Parallel Interrogation of &amp;#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Zeghal, Manel; Laroche, Geneviève; Giguère, Patrick M.

doi:10.3791/60823

Cited by 10 publications

(24 citation statements)

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“…The PRESTO-Tango GPCR assay possesses unique and advantageous features that enable the simultaneous testing of a set of amiloride derivatives at all class A GPCRs. 28 , 29 The difficulty in screening the entire druggable GPCRome in parallel is mainly due to the inherent diversity of G protein signal-transduction cascades. The measurement of G protein-independent β-arrestin recruitment provides a universal assay platform, as nearly all tested GPCRs can induce arrestin translocation.…”

Section: Resultsmentioning

confidence: 99%

“…The PRESTO-Tango GPCR assay possesses unique and advantageous features that enable the simultaneous testing of a set of amiloride derivatives at all class A GPCRs. , The difficulty in screening the entire druggable GPCRome in parallel is mainly due to the inherent diversity of G protein signal-transduction cascades. The measurement of G protein-independent β-arrestin recruitment provides a universal assay platform, as nearly all tested GPCRs can induce arrestin translocation. ,, We selected to test 5-( N , N -dimethyl)amiloride (DMA), 5-( N -methyl- N -isobutyl)amiloride (MIA), 5-( N -ethyl- N -isopropyl)-amiloride (EIPA), 5-( N , N -hexamethylene)amiloride (HMA), and the sodium-hydrogen exchanger isoform 1 (NHE1) inhibitor zoniporide.…”

Section: Resultsmentioning

confidence: 99%

“…To obtain better pharmacological profiles of these drugs at GPCRs, we decided to perform reverse pharmacology by screening a nontoxic concentration of the selected inhibitors. The Presto-Tango is a unique open resource that allows simultaneous testing of most class A GPCRs using β-arrestin2 recruitment to measure receptor activity. , This signal amplification platform is extremely sensitive and allows the detection of weak partial agonists, which usually run undetected by most assays. As a reporter assay, stimulation is performed over 16 h, conducive to increased sensitivity and minimization of the temporal aspect of drug action and interaction.…”

Section: Discussionmentioning

confidence: 99%

“…Data of three independent experiments ( n = 3) performed in quadruplicate are presented as Relative Light Unit (RLU) or normalized as indicated in figure legends. Parallel interrogation was performed as previously published by us, 29 with the exception that custom-made DMEM (Wisent, Inc., QC, Canada) was used when different sodium concentrations were tested. Na + -Free DMEM was adjusted with the desired NaCl concentration and compensated with choline chloride for a final concentration of 140 mM ion + Cl – (final osmolarity of 337 mOsm/kg).…”

Section: Methodsmentioning

confidence: 99%

“…On the next day, media and drug solutions were removed, and 20 μL per well of homemade Glo reagent (108 mM Tris−HCl; 42 mM Tris-Base, 75 mM NaCl, 3 mM MgCl2, 5 mM dithiothreitol (DTT), 0.2 mM coenzyme A, 0.14 mg/mL Dluciferin, 1.1 mM adenosine triphosphate (ATP), 0.25% v/v Triton X-100, 2 mM sodium hydrosulfite) was added. The plates were incubated for 10 min at room temperature in the dark before counting using a Hidex Sense Beta Plus (Gamble 29 with the exception that custom-made DMEM (Wisent, Inc., QC, Canada) was used when different sodium concentrations were tested. Na + -Free DMEM was adjusted with the desired NaCl concentration and compensated with choline chloride for a final concentration of 140 mM ion + Cl − (final osmolarity of 337 mOsm/kg).…”

Section: ■ Conclusionmentioning

confidence: 99%

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Functional Characterization of Sodium Channel Inhibitors at the Delta-Opioid Receptor

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Existing pharmacotherapies acting on the opioid receptor system have been extensively used to treat chronic pain and addictive disorders. Nevertheless, the adverse side effects associated with opioid therapy underscore the need for concerted measures to develop safer analgesics. A promising avenue of research stems from the characterization of a sodium-dependent allosteric regulation site housed within the delta-opioid receptor and several other G protein-coupled receptors (GPCRs), thereby revealing the presence of a cluster of sodium and water molecules lodged in a cavity thought to be present only in the inactive conformation of the receptor. Studies into the structure–function relationship of said pocket demonstrated its critical involvement in the functional control of GPCR signaling. While the sodium pocket has been proposed to be present in the majority of class A GPCRs, the shape of this allosteric cavity appears to have significant structural variation among crystallographically solved GPCRs, making this site optimal for the design of new allosteric modulators that will be selective for opioid receptors. The size of the sodium pocket supports the accommodation of small molecules, and it has been speculated that promiscuous amiloride and 5′-substituted amiloride-related derivatives could target this cavity within many GPCRs, including opioid receptors. Using pharmacological approaches, we have described the selectivities of 5′-substituted amiloride-related derivatives, as well as the hitherto undescribed activity of the NHE1 inhibitor zoniporide toward class A GPCRs. Our investigations into the structural features of the delta-opioid receptor and its ensuing signaling activities suggest a bitopic mode of overlapping interactions involving the orthosteric site and the juxtaposed Na + pocket, but only at the active or partially active opioid receptor.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: ■ Conclusionmentioning

confidence: 99%

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Development of a V5-tag–directed nanobody and its implementation as an intracellular biosensor of GPCR signaling

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et al. 2023

Journal of Biological Chemistry

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Beyond the Nucleus: Plastic Chemicals Activate G Protein-Coupled Receptors

McPartland,

Stevens,

Bartosova

et al. 2024

Environ. Sci. Technol.

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G protein-coupled receptors (GPCRs) are central mediators of cell signaling and physiological function. Despite their biological significance, GPCRs have not been widely studied in the field of toxicology. Herein, we investigated these receptors as novel targets of plastic chemicals using a high-throughput drug screening assay with 126 human non-olfactory GPCRs. In a first-pass screen, we tested the activity of triphenol phosphate, bisphenol A, and diethyl phthalate, as well as three real-world mixtures of chemicals extracted from plastic food packaging covering all major polymer types. We found 11 GPCR-chemical interactions, of which the chemical mixtures exhibited the most robust activity at adenosine receptor 1 (ADORA1) and melatonin receptor 1 (MTNR1A). We further confirm that polyvinyl chloride and polyurethane products contain ADORA1 or MTNRA1 agonists using a confirmatory secondary screen and pharmacological knockdown experiments. Finally, an analysis of the associated gene ontology terms suggests that ADORA1 and MTNR1A activation may be linked to downstream effects on circadian and metabolic processes. This work highlights that signaling disruption caused by plastic chemicals is broader than that previously believed and demonstrates the relevance of nongenomic pathways, which have, thus far, remained unexplored.

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Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Cited by 10 publications

References 0 publications

Functional Characterization of Sodium Channel Inhibitors at the Delta-Opioid Receptor

Functional Characterization of Sodium Channel Inhibitors at the Delta-Opioid Receptor

Development of a V5-tag–directed nanobody and its implementation as an intracellular biosensor of GPCR signaling

Beyond the Nucleus: Plastic Chemicals Activate G Protein-Coupled Receptors

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