OptoGluNAM4.1, a Photoswitchable allosteric antagonist for real-time control of mGlu4 receptor activity

OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system

Shining light on an mGlu5 photoswitchable NAM: A theoretical perspective

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality

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

Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlSynthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution

Development of light-modulated allosteric ligands for remote, non-invasive control of neuronal receptors

In the brain events happen in the scale of milliseconds, and the fine processes of neurons and neuroglia are highly compartmentalized at a microscopic level. These exclusive features of the brain define extremely precise temporal and spatial patterns of cellular activity, which are of fundamental importance for its proper functioning, because they allow the fast processing, sorting, integration, and flow of information with high reproducibility and precision. To gain deeper understanding of how these patterns are organized in time and space, we need new tools that overcome the spatiotemporal limitations of the currently available techniques. Recently, neurobiology was revolutionized by the idea of using light to control neuronal proteins remotely with millisecond- and micrometer-precision, which led to the development of a new field of study called optogenetics. After more than a decade, the control of protein function with light has gone far beyond optogenetics and its need of genetic manipulations. Optopharmacology is now gaining importance because it is less invasive and suitable for controlling endogenous proteins with light, and each year compounds with enhanced photochemical and pharmacological properties are developed. This thesis reviews the optopharmacological tools developed so far to study a family of neuronal proteins called metabotropic glutamate (mGlu) receptors. We are interested in these proteins because they participate in neurotransmission, and are link to neuropathologies when dysfunctional. Thanks to pioneering advances in probing these receptors with light, many features of mGlu signaling have been unraveled, and it is now emerging that these receptors follow activation mechanisms different from those initially foreseen. Still, it is not clear what their exact kinetics are, or which are the functional consequences of temporal and spatial patterns of activity – which are widespread both among brain structures and across evolution. Despite the fundamental relevance of mGlu receptors to brain computing in physiology and disease, the mechanisms that govern their functioning are still partially understood, and this is mainly due to the scarcity of tools to activate mGlu receptors with spatiotemporal precision. The aim of this thesis was to expand the toolbox of optical switches to activate with light mGlu receptors, with special interest in respecting the physiological context of their activation. For that purpose, we discarded approaches based on genetic engineering of receptors, as well as irreversible uncaging of compounds. We preferred the use of optopharmacology, and specifically applied it to allosteric modulators, which display higher selectivity and more physiological activation than orthosteric ligands. This objective implied technical challenges due to the structural restrictions of mGlu allosteric binding pockets, but at the same time offered high gains to spatiotemporally control these receptors both in therapeutic and basic research applications. From these premises, we: 1. developed the first light-regulated allosteric modulators targeting metabotropic glutamate receptors. The molecular design, in vitro and in vivo characterization of alloswitch-1 and G4optoNAM are presented in Chapters 1-2. 2. expanded the knowledge about this new class of compounds through a library of compounds derived from alloswitch-1, and present the inferred data about structure-activity relationship and optimal optopharmacological characteristics for allosteric photoswitches of mGlu receptors (Chapter 3). 3. demonstrated the functional photoisomerization of alloswitch-1 by using two-photon stimulation, with the aim of exploring the resolution limits of reversible optical switches (Chapter 4). Overall, this thesis shows for the first time the design and characterization of optical switches for the allosteric and remote control of endogenous mGlu receptors in vitro and in vivo with light. This advance broadens the availability of optical tools in research to manipulate mGlu receptors with high temporal and spatial resolution, and represents a step forward in innovative opportunities to treat neuropathologies with light.En el sistema nerviós els esdeveniments es desenvolupen en l’escala temporal dels milisegons, i els processos que tenen lloc en neurones i cèl·lules de la glia presenten compartimentalitzacions microscòpiques. Aquesta organització determina uns patrons d’activitat ben definits temporal i espacialment, els quals permeten el precís funcionament del sistema nerviós per tal de transmetre, integrar i processar la informació d’una forma rapida i especifica. Per entendre millor el modus operandi del cervell en el temps i l’espai, calen noves eines que permetin superar les limitacions espaitemporals de les tecnologies existents per l’observació passiva o l’activa manipulació del sistema nerviós. Una de les estratègies més rapides i precises per activar e inactivar proteïnes neuronals es basa en la seva fotosensibilització, per tal de poder-les controlar mitjançant la precisió espai-temporal incomparable que la llum ofereix. Aquesta tesi fa un resum de les eines òptiques disponibles per detectar (sensors) e induir (commutadors) l’activitat d’una família de proteïnes neuronals denominades receptors metabotropics de glutamat (mGlu). Estem interessats en aquests receptors per la importància que tenen com moduladors de la neurotransmissió, i el seu rol en el desenvolupament de neuropatologies. L’objectius de la tesi fou desenvolupar eines optofarmacològiques pel control òptic i reversible dels receptors mGlu amb llum, considerant els grans avantatges d’especificitat espaitemporal que ofereix el fotocontrol de proteïnes i l’escassetat de tals eines. El primer capítol descriu el disseny, la síntesi i la caracterització d’alloswitch-1, el primer fotocommutador al·lostèric capaç d’activar receptors mGlu amb llum de forma reversible. El segon capítol il·lustra la caracterització de G4optoNAM, un fotocommutador al·lostèric actiu en receptors mGlu4. El tercer capítol recull una llibreria de compostos derivats del precursor alloswitch-1 amb diverses substitucions químiques, que presenten característiques fotofísiques i optofarmacològiques variades. Al quart i últim capítol demostrem la capacitat dels alloswitches de fotoisomeritzar amb il·luminació micromètrica amb un làser multifotó. La nostra capacitat d’expandir el ventall d’eines optofarmacològiques que permeten un control farmacològic de receptors neuronals amb llum, de forma remota i no invasiva, ha aportat a la comunitat científica noves metodologies farmacològiques per a l’estudi de la fisiopatologia del sistema nerviós

