Novel Inhibitory Site Revealed by XAP044 Mode of Action on the Metabotropic Glutamate 7 Receptor Venus Flytrap Domain.

Metabotropic glutamate (mGlu) receptors play a key role in modulating most synapses in the brain. The mGlu7 receptors inhibit presynaptic neurotransmitter release and offer therapeutic possibilities for post-traumatic stress disorders or epilepsy. Screening campaigns provided mGlu7-specific allosteric modulators as the inhibitor XAP044 (Gee et al. J. Biol. Chem. 2014). In contrast to other mGlu receptor allosteric modulators, XAP044 does not bind in the transmembrane domain but to the extracellular domain of the mGlu7 receptor and not at the orthosteric site. Here, we identified the mode of action of XAP044, combining synthesis of derivatives, modeling and docking experiments, and mutagenesis. We propose a unique mode of action of these inhibitors, preventing the closure of the Venus flytrap agonist binding domain. While acting as a noncompetitive antagonist of L-AP4, XAP044 and derivatives act as apparent competitive antagonists of LSP4-2022. These data revealed more potent XAP044 analogues and new possibilities to target mGluRs.

A "double-edged" role for type-5 metabotropic glutamate receptors in pain disclosed by light-sensitive drugs.

Knowing the site of drug action is important to optimize effectiveness and address any side effects. We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.

Optical Control of Adenosine A2A Receptor Using Istradefylline Photosensitivity.

In recent years, there has been growing interest in the potential therapeutic use of inhibitors of adenosine A2A receptors (A2AR) for the treatment of neurodegenerative diseases and cancer. Nevertheless, the widespread expression of A2AR throughout the body emphasizes the importance of temporally and spatially selective ligands. Photopharmacology is an emerging strategy that utilizes photosensitive ligands to attain high spatiotemporal precision and regulate the function of biomolecules using light. In this study, we combined photochemistry and cellular and in vivo photopharmacology to investigate the light sensitivity of the FDA-approved antagonist istradefylline and its potential use as an A2AR photopharmacological tool. Our findings reveal that istradefylline exhibits rapid trans-to-cis isomerization under near-UV light, and prolonged exposure results in the formation of photocycloaddition products. We demonstrate that exposure to UV light triggers a time-dependent decrease in the antagonistic activity of istradefylline in A2AR-expressing cells and enables real-time optical control of A2AR signaling in living cells and zebrafish. Together, these data demonstrate that istradefylline is a photoinactivatable A2AR antagonist and that this property can be utilized to perform photopharmacological experiments in living cells and animals.

Photopharmacological manipulation of amygdala metabotropic glutamate receptor mGlu4 alleviates neuropathic pain.

Neuropathic pain is a common health problem resulting in exacerbated response to noxious and non noxious stimuli, as well as impaired emotional and cognitive responses. Unfortunately, neuropathic pain is also one of the most difficult pain syndromes to manage, highlighting the importance of better understanding the brain regions and neuromodulatory mechanisms involved in its regulation. Among the many interconnected brain areas which process pain, the amygdala is known to play an important role in the integration of sensory and emotional pain signals. Here we questioned the ability of a recently identified neuromodulatory mechanism associated with the metabotropic glutamate receptors mGlu4 in the amygdala to modulate neuropathic pain. In a murine model of peripheral mononeuropathy, we demonstrate that pharmacological activation of amygdala mGlu4 efficiently alleviates sensory and depressive-like symptoms in both male and female mice. Moreover, we reveal a differential modulation of these symptoms. Activating mGlu4 in the contralateral amygdala relative to the side of the mononeuropathy, is necessary and sufficient to relieve both sensory and depressive-like symptoms, while ipsilateral activation solely reduces depressive-like symptoms. Furthermore, using photopharmacology, a recent strategy allowing precise photocontrol of endogenous proteins, we further demonstrate the dynamic alleviation of neuropathic pain through light-dependent facilitation of mGlu4 by a photoswitchable positive allosteric modulator. Finally, coupling photopharmacology and analgesic conditioned place preference, we show a significant pain-reducing effect of mGlu4 activation. Taken together, these data highlight the analgesic potential of enhancing amygdala mGlu4 activity to counteract neuropathy reinforcing its therapeutic interest for the treatment of pathological pain.

Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

[Recent advances in the structural biology of the class C G protein-coupled receptors: The metabotropic Glutamate receptor 5]. / Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5.

