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2.
Nat Commun ; 13(1): 7109, 2022 11 19.
Article in English | MEDLINE | ID: mdl-36402762

ABSTRACT

Carvedilol is among the most effective ß-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of ß1-adrenoceptors, arrestin-biased signalling via ß2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through ß2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the ß-adrenoceptor system.


Subject(s)
Adrenergic beta-Antagonists , Myocardial Infarction , Humans , Carvedilol/pharmacology , Adrenergic beta-Antagonists/pharmacology , Receptors, Adrenergic, beta-2/genetics , Myocardial Infarction/drug therapy
3.
Nat Commun ; 11(1): 3033, 2020 06 19.
Article in English | MEDLINE | ID: mdl-32561830

ABSTRACT

Endogenous opioid peptides and prescription opioid drugs modulate pain, anxiety and stress by activating opioid receptors, currently classified into four subtypes. Here we demonstrate that ACKR3/CXCR7, hitherto known as an atypical scavenger receptor for chemokines, is a broad-spectrum scavenger of opioid peptides. Phylogenetically, ACKR3 is intermediate between chemokine and opioid receptors and is present in various brain regions together with classical opioid receptors. Functionally, ACKR3 is a scavenger receptor for a wide variety of opioid peptides, especially enkephalins and dynorphins, reducing their availability for the classical opioid receptors. ACKR3 is not modulated by prescription opioids, but we show that an ACKR3-selective subnanomolar competitor peptide, LIH383, can restrain ACKR3's negative regulatory function on opioid peptides in rat brain and potentiate their activity towards classical receptors, which may open alternative therapeutic avenues for opioid-related disorders. Altogether, our results reveal that ACKR3 is an atypical opioid receptor with cross-family ligand selectivity.


Subject(s)
Opioid Peptides/chemistry , Receptors, CXCR/metabolism , Analgesics, Opioid/chemistry , Animals , Brain/metabolism , Cell Line, Tumor , Central Nervous System/drug effects , Chemokines/metabolism , Humans , Ligands , MAP Kinase Signaling System , Male , Phosphorylation , Rats , Rats, Wistar , Signal Transduction , Structure-Activity Relationship , beta-Arrestin 1/metabolism
4.
Chemistry ; 26(55): 12615-12623, 2020 Oct 01.
Article in English | MEDLINE | ID: mdl-32428383

ABSTRACT

The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative BIM-46174 and its dimeric form BIM-46187 (1) are heterocyclized dipeptides that belong to the very few cell-permeable compounds known to preferentially silence Gαq proteins. To explore the chemical space of Gαq inhibitors of the BIM chemotype, a combinatorial approach was conducted towards a library of BIM molecules. This library was evaluated in a second messenger-based fluorescence assay to analyze the activity of Gαq proteins through the determination of intracellular myo-inositol 1-phosphate. Structure-activity relationships were deduced and structural requirements for biological activity obtained, which were (i) a redox reactive thiol/disulfane substructure, (ii) an N-terminal basic amino group, (iii) a cyclohexylalanine moiety, and (iv) a bicyclic skeleton. Active compounds exhibited cellular toxicity, which was investigated in detail for the prototypical inhibitor 1. This compound affects the structural cytoskeletal dynamics in a Gαq/11 -independent manner.


Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11 , Pyrazines , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Ligands , Pyrazines/pharmacology , Structure-Activity Relationship
5.
Medchemcomm ; 10(10): 1838-1843, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-32180917

ABSTRACT

The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative BIM-46174 has received attention as Gαq inhibitor. We conducted structural reductions to monocyclic and bicyclic substructures to explore the chemical space of BIM fragments and to gain insights into the pharmacophore of BIM-type Gαq inhibitors. Two piperazin-2-one-containing fragments and a small library of bicyclic lactams featuring fused pyrazine and diazepine rings were synthesized and evaluated. The results of a second messenger-based cellular assay indicate that the entire BIM structure is required for efficient Gαq inhibition.

6.
Sci Signal ; 11(559)2018 12 04.
Article in English | MEDLINE | ID: mdl-30514810

ABSTRACT

Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the ß-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.


