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1.  Reversible Inhibitors of Regulators of G-protein Signaling Identified in a High-throughput Cell-based Calcium Signaling Assay 
Cellular signalling  2013;25(12):10.1016/j.cellsig.2013.09.007.
Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson’s disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with Doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1,365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100 μM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.
doi:10.1016/j.cellsig.2013.09.007
PMCID: PMC3848259  PMID: 24041654
G-protein coupled receptors; M3 muscarinic acetylcholine receptor; Regulator of G-protein signaling; Small molecule inhibitor; High-throughput screen
2.  Conformational Dynamics of a Regulator of G-protein Signaling Protein Reveals a Mechanism of Allosteric Inhibition by a Small Molecule 
ACS chemical biology  2013;8(12):10.1021/cb400568g.
Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 which covalently binds to cysteine residues on RGS4. We apply temperature-accelerated molecular dynamics (TAMD) to carry out enhanced conformational sampling of apo RGS4 structures, and consistently find that the α5-α6 helix pair of RGS4 can spontaneously span open-like conformations, allowing binding of CCG-50014 to the buried side-chain of Cys95. Both NMR experiments and MD simulations reveal chemical shift perturbations in residues in the vicinity of inhibitor binding site as well as in the RGS4-Gα binding interface. Consistent with a loss of G-protein binding, GAP activity, and allosteric mechanism of action of CCG-50014, our simulations of the RGS4-Gα complex in the presence of inhibitor suggest a relatively unstable protein-protein interaction. These results have potential implications for understanding how the conformational dynamics among RGS proteins may play a key role in the sensitivity of inhibitors.
doi:10.1021/cb400568g
PMCID: PMC3876963  PMID: 24093330
3.  Microfluidic interrogation and mathematical modeling of multi-regime calcium signaling dynamics 
Through microfluidic interrogation we analyzed real-time calcium responses of HEK293 cells stimulated with short pulses of the M3 muscarinic receptor ligand carbachol in two different concentration regimes. Lower ligand concentrations elicit oscillatory calcium signals while higher concentrations trigger a rapid rise that eventually settles down at a steady-state slightly above pre-stimulus levels, referred to as an acute signal. Cells were periodically pulsed with carbachol at these two concentration regimes using a custom-made microfluidic platform, and the resulting calcium signals were measured with a single fluorescent readout. Pulsed stimulations at these two concentration regimes resulted in multiple types of response patterns that each delivered complementary information about the M3 muscarinic receptor signaling pathway. These multiple types of calcium response patterns enabled development of a comprehensive mathematical model of multi-regime calcium signaling. The resulting model suggests that dephosphorylation of deactivated receptors is rate limiting for recovery of calcium signals in the acute regime (high ligand concentration), while calcium replenishment and IP3 production determine signal recovery in the oscillatory regime (low ligand concentration). This study not only provides mechanistic insight into multi-regime signaling of the M3 muscarinic receptor pathway, but also provides a general strategy for analyzing multi-regime pathways using only one fluorescent readout.
doi:10.1039/c3ib40032h
PMCID: PMC3704220  PMID: 23732791
4.  Optimization of Novel Nipecotic Bis(amide) Inhibitors of the Rho/MKL1/SRF Transcriptional Pathway as Potential Anti-metastasis Agents 
CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthesis of conformationally restricted analogs (e.g. 2) with improved selectivity for inhibiting invasion vs acute cytotoxicity. In this study we conducted a survey of aromatic substitution with the goal of improving physicochemical parameters (e.g. ClogP, MW) for future efficacy studies in vivo. Two new compounds were identified that attenuated cytotoxicity even further, and were 4-fold more potent than 2 at inhibiting PC-3 cell migration in a scratch wound assay. One of these (8a, CCG-203971, IC50 = 4.2 μM) was well tolerated in mice for 5 days at 100 mg/kg/day i.p., and was able to achieve plasma levels exceeding the migration IC50 for up to 3 hours.
doi:10.1016/j.bmcl.2013.04.080
PMCID: PMC3707396  PMID: 23707258
5.  Conditional disruption of interactions between Gαi2 and regulator of G protein signaling (RGS) proteins protects the heart from ischemic injury 
Background
Regulator of G protein signaling (RGS) proteins suppress G protein coupled receptor signaling by catalyzing the hydrolysis of Gα-bound guanine nucleotide triphosphate. Transgenic mice in which RGS-mediated regulation of Gαi2 is lost (RGS insensitive Gαi2G184S) exhibit beneficial (protection against ischemic injury) and detrimental (enhanced fibrosis) cardiac phenotypes. This mouse model has revealed the physiological significance of RGS/Gαi2 interactions. Previous studies of the Gαi2G184S mutation used mice that express this mutant protein throughout their lives. Thus, it is unclear whether these phenotypes result from chronic or acute Gαi2G184S expression. We addressed this issue by developing mice that conditionally express Gαi2G184S.
