Multiplexed high-throughput screen for 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: 711
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 (F/P ratio between 2 and 3, supplied by project collaborator). 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 PE-Cy5 (680/30 nanom), APC (665/20 nanom) and APC-Cy7 (FL9, 750 nanom LP) channel at 488 nanom or 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.
This design allows all 5 RGS proteins to be assayed in one multiplex, since each bead set is associated with a unique optical address that is also coupled to a unique RGS protein. The bead sets are distinguished by distance emission characteristics at 750+ nanom with excitation at 635 nm. For instance, bio-RGS4 might be noncovalently coupled to red level 1 beads, whereas bio-RGS7 might be coupled to red level 2 beads, and so forth. 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 streptavidin-only bead control (no associated bio-RGS protein) is incorporated into each well as a scavenger as well as a fluorescence indicator to resolve inherent fluorescent properties (at 530 nanom emission) of the test compounds.
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 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 in IDLeQuery 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 no PUBCHEM_ACTIVITY_SCORE is assigned. In this set of 218,702 compounds, there are only 252 missing compounds.
RGS proteins from different batches exhibit different Galphao binding capacity. When the mean of the measured MCF from the positive control beads is less than 2 times the mean of the negative control bead set, and very close to the signal from the Scavenger bead set, the results for that particular protein is "inconclusive", thus the target protein in that plate is considered Inactive (see column titled PUBCHEM_ASSAYDATA_COMMENT). The PUBCHEM_ACTIVITY_OUTCOME of an Inactive RGS Protein is "inconclusive", and a PUBCHEM_ACTIVITY_SCORE of 0 is automatically assigned. Significant amounts of inactive protein RGS19 and RGS7 were found in this multiplexing assay.
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. In this set of 218,702 compounds, 456 of them were flagged as potentially fluorescent.
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.
PUBCHEM_ACTIVITY_SCORE corresponds directly with the percent of regulation of the compound (|%Reg|), where |%Reg| is the absolute value of RV minus 100. The maximum PUBCHEM_ACTIVITY_SCORE of 100 for primary screening is given when |%Reg| is greater than 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.
RV cutoff rates were employed to determine the activity of test compounds. For targets RGS4 and RGS7, compounds were considered "Active" if the |%Reg| was greater than 50; for target RGS8, compounds were considered "Active" if the |%Reg| was greater than 40. Due to a compound library dependent "edge effect" phenomena, target RGS16 was analyzed differently. For the majority of the data set, compounds with a |%Reg| of greater than 75 were considered "Active". Data from compounds located in rows A and P of 504 plates were assigned a special filter (see column titled PUBCHEM_ASSAYDATA_COMMENT), and only those with RV minus 100 (not absolute value of %Reg) less than 0 or greater than 135 were considered "Active". Active compounds include both activators - where %Reg is positive - and inhibitors - where %Reg is negative (see column titled ACTIVITY). From the 218,702 screened compounds, a total of 1908 compounds were chosen for further investigation as potential regulators of all multiplexed RGS proteins. Approximately 700-800 hit compounds per protein target (~0.3% hit rate) were identified. Of those, 63 compounds are RGS4 specific, 48 are RGS7 specific, 179 are RGS8 specific and 399 are considered RGS16 specific.
RGS bead populations are distinguished using the mean channel fluorescence from FL9 (red) channel, which can potentially be affected by red fluorescent compounds, especially the bead set that has the lowest FL9 intensity. When there are significantly more events detected from the second dimmest bead set then the rest of the bead set, and less than 25 events are detected for the dimmest bead set, the compound is flagged as "Potential Red Fluorescent Compound" (see column titled PUBCHEM_ASSAYDATA_COMMENT). In this set of 218,702 compounds, only four compounds were flagged as potential red fluorescent compounds.
The Z prime score for the plates are listed in column titled Z_Prime_Value. For the entire library, average Z prime score for target RGS4 is 0.72, with a 0.2 standard deviation; for target RGS7, when excluding inactive proteins, Z prime score is 0.72 (0.12 for the complete library), the standard deviation is 0.18; for target RGS8, Z prime score is 0.72 and standard deviation is 0.19; for target RGS16, due to the library dependent "edge effect", the Z prime score is 0.3 with a 0.68 standard deviation; and for target RGS19, Z prime score is off limit because of the poor activity of the protein.
For easy user indexing, the PubChem Activity Scores (see column titled Ri_ACTIVITY_SCORE) for the same compound on other targets as well as comments about whether the compound is an activator/inhibitor to target RGSi, and whether target RGSi is an Inactive target (see column Ri_ASSAY_COMMENT) are also included in the file, where "i" can be 4, 7, 8, 16, or 19.
Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry, RGS4, RGS7, RGS8, RGS16, RGS19, multiplex bead-based screening
** Test Concentration.
Data Table (Concise)