“Photo‐Adrenalines”: Photoswitchable β₂‐Adrenergic Receptor Agonists as Molecular Probes for the Study of Spatiotemporal Adrenergic Signaling

Xu X., Shonberg J., Kaindl J., Clark M.J., Stößel A., Maul L., Mayer D., Hübner H., Hirata K., Venkatakrishnan A.J., Dror R.O., Kobilka B.K., Sunahara R.K., Liu X., Gmeiner P.

AbstractG protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the β1 and β2 adrenergic receptors (β1AR and β2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the β2AR over the β1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the β2AR, as well as a less stable binding pocket for constrained epinephrine in the β1AR. The differences in the amino acid sequence of the extracellular vestibule of the β1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the β2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

Bosma R., Dijon N.C., Zheng Y., Schihada H., Hauwert N.J., Shi S., Arimont M., Riemens R., Custers H., van de Stolpe A., Vischer H.F., Wijtmans M., Holliday N.D., Kuster D.W., Leurs R.

Summary In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors β1-AR and β2-AR, applying an azologization strategy to the first-generation beta-blocker propranolol. The resulting compounds (Opto-prop-1, -2, -3) have good photochemical properties with high levels of light-induced trans-cis isomerization (>94%) and good thermal stability (t1/2 > 10 days) of the resulting cis-isomer in an aqueous buffer. Upon illumination with 360-nm light to PSScis, large differences in binding affinities were observed for photoswitchable compounds at β1-AR as well as β2-AR. Notably, Opto-prop-2 (VUF17062) showed one of the largest optical shifts in binding affinities at the β2-AR (587-fold, cis-active), as recorded so far for photoswitches of G protein-coupled receptors. We finally show the broad utility of Opto-prop-2 as a light-dependent competitive antagonist of the β2-AR as shown with a conformational β2-AR sensor, by the recruitment of downstream effector proteins and functional modulation of isolated adult rat cardiomyocytes.

Duran‐Corbera A., Faria M., Ma Y., Prats E., Dias A., Catena J., Martinez K.L., Raldua D., Llebaria A., Rovira X.

New azobenzene-based molecular photoswitches active for the therapeutic β1-adrenoceptor (β1-AR) are presented. The most promising candidate, named pAzo-2, has a potency and β1/β2 selectivity similar to approved beta blockers. Importantly, pAzo-2 is compatible with imaging techniques and its potential as a cardioselective light-controlled drug has been validated by the reversible photomodulation of the cardiac rhythm in living zebrafish larvae.

Gerwe H., He F., Pottie E., Stove C., Decker M.

Classical psychedelics are a group of hallucinogens which trigger non-ordinary states of consciousness through activation of the 5-HT2A receptor (5-HT2A R) in the brain. However, the exact mechanism of how 5-HT2A R agonism alters perception remains elusive. When studying receptor signaling, tools which work at the same spatiotemporal resolution as the receptor are exceptionally useful. To create such a tool, we designed a set of photoswitchable ligands based on the classical psychedelic N,N-dimethyltryptamine (DMT). By incorporation of the DMT-indole ring into the photoswitchable system, we obtained red-shifted ligands which can be operated by visible light. Among these azo-DMTs, compound 2 h ("Photo-DMT") stands out as its cis isomer exhibits DMT like activity while the trans isomer acts as weak partial agonist. Such a cis-on "efficacy switch" substantially expands the pharmacological toolbox to investigate the complex mechanisms of 5-HT2A R signaling.

Wijtmans M., Josimovic I., Vischer H.F., Leurs R.

The field of photopharmacology of Class A GPCR ligands has recently attracted attention. In this review we analyze 31 papers on currently available photoswitchable ligands for Class A GPCRs. Using the six most recurring terms of all combined paper abstracts, one can extract the overarching goal of this area of research: "Photoswitchable ligands control receptor activity with light" (represented in the TOC graphic). We analyze the design, photochemistry and pharmacology of the photoswitchable ligands. Trends, challenges and limitations will be discussed. A number of efficient photoswitchable ligands that allow optical modulation of GPCR function in various in vitro assays are presented. Moreover, optical modulation of in vivo GPCR function is within reach and the first reports to this end are highlighted.

Gusach A., Maslov I., Luginina A., Borshchevskiy V., Mishin A., Cherezov V.

• GPCRs are highly dynamic proteins that sample multiple conformational states. • Dynamic behavior of GPCRs can be described using the concept of energy landscapes. • Understanding signaling mechanisms requires both structure and dynamics. • New approaches uncover GPCR dynamics in population and at a single molecule level. G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins that are involved in regulation of sensory and physiological processes and implicated in many diseases. The last decade revolutionized the GPCR field by unraveling multiple high-resolution structures of many different receptors in complexes with various ligands and signaling partners. A complete understanding of the complex nature of GPCR function is, however, impossible to attain without combining static structural snapshots with information about GPCR dynamics obtained by complementary spectroscopic techniques. As illustrated in this review, structure and dynamics studies are now paving the way for understanding important questions of GPCR biology such as partial and biased agonism, allostery, oligomerization, and other fundamental aspects of GPCR signaling.