Class C GPCRs, that include metabotropic glutamate receptors (mGlu), taste receptors, GABAB receptor and Calcium-sensing receptor, are unusual in terms of their molecular architecture and allosteric regulation. They all form obligatory dimers, dimerization being fundamental for their function. More specifically, the mGlu are activated by the main excitatory neurotransmitter, L-glutamate. mGlu activation by glutamate binding in the venus flytrap domain (VFT) triggers conformational changes that are transmitted, through the Cystein-Rich Domain (CRD), to the conserved fold of 7 transmembrane helices (7TM), that couples to intracellular G protein. mGlu activity can also be allosterically modulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7TM. Recent progress in cryo-electron microscopy (cryoEM) has allowed unprecedented advances in deciphering the structural and molecular basis of their activation mechanism. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. The diversity of inactive conformations for the class C was unexpected but allows PAM stabilising a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. Here we present and discuss recent structural characterisation of mGlu receptors, highlighting findings that make the class C of GPCR unique. Understanding the structural basis of mGlu dimer signaling represents a landmark achievement and paves the way for structural investigation of GPCR dimer signaling in general. Structural information will open new avenues for structure-based drug design.

Title: Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5. Abstract: La classe C des Récepteurs Couplés aux Protéines G (RCPG) comprend plusieurs membres aux fonctions physiologiques importantes comme par exemple les récepteurs des principaux neurotransmetteurs excitateurs (glutamate) et inhibiteurs (GABA) du système nerveux, les récepteurs des goûts umami et sucré et les récepteurs sensibles au calcium. Ces récepteurs possèdent une architecture moléculaire particulière, caractérisée par la présence d'un large domaine extracellulaire (ECD) relié à un domaine membranaire composé de 7 hélices transmembranaires (7TM). De plus, ils forment tous des dimères obligatoires, la dimérisation étant fondamentale pour leur fonction. La fixation d'agoniste dans l'ECD induit l'activation du récepteur. L'activité des agonistes peut être modulée de manière allostérique par des modulateurs positifs (PAM) ou négatifs (NAM), se liant au domaine 7TM. Il est important de comprendre comment les changements de conformation induits par la liaison des agonistes au sein du domaine extracellulaire sont transmis au domaine transmembranaire mais aussi de comprendre les bases structurales et moléculaires de la régulation allostérique des récepteurs de la classe C. Les progrès récents de la microscopie électronique en conditions cryogéniques (cryoEM) ont permis des avancées sans précédent dans le décryptage des bases structurelles et moléculaires des mécanismes d'activation des RCPG de classe C, et notamment du récepteur métabotropique du glutamate de type 5 (mGlu5). Le glutamate entraîne une fermeture et un changement d'orientation des domaines extracellulaires qui induit un mouvement important entre les sous-unités, rapprochant les 7TM et stabilisant la conformation active du récepteur. La diversité de conformations inactives pour les récepteurs de la classe C était inattendue mais propice à une activation possible par des PAM. Ces derniers stabilisent une conformation active des 7TM, indépendante des changements conformationnels induits par les agonistes, représentant un mode alternatif d'activation des récepteurs mGlu. Nous présentons et discutons ici les caractérisations structurales récentes des récepteurs de classe C, en soulignant les résultats qui rendent cette famille de récepteurs unique. La compréhension de la base structurelle de la signalisation des dimères de mGlu représente une réalisation historique et ouvre la voie à l'analyse de la signalisation des dimères de RCPG en général. Ces analyses structurales devraient également ouvrir de nouvelles voies pour la conception de médicaments ciblant cette famille de récepteurs qui sont aussi des cibles thérapeutiques.

International Union of Basic and Clinical Pharmacology. CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors.

