University of New Mexico Assay Overview:
Assay Support: NIH R21NS057014
HTS to identify small molecule regulators of RGS family protein interactions
PI: Larry Sklar, Ph.D., Richard Neubig, Ph.D

Assay Implementatiion: Yang Wu Ph.D, Mark Hynes Ph.D, Anna Waller Ph.D, Mark Carter MS

Assay Background and Significance:

This report summarizes the series of assays used to identify small molecule modulators of regulators of G protein signaling (RGS) family protein interactions via the G protein alpha o (G alpha o) subunit and the following 5 RGS family members: RGS4, RGS7, RGS16, and RGS19.

The initial assay was a multiplex high throughput screen (HTS) of a 218,538-member compound collection from the NIH Molecular Libraries Small Molecule Repository (MLSMR). Each component of the multiplex assay consists of a streptavidin functionalized polystyrene bead, a biotinylated RGS-fusion protein target (bio-RGS, five total), and a fluorescent probe, AlexaFluor488-labeled Galphao protein (F/P ratio between 2 and 3). 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 nanometer), APC (665/20 nanometer) and APC-Cy7 (FL9, 750 nanometer LP) channel at 488 nanometer or 635 nanometer excitation (Spherotech product numbers SVPAK-5067-5B). 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+ nanometer with excitation at 635 nanometer.


From the 218,702 screened compounds, a total of 1629 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.

The current report assembles the list of compounds that demonstrated activity across all RGS assays.

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, AlexaFluo488-Galphao fusion protein (AF-Galphao, F/P ratio between 2 and 3, supplied by project collaborator). Bead sets are coated with individual bio-RGS proteins in BCBL buffer (PBS, pH8.0, 0.1% BSA) and incubated overnight at 4 degrees C under mild vortexing. AF-Galphao are diluted in AMF buffer(50 milliM MgCl2, 50 microM AlCl3, 50 milliM NaF, 10 milliM GDP in FB) 10 minutes before adding to the bead to form the activated AF-Galphao-GDP-AlF4-complex, which binds RGS proteins in high affinity.

The mulitplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in BCBL buffer for 20 minutes before adding the appropriate bio-RGS fusion protein. The bead sets (Spherotech product numbers SVPAK-5067-5B) have similar size (~ 5 micron diameter) and are distinguished by distance emission characteristics at 750+ nanometer with excitation at 635 nanometer. Thus, bio-RGS4 might be noncovalently coated onto red level 1 beads, bio-RGS7 onto red level 2 beads, etc. The 5 bead sets (each with bound protein) and an uncoated bead set (Scavenger beads, see below) are centrifuged separately, washed once in BCBL, followed by another wash in FB(50 milliM HEPES, 100 milliM NaCl, 0.1% Lubrol, and 0.1% BSA, pH 8.0) then diluted in FB and combined just before loading into assay plates to minimize bead-protein dissociation before the assay begins. The streptavidin-only bead control (no associated bio-RGS protein) is incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nanometer emission) of the test compounds. Bead density of all six bead sets is approximately the same.

The assay is conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 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 on each plate (Positive Control Beads). Plates are incubated under mild vortexing for 30-90 min 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 Negative Control Beads in 7 nanomolar 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 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 nanometer (FL1) and 750+ nanometer (FL9) are collected on a Cyan Flow Cytometer (Dako). Analysis is performed using time-resolved acquisition into a single data file and analysis using 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 FL9 emission distinguishes the beads coated with different proteins, and the median channel fluorescence (MCF) per bead population is calculated.

Summary and Sequence of Assays

MLSMR analysis

HTS RGS4-Galphao: AID 1415
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >50% regulation (activator or inhibitor)
# evaluated: 218,538
# active: 711

HTS RGS8-Galphao: AID 1423
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >40% regulation (activator or inhibitor)
# evaluated: 218,538
# active: 750

HTS RGS7-Galphao: AID 1439
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >50% regulation (activator or inhibitor)
# evaluated: 218,538
# active: 770

HTS RGS19-Galphao: AID 1440
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion:
# evaluated: 218,538
# active: 0

HTS RGS16-Galphao: AID 1441
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >75% regulation (activator or inhibitor), >100% regulation for compounds assessed by annotated special filter set
# evaluated: 218,538
# active: 826

HTS Counter screen for biotinylated proteins binding to avidin beads: AID 1783
Method: flow cytometry, single plex bead-based, fluorescent-biotin binding to avidin beads
Test concentration: 10 microM
Activity criterion: Percent inhibition > 50%
# evaluated: 1658
# active: 0

HTS Profiling compound fluorescence on Avidin Beads: AID 1775
Method: flow cytometry, multiplex bead-based, calibration beads
Test concentration: 10 microM
Activity criterion: kMESF > 47 (kMESF: kilo Molescules of Equivalent Soluble Fluorophore)
# evaluated: 217462
# active: 825

HTS Single point concentration confirmation for RGS19-Galphao: AID 1841
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >50% regulation (activator or inhibitor)
# evaluated: 1658
# active: 89

HTS Single point concentration confirmation for RGS4-Galphao: AID 1840
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >26% regulation (activator or inhibitor)
# evaluated: 1658
# active: 73

HTS Single point concentration confirmation for RGS16-Galphao: AID 1838
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >26% regulation (activator or inhibitor)
# evaluated: 1658
# active: 75

HTS Single point concentration confirmation for RGS7-Galphao: AID 1837
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >25% regulation (activator or inhibitor)
# evaluated: 1658
# active: 129

HTS Single point concentration confirmation for RGS8-Galphao: AID 1836
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 10 microM
Activity criterion: >25% regulation (activator or inhibitor)
# evaluated: 1658
# active: 48

HTS Dose response, multiplexed for RGS7-Galphao: AID 1871
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 30 microM to 9 nanoM in 9 dilutions
Activity criterion: EC50 < or = 30 microM
# evaluated: 185
# active: 30

HTS Dose respone, multiplexed for RGS16-Galphao: AID 1888
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 30 microM to 9 nanoM in 9 dilutions
Activity criterion: EC50 < or = 30 microM
# evaluated: 185
# active: 53

HTS Dose response, multiplexed for RGS19-Galphao: AID 1884
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 30 microM to 9 nanoM in 9 dilutions
Activity criterion: EC50 < or = 30 microM
# evaluated: 185
# active: 54

HTS Dose respone, multiplexed for RGS8-Galphao: AID 1869
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 30 microM to 9 nanoM in 9 dilutions
Activity criterion: EC50 < or = 30 microM
# evaluated: 185
# active: 50

HTS Dose response, multiplexed for RGS4-Galphao: AID 1872
Method: flow cytometry, multiplex bead-based, ligand binding competition assay
Test concentration: 30 microM to 9 nanoM in 9 dilutions
Activity criterion: EC50 < or = 30 microM
# evaluated: 185
# active: 27

AID: 2295, 2296, 2298, 2307, 2311
SAR series of compounds tested for activity on RGS4, RGS7, RGS16, RGS8, and RGS19

AID: 2826, 2827, 2828, 2829
Additional SAR series of compounds tested for activity on RGS4, RGS7, RGS16, and RGS8

Abbreviations: milliM for millimolar, microM for micromolar, nanoM for nanomolar

Grant Number: NIH R21NS057014