SAR)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-01-26
Hold-until Date: 2010-06-15
Modify Date: 2010-06-15

Data Table ( Complete ):           Active    All
BioActive Compounds: 18
Depositor Specified Assays
880qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)confirmatoryqHTS Assay for Inhibitors of RGS GAP Activity (Red Fluorophore).
879qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Green Fluorophore)confirmatoryqHTS Assay for Inhibitors of RGS GAP Activity (Green Fluorophore).
503Compound Screen Assay, Human RGS18screeningCompound Screen Assay, Human RGS18. Source: SGCOxCompounds (RGS18 Compound Screen)
1415Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.screeningHTS flow cytometric screening of RGS4
1504Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions.summary
University of New Mexico Assay Overview:
Assay Support: NIH R21NS057014
HTS to identify small molecule regulators of RGS family protein interactions
PI: Richard Neubig, Ph.D.
Assay Implementation: Yang Wu Ph.D., Mark Haynes Ph.D., Anna Waller Ph.D., Mark Carter MS
Target Team Leader for the Center: Larry Sklar, Ph.D., (

Assay Background and Significance:

Regulators of G protein signaling (RGS) proteins are a diverse set of intracellular proteins that modulate G protein-coupled receptor (GPCR) signaling [Neitzel and Hepler, 2006]. Their diversity is a result of their localized tissue distribution as well as their preferential regulation of a particular subunit of G protein (Galpha) [Zhong and Neubig, 2001; Neubig and Siderovski, 2002]. Following activation by ligand-bound GPCRs, the Galpha subunit undergoes rapid GTP - GDP exchange, and subsequently dissociates from both the GPCR and the G protein beta-gamma subunit (Gbg). Active GTP-bound Galpha (Galpha-GTP) and Gbg further modulate the activity of a number of down-stream intracellular effectors [Cabrera-Vera et al., 2003]. The duration of G protein signaling is determined by GTP hydrolysis as well as by the re-association of inactive Galpha-GDP with Gbg. Gilman's early work with G-proteins suggested that their intrinsic GTPase activity, typically 2-5 per minute, could not account for the known speed of GPCR signal resolution [Gilman, 1987]. This discrepancy was resolved after the discovery of GTPase Accelerating Proteins (GAPs), of which RGS proteins are a subset. The RGS proteins bind directly to Galpha-GTP, accelerate the rate of GTP hydrolysis, and shorten the lifetime of active G proteins up to several thousand fold [Mukhopadhyay and Ross, 1999; Lan, et al, 2000]. Thus, RGS-Galpha interactions are central to the down-stream regulation of GPCR signaling events. Since GPCRs control numerous physiologic processes in diverse tissues, including brain, heart, liver, and lung, modulation of the RGS/G protein interaction has become an attractive target for drug discovery. Assays based on RGS binding to fluorescently labeled Galpha have recently been described in which immobilized RGS proteins are incubated with labeled Galpha [Roman et al., 2007]. Here we describe a further modification of this basic design where an AlexaFluor-488 conjugated Galphao (AF-Galphao) is incubated with RGS proteins that are immobilized onto microspheres. Binding interactions between AF-Galphao and RGS is subsequently assessed by flow cytometer. This design provides the basis for development of fluorescence-based assays amenable to high throughput flow cytometry screening. We have multiplexed this assay using differentially labeled microspheres and used it to identify small molecule regulators of the RGS-Galphao interaction using the following 5 RGS family members: RGS4, RGS7, RGS8, RGS16, and RGS19.
Protocol used for the dose response assay is similar to the one used for screening, with the exception that compounds were tested at 9 different concentrations. Each component of the multiplex assay consists of a streptavidin functionalized polystyrene bead, a biotinylated RGS-fusion protein target (bio-RGS, five total, supplied by project collaborator), and a fluorescent probe, AlexaFluor488-labeled Galphao protein. Six bead sets are used, including one unlabeled bead set and five sets that are labeled to different intensities with red fluorescence that are fluorescent in APC-Cy7 (FL9, 750 nanom LP) channel at 635 nanom excitation (Spherotech product numbers SVPAK-5067-5B). Individual bead sets are coupled with a single bio-RGS protein by mixing beads and bio-RGS in bead coupling buffer (BCB; PBS, pH8.0 supplemented with 0.1% BSA). The mixture is then incubated overnight at 4oC under mild vortexing. The 5 bead sets (each with a bound protein) and an uncoated bead set (Scavenger beads, see below) are centrifuged separately, washed once in BCB, followed by another wash in flow buffer (FB; 50 milliM HEPES, 100 milliM NaCl, 0.1% Lubrol, and 0.1% BSA, pH 8.0). The bead sets are then resuspended in FB and stored on ice until the plates are assembled using the Biomek FXp, Automated work station. Before incubation with bead-coupled RGS proteins, AF-Galphao is incubated for 10 minutes at room temperature in AMF buffer (50 milliM MgCl2, 50 microM AlCl3, 50 milliM NaF, 10 milliM GDP in FB). This incubation produces the activated AF-Galphao-GDP-AlF4-complex that binds RGS proteins with high affinity.

