Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening

Echeverría, Emiliana Beatriz; Rueda, Ana Julia Velez; Cabrera, Maia; Juritz, Ezequiel; Burghi, Valeria; Fabián, Lucas; Davio, Carlos; Menna, Pablo Lorenzano; Fernández, Natalia Cristina

doi:10.1016/j.lfs.2019.116872

Cited by 5 publications

(12 citation statements)

References 63 publications

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“…Results from co‐immunoprecipitation experiments showed that C3Z392 and C5Z299 significantly diminished the proportion of GRK2 that co‐immunoprecipitated with Gαs, suggesting that, as expected, inhibitors` mechanism of action consists of avoiding PPI between GRK2 and Gαs (Figure 4B). Also, the involvement of the Gα subunit in the translocation of GRK2 to the plasma membrane in an active signaling environment has been previously demonstrated 27 . Herein, the co‐expression of RH‐GRK2 with a receptor coupled to Gαs, such as H2R, led to a redistribution of RH‐GRK2 from the cytosol to the plasma membrane (Figure 3A) that was hampered by inhibitors C2Z858, C3Z392, and C5Z299 (Figure 3B).…”

Section: Discussionsupporting

confidence: 53%

“…In a previous report, we described a DBVS approach based on the RH domain of GRK2 as a molecular target to search for potential inhibitors of the interaction between GRK2 and Gα protein. From the hit list purchased from the supplier Enamine, we found a set of molecules able to enhance calcium signaling (Gαq‐coupled) 27 . In the present work, we aimed to test the hit list on their ability to inhibit GRK2 action over Gαs signaling.…”

Section: Resultsmentioning

confidence: 99%

“…We identified inhibitors of the desensitization of a Gαq coupled receptor. 27 In this report, we identified candidates that inhibited the desensitization of a Gαs coupled receptor, GRK2 translocation to the plasma membrane, and GRK2-Gαs coimmunoprecipitation. This will contribute to obtaining novel drug candidates with the ability to interfere with phosphorylation independent actions of GRK2 that may be useful for the treatment of pathological conditions where this protein plays a key role.…”

Section: Introductionmentioning

confidence: 97%

“…In a previous publication, we showed the results of a docking‐based virtual screening (DBVS) directed to the less explored RH domain of GRK2. We identified inhibitors of the desensitization of a Gαq coupled receptor 27 . In this report, we identified candidates that inhibited the desensitization of a Gαs coupled receptor, GRK2 translocation to the plasma membrane, and GRK2‐Gαs coimmunoprecipitation.…”

Section: Introductionmentioning

confidence: 99%

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Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling

Echeverría

Ripoll

Fabián

et al. 2022

Pharmacology Res & Perspec

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G protein‐coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2‐mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates’ ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation‐independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

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Section: Discussionsupporting

confidence: 53%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling

Echeverría

Ripoll

Fabián

et al. 2022

Pharmacology Res & Perspec

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“…We have previously reported that the regulator of G-protein signaling (RGS) homology domain of GRK2 (RH) is sufficient to mediate the desensitization of a Gas coupled GPCR, the histamine type 2 receptor (H2R) (Fernandez et al, 2011). Moreover, RH has been involved in phosphorylation independent signaling attenuation of several GPCRs (Sallese et al, 2000;Usui et al, 2000;Reiter et al, 2001;Dhami et al, 2002;Iwata et al, 2005;Namkung et al, 2009;Fernandez et al, 2011;Echeverria et al, 2019). When cells are stimulated with 10µM BRL37344 a direct interaction between wild type GRK2 and HA-Gas is observed by coimmunoprecipitation assay ( Figure 4A).…”

Section: Rgs-mediated Regulation Of Human B3ar In Hek293t Transfectedmentioning

confidence: 96%

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor

et al. 2020

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G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although b3-adrenergic receptor (b3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce b3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of b3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human b3AR presented a short-term desensitization of cAMP response stimulated by the b3AR agonist, BRL37344, and not by forskolin. We found that b3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased b3AR desensitization. Consistently, stimulation of b3AR increased interaction between GRK2 and Gas subunit. Furthermore, in rat cardiomyocytes endogenously expressing b3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the b3AR in heart function and disease.

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Biased agonism at histamine H1 receptor: Desensitization, internalization and MAPK activation triggered by antihistamines

Burghi

Echeverría

Zappia

et al. 2021

European Journal of Pharmacology

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Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening

Cited by 5 publications

References 63 publications

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor

Biased agonism at histamine H1 receptor: Desensitization, internalization and MAPK activation triggered by antihistamines

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