Duran-Corbera A., Catena J., Otero-Viñas M., Llebaria A., Rovira X.

β2-Adrenoceptors (β2-AR) are prototypical G-protein-coupled receptors and important pharmacological targets with relevant roles in physiological processes and diseases. Herein, we introduce Photoazolol-1-3, a series of photoswitchable azobenzene β2-AR antagonists that can be reversibly controlled with light. These new photochromic ligands are designed following the azologization strategy, with a p-acetamido azobenzene substituting the hydrophobic moiety present in many β2-AR antagonists. Using a fluorescence resonance energy transfer (FRET) biosensor-based assay, a variety of photopharmacological properties are identified. Two of the light-regulated molecules show potent β2-AR antagonism and enable a reversible and dynamic control of cellular receptor activity with light. Their photopharmacological properties are opposite, with Photoazolol-1 being more active in the dark and Photoazolol-2 demonstrating higher antagonism upon illumination. In addition, we provide a molecular rationale for the interaction of the different photoisomers with the receptor. Overall, we present innovative tools and a proof of concept for the precise control of β2-AR by means of light.

Velmurugan B.K., Baskaran R., Huang C.

Human G protein-coupled receptors (GPCRs), especially adrenoceptors, play a crucial role in maintaining important physiological activities including cardiovascular and pulmonary functions. Among all adrenoceptors, β-adrenoceptors are the best characterized GPCRs and possess distinctive features as drug targets. Similarly, ligands that activate/deactivate β-adrenoceptors also hold a significant position in the field of biomarker identification and drug discovery. Several studies regarding molecular characterization of the β-adrenoceptor ligands have revealed that ligands with abilities to inhibit basal or intrinsic receptor activity or prevent receptor desensitization are particularly important to efficiently manage detrimental health conditions, including chronic heart failure, asthma, chronic obstructive pulmonary disease, obesity, and diabetes. Given the importance of β-adrenoceptors as molecular targets for many pathological conditions, this review aims to provide a detailed insight on the structural and functional aspects of β-adrenoceptors, with a particular emphasis on their importance as biomarkers and therapeutic targets.

Ricart-Ortega M., Font J., Llebaria A.

New technologies for spatial and temporal remote control of G protein-coupled receptors (GPCRs) are necessary to unravel the complexity of GPCR signalling in cells, tissues and living organisms. An effective approach, recently developed, consists on the design of light-operated ligands whereby light-dependent GPCR activity regulation can be achieved. In this context, the use of light provides an advantage as it combines safety, easy delivery, high resolution and it does not interfere with most cellular processes. In this review we summarize the most relevant successful achievements in GPCR photopharmacology. These recent findings constitute a significant advance in research studies on the molecular dynamics of receptor activation and their physiological roles in vivo. Moreover, these molecules hold potential toward clinical uses as light-operated drugs, which can overcome some of the problems of conventional pharmacology.

Schihada H., Vandenabeele S., Zabel U., Frank M., Lohse M.J., Maiellaro I.

G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α2A-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β2-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands. Hannes Schihada et al. report the design of 11 BRET-based biosensors that allow for quantification of GPCR ligand-binding dynamics in a micro-titer format. The biosensors achieve higher dynamic range and sensitivity than FRET-based biosensors and their design can be extended to the study of other receptor types.

Hüll K., Morstein J., Trauner D.

Synthetic photoswitches have been known for many years, but their usefulness in biology, pharmacology, and medicine has only recently been systematically explored. Over the past decade photopharmacology has grown into a vibrant field. As the photophysical, pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biological targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), soluble proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, we provide an overview of photopharmacology using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. We discuss the scope and limitations of this approach to study biological function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.

Stricker L., Böckmann M., Kirse T.M., Doltsinis N.L., Ravoo B.J.

Getting the green light! Substituted arylazopyrazoles (AAPs) have been investigated as supramolecular photoswitches in aqueous solution. Selective photostationary states (PSSs) and improved binding affinities to β-cyclodextrin have been determined. The experimental findings are supported by results from DFT calculations.

Dolles D., Strasser A., Wittmann H., Marinelli O., Nabissi M., Pertwee R.G., Decker M.

AbstractThe hCB2R plays an important in the immune system and is centrally expressed in microglia. The hCB2R activated by agonists hold great therapeutic potential, e.g., in neuroinflammation. It is currently not yet elucidated how pathophysiological processes are mediated by the hCB2R. Here, photochromic affinity switches based on a drugable benzimidazole core through azologization and computational studies are developed. Structure‐activity relationships (SARs) lead to compounds with high selectivity over hCB1R that can be reversibly switched to a higher affinity cis‐form proved on the receptor level by radioligand binding studies and translating into an affinity change in a functional GTPγS assay. cAMP ELISA and the change in expression level of two genes regulated by CREB proves that the compounds act as partial agonists.

Calbo J., Weston C.E., White A.J., Rzepa H.S., Contreras-García J., Fuchter M.J.

Photoswitchable compounds, which can be reversibly switched between two isomers by light, continue to attract significant attention for a wide array of applications. Azoheteroarenes represent a relatively new but understudied type of photoswitch, where one of the aryl rings from the conventional azobenzene class has been replaced with a five-membered heteroaromatic ring. Initial studies have suggested the azoheteroarenes-the arylazopyrazoles in particular-to have excellent photoswitching properties (quantitative switching and long Z isomer half-life). Here we present a systematic computational and experimental study to elucidate the origin of the long thermal half-lives and excellent addressability of the arylazopyrazoles, and apply this understanding to determine important structure-property relationships for a wide array of comparable azoheteroaryl photoswitches. We identify compounds with Z isomer half-lives ranging from seconds to hours, to days and to years, and variable absorption characteristics, all through tuning of the heteraromatic ring. Conformation perhaps plays the largest role in determining such properties: the compounds with the longest isomerization half-lives adopt a T-shaped ground state Z isomer conformation and proceed through a T-shaped isomerization pathway, whereas the most complete photoswitching is achieved for compounds that have a twisted (rather than T-shaped) Z isomer conformation. By balancing these factors, we report a new azopyrazole 3pzH, which can be quantitatively switched to its Z isomer (>98%) with 355 nm irradiation, near-quantitatively (97%) switched back to the E isomer with 532 nm irradiation, and has a very long half-life for thermal isomerization (t1/2 = 74 d at 25 °C). Given the large tunability of their properties, the predictive nature of their performance, and the other functional opportunities afforded by usage of a heteroaromatic system, we believe the azoheteroaryl photoswitches to have huge potential in a wide range of optically addressable applications.

Billington C.K., Penn R.B., Hall I.P.

History suggests β agonists, the cognate ligand of the β2 adrenoceptor, have been used as bronchodilators for around 5,000 years, and β agonists remain today the frontline treatment for asthma and chronic obstructive pulmonary disease (COPD). The β agonists used clinically today are the products of significant expenditure and over 100 year’s intensive research aimed at minimizing side effects and enhancing therapeutic usefulness. The respiratory physician now has a therapeutic toolbox of long acting β agonists to prophylactically manage bronchoconstriction, and short acting β agonists to relieve acute exacerbations. Despite constituting the cornerstone of asthma and COPD therapy, these drugs are not perfect; significant safety issues have led to a black box warning advising that long acting β agonists should not be used alone in patients with asthma. In addition there are a significant proportion of patients whose asthma remains uncontrolled. In this chapter we discuss the evolution of β agonist use and how the understanding of β agonist actions on their principal target tissue, airway smooth muscle, has led to greater understanding of how these drugs can be further modified and improved in the future. Research into the genetics of the β2 adrenoceptor will also be discussed, as will the implications of individual DNA profiles on the clinical outcomes of β agonist use (pharmacogenetics). Finally we comment on what the future may hold for the use of β agonists in respiratory disease.

Wang A., Since M., Dallemagne P., Rochais C.

ABSTRACTAlzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive onset of symptoms, including memory loss, accompanied by other neurological impairments. This progression is attributed to the deterioration of neuronal connections and a decrease in neurotransmission. Although this phenomenon has been extensively studied in the cholinergic system, it also affects other neurobiological pathways, particularly adrenergic transmission. In this context, the use of agonists, in particular, β2‐adrenergic receptor (β2AR) agonists, may represent a promising therapeutic approach. After reviewing the main pharmacological aspects related to these receptors, we will first present the different existing modulators and their peripheral effects. We will then analyze the results of studies investigating their use in disease models. Finally, we will discuss the conditions and prospects for the development of a new treatment for Alzheimer's disease using a β2AR agonist.

Shi S., Cao Y., Wijtmans M., Vischer H.F., Leurs R.

ABSTRACTAdrenoceptors (ARs) play a vital role in various physiological processes and are key therapeutic targets. The advent of optical control techniques, including optogenetics and photopharmacology, offers the potential to modulate AR signaling with precise temporal and spatial resolution. In this review, we summarize the latest advancements in the optical control of AR signaling, encompassing optogenetics, photocaged compounds, and photoswitchable compounds. We also discuss the limitations of current tools and provide an outlook on the next generation of optogenetic and photopharmacological tools. These emerging optical technologies not only enhance our understanding of AR signaling but also pave the way for potential therapeutic developments.

