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BioAssay: AID 1504

Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions.

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. ..more
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 Tested Compounds
 Tested Compounds
All(1629)
 
 
Active(1629)
 
 
 Tested Substances
 Tested Substances
All(1629)
 
 
Active(1629)
 
 
AID: 1504
Data Source: NMMLSC (UNM_RGS Multiplex Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-01-12
Modify Date: 2010-05-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1629
Related Experiments
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AIDNameTypeComment
503Compound Screen Assay, Human RGS18Screeningdepositor-specified cross reference: Compound Screen Assay, Human RGS18##Source: SGCOxCompounds (RGS18 Compound Screen)
879qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Green Fluorophore)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of RGS GAP Activity (Green Fluorophore)Source: NIH Chemical Genomics Cente
880qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of RGS GAP Activity (Red Fluorophore)Source: NIH Chemical Genomics Center(
1415Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.Screeningdepositor-specified cross reference: HTS RGS4-Galphao
1423Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao.Screeningdepositor-specified cross reference: HTS RGS8-Galphao
1439Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao.Screeningdepositor-specified cross reference: HTS RGS7-Galphao
1440Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao.Screeningdepositor-specified cross reference: HTS RGS19-Galphao
1441Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao.Screeningdepositor-specified cross reference: HTS RGS16-Galphao
1775Profiling compound fluorescence on Avidin Beads with 488 nm excitation and 530 nm emissionOtherdepositor-specified cross reference: HTS Profiling compound fluorescence on Avidin Beads
1783Counter screen for biotinylated proteins binding to avidin beadsScreeningdepositor-specified cross reference: HTS Counter screen for biotinylated proteins binding to avidin beads
1836Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao.Screeningdepositor-specified cross reference: HTS Single point concentration confirmation for RGS8-Galphao
1837Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao.Screeningdepositor-specified cross reference: HTS Single point concentration confirmation for RGS7-Galphao
1838Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao.Screeningdepositor-specified cross reference: HTS Single point concentration confirmation for RGS16-Galphao
1840Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.Screeningdepositor-specified cross reference: HTS Single point concentration confirmation for RGS4-Galphao
1841Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao.Screeningdepositor-specified cross reference: HTS Single point concentration confirmation for RGS19-Galphao
1869Dose respone, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao.Confirmatorydepositor-specified cross reference: HTS Dose respone, multiplexed for RGS8-Galphao
1871Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao.Confirmatorydepositor-specified cross reference: HTS Dose response, multiplexed for RGS7-Galphao
1872Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.Confirmatorydepositor-specified cross reference: HTS Dose response, multiplexed for RGS4-Galphao
1884Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao.Confirmatorydepositor-specified cross reference: HTS Dose response, multiplexed for RGS19-Galphao
1888Dose respone, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao.Confirmatorydepositor-specified cross reference: HTS Dose respone, multiplexed for RGS16-Galphao
1993FPR-induced VLA-4 exposure in U937 cellsScreeningdepositor-specified cross reference
2295Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao for SAR compoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2296Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2298Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2307Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2311Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2826Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao with additional round of SAR compounds.Confirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2827Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao with additional round of SAR compoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2828Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao with additional round of SAR compoundsConfirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
2829Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao with additional round of SAR compounds.Confirmatorydepositor-specified cross reference: Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protei
Description:
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.
Protocol
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
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_RGS4Textual annotation of whether the substance is an inhibitor or an activator for RGS4.String
2RESPONSE_VALUE_RGS4 (10μM**)Percent response for RGS4 calculated with linear normalized Negative Controls and Positive Controls, a value of 100 represents the compound has no impact to the target-Gao protein interaction.Float%
3Z_Prime_Value_RGS4Plate-based Z Prime Value calculated using the mean of all Positive Controls of the plate and Negative Controls as an indication of quality and consistency of the assay.Float
4ACTIVITY_RGS7Textual annotation of whether the substance is an inhibitor or an activator for RGS7.String
5RESPONSE_VALUE_RGS7 (10μM**)Percent response for RGS7 calculated with linear normalized Negative Controls and Positive Controls, a value of 100 represents the compound has no impact to the target-Gao protein interaction.Float%
6Z_Prime_Value_RGS7Plate-based Z Prime Value calculated using the mean of all Positive Controls of the plate and Negative Controls as an indication of quality and consistency of the assay.Float
7ACTIVITY_RGS8Textual annotation of whether the substance is an inhibitor or an activator for RGS8.String
8RESPONSE_VALUE_RGS8 (10μM**)Percent response for RGS8 calculated with linear normalized Negative Controls and Positive Controls, a value of 100 represents the compound has no impact to the target-Gao protein interaction.Float%
9Z_Prime_Value_RGS8Plate-based Z Prime Value calculated using the mean of all Positive Controls of the plate and Negative Controls as an indication of quality and consistency of the assay.Float
10ACTIVITY_RGS16Textual annotation of whether the substance is an inhibitor or an activator for RGS16.String
11RESPONSE_VALUE_RGS16 (10μM**)Percent response for RGS16 calculated with linear normalized Negative Controls and Positive Controls, a value of 100 represents the compound has no impact to the target-Gao protein interaction.Float%
12Z_Prime_Value_RGS16Plate-based Z Prime Value calculated using the mean of all Positive Controls of the plate and Negative Controls as an indication of quality and consistency of the assay.Float
13ACTIVITY_RGS19Textual annotation of whether the substance is an inhibitor or an activator for RGS19.String
14RESPONSE_VALUE_RGS19 (10μM**)Percent response for RGS19 calculated with linear normalized Negative Controls and Positive Controls, a value of 100 represents the compound has no impact to the target-Gao protein interaction.Float%
15Z_Prime_Value_RGS19Plate-based Z Prime Value calculated using the mean of all Positive Controls of the plate and Negative Controls as an indication of quality and consistency of the assay.Float

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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