Small Molecule Tools to Study Cellular Target Engagement for the Intracellular Allosteric Binding Site of GPCRs

Huber, Max E.; Toy, Lara; Schmidt, Maximilian F.; Weikert, Dorothée; Schiedel, Matthias

doi:10.1002/chem.202202565

Cited by 7 publications

(4 citation statements)

References 40 publications

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“…4b ). The most proximal known GPCR allosteric site is the highly conserved intracellular allosteric site observed for the β2AR, CCR, and CXCR receptors 43 , 44 , which neighbors, but does not overlap with that of compound 1. This site is flanked by helices 1, 2, 3, 6, and 7 and, like all other known allosteric inverse agonists, stabilizes the “inward” conformation of TM6 as its means of blocking Gα association, as exemplified by the structure of CCR9 bound to the inverse agonist vercirnon, another sulfonamide (PDB: 5LWE 45 ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

Lees,

Dias,

Rajamohan

et al. 2023

Nat Commun

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GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.

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Section: Resultsmentioning

confidence: 99%

An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

Lees,

Dias,

Rajamohan

et al. 2023

Nat Commun

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show abstract

“…4b), the site described here is novel. The most proximal known GPCR allosteric site is the highly conserved intracellular allosteric site observed for the β2AR, CCR, and CXCR receptors 42,43 , which neighbors, but does not overlap with that of compound 1. This site is flanked by helices 1, 2, 3, 6, and 7 and, like all other known allosteric inverse agonists, stabilizes the "inward" conformation of TM6 as its means of blocking Gα association, as exemplified by the structure of CCR9 bound to the inverse agonist vercirnon, another sulfonamide (PDB: 5LWE 44 ) (Fig.…”

Section: A Novel Inverse Agonist Mechanismmentioning

confidence: 99%

An inverse agonist of orphan receptor GPR61 reveals a novel allosteric mechanism

Lees

Dias

Rajamohan

et al. 2023

Preprint

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GPR61 is a biogenic amine receptor-related orphan GPCR associated with phenotypes relating to appetite and thus, is of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study its structure and function. Here, we report the first ever structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its unprecedented allosteric site and mode of action. These findings offer key mechanistic insights into an orphan GPCR, while providing both a new structural framework and tool compound to support further studies of GPR61 function and modulation.

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“…radioligands are often not well-suited for continuous assay readouts, the detection of low-affinity binders, and cellular target engagement studies investigating their binding to intracellular target sites. 46 Recently, we and others reported the development of fluorescent tracers targeting the IABS of CCR2, CCR9, and CXCR2. 44,45,47,48 These molecular tools were successfully applied for cell-free and cellular binding studies using the nonradioactive NanoBRET technology.…”

mentioning

confidence: 99%

“…However, radioligand binding assays are accompanied by some disadvantages, such as high infrastructure requirements according to radiation protection measures, the production of radioactive waste, and often laborious (heterogeneous) assay protocols, including washing steps for removing the unbound radioligand prior to the assay readout. The latter is also an important reason why radioligands are often not well-suited for continuous assay readouts, the detection of low-affinity binders, and cellular target engagement studies investigating their binding to intracellular target sites . Recently, we and others reported the development of fluorescent tracers targeting the IABS of CCR2, CCR9, and CXCR2. ,,, These molecular tools were successfully applied for cell-free and cellular binding studies using the nonradioactive NanoBRET technology.…”

mentioning

confidence: 99%

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1

Toy,

Huber,

Lee

et al. 2024

ACS Pharmacol. Transl. Sci.

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In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.

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Small Molecule Tools to Study Cellular Target Engagement for the Intracellular Allosteric Binding Site of GPCRs

Cited by 7 publications

References 40 publications

An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

An inverse agonist of orphan receptor GPR61 reveals a novel allosteric mechanism

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1

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