Genome-wide screening for the G-protein-coupled receptor (GPCR) pathway-related therapeutic gene RGS19 (regulator of G protein signaling 19) in bladder cancer.

Bladder cancer is one of the most severe genitourinary cancers, causing high morbidity worldwide. However, the underlying molecular mechanism is not clear, and it is urgent to find target genes for treatment. G-protein-coupled receptors are currently a target of high interest for drug design. Thus, we aimed to identify a target gene-related to G-protein-coupled receptors for therapy. We used The Cancer Genome Atlas (TCGA) and DepMap databases to obtain the expression and clinical data of RGS19. The results showed that RGS19 was overexpressed in a wide range of tumor, especially bladder cancer. We also explored its effect on various types of cancer. High expression of RGS19 was also shown to be significantly associated with poor prognosis. Cell models were constructed for cell cycle detection. shRGS19 can halt the cell cycle at a polyploid point. RGS19 is a G-protein-coupled receptor signaling pathway-related gene with a significant effect on survival. We chose RGS19 as a therapeutic target gene in bladder cancer. The drug GSK1070916 was found to inhibit the effect of RGS19 via cell rescue experiments in vitro.

Impaired regulation of heart rate and sinoatrial node function by the parasympathetic nervous system in type 2 diabetic mice.

Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.

The Antipsychotic Drug Clozapine Suppresses the RGS4 Polyubiquitylation and Proteasomal Degradation Mediated by the Arg/N-Degron Pathway.

Although diverse antipsychotic drugs have been developed for the treatment of schizophrenia, most of their mechanisms of action remain elusive. Regulator of G-protein signaling 4 (RGS4) has been reported to be linked, both genetically and functionally, with schizophrenia and is a physiological substrate of the arginylation branch of the N-degron pathway (Arg/N-degron pathway). Here, we show that the atypical antipsychotic drug clozapine significantly inhibits proteasomal degradation of RGS4 proteins without affecting their transcriptional expression. In addition, the levels of Arg- and Phe-GFP (artificial substrates of the Arg/N-degron pathway) were significantly elevated by clozapine treatment. In silico computational model suggested that clozapine may interact with active sites of N-recognin E3 ubiquitin ligases. Accordingly, treatment with clozapine resulted in reduced polyubiquitylation of RGS4 and Arg-GFP in the test tube and in cultured cells. Clozapine attenuated the activation of downstream effectors of G protein-coupled receptor signaling, such as MEK1 and ERK1, in HEK293 and SH-SY5Y cells. Furthermore, intraperitoneal injection of clozapine into rats significantly stabilized the endogenous RGS4 protein in the prefrontal cortex. Overall, these results reveal an additional therapeutic mechanism of action of clozapine: this drug posttranslationally inhibits the degradation of Arg/N-degron substrates, including RGS4. These findings imply that modulation of protein post-translational modifications, in particular the Arg/N-degron pathway, may be a novel molecular therapeutic strategy against schizophrenia.

Regulator of G protein signaling 17 represents a novel target for treating cisplatin induced hearing loss.

Regulators of G protein signaling (RGS) accelerate the GTPase activity of G proteins to enable rapid termination of the signals triggered by G protein-coupled receptors (GPCRs). Activation of several GPCRs, including cannabinoid receptor 2 (CB2R) and adenosine A1 receptor (A1AR), protects against noise and drug-induced ototoxicity. One such drug, cisplatin, an anticancer agent used to treat various solid tumors, produces permanent hearing loss in experimental animals and in a high percentage of cancer patients who undergo treatments. In this study we show that cisplatin induces the expression of the RGS17 gene and increases the levels of RGS17 protein which contributes to a significant proportion of the hearing loss. Knockdown of RGS17 suppressed cisplatin-induced hearing loss in male Wistar rats, while overexpression of RGS17 alone produced hearing loss in vivo. Furthermore, RGS17 and CB2R negatively regulate the expression of each other. These data suggest that RGS17 mediates cisplatin ototoxicity by uncoupling cytoprotective GPCRs from their normal G protein interactions, thereby mitigating the otoprotective contributions of endogenous ligands of these receptors. Thus, RGS17 represents a novel mediator of cisplatin ototoxicity and a potential therapeutic target for treating hearing loss.

Fragment-Based Nuclear Magnetic Resonance Screen against a Regulator of G Protein Signaling Identifies a Binding "Hot Spot".

