qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)
G-protein-coupled receptors (GPCRs) are major targets for drug discovery. The regulator of G-protein signaling (RGS)-protein family has important roles in GPCR signal transduction. RGS proteins contain a conserved RGS-box, which is often accompanied by other signaling regulatory elements. RGS proteins accelerate the deactivation of G proteins to reduce GPCR signaling; however, some also have an more ..
BioActive Compounds: 1007
Assay Submitter: Siderovski, David P, University of North Carolina at Chapel Hill
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
NIH Grant: NS053754-01
G-protein-coupled receptors (GPCRs) are major targets for drug discovery. The regulator of G-protein signaling (RGS)-protein family has important roles in GPCR signal transduction. RGS proteins contain a conserved RGS-box, which is often accompanied by other signaling regulatory elements. RGS proteins accelerate the deactivation of G proteins to reduce GPCR signaling; however, some also have an effector function and transmit signals. Combining GPCR agonists with RGS inhibitors should potentiate responses, and could markedly increase the agonist's regional specificity. The diversity of RGS proteins with highly localized and dynamically regulated distributions in brain makes them attractive targets for pharmacotherapy of central nervous system disorders. Inhibitors of the RGS:GPCR interaction should prove useful as small molecule tools in this research field. A complex of Gai1 and fluorescently labeled G-alpha-binding peptide (the "GoLoco motif") derived from RGS12 is incubated with library members. Inhibitors of the binding are detected by a decrease in the fluorescence polarization (FP) of the fluorophore. Protein was supplied by Prof. David Siderovski, University of North Carolina at Chapel Hill.
Assay Buffer: 10 mM Tris-HCl, 150 mM NaCl, 0.1 mM GDP (prepared fresh), and 0.05% NP40 at pH 7.5.
Controls: 15 nM Rhodamine labeled peptide dispensed into columns 3 and 4 to generate negative control (full inhibition of binding, -100 % activity).
15 nM Rhodamine labeled peptide/25 nM protein in columns 1, 2, 5 - 48. Titration of unlabeled peptide control ([H]TMGEEDFFDLLAKSQSKRMDDQRVDLAG[NH2], custom synthesized and HPLC-purified by Tufts University Core Facility) from 20 mM, then 1:2 dilution, 16-point in duplicate, pin-transferred to column 2, rows 1 to 32. Column 1 is neutral.
Rhodamine-labeled peptide (Rhodamine-DEAEEFFELISKAQSNRADDQRGLLRKEDLVLPEFLR-NH2) was custom-synthesized and HPLC-purified by Invitrogen (Carlsbad, CA).
Four uL of reagents were dispensed to 1536-well Greiner black solid-bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 10 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using BODIPY for the rhodamine-labeled decapeptide probe. During dispense, reagent bottles were kept submerged into 4 deg C water bath and all liquid lines were covered with aluminum foil to minimize probe and protein degradation. All screening operations were performed on a Kalypsys robotic system (Kalypsys, Inc., San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms.
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, rgs, rgs12, regulator of G-protein signalling 12, GTPase accelerating protein
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
** Test Concentration.
Data Table (Concise)