Novel Peptide Ligands of RGS4 from a Focused One‐Bead, One‐Compound Library

Roof, Rebecca A.; Sobczyk-Kojiro, Katarzyna; Turbiak, Anjanette J.; Roman, David L.; Pogozheva, Irina D.; Blazer, Levi L.; Neubig, Richard R.; Mosberg, Henry I.

doi:10.1111/j.1747-0285.2008.00687.x

Cited by 28 publications

(31 citation statements)

References 45 publications

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“…As RGS proteins have received increasing attention as novel drug targets, we and others have been successful in developing small molecule RGS inhibitors (Roman et al, 2007;Roof et al, 2008;Blazer et al, 2010Blazer et al, , 2011Blazer et al, , 2015Turner et al, 2012). However, for many RGS proteins, including RGS2, increasing function would have greater therapeutic potential.…”

Section: Discussionmentioning

confidence: 99%

Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury

Sjögren

Parra

Atkins

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Regulator of G protein signaling (RGS) proteins have emerged as novel drug targets since their discovery almost two decades ago. RGS2 has received particular interest in cardiovascular research due to its role in regulating G q signaling in the heart and vascular smooth muscle. RGS22/2 mice are hypertensive, prone to heart failure, and display accelerated kidney fibrosis. RGS2 is rapidly degraded through the proteasome, and human mutations leading to accelerated RGS2 protein degradation correlate with hypertension. Hence, stabilizing RGS2 protein expression could be a novel route in treating cardiovascular disease. We previously identified cardiotonic steroids, including digoxin, as selective stabilizers of RGS2 protein in vitro. In the current study we investigated the functional effects of digoxin-mediated RGS2 protein stabilization in vivo. Using freshly isolated myocytes from wild-type and RGS22/2 mice treated with vehicle or low-dose digoxin (2 mg/kg/day for 7 days) we demonstrated that agonist-induced cAMP levels and cardiomyocyte contractility was inhibited by digoxin in wild-type but not in RGS2 2/2 mice. This inhibition was accompanied by an increase in RGS2 protein levels in cardiomyocytes as well as in whole heart tissue. Furthermore, digoxin had protective effects in a model of cardiac injury in wild-type mice and this protection was lost in RGS2 2/2 mice. Digoxin is the oldest known therapy for heart failure; however, beyond its activity at the Na 1 /K 1 -ATPase, the exact mechanism of action is not known. The current study adds a novel mechanism, whereby through stabilizing RGS2 protein levels digoxin could exert its protective effects in the failing heart.

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Section: Discussionmentioning

confidence: 99%

Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury

Sjögren

Parra

Atkins

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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“…All proteins were expressed in and harvested from BL21-DE3 Escherichia coli via standard transformation, growth, and lysis protocols (Lee et al, 1994;Lan et al, 1998Lan et al, , 2000Roman et al, 2007;Roof et al, 2008). Histidine-tagged RGS4 was purified over a Ni-NTA affinity column (QIAGEN) followed by cation exchange chromatography and size exclusion chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…There have been several reported peptide inhibitors of RGS4 and related family members (Roof et al, 2006(Roof et al, , 2008Wang et al, 2008) and one disclosed small-molecule inhibitor (Roman et al, 2007). Because of the physical properties of the peptides, none of them function in a cellular environment unless they are introduced intracellularly [e.g., by dialysis via a patch pipette (Roof et al, 2006)].…”

Section: Introductionmentioning

confidence: 99%

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Blazer¹,

Roman²,

Chung³

et al. 2010

Mol Pharmacol

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Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((, inhibit the interaction between RGS4 and G␣ o with an IC 50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 Ͼ 19 ϭ 16 Ͼ 8 Ͼ Ͼ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to G␣ o . On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

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“…The unique peptide chain on each bead is typically identified by Edman degradation. OBOC peptide libraries have been used to identify cyclic peptides that bind to breast cancer cell lines, 5 cyclic peptides that interact with the somatostatin receptor, which is overexpressed in neuroendocrine tumours, 6 linear peptides that interact with integrins, 7 inhibitors of RGS proteins, 8 and small peptides containing unnatural amino acids that inhibit ␣␤ protein aggregation. 9 However, because imaging agents require the addition of a radionuclide to provide visualization and traditional peptide libraries do not take into account the presence of an imaging entity, the potential of peptide libraries in imaging agent development is not fully realized.…”

Section: Introductionmentioning

confidence: 99%

The development of organometallic OBOC peptide libraries and sequencing of N-terminal rhenium(I) tricarbonyl-containing peptides utilizing MALDI tandem mass spectrometry

CruickshankDana

Luyt

2015

Can. J. Chem.

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The development of peptide-based imaging agents through screening of large peptide libraries is hindered by the additional requirement of a radionuclide−chelator complex that can negatively affect the binding properties of the peptide. Herein, we report N-terminal rhenium(I)tricarbonyl OBOC (one-bead, one-compound) peptide libraries for use in the direct screening of potential imaging agents. The rhenium(I) tricarbonyl is incorporated directly in the library as an imaging entity surrogate to account for the presence of a technetium-99m radionuclide chelate. The identification of unknown organometallic peptides on single beads is successfully accomplished through MALDI tandem mass spectrometry, preceded by a systematic investigation of the effects of a variety of N-terminal rhenium(I) tricarbonyl chelates on peptide fragmentation patterns.Résumé : Le développement d'agents d'imagerie peptidiques, en passant au crible de grandes bibliothèques de peptides, est freiné par la nécessité d'utiliser un complexe radionucléide−chélateur qui peut affecter négativement les propriétés de liaison du peptide. Nous traitons ici de bibliothèques de peptides élaborées à l'aide de la méthode OBOC (une bille, un composé) et comportant le rhénium(I)tricarbonyl N-terminal, utilisées dans la sélection directe d'agents d'imagerie potentiels. Le rhénium(I) tricarbonyl est directement inséré dans la bibliothèque comme substitut d'élément d'imagerie pour rendre compte de la présence d'un chélate radionucléide de technétium-99m. L'identification de peptides organométalliques inconnus sur des billes séparées est réalisée avec succès au moyen de la MALDI couplée à la spectrométrie de masse, précédée d'une étude systématique des effets de différents chélates de rhénium(I) tricarbonyl N-terminal sur les schémas de fragmentation peptidique. [Traduit par la Rédaction]

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Novel Peptide Ligands of RGS4 from a Focused One‐Bead, One‐Compound Library

Cited by 28 publications

References 45 publications

Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury

Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

The development of organometallic OBOC peptide libraries and sequencing of N-terminal rhenium(I) tricarbonyl-containing peptides utilizing MALDI tandem mass spectrometry

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