Chemical Genetics Reveals an RGS/G-Protein Role in the Action of a Compound

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation

SBI2 HCS/HCA 3D Imaging: Best Practices and Unmet Needs Colloquium

In this, the third annual SBI2 conference, the afternoon of the opening day was dedicated to short presentations and discussions revolving around HCS/HCA 3D Imaging: Best Practices and Unmet Needs. The growing interest in conducting assays in 3D is driven by the realization that there is often a disconnect between observations made with cells grown in 2D in a tissue culture plate and biology occurring in an organism which occurs in context of other cell types and a complex extracellular matrix. This is apparent when you look at the high failure rate of early candidates in the drug discovery process. The push towards 3D assay systems is an attempt to increase clinical relevance, while at the same time preserving an acceptable level of throughput to evaluate potential clinical candidates. With growing scientific interest in 3D biology imaging-based assays are emerging as an obvious choice to measure complex phenotypes and move towards the realization and quantification of the concept of an “organ in a dish”

Amino Acid Substitutions in Mutants of the Yeast G-αq Protein Gpa1

<p>A theoretical three-dimensional structure of the yeast Gpa1 G-α protein in complex with the Ste4 protein (G-β) is shown. The position of four amino acid substitutions with phenotypes of interest is indicated by circles. Two alpha helices are indicated by yellow highlighting of the protein backbone. In higher eukaryotes, these helices are considered to form the interface with G-αq downstream effector proteins. Three mutations affecting adaptation to mating pheromone lie on this face: E355K and E364K both hyper-adapt while the M362I allele described in this work is hypo-adaptive. For reference, the position of a mutation affecting sensitivity to RGS GAP activity, G302S, is also shown.</p

Analysis of Molecular Signaling Events Controlling Muscular Contraction in C. elegans

<p>The diagram shows the molecular components of the signaling pathways downstream of the muscarinic GPCRs in <i>C. elegans</i>. Alleles of genes encoding proteins in the pathway were tested for their effect on the Egl-d phenotype caused by treatment with BMS-192364. For the pathway members indicated by stars, certain alleles altered the response to treatment with BMS-192364. Specifically, resistance to the Egl-d phenotype was conferred by two gain-of-function alleles of <i>egl-19</i> and one of <i>egl-30,</i> and by two loss-of-function alleles of <i>eat-16</i> and one of <i>goa-1</i>.</p

The Yeast <i>gpa1-M362I</i> Mutant Allele Causes a Hypo-Adaptation Phenotype

<div><p>Images show the growth of a monolayer of yeast cells around a paper disc containing alpha factor, the peptide ligand for the Ste3 GPCR. A zone of growth inhibition is visible as a “halo” around each disc.</p><p>(A) Yeast strain contains a wild-type G-αq gene <i>(GPA1)</i> and has a chromosomal deletion of the <i>SST2</i> gene, encoding an RGS protein.</p><p>(B) Yeast strain contains a wild-type G-αq gene <i>(GPA1)</i> and a chromosomal deletion of the <i>SST2</i> gene, but carries wild-type <i>SST2</i> on a plasmid.</p><p>(C) Yeast strain contains a mutant G-αq gene <i>(gpa1-M362I)</i> and has a chromosomal deletion of the <i>SST2</i> gene, encoding an RGS protein.</p><p>(D) Yeast strain contains a mutant G-αq gene <i>(gpa1-M362I)</i> and a chromosomal deletion of the <i>SST2</i> gene, but carries wild-type <i>SST2</i> on a plasmid.</p></div

Models for Mechanism of Action

<p>Four models of small-molecule action are presented. In model 1, the small-molecule acts directly and uniquely as an antagonist of G-αq. In model 2 the small-molecule acts directly and uniquely as an agonist of the RGS protein's GAP activity. In models 3 and 4 (our preferred models), the small molecule interacts with both the RGS protein and G-αq, leading to an increased affinity of RGS for the complex and/or an “abortive” complex (failure of G-αq to recycle). All models lead to reduction of the GPCR signal through the activated G-αq.</p

Effect of BMS-192364 in Combination with Other Modulators of Calcium Signaling

<div><p>The graphs display fluorescence intensity measurements for HEK293 cells preloaded with Fluo-4 then stimulated with the muscarinic GPCR agonist carbachol at 100 μM. Five baseline fluorescence measurements were taken prior to the injection of carbachol. Where indicated, BMS-195270 or BMS-192364 (100 μM) were added 15 min prior to the carbachol stimulation. The timing of carbachol addition is indicated by a black arrowhead.</p><p>(A) Where indicated, cells were pre-incubated for 15 min with the calcium channel blocker niguldipine (100 μM).</p><p>(B) Where indicated, cells were pre-incubated for 24 h with the G-protein antagonist pertussis toxin (150 ng/ml).</p><p>(C) The treated cells are overexpressing the G-αq mutant allele G188S, which is known to be insensitive to RGS GAP activity.</p></div

Effect of Small Molecules on Wild-Type and Mutant C. elegans

<div><p>(A) The gonad/vulval region of wild-type worms is shown. In the left panel, black arrows indicate the normal, organized array of early stage eggs. The right panel shows a worm treated with BMS-192364 at 0.3 mM. The white arrows indicate late stage eggs that have been retained in the gonad.</p><p>(B) Dose-response curve for BMS-192364, showing effect on egg laying in C. elegans. The percentage of worms displaying an Egl-d phenotype was determined by counting the number of “commas” contained within the animal.</p><p>(C) Quantification of the Egl-d phenotype in four C. elegans mutant strains—<i>ep271, ep272, ep273,</i> and <i>ep275—</i>that were identified in a screen for resistance to the small molecule. Black bars, no treatment. Grey bars, worms treated with BMS-192364 at 0.4 mM.</p><p>(D) Table showing identity of the affected gene in <i>C. elegans–</i>resistant mutant strains, the amino acid changes, and predicted effect on protein function.</p></div