Subject(s)
Cell Proliferation , Frizzled Receptors/metabolism , Pancreatic Neoplasms/pathology , Wnt-5a Protein/metabolism , Calcium/metabolism , Diglycerides/metabolism , Frizzled Receptors/chemistry , HEK293 Cells , Humans , Pancreatic Neoplasms/metabolism , Protein Conformation , Protein Kinase C/metabolism , Signal Transduction , Tumor Cells, Cultured , Wnt-5a Protein/chemistry
7.
Thromb Haemost ; 118(10): 1803-1814, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30235481

ABSTRACT

Heparanase (HPSE) is an endo-ß-D-glucuronidase that cleaves heparan sulphate (HS) chains of proteoglycans (HSPGs). Besides a remodelling of the extracellular matrix, HPSE increases the bioavailability of pro-angiogenic mediators, such as HS-associated vascular endothelial growth factor (VEGF), thereby contributing to metastatic niche formation. Notably, HPSE also induces release of VEGF from tumour cells independent of its enzymatic activity, but the underlying molecular mechanisms remain unresolved. We found that exogenous addition of latent HPSE stimulates VEGF release from human MV3 melanoma cells. The same effect was noted upon direct stimulation of thrombin receptor (protease-activated receptor 1 [PAR-1]) by Thrombin Receptor Activator Peptide 6 (TRAP-6). The matricellular ligand cysteine-rich 61 protein (Cyr61) was identified as pathway component since Cyr61 knockdown in MV3 cells abolished the VEGF release by TRAP-6 and HPSE. Since both TRAP-6 and HPSE mediated an up-regulation of phosphorylated focal adhesion kinase, which could be blocked by antagonizing PAR-1, we postulated a crosstalk between latent HPSE and PAR-1 in promoting pro-angiogenic pathways. To test this hypothesis at a molecular level, we applied dynamic mass redistribution (DMR) technique measuring intracellular mass relocation as consequence of direct receptor activation. Indeed, latent HPSE evoked a concentration-dependent DMR signal in MV3 cells as TRAP-6 did. Both could be modulated by targeting G-protein receptor signalling in general or by the PAR-1 inhibitor RWJ 56110. Using cells devoid of cell surface HS synthesis, we could confirm HPSE effects on PAR-1, independent of HSPG involvement. These data indicate, for the first time, a crosstalk between latent HPSE, thrombin receptor activation and G-protein signalling in general.


Subject(s)
Angiogenesis Inducing Agents/metabolism , Cysteine-Rich Protein 61/metabolism , Glucuronidase/metabolism , Heparitin Sulfate/metabolism , Melanoma/metabolism , Peptide Fragments/metabolism , Receptor, PAR-1/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cysteine-Rich Protein 61/genetics , Humans , Melanoma/pathology , Neoplasm Metastasis , RNA, Small Interfering/genetics , Receptor Cross-Talk , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism
8.
ChemMedChem ; 13(16): 1634-1643, 2018 08 20.
Article in English | MEDLINE | ID: mdl-29873888

ABSTRACT

Direct targeting of intracellular Gα subunits of G protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known Gq α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of Gq in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of Gq and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than Gq .


Subject(s)
Depsipeptides/pharmacology , Enzyme Inhibitors/pharmacology , GTP-Binding Protein alpha Subunits, Gq-G11/antagonists & inhibitors , Animals , Binding Sites , CHO Cells , Cattle , Cricetulus , Depsipeptides/chemical synthesis , Depsipeptides/chemistry , Depsipeptides/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , HEK293 Cells , Humans , Magnetic Resonance Spectroscopy , Mice , Molecular Docking Simulation , Molecular Structure , Protein Binding , Rats , Structure-Activity Relationship
9.
Nat Commun ; 9(1): 341, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29362459

ABSTRACT

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.


Subject(s)
GTP-Binding Proteins/genetics , MAP Kinase Signaling System , Receptors, G-Protein-Coupled/metabolism , beta-Arrestin 1/metabolism , beta-Arrestin 2/metabolism , CRISPR-Cas Systems , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits, G12-G13/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Proteins/metabolism , Gene Knockout Techniques , HEK293 Cells , Humans , Phosphorylation , Signal Transduction , beta-Arrestins/metabolism
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