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Determinants of Ligand Subtype-Selectivity at α-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR


Yong, Kelvin J; Vaid, Tasneem M; Shilling, Patrick J; Wu, Feng-Jie; Williams, Lisa M; Deluigi, Mattia; Plückthun, Andreas; Bathgate, Ross A D; Gooley, Paul R; Scott, Daniel J (2018). Determinants of Ligand Subtype-Selectivity at α-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR. ACS Chemical Biology, 13(4):1090-1102.

Abstract

α- and α-adrenoceptors (α-AR and α-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α-AR and α-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α-AR and α-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α-AR over α-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.

Abstract

α- and α-adrenoceptors (α-AR and α-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α-AR and α-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α-AR and α-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α-AR over α-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biochemistry
Life Sciences > Molecular Medicine
Language:English
Date:20 April 2018
Deposited On:12 Jun 2018 14:07
Last Modified:29 Jul 2020 07:19
Publisher:American Chemical Society (ACS)
ISSN:1554-8929
OA Status:Closed
Publisher DOI:https://doi.org/10.1021/acschembio.8b00191
PubMed ID:29537256

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