A High Throughput Screen for RGS Proteins Using Steady State Monitoring of Free Phosphate Formation

Abstract

G-protein coupled receptors are a diverse group that are the target of over 50% of marketed drugs. Activation of these receptors results in the exchange of bound GDP for GTP in the Gα subunit of the heterotrimeric G-protein. The Gα subunit dissociates from the β/γ subunits and both proceed to affect downstream signaling targets. The signal terminates by the hydrolysis of GTP to GDP and is temporally regulated by Regulators of G-protein Signaling (RGS) proteins that act as GTPase Activating Proteins (GAPs). This makes RGS proteins potentially desirable targets for “tuning” the effects of current therapies as well as developing novel pharmacotherapies. Current methods for evaluating RGS activity depend on laborious and/or expensive techniques. In this study we developed a simple and inexpensive assay for the steady state analysis of RGS protein GAP activity, using RGS4, RGS8 and RGS17 as models. Additionally, we report the use of RGS4 as a model for high throughput assay development. After initial setup, this assay can be conducted in a highly parallel fashion with a read time of less than 8 minutes for a 1536-well plate. The assay exhibited a robust Z-factor of 0.6 in a 1536-well plate. We conducted a pilot screen for inhibitors using a small, 2320 compound library. From this screen, 13 compounds were identified as compounds for further analysis. The successful development of this assay for high-throughput screening provides a low cost, high speed, simple method for assessing RGS protein activity.

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

“…The ultimate colorimetric complex is formed after the formation of phosphomolybdate, indicating enough time is required for full color development [23]. Hence, the staining time was optimized (Fig.…”

Regulators of G-Protein-Signaling Proteins: Negative Modulators of G-Protein-Coupled Receptor Signaling

“…RGS17 is ubiquitously expressed, from yeast to mammals but it is expressed in few tissues and organs (Bodle et al, 2013;Jung et al, 2012;Mackie and Roman, 2011;Monroy et al, 2013b;Mosakhani et al, 2013;Zhang et al, 2012a). It is expressed mainly in brain areas heavily innervated with dopamine, such as the nucleus accumbens and striatum (Stanwood et al, 2006) and high significance in the cerebellum (Larminie et al, 2004) and in the central nervous system, in the hypothalamus, midbrain, and pons-medulla (Garzon et al, 2005a).…”

Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism

“…It is generally known that GDP/GTP exchange is the rate-limiting step in multiple turnover measurements of G␣ isoforms (9 -12). Therefore, beside steady-state assays using ␥-32 P labeling (13) or malachite green (14), pre-steady-state assays are used to characterize the hydrolysis reaction of G␣ isoforms (15)(16)(17)(18). We present here for the first time single turnover measurements of G␣ i1 using time-resolved FTIR spectroscopy, an ultrasensitive method that can be applied in solution and has been successfully used for photoactivable proteins like bacteriorhodopsin (19,20), channelrhodopsin (21), and other rhodopsins (22).…”