Metabotropic glutamate (mGlu) receptors respond to glutamate, the major excitatory neurotransmitter in the mammalian brain, mediating a modulatory role that is critical for higher-order brain functions such as learning and memory. Since the first mGlu receptor was cloned in 1992, eight subtypes have been identified along with many isoforms and splice variants. The mGlu receptors are transmembrane-spanning proteins belonging to the class C G protein-coupled receptor family and represent attractive targets for a multitude of central nervous system disorders. Concerted drug discovery efforts over the past three decades have yielded a wealth of pharmacological tools including subtype-selective agents that competitively block or mimic the actions of glutamate or act allosterically via distinct sites to enhance or inhibit receptor activity. Herein, we review the physiologic and pathophysiological roles for individual mGlu receptor subtypes including the pleiotropic nature of intracellular signal transduction arising from each. We provide a comprehensive analysis of the in vitro and in vivo pharmacological properties of prototypical and commercially available orthosteric agonists and antagonists as well as allosteric modulators, including ligands that have entered clinical trials. Finally, we highlight emerging areas of research that hold promise to facilitate rational design of highly selective mGlu receptor-targeting therapeutics in the future. SIGNIFICANCE STATEMENT: The metabotropic glutamate receptors are attractive therapeutic targets for a range of psychiatric and neurological disorders. Over the past three decades, intense discovery efforts have yielded diverse pharmacological tools acting either competitively or allosterically, which have enabled dissection of fundamental biological process modulated by metabotropic glutamate receptors and established proof of concept for many therapeutic indications. We review metabotropic glutamate receptor molecular pharmacology and highlight emerging areas that are offering new avenues to selectively modulate neurotransmission.

Mechanistic Insights into Light-Driven Allosteric Control of GPCR Biological Activity.

G protein-coupled receptors (GPCR), including the metabotrobic glutamate 5 receptor (mGlu5), are important therapeutic targets and the development of allosteric ligands for targeting GPCRs has become a desirable approach toward modulating receptor activity. Traditional pharmacological approaches toward modulating GPCR activity are still limited since precise spatiotemporal control of a ligand is lost as soon as it is administered. Photopharmacology proposes the use of photoswitchable ligands to overcome this limitation, since their activity can be reversibly controlled by light with high precision. As this is still a growing field, our understanding of the molecular mechanisms underlying the light-induced changes of different photoswitchable ligand pharmacology is suboptimal. For this reason, we have studied the mechanisms of action of alloswitch-1 and MCS0331; two freely diffusible, mGlu5 phenylazopyridine photoswitchable negative allosteric modulators. We combined photochemical, cell-based, and in vivo photopharmacological approaches to investigate the effects of trans-cis azobenzene photoisomerization on the functional activity and binding ability of these ligands to the mGlu5 allosteric pocket. From these results, we conclude that photoisomerization can take place inside and outside the ligand binding pocket, and this leads to a reversible loss in affinity, in part, due to changes in dissociation rates from the receptor. Ligand activity for both photoswitchable ligands deviates from high-affinity mGlu5 negative allosteric modulation (in the trans configuration) to reduced affinity for the mGlu5 in their cis configuration. Importantly, this mechanism translates to dynamic and reversible control over pain following local injection and illumination of negative allosteric modulators into a brain region implicated in pain control.

Development and validation of a mass spectrometry binding assay for mGlu5 receptor.

Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites. Graphical abstract.

Strategies and considerations of G-protein-coupled receptor photopharmacology.

G-protein-coupled receptor (GPCR) pharmacology tends to be complex and at times poorly understood. This has led to the development of GPCR-targeting agents that often demonstrate poor pharmacokinetic properties and poor selectivity for their target receptors. One approach that is emerging as a means of addressing these limitations is the use of molecules whose activity can be controlled by light. Photopharmacology involves the incorporation of a photoswitch into the structure of a given compound, cage or linker and following irradiation with light, undergoes a structural rearrangement, which changes its biological activity. The use of light-regulated ligands offers the opportunity to modulate and understand GPCR signaling in a more spatiotemporal manner than classical pharmacological approaches. In this chapter we will discuss some of the advancements that have been made in photopharmacology, particularly in developing photoswitchable ligands that target class A GPCRs, e.g., muscarinic acetylcholine receptors, class B GPCRs, e.g., glucagon-like peptide-1 receptor, and class C GPCRs, e.g., metabotrobic glutamate receptors. Given the intricacy of GPCR pharmacology, this chapter will also discuss some of the challenges the field faces when designing photopharmacological tools. Furthermore, it will propose that it is with a full appreciation of the spectrum of pharmacological and pharmacokinetic properties of photoswitchable ligands that research will be better placed to develop ligands with a reduced risk of failure during preclinical progression. This will likely enable photopharmacological approaches to continue to find novel applications and offer new perspectives in understanding (patho)physiology to ultimately inform future GPCR drug discovery efforts.

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3.

Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl- ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive "chloride-lock" network. Indeed, Cl- ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors' large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl-mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl- ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.