After preparation, the bio-RGS coupled bead sets and the uncoated Scavenger bead set are further diluted in FB, combined and loaded into 384-well microplates using the Biomek liquid handling workstation. The assay is conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture containing ~3,000 beads from each bead set, 0.1 microliters of test compound, and 5 microliters of 14 nanoM AF-Galphao-GDP-AlF4 in FB). Final assay concentration of AF-Galphao-GDP-AlF4 is 7 nanoM. The test compounds are arranged in serial dilutions of 1:3 starting at 30 microM concentrations and ending at 9 nanoM. Controls, which contain the same bead mixture and AF-Galphao-GDP-AlF4 but no test compound, are located in columns 1 and 2 of each plate (Positive Control Beads). Plates are incubated under mild vortexing for 30-90 minutes at room temperature.

Specificity of AF-Galphao-GDP-AlF4 binding is determined with a Negative Control using mixture of blank (no RGS) red color-coded streptavidin bead sets because there are no known universal blocking peptides for all 5 RGS proteins. The AF-Galphao Negative Control is run daily as a separate single tube assay by incubating the blank bead sets in 7 nanoM of AF-Galphao-GDP-AlF4 under mild vortex for 30-90 min.

Sample analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt(R) system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nanom (FL1) and 750+ nanom (FL9) are collected on a Cyan Flow Cytometer (Dako). Time-resolved data is acquired as a single data file that is subsequently analyzed using HyperView(R) software (software developed inhouse by Dr. Bruce Edwards) that merges flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed to produce annotated fluorescence summary data for each well. The parsed data are then processed through a Microsoft Excel template file, constructed specifically for the assay, which segregates data from each RGS-target protein 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. Additional gating based on FL9 (red) emission distinguishes the beads coated with different proteins. Per bead population, the median channel fluorescence (MCF) from FL1 (green) channel is calculated for analysis of AlexaFluor488-labeled Galphao binding.


In order to get a significant measurement of a compound's effect on a particular RGS protein, 25 beads with that particular protein bound needs to be collected from a well. When less than 25 beads are counted, the result for that protein is considered missing. Compounds from missing wells are given a PUBCHEM_ACTIVITY_OUTCOME = 4 and PUBCHEM_ACTIVITY_SCORE =0.

When the measured emission is potentially attributed to the innate fluorescence of the compound, these compounds are flagged as "Possible_Fluorescent_Compound" (see column titled PUBCHEM_ASSAYDATA_COMMENT). Assessment of fluorescence is made by comparing the influence of compound fluorescence on the scavenger beads in one well. In the presence of fluorescent compound, percent response of Scavenger at 30 microM was greater than 250%.