Panfilov M.A., Starodubtseva E.S., Karogodina T.Y., Vorob’ev A.Y., Moskalensky A.E.

Engineered light-sensitive molecules offer a sophisticated toolkit for the manipulation of biological systems with both spatial and temporal precision. Notably, artificial “caged” compounds can activate specific receptors solely in response to light exposure. However, the uncaging process can lead to the formation of potentially harmful byproducts. For example, the photochemical release of adrenaline (epinephrine) is accompanied by the formation of adrenochrome, which has neuro- and cardiotoxic effects. To investigate this effect in detail, we synthesized and compared two “caged” epinephrine analogs. The first was a classical compound featuring an ortho-nitrobenzyl protecting group attached to the amino group of epinephrine. The second analog retained the ortho-nitrobenzyl group but included an additional carbamate linker. The photolysis of both compounds was conducted under identical conditions, and the resulting products were analyzed using UV–Vis spectroscopy, chromatography, and NMR techniques. Surprisingly, while the classical compound led to the formation of adrenochrome, the carbamate-type caged epinephrine did not produce this byproduct, resulting in the clean release of the active substance. Subsequently, we assessed the novel compound in an in vitro platelet activation assay. The results demonstrated that the uncaging of epinephrine significantly enhances platelet activation, making it a valuable tool for advanced signaling studies.

Starodubtseva E.S., Karogodina T.Y., Panfilov M.A., Sheven D.G., Selyutina O.Y., Vorob’ev A.Y., Moskalensky A.E.

Control of biological activity with light is a fascinating idea. “Caged” compounds, molecules modified with photolabile protecting group, are one of the instruments for this purpose. Adrenergic receptors are essential regulators of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. These receptors are largely used as pharmacologic targets. Photolabile “caged” analogs of adrenergic receptor agonists has been reported more than 30 years ago. We report that the photolysis of epinephrine analogs, apart from liberation of the epinephrine, is accompanied by a formation of significant amount of adrenochrome, a compound with neuro- and cardiotoxic effect.

Gerwe H., Schaller E., Sortino R., Opar E., Martínez -Tambella J., Bermudez M., Lane J.R., Gorostiza P., Decker M.

AbstractThe field of G protein‐coupled receptor (GPCR) research has greatly benefited from the spatiotemporal resolution provided by light controllable, i.e., photoswitchable ligands. Most of the developed tools have targeted the Rhodopsin‐like family (Class A), the largest family of GPCRs. However, to date, all such Class A photoswitchable ligands were designed to act at the orthosteric binding site of these receptors. Herein, we report the development of the first photoswitchable allosteric modulators of Class A GPCRs, designed to target the M1 muscarinic acetylcholine receptor. The presented benzyl quinolone carboxylic acid (BQCA) derivatives, Photo‐BQCisA and Photo‐BQCtrAns, exhibit complementary photopharmacological behavior and allow reversible control of the receptor using light as an external stimulus. This makes them valuable tools to further investigate M1 receptor signaling and a proof of concept for photoswitchable allosteric modulators at Class A receptors.

Gerwe H., Schaller E., Sortino R., Opar E., Martínez -Tambella J., Bermudez M., Lane J.R., Gorostiza P., Decker M.

AbstractDas Forschungsgebiet der G‐Protein‐gekoppelten Rezeptoren (GPCRs) hat stark vom Gewinn an räumlich‐zeitlicher Auflösung profitiert, der mit der Entwicklung licht‐kontrollierbarer und ‐schaltbarer Verbindungen einhergeht. Die meisten der synthetisierten molekularen Werkzeuge adressieren die Klasse A GPCRs (Rhodopsin‐ähnlich), die größte Subfamilie dieser Rezeptorklasse. All diese molekularen Werkzeuge wurden entworfen, um die orthosterische Bindungsstelle der GPCRs zu adressieren. In dieser Arbeit berichten wir über die ersten photoschaltbaren allosterischen Modulatoren an Klasse A GPCRs, die entwickelt wurden, um den muscarinischen Acetylcholinrezeptor Subtyp 1 (M1) zu aktivieren. Die dargestellten Derivate der Benzylchinoloncarbonsäure (BQCA), nämlich Photo‐BQCisA and Photo‐BQCtrAns, zeigen komplementäres photopharmakologisches Verhalten und erlauben die reversible Kontrolle über den M1R mittels Licht als externem Stimulus. Dieses Verhalten lässt diese Substanzen wertvoll erscheinen, etwa um das M1R‐Signalling zu untersuchen und zudem dient diese Arbeit als Machbarkeitsstudie für die Darstellung anderer photoschaltbarer Modulatoren für Klasse A‐Rezeptoren