Profiling of orthosteric and allosteric group-III metabotropic glutamate receptor ligands on various G protein-coupled receptors with Tag-lite® assays.

Group-III metabotropic glutamate (mGlu) receptors are important synaptic regulators and are potential druggable targets for Parkinson disease, autism and pain. Potential drugs include orthosteric agonists in the glutamate binding extracellular domain and positive allosteric modulators interacting with seven-pass transmembrane domains. Orthosteric agonists are rarely completely specific for an individual group-III mGlu subtype. Furthermore they often fail to pass the blood-brain barrier and they constitutively activate their target receptor. These properties limit the potential therapeutic use of orthosteric agonists. Allosteric modulators are more specific and maintain the biological activity of the targeted receptor. However, they bind in a hydrophobic pocket and this limits their bio-availability and increases possible off-target action. It is therefore important to characterize the action of potential drug targets with a multifaceted and deeply informative assay. Here we aimed at multifaceted deep profiling of the effect of seven different agonists, and seven positive allosteric modulators on 34 different G protein-coupled receptors by a Tag-lite® assay. Our results did not reveal off-target activity of mGlu orthosteric agonists. However, five allosteric modulators had either positive or negative effects on non-cognate G protein-coupled receptors. In conclusion, we demonstrate the power of the Tag-lite® assay for potential drug ligand profiling on G protein-coupled receptors and its potential to identify positive allosteric compounds.

Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites.

A group III metabotropic glutamate (mGlu) receptor agonist (PCEP) was identified by virtual HTS. This orthosteric ligand is composed by an l-AP4-derived fragment that mimics glutamate and a chain that binds into a neighboring pocket, offering possibilities to improve affinity and selectivity. Herein we describe a series of derivatives where the distal chain is replaced by an aromatic or heteroaromatic group. Potent agonists were identified, including some with a mGlu4 subtype preference, e.g., 17m (LSP1-2111) and 16g (LSP4-2022). Molecular modeling suggests that aromatic functional groups may bind at either one of the two chloride regulatory sites. These agonists may thus be considered as particular bitopic/dualsteric ligands. 17m was shown to reduce GABAergic synaptic transmission at striatopallidal synapses. We now demonstrate its inhibitory effect at glutamatergic parallel fiber-Purkinje cell synapses in the cerebellar cortex. Although these ligands have physicochemical properties that are markedly different from typical CNS drugs, they hold significant therapeutic potential.

Shedding light on metabotropic glutamate receptors using optogenetics and photopharmacology.

Metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors activated by glutamate, the main excitatory neurotransmitter of the mammalian central nervous system. These receptors are considered as potential therapeutic targets in many neurological diseases but a better understanding of their complex molecular dynamics and of their role in the normal and pathological functioning of the brain is still required. Manipulating mGluRs with high spatial and temporal precision holds great promise for deciphering their physiological and pathological functions. This article reviews several recently developed optogenetic and photopharmacological solutions for the optical control of mGluRs and their applications, from the study of the molecular dynamics of receptor activation to the study of their roles in vivo.

Emerging Trends in Pain Modulation by Metabotropic Glutamate Receptors.

Pain is an essential protective mechanism meant to prevent tissue damages in organisms. On the other hand, chronic or persistent pain caused, for example, by inflammation or nerve injury is long lasting and responsible for long-term disability in patients. Therefore, chronic pain and its management represents a major public health problem. Hence, it is critical to better understand chronic pain molecular mechanisms to develop innovative and efficient drugs. Over the past decades, accumulating evidence has demonstrated a pivotal role of glutamate in pain sensation and transmission, supporting glutamate receptors as promising potential targets for pain relieving drug development. Glutamate is the most abundant excitatory neurotransmitter in the brain. Once released into the synapse, glutamate acts through ionotropic glutamate receptors (iGluRs), which are ligand-gated ion channels triggering fast excitatory neurotransmission, and metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors modulating synaptic transmission. Eight mGluRs subtypes have been identified and are divided into three classes based on their sequence similarities and their pharmacological and biochemical properties. Of note, all mGluR subtypes (except mGlu6 receptor) are expressed within the nociceptive pathways where they modulate pain transmission. This review will address the role of mGluRs in acute and persistent pain processing and emerging pharmacotherapies for pain management.

Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer.

GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer.

Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator.

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.

Illuminating Phenylazopyridines To Photoswitch Metabotropic Glutamate Receptors: From the Flask to the Animals.

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity.