Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein ␣-subunits (G␣) and thus shorten the time course and reduce the magnitude of G-protein ␣-and ␤␥-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess G␣-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1,sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/G␣ o binding with 3 to 5 M potency. It binds to RGS4, inhibits RGS4 stimulation of G␣ o GTPase activity in vitro, and prevents RGS4 regulation of -opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/G␣ protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

Converging lines of evidence indicate that elevations in synaptic dopamine levels play a pivotal role in the reinforcing effects of cocaine, which are associated with its abuse liability. This evidence has led to the exploration of dopamine receptor blockers as pharmacotherapy for cocaine addiction. While neither D1 nor D2 receptor antagonists have proven effective, medications acting at two other potential targets, D3 and D4 receptors, have yet to be explored for this indication in the clinic. Buspirone, a 5-HT1A partial agonist approved for the treatment of anxiety, has been reported to also bind with high affinity to D3 and D4 receptors. In view of this biochemical profile, the present research was conducted to examine both the functional effects of buspirone on these receptors and, in non-human primates, its ability to modify the reinforcing effects of i.v. cocaine in a behaviourally selective manner. Radioligand binding studies confirmed that buspirone binds with high affinity to recombinant human D3 and D4 receptors (~98 and ~29 nM respectively). Live cell functional assays also revealed that buspirone, and its metabolites, function as antagonists at both D3 and D4 receptors. In behavioural studies, doses of buspirone that had inconsistent effects on food-maintained responding (0.1 or 0.3 mg/kg i.m.) produced a marked downward shift in the dose–effect function for cocaine-maintained behaviour, reflecting substantial decreases in self-administration of one or more unit doses of i.v. cocaine in each subject. These results support the further evaluation of buspirone as a candidate medication for the management of cocaine addiction.

N-(3-fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)-butyl)-aryl carboxamides were prepared and evaluated for binding and function at dopamine D3 (D3R) and D2 receptors (D2R). In this series, we discovered some of the most D3R selective compounds reported to date, (e.g. 8d and 8j >1000-fold D3R-selective over D2R.) In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized and while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced binding affinities at D3R by >100-fold (e.g. D3RKi for 15b = 393 v. for 8j = 2.6 nM) resulting in compounds with significantly reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine class of compounds and further reveals a point of separation between structure-activity relationships at D3R and D2R.

Hydrolysis of fluorescent GTP analogues BODIPY® FL guanosine 5-O-(thiotriphosphate) (BGTP␥S) and BODIPY® FL GTP (BGTP)by G␣ i1 and G␣ o was characterized using on-line capillary electrophoresis laser-induced fluorescence assays in order that changes in substrate, substrate-enzyme complex, and product could be monitored separately. Nanomolar RGS increased the rate of enzyme product formation (BODIPY® FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudosingle-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38 -81% by 40 M YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that G␣ subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTP␥S hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe. G proteins1 are involved in many important physiological processes. Ligands binding to cell surface receptors signaling through G protein pathways (like G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs)) induce the G protein to exchange GDP for GTP, activating the protein. Activated G protein can transmit signals to downstream effectors such as adenylyl cyclase, phospholipases, ion channels, or cGMP phosphodiesterase, which ultimately result in cellular responses initiated by the extracellular ligand stimulus. Signaling is ceased by the hydrolysis of GTP to GDP via the intrinsic GTPase activity of the G protein (reviewed in Refs. 1 and 2). G protein deactivation can be accelerated by interactions with GTPase-activating proteins (GAPs) such as regulators of G protein signaling (RGS) (3, 4).Most studies of G protein-mediated signaling have relied on utilization of guanine nucleotide analogues that both behave similarly to the native species and have been modified such that they can be sensitively detected. The most common modification involves insertion of radiolabeled isotopes of phosphorous or sulfur to yield [␥-32 P]GTP and [␥-35 S]GTP␥S, which can then be used in filter-binding assays to measure GTPase activity and the extent of nucleotide binding, respectively. More recently, fluorescent guanine nucleotide derivatives N-methyl-3Ј-O-anthranoyl (MANT) and BODIPY® have been developed (5, 6), allowing for G protein assays with greater sensitivity than previous fluorescence assays that relied on detection of changes in intrinisic tryptophan fluorescence. A number of studies have reported on use of these fluorescent GTP derivatives with low molecular weight, Ras-like G proteins. In general, the binding affinities and rate constants reported for MANT analogues of GTP and GDP are within a factor of 2 of those for unmodified guanine nucleotides (7), rendering these probes useful for studies of basal and GAP-stimulated GTPase activity (8 -13) and interactions with guanine nucleotide exchange factors (14...