Regulator of G protein signaling (RGS) proteins have attracted attention as a result of their primary role in directing the specificity as well as the temporal and spatial aspects of G protein-coupled receptor signaling. In addition, alterations in RGS protein expression have been observed in a number of disease states, including certain cancers. In this area, RGS17 is of particular interest. It has been demonstrated that, while RGS17 is expressed primarily in the central nervous system, it has been found to be inappropriately expressed in lung, prostate, breast, cervical, and hepatocellular carcinomas. Overexpression of RGS17 leads to dysfunction in inhibitory G protein signaling and an overproduction of the intracellular second messenger cAMP, which in turn alters the transcription patterns of proteins known to promote various cancer types. Suppressing RGS17 expression with RNA interference (RNAi) has been found to decrease tumorigenesis and sufficiently prevents cancer cell migration, leading to the hypothesis that pharmacological blocking of RGS17 function could be useful in anticancer therapies. We have identified small-molecule fragments capable of binding the RGS homology (RH) domain of RGS17 by using a nuclear magnetic resonance fragment-based screening approach. By chemical shift mapping of the two-dimensional 15 N,1 H heteronuclear single quantum coherence (HSQC) spectra of the backbone-assigned 15 N-labeled RGS17-RH, we determined the fragment binding sites to be distant from the Gα interface. Thus, our study identifies a putative fragment binding site on RGS17 that was previously unknown.

Regulator of G Protein Signalling 4 (RGS4) as a Novel Target for the Treatment of Sensorineural Hearing Loss.

We and others have previously identified signalling pathways associated with the adenosine A1 receptor (A1R) as important regulators of cellular responses to injury in the cochlea. We have shown that the "post-exposure" treatment with adenosine A1R agonists confers partial protection against acoustic trauma and other forms of sensorineural hearing loss (SNHL). The aim of this study was to determine if increasing A1R responsiveness to endogenous adenosine would have the same otoprotective effect. This was achieved by pharmacological targeting of the Regulator of G protein Signalling 4 (RGS4). RGS proteins inhibit signal transduction pathways initiated by G protein-coupled receptors (GPCR) by enhancing GPCR deactivation and receptor desensitisation. A molecular complex between RGS4 and neurabin, an intracellular scaffolding protein expressed in neural and cochlear tissues, is the key negative regulator of A1R activity in the brain. In this study, Wistar rats (6-8 weeks) were exposed to traumatic noise (110 dBSPL, 8-16 kHz) for 2 h and a small molecule RGS4 inhibitor CCG-4986 was delivered intratympanically in a Poloxamer-407 gel formulation for sustained drug release 24 or 48 h after noise exposure. Intratympanic administration of CCG-4986 48 h after noise exposure attenuated noise-induced permanent auditory threshold shifts by up to 19 dB, whilst the earlier drug administration (24 h) led to even better preservation of auditory thresholds (up to 32 dB). Significant improvement of auditory thresholds and suprathreshold responses was linked to improved survival of sensorineural tissues and afferent synapses in the cochlea. Our studies thus demonstrate that intratympanic administration of CCG-4986 can rescue cochlear injury and hearing loss induced by acoustic overexposure. This research represents a novel paradigm for the treatment of various forms of SNHL based on regulation of GPCR.

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor.

BACKGROUND: Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS: Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS: We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSION: We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein α-subunits, accelerating GTP hydrolysis. Selective inhibition of RGS proteins increases G-protein activity and may provide unique tissue specificity. Thiadiazolidinones (TDZDs) are covalent inhibitors that act on cysteine residues to inhibit RGS4, RGS8, and RGS19. There is a correlation between protein flexibility and potency of inhibition by the TDZD 4-[(4- fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione (CCG-50014). In the context of a single conserved cysteine residue on the α 4 helix, RGS19 is the most flexible and most potently inhibited by CCG-50014, followed by RGS4 and RGS8. In this work, we identify residues responsible for differences in both flexibility and potency of inhibition among RGS isoforms. RGS19 lacks a charged residue on the α 4 helix that is present in RGS4 and RGS8. Introducing a negative charge at this position (L118D) increased the thermal stability of RGS19 and decreased the potency of inhibition of CCG-50014 by 8-fold. Mutations eliminating salt bridge formation in RGS8 and RGS4 decreased thermal stability in RGS8 and increased potency of inhibition of both RGS4 and RGS8 by 4- and 2-fold, respectively. Molecular dynamics simulations with an added salt bridge in RGS19 (L118D) showed reduced RGS19 flexibility. Hydrogen-deuterium exchange studies showed striking differences in flexibility in the α 4 helix of RGS4, 8, and 19 with salt bridge-modifying mutations. These results show that the α 4 salt bridge-forming residue controls flexibility in several RGS isoforms and supports a causal relationship between RGS flexibility and the potency of TDZD inhibitors. SIGNIFICANCE STATEMENT: Inhibitor potency is often viewed in relation to the static structure of a target protein binding pocket. Using both experimental and computation studies we assess determinants of dynamics and inhibitor potency for three different RGS proteins. A single salt bridge-forming residue determines differences in flexibility between RGS isoforms; mutations either increase or decrease protein motion with correlated alterations in inhibitor potency. This strongly suggests a causal relationship between RGS protein flexibility and covalent inhibitor potency.