Methods
Mice that conditionally express RGS insensitive Gαi2G184S were generated using a floxed minigene strategy. Conditional expression of Gαi2G184S was characterized by reverse transcription polymerase chain reaction and by enhancement of agonist-induced inhibition of cAMP production in isolated cardiac fibroblasts. The impact of conditional RGS insensitive Gαi2G184S expression on ischemic injury was assessed by measuring contractile recovery and infarct sizes in isolated hearts subjected to 30 min ischemia and 2 hours reperfusion.
Results
We demonstrate tamoxifen-dependent expression of Gαi2G184S, enhanced inhibition of cAMP production, and cardioprotection from ischemic injury in hearts conditionally expressing Gαi2G184S. Thus the cardioprotective phenotype previously reported in mice expressing Gαi2G184S does not require embryonic or chronic Gαi2G184S expression. Rather, cardioprotection occurs following acute (days rather than months) expression of Gαi2G184S.
Conclusions
These data suggest that RGS proteins might provide new therapeutic targets to protect the heart from ischemic injury. We anticipate that this model will be valuable for understanding the time course (chronic versus acute) and mechanisms of other phenotypic changes that occur following disruption of interactions between Gαi2 and RGS proteins.
doi:10.1186/2050-6511-15-29
PMCID: PMC4059092  PMID: 24899231
G protein coupled receptors; Ischemia-reperfusion; Cre-LoxP; Mutation; cAMP inhibition; Regulator of G protein signaling; RGS
6.  Gain-of-function mutation in Gnao1: A murine model of epileptiform encephalopathy (EIEE17)? 
Mammalian Genome  2014;25(5-6):202-210.
G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gαo, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1+/G184S) that prevents Go turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41–70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00335-014-9509-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00335-014-9509-z
PMCID: PMC4042023  PMID: 24700286
7.  Toll-like receptor-induced inflammatory cytokines are suppressed by Gain of function or overexpression of Gαi2 protein 
Inflammation  2012;35(5):1611-1617.
Previous studies have implicated a role of Gαi proteins as co-regulators of Toll –like receptor (TLR) activation. These studies largely derived from examining the effect of Gαi protein inhibitors or genetic deletion of Gαi proteins. However the effect of increased Gαi protein function or Gαi protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of Gαi proteins suppresses TLR2 and TLR4 -induced inflammatory cytokines. Novel transgenic mice with genomic “knock-in” of a Regulator of G-protein Signaling (RGS)-insensitive Gnai2 allele (Gαi2 G184S/G184S; GS/GS) were employed. These mice express essentially normal levels of Gαi2 protein, however the Gαi2 is insensitive to its negative regulator RGS thus rendering more sustained Gαi2 protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress Gαi2 protein (Raw Gαi2). Peritoneal macrophages, splenocytes and mouse embryonic fibroblasts (MEF) were isolated from WT and GS/GS mice and were stimulated with LPS, Pam3CSK4 or Poly (I:C). We also subjected WT and GS/GS mice to endotoxic shock (LPS 25mg/kg i.p.) and plasma TNFα and IL-6 production were determined. We found that in vitro LPS and Pam3CSK4 induced TNFα and IL-6 production are decreased in macrophages from GS/GS mice compared with WT mice (p<0.05). In vitro LPS and Pam3CSK4 induced IL-6 production in splenocytes and in vivo LPS induced IL-6 were suppressed in GS/GS mice. Poly (I:C) induced TNFα and IL-6 in vitro demonstrated no difference between GS/GS mice and WT mice. LPS induced IL-6 production was inhibited in MEFs from GS/GS mice similarly to macrophage and splenocytes. In parallel studies, Raw Gαi2 cells also exhibit decreased TNFα and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that Gαi2 proteins are novel negative regulators of TLR activation.
doi:10.1007/s10753-012-9476-z
PMCID: PMC3563060  PMID: 22581266
Gαi protein; TLR signaling; LPS; endotoxemia; inflammatory cytokines
8.  A nanomolar potency small molecule inhibitor of Regulator of G protein Signaling (RGS) proteins† 
Biochemistry  2011;50(15):3181-3192.
Regulators of G-Protein signaling (RGS) proteins are potent negative modulators of signal transduction through G-Protein coupled receptors. They function by binding to activated (GTP-bound) Gα subunits and accelerating the rate of GTP hydrolysis. Modulation of RGS activity by small molecules is an attractive mechanism to fine-tune GPCR signaling for therapeutic and research purposes. Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC50 for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3,000 fold higher than those required to inhibit RGS4 function. It is also >1,000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-ethylmaleimide or iodoacetamide. Analysis of the cysteine reactivity of the compound shows that compound binding to Cys107 in RGS8 inhibits Gα- binding in a manner that can be reversed by cleavage of the compound-RGS disulfide bond. If the compound reacts with Cys160 in RGS8, the adduct induces RGS denaturation and activity cannot be restored by compound removal. The high potency and good selectivity of CCG-50014 make it a useful tool for studying the functional roles of RGS4.
doi:10.1021/bi1019622
PMCID: PMC3090679  PMID: 21329361
RGS protein; Small molecule protein-protein interaction inhibitor; GPCR
9.  Differential Control of Opioid Antinociception to Thermal Stimuli in a Knock-In Mouse Expressing Regulator of G-Protein Signaling-Insensitive Gαo Protein 
Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the μ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, GαoG184S (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.
doi:10.1523/JNEUROSCI.5470-12.2013
PMCID: PMC3740968  PMID: 23467353
10.  Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions 
A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided.
doi:10.1002/0471142956.cy1311s51
PMCID: PMC2849137  PMID: 20069525
RGS; G protein; Protein-Protein Interaction; FCPIA; High Throughput Screening; Multiplexing
11.  The Loss of RGS Protein-Gαi2 Interactions Results in Markedly Impaired Mouse Neutrophil Trafficking to Inflammatory Sites 
Molecular and Cellular Biology  2012;32(22):4561-4571.
Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gαi subunits to exchange GDP for GTP. By limiting the duration that Gαi subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gαi2 interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gαi activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.
doi:10.1128/MCB.00651-12
PMCID: PMC3486189  PMID: 22966200
12.  Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound Library 
Chemical biology & drug design  2008;72(2):111-119.
Regulators of G Protein Signaling (RGS) accelerate GTP hydrolysis by Gα 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, mechanistic evaluation and structure-activity relationships (SAR) of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH2, S-S) (Roof, et al. Chem Biol Drug Des 2006; 67:266-274). Using a focused one-bead, one-compound (OBOC) peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2, (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated Gαo GTPase activity at 25–50 μM. They selectively inhibit RGS4 but not RGS7, RGS16 and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.
doi:10.1111/j.1747-0285.2008.00687.x
PMCID: PMC2917810  PMID: 18637987
One-Bead; One-Compound Library (OBOC); Focused library; Regulators of G-Protein Signaling (RGS); Protein-protein interaction (PPI) inhibitors; Structure-activity relationship (SAR)
13.  Polyplexed FCPIA (Flow Cytometry Protein Interaction Assay): A Novel High Throughput Screening Paradigm For RGS Protein Inhibitors 
Journal of biomolecular screening  2009;14(6):610-619.
Intracellular signaling cascades are a series of regulated protein-protein interactions that may provide a number of targets for potential drug discovery. Here, we examine the interaction of Regulators of G protein signaling (RGS) proteins with the G protein Gαo, using a flow cytometry protein interaction assay (FCPIA). FCPIA accurately measures nanomolar binding constants of this protein-protein interaction, and has been used in high throughput screening. This report focuses on five RGS proteins (4, 6, 7, 8 and 16). In order to increase the content of screens, we assessed high throughput screening of these RGS proteins in multiplex, by establishing binding constants of each RGS with Gαo in isolation, and then in a multiplex format with five RGS proteins present. In order to use this methodology as a higher-content multiplex protein-protein interaction screen, we established Z' factor values for RGS proteins in multiplex of 0.73 to 0.92, indicating this method is suitable for screening using FCPIA. To increase throughput, we also compressed a set of 8,000 compounds by combining 4 compounds in a single assay well. Subsequent deconvolution of the compounds mixtures verified the identification of active compounds at specific RGS targets in our mixtures using the polyplexed FCPIA method.
doi:10.1177/1087057109336590
PMCID: PMC2908316  PMID: 19531661
G protein; RGS; Flow Cytometry; FCPIA; High throughput screening
14.  MScreen: An Integrated Compound Management and High Throughput Screening (HTS) Data Storage and Analysis System 
Journal of biomolecular screening  2012;17(8):1080-1087.
High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open information environment which enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up.
doi:10.1177/1087057112450186
PMCID: PMC3600606  PMID: 22706349
chemoinformatics; data analysis software; open source; high-throughput screening
15.  High-Throughput Screening for Small Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay 
Journal of biomolecular screening  2009;14(2):161-172.
Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
doi:10.1177/1087057108328761
PMCID: PMC2698131  PMID: 19196702
high-throughput screening; fluorescence polarization; RhoGEF; RhoA; LARG; Drug Discovery
16.  Small Molecule Inhibitors of Regulator of G Protein Signalling (RGS) Proteins 
ACS medicinal chemistry letters  2012;3(2):146-150.
Recently regulators of G protein signalling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of GPCRs, the target of so many drugs. Inhibitors of RGS proteins must block a protein-protein interaction (RGS-Gα), but also be cell and, depending on the therapeutic target, blood brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay and this has now been optimised for activity, selectivity and solubility. The newly developed ligands (11b, 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilisation activity of the lead 1a and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.
doi:10.1021/ml200263y
PMCID: PMC3285107  PMID: 22368763
RGS4; RGS protein; thiadiazolidinone; GPCR; protein-protein interaction
17.  Small Molecule Inhibitors of Regulators of G Protein Signaling (RGS) Proteins 
ACS Medicinal Chemistry Letters  2011;3(2):146-150.
Recently, regulators of G protein signaling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of G protein-coupled receptors, the target of so many drugs. Inhibitors of RGS proteins must block a protein–protein interaction (RGS-Gα) but also be cell and, depending on the therapeutic target, blood–brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay, and this has now been optimized for activity, selectivity, and solubility. The newly developed ligands (11b and 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilization activity of the lead 1a, and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.
doi:10.1021/ml200263y
PMCID: PMC3285107  PMID: 22368763
RGS4; RGS protein; thiadiazolidinone; GPCR; protein−protein interaction
18.  And the winner is … RGS4! 
Circulation research  2008;103(5):444-446.
doi:10.1161/CIRCRESAHA.108.183384
PMCID: PMC2712674  PMID: 18757831
Arrhythmia; Automaticity; Mouse mutants; Potassium channels; G proteins
19.  Gαi2-mediated protection from ischaemic injury is modulated by endogenous RGS proteins in the mouse heart 
Cardiovascular Research  2011;91(1):45-52.
Aims
Regulator of G protein signalling (RGS) proteins act as molecular ‘off switches’ that terminate G protein signalling by catalyzing the hydrolysis of Gα-bound GTP to GDP. Many different Gαi-coupled receptors have been implicated in the cardioprotective effects of ischaemic preconditioning. However, the role of RGS proteins in modulating cardioprotection has not been previously investigated. We used mice that were homozygous (GS/GS) or heterozygous (GS/+) for a mutation in Gαi2 rendering it RGS-insensitive (G184S) to determine whether interactions between endogenous RGS proteins and Gαi2 modulate Gαi-mediated protection from ischaemic injury.
Methods and results
Langendorff-perfused mouse hearts were subjected to 30 min global ischaemia and 2 h reperfusion. Infarcts in GS/GS (14.5% of area at risk) and GS/+ (22.6% of AAR) hearts were significantly smaller than those of +/+ hearts (37.2% of AAR) and recovery of contractile function was significantly enhanced in GS/GS and GS/+ hearts compared with +/+ hearts. The cardioprotective phenotype was not reversed by wortmannin or U0126 but was reversed by 5-hydroxydecanoic acid and HMR 1098, indicating that RGS-insensitive Gαi2 protects the heart through a mechanism that requires functional ATP-dependent potassium channels but does not require acute activation of extracellular-regulated kinase or Akt signalling pathways.
Conclusions
This is the first study to demonstrate that Gαi2-mediated cardioprotection is suppressed by RGS proteins. These data suggest that RGS proteins may provide novel therapeutic targets to protect the heart from ischaemic injury.
doi:10.1093/cvr/cvr054
PMCID: PMC3112020  PMID: 21349876
Ischaemic preconditioning; Ischaemia reperfusion injury; Regulator of G protein signalling (RGS); Gi2
20.  Complementary Cell-Based High Throughput Screens Identify Novel Modulators of the Unfolded Protein Response 
Journal of Biomolecular Screening  2011;16(8):825-835.
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
doi:10.1177/1087057111414893
PMCID: PMC3374590  PMID: 21844328
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
21.  Gα subunit coordinates with ephrin-B to balance self-renewal and differentiation in neural progenitor cells 
Stem Cells (Dayton, Ohio)  2010;28(9):1581-1589.
