Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.
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 ..
BioActive Compounds: 73
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., (firstname.lastname@example.org)
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.
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). Test compound concentration is 10 microM. 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 IDLeQuery/HyperView 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 Green 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 compound, if the measured MCF from scavenger bead is greater than or equal to 40% of that from the positive control bead set, the compound is considered a potential fluorescent compound.
Sample fluorescence for all the different proteins in a well are corrected for background fluorescence, based on potential systematic trends in data over the entire plate (whole plate trends), normalization is also utilized to calculate the percent regulation of the test compound. The background subtracted and normalized sample fluorescence is referred to as the Response Value of the test compound (RV, see column titled RESPONSE_VALUE). Due to the nature of this assay, instead of using a universal positive control for the whole plate, individual row trends of the controls are evaluated by linear regression. Then based on the plate location of a sample, a calculated Positive Control value (Notated with LinFit) is utilized for calculating the normalized response value. RV of the compound is calculated by the following equation:
RV = 100 x (SampleFL-NCntrl)/(LinFitPCntrl-NCntrl)
where all variables are in units of MCF in FL1 (530 nanom) associated with the protein-coupled bead set. SampleFL is for beads in wells containing test compound, LinFitPCntrl is the calculated value based on row dependent linear regression of wells without test compounds, and NCntrl is from the measurement non-specific AF-labeled Galphao binding in absence of RGS protein. Baseline of RV is 100, and represents a test compound that has no effect on the RGS-Galphao binding. A compound with activating effects would have RV greater than 100 and a compound with inhibitory effects would have RV less than 100.
PUBCHEM_ACTIVITY_SCORE corresponds directly with the percent of regulation of the compound; PUBCHEM_ACTIVITY_SCORE is the absolute value of RV minus 100,|%Reg|. The maximum PUBCHEM_ACTIVITY_SCORE of 100 for primary screening is given when |%Reg| is greater then 100, meaning 100% activation or inhibition effect; the minimum PUBCHEM_ACTIVITY_SCORE of 0 means the substance has no impact on the target RGS-Galphao interaction.
An active compound for RGS4 have a PUBCHEM_ACTIVITY_SCORE greater than 26 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
** Test Concentration.
Data Table (Concise)