The Potential of 19F NMR Application in GPCR Biased Drug Discovery

doi:10.1016/j.tips.2020.11.001

Trends in Pharmacological Sciences

Volume 42, Issue 1, January 2021, Pages 19-30

https://doi.org/10.1016/j.tips.2020.11.001 Get rights and content

Highlights

Studies indicate that G protein-coupled receptor (GPCR) activation is a multistate transition process instead of a simple switch, suggesting the possibility of modulating the GPCR function through tuning of these individual states.
A biased ligand was proposed to specifically target one signaling pathway over others in a multiple-signaling coexistence system. However, despite the progress in structural biology, a detailed mechanism of how this is achieved remains elusive.
Research has indicated that different GPCR conformations interact with different downstream partners, dictating various pharmacological outputs.
¹⁹F NMR has been demonstrated to be extremely promising in delineating GPCR conformations, attributable to its quantitative properties and high sensitivity toward electrostatic changes of the probe microenvironment. Thus, there is tremendous potential for biased drug discovery by establishing a rigorous correlation between individual receptor conformations and the pharmacological consequence upon ligand binding.

Although structure-based virtual drug discovery is revolutionizing the conventional high-throughput cell-based screening system, its limitation is obvious, together with other critical challenges. In particular, the resolved static snapshots fail to represent a full free-energy landscape due to homogenization in structural determination processing. The loss of conformational heterogeneity and related functional diversity emphasize the necessity of developing an approach that can fill this space. In this regard, NMR holds undeniable potential. However, outstanding questions regarding the NMR application remain. This review summarizes the limitations of current drug discovery and explores the potential of ¹⁹F NMR in establishing a conformation-guided drug screening system, advancing the cell- and structure-based discovery strategy for G protein-coupled receptor (GPCR) biased drug screening.

Section snippets

Biased Agonism in the GPCR

Biased agonism is a ligand-based signaling preference [1] observed when multiple signal pathways coexist in a signaling process. Since its introduction in the 1990s [2], the study of biased agonism has overwhelmingly centered on G protein-coupled receptors (GPCRs) (see Glossary) and, in particular, focused on two classical signal pathways: G protein and β-arrestin [3] (Figure 1). As a matter of fact, the concept of biased agonism can be applied to any signaling where the ligand-activated

Lack of Molecular Understanding of Biased Signaling

While significant progress has been made in linking distinct pharmacological phenomena to various ligand bindings and their signaling effectors, such as taking advantage of approaches like bioluminescence resonance energy transfer (BRET) [10,11], the following has not yet been fully established: (i) techniques for quantitative analyses of ligand functional selectivity [12] in order to identify the roles of individual signal pathways in a multisignaling system; (ii) techniques for predicting the

Missing Parts in Current Drug Screening Systems

With differential ligand binding, GPCRs can interact selectively with various intracellular partners, resulting in different downstream signaling and pharmacological effects. Although studies have advanced our understanding of the GPCR activation process, the functional relevance of each individual conformation and their roles in the signaling process remains elusive. The increased number of protein structures and the advancement of computational tools such as pharmacophore models [32],

Limitation of Cell- and Structure-Based Drug Discovery Systems

With the advancement of X-ray crystallography and cryo-electron microscopy (cryo-EM), structural biology has made tremendous progress. So far, over 370 structures of more than 70 GPCRs [48] have been resolved, providing unprecedented structural insights into receptor activation and allostery. Despite these, X-ray and cryo-EM are unable to elucidate dynamics of individual proteins or PPIs. Structural snapshots cannot capture a continuous conformational transition, extant structures associated

Progress of NMR Application in Receptor Conformational Delineation

It has been reported that the fluorine nucleus has a distinctly high gyromagnetic ratio and, thus, greatest sensitivity for NMR, next to tritium and ¹H nuclei [67]. ¹⁹F NMR as a result exhibits a broad scope of chemical shifts over 1000 ppm [68], indicating a remarkable sensitivity to surrounding environmental changes, with potential to detect the subtle electrostatic changes associated with receptor activation. This provides a plausibility for rational design of biased drugs by delineating the

Concluding Remarks and Future Perspectives

The current drug discovery strategy is predominantly based on the measurement of dose-dependent downstream signaling such as the signaling levels of cAMP or Ca²⁺. Therefore, receptor activation is typically described as an on/off two-state switch. As a result of this oversimplification, developed drugs based on the current system tend to overactivate or oversuppress downstream signaling, resulting in concomitant side effects. A detailed mechanistic understanding of correlations between ligand,

Acknowledgments

The article was supported by Nexus Initiative (L.Y.) from University of South Florida (USF) as well as startup funding (L.Y) from the department of Cell Biology, Microbiology and Molecular Biology at USF.

Disclaimer Statement

L.Y. is an independent principal investigator at University of South Florida as well as being affiliated with H. Lee Moffitt Cancer Center and Research Institute. None of the authors have conflicts of interest with their affiliations.

Glossary

Balanced agonist: a drug that increases activities of multiple signaling pathways.
Biased ligand: a drug that increases the activity of a specific signaling pathway.
Cryo-electron microscopy (cryo-EM): a procedure that deep-freezes a sample and uses electrons to make an image of protein structure.
G protein-coupled receptor (GPCR): a class of seven-transmembrane proteins that transmit extracellular to intracellular signals and are triggered by a wide range of factors, including light, compounds, peptides,

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Citation Excerpt :
This difference altered the electrostatic environments that surrounded the probes BTFMA and TET, causing the different observed chemical shifts. Biomolecular dynamics play a major role in the context of ligand binding, receptor activation, allosteric modulation, and biased signaling, and are highly relevant to a specific physiological or pharmacological output (Latorraca et al., 2017; Wang et al., 2021b). So far, GPCR dynamics have been primarily studied computationally using molecular dynamics (MD) simulations (Mafi et al., 2022) due to the challenges of analyzing them experimentally.
Advances in X-ray crystallography and cryoelectron microscopy enabled unprecedented insights into the activation processes of G protein-coupled receptors (GPCRs). However, these static receptor structures provide limited information about dynamics and conformational transitions that play pivotal roles in mediating signaling diversity through the multifaceted interactions between ligands, receptors, and transducers. Developing NMR approaches to probe the dynamics of conformational transitions will push the frontier of receptor science toward a more comprehensive understanding of these signaling processes. Although much progress has been made during the last decades, it remains challenging to delineate receptor conformational states and interrogate the functions of the individual states at a quantitative level. Here we cover the progress of ¹⁹F NMR applications in GPCR conformational and dynamic studies during the past 20 years. Current challenges and limitations of ¹⁹F NMR for studying GPCR dynamics are also discussed, along with experimental strategies that will drive this field forward.
Conformational dynamics in GPCR signaling by NMR
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G-protein-coupled receptors (GPCRs) mediate a wide range of cellular responses to various ligands or stimuli, and are the most important drug targets associated with human diseases. While major advances in GPCR structural biology have greatly deepened our understanding of its activation mechanism, the highly complex changes in the structural dynamics of GPCRs during activation remain underdetermined and their links to physiological functions largely unknown. Solution nuclear magnetic resonance (NMR) spectroscopy is an essential technique that allows the characterization of protein structural dynamics at atomic level, and has been applied in the studies of GPCR structural-function relationship in the past decade. Herein, we summarize a few specific studies in which solution NMR methods were employed and provided novel insights into questions difficult to be addressed by other methods.
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Current resolved structures of GPCRs and G protein complexes provided important insights into G protein activation. However, the binding or dissociation of GPCRs with G protein is instantaneous and highly dynamic in the intracellular environment. The conformational dynamic of G protein still needs to be addressed. In this study, we applied ¹⁹F solution NMR spectroscopy to monitor the conformational changes of G protein upon interact with detergent mimicking membrane and receptor. Our results show that there are two states equilibria in the G_α in apo states. The interaction of G_α with detergents will accelerate this conformational transformation and induce a state that tends to bind to GPCRs. Finally, the G_α proteins presented a fully activation state when they coupled to GPCRs.
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NMR studies on membrane proteins, in particular GPCRs, have exploited 19F labels to investigate conformational changes upon agonist/antagonist binding in this important class of receptors, with the goal to correlate specific receptor conformations to the pharmacological outcome of ligand binding. Indeed, studies of this kind possess an appealing potential for drug screening and drug discovery (see recent reviews by Wang and others [Picard and Prosser, 2021; Wang et al., 2021b]). In most cases, 19F labels are conjugated to naturally present cysteines near the cytoplasmic face of the receptor (Frei et al., 2020) or on strategically introduced Cys residues at positions sensitive to signal transduction.
Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool for probing structure, dynamics, folding, and interactions at atomic resolution. While naturally occurring magnetically active isotopes, such as ¹H, ¹³C, or ¹⁵N, are most commonly used in biomolecular NMR, with ¹⁵N and ¹³C isotopic labeling routinely employed at the present time, ¹⁹F is a very attractive and sensitive alternative nucleus, which offers rich information on biomolecules in solution and in the solid state. This perspective summarizes the unique benefits of solution and solid-state ¹⁹F NMR spectroscopy for the study of biological systems. Particular focus is on the most recent studies and on future unique and important potential applications of fluorine NMR methodology.
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Published by Elsevier Ltd.