Regulators of G-protein signaling (RGS) accelerate guanine triphosphate hydrolysis by Ga-subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design of a constrained peptide inhibitor of RGS4 (1: Ac-ValLys-[Cys-Thr-Gly-Ile-Cys]-Glu-NH 2 , S-S) based on the structure of the Gai switch 1 region but its mechanism of action was not established. In the present study, we show that 1 inhibits RGS4 by mimicking and competing for binding with the switch 1 region of Gai and that peptide 1 shows selectivity for RGS4 and RGS8 versus RGS7. Structure-activity relationships of analogs related to 1 are described that illustrate key features for RGS inhibition. Finally, we demonstrate activity of the methylene dithioether-bridged peptide inhibitor, 2, to modulate muscarinic receptor-regulated potassium currents in atrial myocytes. These data support the proposed mechanism of action of peptide RGS inhibitors, demonstrate their action in native cells, and provide a starting point for the design of RGS inhibitor drugs. When activated by an agonist, a G protein-coupled receptor (GPCR) stimulates exchange of guanine triphosphate (GTP) for guanine diphosphate (GDP) on the Ga-subunit of the G protein, which then undergoes an activating conformational change that allows it and its associated bc-subunit to interact with effector proteins (1). The Ga-subunit inactivates itself by hydrolyzing GTP to GDP followed by reassociation with Gbc. Regulators of G-protein signaling (RGS) proteins are GTPase accelerating proteins (GAPs) for Ga-subunits (1). They bind to the activated Ga protein, stabilize the transition state for GTP hydrolysis without directly interacting with the nucleotide (2), accelerate GTP hydrolysis and inactivation of the G protein, and inhibit cell responses to GPCR signaling.There are 20 classical RGS proteins and at least 16 RGS homology (RH) domains identified in the human genome (3). The RGS proteins have some Ga specificity (1), some receptor specificity (4-7), and unique expression patterns (8-10). Because of this, an RGS inhibitor could selectively potentiate GPCR signaling in specific tissues or brain regions (11,12). Many disease states have been attributed to defects in cell signaling including Parkinson's disease and Alzheimer's disease (13). An RGS inhibitor that could, for example, increase dopamine or acetylcholine responses in specific brain regions may have significant therapeutic potential on its own or might enhance effects of existing agonists in a tissue-specific manner to reduce side-effects (11,12). RGS proteins have also been proposed as therapeutic targets in the treatment of diabetes (14), opioid tolerance (3), heart failure (15), asthma (16), and cancer (17).The RGS proteins are divided into several families based on the homology of the 120 amino acid RGS domain as well as the presence or absence of other domains (1)....

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The D3 dopamine receptor represents an important target in drug addiction in that reducing receptor activity may attenuate the self-administration of drugs and/or disrupt drug or cue-induced relapse. Medicinal chemistry efforts have led to the development of D3 preferring antagonists and partial agonists that are >100-fold selective vs. the closely related D2 receptor, as best exemplified by extended-length 4-phenylpiperazine derivatives. Based on the D3 receptor crystal structure, these molecules are known to dock to two sites on the receptor where the 4-phenylpiperazine moiety binds to the orthosteric site and an extended aryl amide moiety docks to a secondary binding pocket. The bivalent nature of the receptor binding of these compounds is believed to contribute to their D3 selectivity. In this study, we examined if such compounds might also be “bitopic” such that their aryl amide moieties act as allosteric modulators to further enhance the affinities of the full-length molecules for the receptor. First, we deconstructed several extended-length D3-selective ligands into fragments, termed “synthons”, representing either orthosteric or secondary aryl amide pharmacophores and investigated their effects on D3 receptor binding and function. The orthosteric synthons were found to inhibit radioligand binding and to antagonize dopamine activation of the D3 receptor, albeit with lower affinities than the full-length compounds. Notably, the aryl amide-based synthons had no effect on the affinities or potencies of the orthosteric synthons, nor did they have any effect on receptor activation by dopamine. Additionally, pharmacological investigation of the full-length D3-selective antagonists revealed that these compounds interacted with the D3 receptor in a purely competitive manner. Our data further support that the 4-phenylpiperazine D3-selective antagonists are bivalent and that their enhanced affinity for the D3 receptor is due to binding at both the orthosteric site as well as a secondary binding pocket. Importantly, however, their interactions at the secondary site do not allosterically modulate their binding to the orthosteric site.

J. Neurochem. (2009) 112, 1026–1034. Abstract Regulators of G protein signaling (RGS) proteins act as GTPase‐accelerating protein to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu‐opioid signaling, C6 cells stably expressing a mu‐opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu‐opioid inhibition of forskolin‐stimulated adenylyl cyclase by [d‐Ala2,N‐Me‐Phe4,Gly‐ol]‐enkephalin (DAMGO), a mu‐specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a twofold loss in DAMGO potency but had no effect in cells expressing RGS‐insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 as a result of weak physical association with Gαi2 and minimal GTPase‐accelerating protein activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein‐coupled receptor signaling.