Development of R7BP inhibitors through cross-linking coupled mass spectrometry and integrated modeling.

Protein-protein interaction (PPI) networks are known to be valuable targets for therapeutic intervention; yet the development of PPI modulators as next-generation drugs to target specific vertices, edges, and hubs has been impeded by the lack of structural information of many of the proteins and complexes involved. Building on recent advancements in cross-linking mass spectrometry (XL-MS), we describe an effective approach to obtain relevant structural data on R7BP, a master regulator of itch sensation, and its interfaces with other proteins in its network. This approach integrates XL-MS with a variety of modeling techniques to successfully develop antibody inhibitors of the R7BP and RGS7/Gß5 duplex interaction. Binding and inhibitory efficiency are studied by surface plasmon resonance spectroscopy and through an R7BP-derived dominant negative construct. This approach may have broader applications as a tool to facilitate the development of PPI modulators in the absence of crystal structures or when structural information is limited.

Role of hippocampal 5-HT1A receptors in the antidepressant-like phenotype of mice expressing RGS-insensitive Gαi2 protein.

A single base mutation in the Gαi2 protein (G184S) renders this Gα subunit insensitive to the negative modulatory effects of Regulator of G-protein Signaling (RGS) proteins. Mice expressing this RGS insensitive (RGSi) variant of Gαi2 (RGSi Gαi2) display a spontaneous antidepressant-like phenotype that is reversible by treatment with the 5-HT1A receptor (5-HT1AR) antagonist WAY100635. Here we test the hypothesis that increased activity of 5-HT1ARs in the hippocampus of RGSi Gαi2 knock-in mice is responsible for the expression of the observed antidepressant-like behavior. We administered the 5-HT1AR antagonist WAY100635 or the agonist 8-OH-DPAT via bilateral intra-hippocampal infusion cannulae and evaluated antidepressant-like behavior using the tail suspension test (TST). WAY100635 reversed the antidepressant-like phenotype of the RGSi Gαi2 knock-in mice and 8-OH-DPAT produced an antidepressant-like response in wild type mice that was blocked by systemic WAY100635. Furthermore, intra-hippocampal infusion of the RGS19/4 inhibitor CCG-203769 produced an antidepressant-like effect in female mice. Ex-vivo slice recording confirmed the 5-HT1AR-mediated decrease in hippocampal CA1 pyramidal neuron excitability was enhanced in the RGSi Gαi2 knock-in mice. There was no change in hippocampal 5-HT1AR expression as measured by ligand binding but there was a compensatory reduction in Gαi proteins. The findings demonstrate that RGS protein control of hippocampal 5-HT1AR signaling is necessary and sufficient to account for the antidepressant-like phenotype in the RGSi Gαi2 knock-in mice and that RGS proteins highly expressed in the hippocampus should be investigated as targets for novel antidepressant therapies.

Regulator of G-Protein Signaling 4 (RGS4) Controls Morphine Reward by Glutamate Receptor Activation in the Nucleus Accumbens of Mouse Brain.

Crosstalk between G-protein signaling and glutamatergic transmission within the brain reward circuits is critical for long-term emotional effects (depression and anxiety), cravings, and negative withdrawal symptoms associated with opioid addiction. A previous study showed that Regulator of G-protein signaling 4 (RGS4) may be implicated in opiate action in the nucleus accumbens (NAc). However, the mechanism of the NAc-specific RGS4 actions that induce the behavioral responses to opiates remains largely unknown. The present study used a short hairpin RNA (shRNA)-mediated knock-down of RGS4 in the NAc of the mouse brain to investigate the relationship between the activation of ionotropic glutamate receptors and RGS4 in the NAc during morphine reward. Additionally, the shRNA-mediated RGS4 knock-down was implemented in NAc/striatal primary-cultured neurons to investigate the role that striatal neurons have in the morphine-induced activation of ionotropic glutamate receptors. The results of this study show that the NAc-specific knockdown of RGS4 significantly increased the behaviors associated with morphine and did so by phosphorylation of the GluR1 (Ser831) and NR2A (Tyr1325) glutamate receptors in the NAc. Furthermore, the knock-down of RGS4 enhanced the phosphorylation of the GluR1 and NR2A glutamate receptors in the primary NAc/striatal neurons during spontaneous morphine withdrawal. These findings show a novel molecular mechanism of RGS4 in glutamatergic transmission that underlies the negative symptoms associated with morphine administration.