Trends in Pharmacological Sciences

ReviewThe Potential of 19F NMR Application in GPCR Biased Drug Discovery

Highlights

Section snippets

Biased Agonism in the GPCR

Lack of Molecular Understanding of Biased Signaling

Missing Parts in Current Drug Screening Systems

Limitation of Cell- and Structure-Based Drug Discovery Systems

Progress of NMR Application in Receptor Conformational Delineation

Concluding Remarks and Future Perspectives

Acknowledgments

Disclaimer Statement

Glossary

Genomics

iScience

Bioorg. Med. Chem. Lett.

Cell

Methods

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Mol. Biol.

Trends Pharmacol. Sci.

Cell

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J. Biol. Chem.

Biochim. Biophys. Acta, Proteins Proteomics

Structure

J. Biol. Chem.

J. Struct. Biol.

Methods Cell Biol.

Curr. Opin. Struct. Biol.

Biased signalling: from simple switches to allosteric microprocessors

Nat. Rev. Drug Discov.

Contribution of ligand structure to activation of alpha 2-adrenergic receptor subtype coupling to Gs

Mol. Pharmacol.

Independent-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

Proc. Natl. Acad. Sci. U. S. A.

Dynamic bias and its implications for GPCR drug discovery

Nat. Rev. Drug Discov.

Quantifying ligand bias at seven-transmembrane receptors

Mol. Pharmacol.

Identification of the first marine-derived opioid receptor “balanced” agonist with a signaling profile that resembles the endorphins

ACS Chem. Neurosci.

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors

Sci. Signal.

Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells

Nat. Protoc.

NanoBRET: the bright future of proximity-based assays

Front. Bioeng. Biotechnol.

Biased agonism in drug discovery – is it too soon to choose a path?

Mol. Pharmacol.

A pluridimensional view of biased agonism

Mol. Pharmacol.

Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on drug discovery

Pharmacol. Rev.

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

Nature

Activation of the A2A adenosine G-protein-coupled receptor by conformational selection

Nature

Mechanistic insights into allosteric regulation of the A2A adenosine G-protein-coupled receptor by physiological cations

Nat. Commun.

Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer

J. Am. Chem. Soc.

The role of ligands on the equilibria between functional states of a G protein-coupled receptor

J. Am. Chem. Soc.

Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR

Science

β2-adrenergic receptor activation by agonists studied with 19F NMR spectroscopy

Angew. Chem. Int. Ed.

A2A adenosine receptor functional states characterized by 19F-NMR

Proc. Natl. Acad. Sci. U. S. A.

Structure and dynamics of GPCR signaling complexes

Nat. Struct. Mol. Biol.

Ligand-dependent modulation of G protein conformation alters drug efficacy

Cell

Signalling bias in new drug discovery: detection, quantification and therapeutic impact

Nat. Rev. Drug Discov.

Structural features for functional selectivity at serotonin receptors

Science

A new pharmacophore model for the design of sigma-1 ligands validated on a large experimental dataset

Review
The Potential of ¹⁹F NMR Application in GPCR Biased Drug Discovery

Activation of the A_2A adenosine G-protein-coupled receptor by conformational selection

Mechanistic insights into allosteric regulation of the A_2A adenosine G-protein-coupled receptor by physiological cations

Biased signaling pathways in β₂-adrenergic receptor characterized by ¹⁹F-NMR

β₂-adrenergic receptor activation by agonists studied with ¹⁹F NMR spectroscopy

A_2A adenosine receptor functional states characterized by ¹⁹F-NMR