Percent response is calculated per plate basis for each compound concentration by the following equation:
%Response = 100 x (SampleFL-NCntrl)/(PCntrl-NCntrl)
where all variables are in units of median channel fluorescence (MCF) in FL1 (530 nanom) associated with the protein-coupled bead set. PCntrl is the plate average of wells with DMSO, and NCntrl is from the measurement non-specific AF-labeled Galphao binding in absence of RGS protein. Baseline of %Response is 100, and represents a test compound that has no effect on the RGS-Galphao binding. A compound with activating effects would have %Response greater than 100 and a compound with inhibitory effects would have %Response less than 100.

The %Response data of duplicate values from each compound concentration 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. Curves were fitted for both activators and inhibitors, thus fit statistics were used to determine the concentration of added test compound that effected fluorescent ligand binding by 50 percent (EC50, microM) with 95% confidence intervals of the estimated EC50 value, Hillslope, and the correlation coefficient (r squared) indicative of goodness-of-fit. For compounds with significant inhibitory affects, Ki values were calculated from the EC50 values based on the following equation:
Ki = EC50/(1 + [AF-Galphao]/Kd
where [AF-Galphao] is the assay concentration of fluorescently labeled Galphao of 7 nanoM and Kd is the specific targets affinity for Galphao. For RGS4 that value had been determined to be 40 nanoM. Note that this equation is theoretically only applicable to dose response curves of absolute value of Hillslopes equal to 1, however the calculations of Ki's reported did not take into account the Hillslope.

PUBCHEM_ACTIVITY_SCORE were calculated based on an EC50 cutoff of 30 microM, by using the following equations:
SCORE = 100*(1-EC50/30 )
And compounds were demeaned active if the EC50 is equal to or less than 30 microM. Thus an active compound have a PUBCHEM_ACTIVITY_SCORE greater than 0 and the type of active compound (Activator or Inhibitor) is listed in column 'Activity'.

Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, RGS4, RGS7, RGS8, RGS16, RGS19, multiplex bead-based screening
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_QUALIFIERQualifier for the estimated EC50 value.String
2EC50_MICROM*Estimated EC50 value in microM from dose response fittingFloatμM
3STD_EC50Standard deviation associated with the estimated EC50 value.FloatμM
4KI_MICROMKi or inhibitor affinity calculated from EC50 for compounds exhibiting inhibitory effectsFloatμM
5HILLSLOPEEstimated Hillslope from fitting of the dose response dataFloat
6PERCENT_SPANPercent response difference between maximum and minimum sample concentrations.Float%
7RSQRResidual squared from the sigmoidal curve fitting of the dose response data.Float
8N_POINTSNumber of data points used during the data fitting, spread over 9 different concentrations.Integer
9RESPONSE_TYPEAnnotation of type of response exhibited by test compound.String
10AVERESPONSE_0.003_MICROM (0.003uM) (0.003μM**)Average percent response at 0.003 microM test compound.Float%
11AVERESPONSE_0.009_MICROM (0.009uM) (0.009μM**)Average percent response at 0.009 microM test compound.Float%
12AVERESPONSE_0.031_MICROM (0.031uM) (0.031μM**)Average percent response at 0.031 microM test compound.Float%
13AVERESPONSE_0.10_MICROM (0.10uM) (0.1μM**)Average percent response at 0.10 microM test compound.Float%
14AVERESPONSE_0.31_MICROM (0.31uM) (0.31μM**)Average percent response at 0.31 microM test compound.Float%
15AVERESPONSE_1.1_MICROM (1.1uM) (1.1μM**)Average percent response at 1.1 microM test compound.Float%
16AVERESPONSE_3.1_MICROM (3.1uM) (3.1μM**)Average percent response at 3.1 microM test compound.Float%
17AVERESPONSE_10_MICROM (10uM) (10μM**)Average percent response at 10 microM test compound.Float%
18AVERESPONSE_31.5_MICROM (31.5uM) (31.5μM**)Average percent response at 31.5 microM test compound.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: NIH R21NS057014

Data Table (Concise)