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July 01, 2011 (Vol. 31, No. 13)

Modulation of G-Protein Coupled Receptors

Novel Approach Targets RGS Proteins Using Engineered Gα Proteins and Transcreener® GDP Assay

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    Figure 2. RGScreen Assay relies on proprietary Gα variants with altered GTPase kinetics. Normally, GDP dissociates from isolated Gα proteins very slowly so RGS GAP effects are not detectable using GTPase assays. We have mutated Gαi proteins so that GDP dissociation is no longer rate limiting, yet the proteins still serve as functional substrates in RGS protein GAP reactions. GDP is detected using the Transcreener GDP Assay.

    BellBrook Labs’ RGScreen™ is a biochemical HTS assay that detects RGS GAP catalytic activity (Figure 2). There are two key components to the approach: 1) altering the relative rates of Gα GTPase and GDP dissociation so that GDP dissociation is no longer rate limiting allows for the use of steady-state enzymatic assays for monitoring changes in Gα GTPase activity, and 2) selective immunodetection of GDP using the Transcreener® GDP Assay enables homogenous, fluorescence-based detection of Gα GTPase activity in a multiwell format. In combination, these developments enable direct detection of RGS-catalyzed stimulation of Gα GTP hydrolysis in a robust HTS format.

    We used well-characterized mutant variants of the Gαi1 and orthologous mutations from related Gα proteins to overcome the disparity between GDP dissociation and GTPase activity. The GTPase activity of the mutated Gα proteins was stimulated six- to tenfold by known interacting RGS protein, demonstrating the ability to directly measure RGS protein GAP activity using steady-state GTP hydrolysis assays.

    Importantly, there was no stimulation of GTPase activity by noninteracting RGS domains, demonstrating that the selectivity of the Gα/RGS domain interaction was not altered by the mutations. Moreover, independent binding studies confirmed that the mutated Gα proteins interacted with RGS proteins with the expected specificity.

    The double mutant strategy has thus far been successfully applied to three Gα proteins in addition to Gαi1 and also used to measure the GAP activity of four members of the RGS family. We performed a pilot screen of RGS4 protein with a 960 compound library of bioactives in 384-well format. The assay window was more than 100 mP, and the data quality was very high, resulting in a Z’ value of 0.83 and a Z factor or 0.71.

    By including control wells containing Gα but lacking RGS protein, we simultaneously counterscreened for Gα inhibitors. Of the 960 compounds in the library, 17 were initially considered hits, and 10 of these were excluded on the basis of the counterscreen resulting in an RGS4-specific hit rate of 0.7%.

    More recently, we completed 50,000 compound screens of two RGS proteins with similar assay quality statistics and hit rates.

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