Proper development of the mammalian brain requires that neural progenitor cells balance self-renewal and differentiation under precise temporal and spatial regulation, but the underlying mechanisms are not well understood. In this study, we identify Gα subunit as a positive regulator of mammalian neurogenesis, working with the RGS-mediated ephrin-B signaling pathway as two opposing forces to maintain a balance between self-renewal and differentiation in the developing mouse cerebral cortex. Multiple Gαi subunits are expressed by cortical neural progenitor cells during the course of cortical neurogenesis. Activation of Gαi signaling, through in utero electroporation mediated expression of wild-type and constitutively active Gαi subunits, counteracts the function of ephrin-B in cortical neural progenitors to induce differentiation. Genetic knock-in of an RGS-insensitive G184SGαi2 causes early cell cycle exit and a reduction of cortical neural progenitor cells and leads to a defect in the production of late born cortical neurons, similar to what is observed in mutant mice with deficiency in ephrin-B reverse signaling pathway. This study reveals a role of Gα subunit in mammalian neurogenesis and uncovers a developmental mechanism, coordinated by the Gα and ephrin-B signaling pathways, for control of the balance between self-renewal and differentiation in neural progenitor cells.
doi:10.1002/stem.474
PMCID: PMC3265139  PMID: 20629171
Gα subunit; Ephrin-B/RGS signaling; neural progenitor cells; cortical neurogenesis; self-renewal and differentiation
22.  Isoflurane-Induced Changes in Righting Response and Breathing are Modulated by RGS Proteins 
Anesthesia and analgesia  2009;109(5):1500-1505.
Background
Recent evidence suggests that G protein coupled receptors, especially those linked to Gαi, contribute to the mechanisms of anesthetic action. Regulator of G protein signaling (RGS) proteins bind to activated Gαi and inhibit its signal transduction. Genomic knock-in mice with an RGS-insensitive Gαi2 G184S (Gαi2 GS) allele exhibit enhanced Gαi2 signaling and provide a novel approach for investigating the role of Gαi2 signaling and RGS proteins in general anesthesia.
Methods
Homozygous Gαi2 GS/GS and wild type (WT) mice were anesthetized with isoflurane and time (s) to loss and resumption of righting response was quantified. During recovery from isoflurane anesthesia breathing was quantified in a plethysmography chamber for both lines of mice.
Results
Gαi2 GS/GS mice required significantly less time for loss of righting and significantly more time for resumption of righting than WT mice. During recovery from isoflurane anesthesia, Gαi2 GS/GS mice exhibited significantly greater respiratory depression. Poincaré analyses show that GS/GS mice have diminished respiratory variability compared to WT mice.
Conclusion
Modulation of Gαi2 signaling by RGS proteins alters loss and resumption of wakefulness, and state-dependent changes in breathing.
doi:10.1213/ANE.0b013e3181ba7815
PMCID: PMC3152726  PMID: 19843788
23.  GPCR-OKB: the G Protein Coupled Receptor Oligomer Knowledge Base 
Bioinformatics  2010;26(14):1804-1805.
Summary: Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers.
Availability and Implementation: The GPCR-OKB web application is freely available at http://www.gpcr-okb.org
Contact: marta.filizola@mssm.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq264
PMCID: PMC2894509  PMID: 20501551
24.  Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by RGS proteins 4 or 8 and 7 in permeabilised C6 cells is Gα subtype dependent 
Journal of neurochemistry  2009;112(4):1026-1034.
Regulators of G protein signaling (RGS) proteins act as GTPase accelerating proteins (GAPs) 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 (AC) by DAMGO, a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a two-fold 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 due to weak physical association with Gαi2 and minimal GAP 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 AC and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.
doi:10.1111/j.1471-4159.2009.06519.x
PMCID: PMC2947325  PMID: 20002516
RGS proteins; mu-opioid; adenylyl cyclase; permeabilization; Gα proteins
25.  Design, synthesis and prostate cancer cell-based studies of analogs of the Rho/MKL1 transcriptional pathway inhibitor, CCG-1423 
We recently identified bis(amide) CCG-1423 (1) as a novel inhibitor of RhoA/C-mediated gene transcription that is capable of inhibiting invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. An initial structure-activity relationship study focusing on bioisosteric replacement of the amides and conformational restriction identified two compounds, 4g and 8, with improved selectivity for inhibition of RhoA/C-mediated gene transcription and attenuated cytotoxcity relative to 1. Both compounds were also capable of inhibiting cell invasion with equal efficacy to 1 but with less attendant cytotoxicity.
doi:10.1016/j.bmcl.2009.11.056
PMCID: PMC2818594  PMID: 19963382

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