Suppression of RGSz1 function optimizes the actions of opioid analgesics by mechanisms that involve the Wnt/ß-catenin pathway.

Regulator of G protein signaling z1 (RGSz1), a member of the RGS family of proteins, is present in several networks expressing mu opioid receptors (MOPRs). By using genetic mouse models for global or brain region-targeted manipulations of RGSz1 expression, we demonstrated that the suppression of RGSz1 function increases the analgesic efficacy of MOPR agonists in male and female mice and delays the development of morphine tolerance while decreasing the sensitivity to rewarding and locomotor activating effects. Using biochemical assays and next-generation RNA sequencing, we identified a key role of RGSz1 in the periaqueductal gray (PAG) in morphine tolerance. Chronic morphine administration promotes RGSz1 activity in the PAG, which in turn modulates transcription mediated by the Wnt/ß-catenin signaling pathway to promote analgesic tolerance to morphine. Conversely, the suppression of RGSz1 function stabilizes Axin2-Gαz complexes near the membrane and promotes ß-catenin activation, thereby delaying the development of analgesic tolerance. These data show that the regulation of RGS complexes, particularly those involving RGSz1-Gαz, represents a promising target for optimizing the analgesic actions of opioids without increasing the risk of dependence or addiction.

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors.

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Screen Targeting Lung and Prostate Cancer Oncogene Identifies Novel Inhibitors of RGS17 and Problematic Chemical Substructures.

Regulator of G protein signaling (RGS) proteins temporally regulate heterotrimeric G protein signaling cascades elicited by G protein-coupled receptor activation and thus are essential for cell homeostasis. The dysregulation of RGS protein expression has been linked to several pathologies, spurring discovery efforts to identify small-molecule inhibitors of these proteins. Presented here are the results of a high-throughput screening (HTS) campaign targeting RGS17, an RGS protein reported to be inappropriately upregulated in several cancers. A screen of over 60,000 small molecules led to the identification of five hit compounds that inhibit the RGS17-Gαo protein-protein interaction. Chemical and biochemical characterization demonstrated that three of these hits inhibited the interaction through the decomposition of parent compound into reactive products under normal chemical library storage/usage conditions. Compound substructures susceptible to decomposition are reported and the decomposition process characterized, adding to the armamentarium of tools available to the screening field, allowing for the conservation of resources in follow-up efforts and more efficient identification of potentially decomposed compounds. Finally, analogues of one hit compound were tested, and the results establish the first ever structure-activity relationship (SAR) profile for a small-molecule inhibitor of RGS17.

Regulating the regulators: Epigenetic, transcriptional, and post-translational regulation of RGS proteins.

Regulators of G protein signaling (RGS) are a family of proteins classically known to accelerate the intrinsic GTPase activity of G proteins, which results in accelerated inactivation of heterotrimeric G proteins and inhibition of G protein coupled receptor signaling. RGS proteins play major roles in essential cellular processes, and dysregulation of RGS protein expression is implicated in multiple diseases, including cancer, cardiovascular and neurodegenerative diseases. The expression of RGS proteins is highly dynamic and is regulated by epigenetic, transcriptional and post-translational mechanisms. This review summarizes studies that report dysregulation of RGS protein expression in disease states, and presents examples of drugs that regulate RGS protein expression. Additionally, this review discusses, in detail, the transcriptional and post-transcriptional mechanisms regulating RGS protein expression, and further assesses the therapeutic potential of targeting these mechanisms. Understanding the molecular mechanisms controlling the expression of RGS proteins is essential for the development of therapeutics that indirectly modulate G protein signaling by regulating expression of RGS proteins.

Evaluation of the Selectivity and Cysteine Dependence of Inhibitors across the Regulator of G Protein-Signaling Family.

Since their discovery more than 20 years ago, regulators of G protein-signaling (RGS) proteins have received considerable attention as potential drug targets because of their ability to modulate Gα activity. Efforts to identify small molecules capable of inhibiting the protein-protein interactions between activated Gα subunits and RGS proteins have yielded a substantial number of inhibitors, especially toward the well studied RGS4. These efforts also determined that many of these small molecules inhibit the protein-protein interactions through covalent modification of cysteine residues within the RGS domain that are located distal to the Gα-binding interface. As some of these cysteine residues are highly conserved within the RGS family, many of these inhibitors display activity toward multiple RGS family members. In this work, we sought to determine the selectivity of these small-molecule inhibitors against 12 RGS proteins, as well as against the cysteine-null mutants for 10 of these proteins. Using both biochemical and cell-based methods to assess Gα-RGS complex formation and Gα enzymatic activity, we found that several previously identified RGS4 inhibitors were active against other RGS members, such as RGS14, with comparable or greater potency. Additionally, for every compound tested, activity was dependent on the presence of cysteine residues. This work defines the selectivity of commercially available RGS inhibitors and provides insight into the RGS family members for which drug discovery efforts may be most likely to succeed.

Absence of regulator of G-protein signaling 4 does not protect against dopamine neuron dysfunction and injury in the mouse 6-hydroxydopamine lesion model of Parkinson's disease.

Regulator of G-protein signaling 4 (RGS4), a member of the RGS family of proteins that inactivate G-proteins, has gained interest as a potential drug target for neurological disorders, such as epilepsy and Parkinson's disease (PD). In the case of PD, the main current options for alleviating motor symptoms are dopamine replacement therapies, which have limitations because of side effects and reduced effectiveness over the long term. Research on new nondopaminergic PD drug targets has indicated that inhibition of RGS4 could be an effective adjuvant treatment option. The effectiveness of RGS4 inhibition for an array of PD-linked functional and structural neuroprotection end points has not yet been demonstrated. Here, we use the 6-hydroxydopamine (6-OHDA) lesioning model of the nigrostriatal pathway in mice to address this question. We observe, using a battery of behavioral and pathological measures, that mice deficient for RGS4 are not protected from 6-OHDA-induced injury and show enhanced susceptibility in some measures of motor function. Our results suggest that inhibition of RGS4 as a nondopaminergic target for PD should be approached with caution.

Epigenetic regulation of RGS2 (Regulator of G-protein signaling 2) in chemoresistant ovarian cancer cells.

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.

The evolution of regulators of G protein signalling proteins as drug targets - 20 years in the making: IUPHAR Review 21.

Regulators of G protein signalling (RGS) proteins are celebrating the 20th anniversary of their discovery. The unveiling of this new family of negative regulators of G protein signalling in the mid-1990s solved a persistent conundrum in the G protein signalling field, in which the rate of deactivation of signalling cascades in vivo could not be replicated in exogenous systems. Since then, there has been tremendous advancement in the knowledge of RGS protein structure, function, regulation and their role as novel drug targets. RGS proteins play an important modulatory role through their GTPase-activating protein (GAP) activity at active, GTP-bound Gα subunits of heterotrimeric G proteins. They also possess many non-canonical functions not related to G protein signalling. Here, an update on the status of RGS proteins as drug targets is provided, highlighting advances that have led to the inclusion of RGS proteins in the IUPHAR/BPS Guide to PHARMACOLOGY database of drug targets.

A new model of nerve injury in the rat reveals a role of Regulator of G protein Signaling 4 in tactile hypersensitivity.

Tactile hypersensitivity is one of the most debilitating symptoms of neuropathic pain syndromes. Clinical studies have suggested that its presence at early postoperative stages may predict chronic (neuropathic) pain after surgery. Currently available animal models are typically associated with consistent tactile hypersensitivity and are therefore limited to distinguish between mechanisms that underlie tactile hypersensitivity as opposed to mechanisms that protect against it. In this study we have modified the rat model of spared nerve injury, restricting the surgical lesion to a single peripheral branch of the sciatic nerve. This modification reduced the prevalence of tactile hypersensitivity from nearly 100% to approximately 50%. With this model, we here also demonstrated that the Regulator of G protein Signaling 4 (RGS4) was specifically up-regulated in the lumbar dorsal root ganglia and dorsal horn of rats developing tactile hypersensitivity. Intrathecal delivery of the RGS4 inhibitor CCG63802 was found to reverse tactile hypersensitivity for a 1h period. Moreover, tactile hypersensitivity after modified spared nerve injury was most frequently persistent for at least four weeks and associated with higher reactivity of glial cells in the lumbar dorsal horn. Based on these data we suggest that this new animal model of nerve injury represents an asset in understanding divergent neuropathic pain outcomes, so far unravelling a role of RGS4 in tactile hypersensitivity. Whether this model also holds promise in the study of the transition from acute to chronic pain will have to be seen in future investigations.