Development of light-modulated allosteric ligands for remote, non-invasive control of neuronal receptors

In the brain events happen in the scale of milliseconds, and the fine processes of neurons and neuroglia are highly compartmentalized at a microscopic level. These exclusive features of the brain define extremely precise temporal and spatial patterns of cellular activity, which are of fundamental importance for its proper functioning, because they allow the fast processing, sorting, integration, and flow of information with high reproducibility and precision. To gain deeper understanding of how these patterns are organized in time and space, we need new tools that overcome the spatiotemporal limitations of the currently available techniques. Recently, neurobiology was revolutionized by the idea of using light to control neuronal proteins remotely with millisecond- and micrometer-precision, which led to the development of a new field of study called optogenetics. After more than a decade, the control of protein function with light has gone far beyond optogenetics and its need of genetic manipulations. Optopharmacology is now gaining importance because it is less invasive and suitable for controlling endogenous proteins with light, and each year compounds with enhanced photochemical and pharmacological properties are developed. This thesis reviews the optopharmacological tools developed so far to study a family of neuronal proteins called metabotropic glutamate (mGlu) receptors. We are interested in these proteins because they participate in neurotransmission, and are link to neuropathologies when dysfunctional. Thanks to pioneering advances in probing these receptors with light, many features of mGlu signaling have been unraveled, and it is now emerging that these receptors follow activation mechanisms different from those initially foreseen. Still, it is not clear what their exact kinetics are, or which are the functional consequences of temporal and spatial patterns of activity – which are widespread both among brain structures and across evolution. Despite the fundamental relevance of mGlu receptors to brain computing in physiology and disease, the mechanisms that govern their functioning are still partially understood, and this is mainly due to the scarcity of tools to activate mGlu receptors with spatiotemporal precision. The aim of this thesis was to expand the toolbox of optical switches to activate with light mGlu receptors, with special interest in respecting the physiological context of their activation. For that purpose, we discarded approaches based on genetic engineering of receptors, as well as irreversible uncaging of compounds. We preferred the use of optopharmacology, and specifically applied it to allosteric modulators, which display higher selectivity and more physiological activation than orthosteric ligands. This objective implied technical challenges due to the structural restrictions of mGlu allosteric binding pockets, but at the same time offered high gains to spatiotemporally control these receptors both in therapeutic and basic research applications. From these premises, we: 1. developed the first light-regulated allosteric modulators targeting metabotropic glutamate receptors. The molecular design, in vitro and in vivo characterization of alloswitch-1 and G4optoNAM are presented in Chapters 1-2. 2. expanded the knowledge about this new class of compounds through a library of compounds derived from alloswitch-1, and present the inferred data about structure-activity relationship and optimal optopharmacological characteristics for allosteric photoswitches of mGlu receptors (Chapter 3). 3. demonstrated the functional photoisomerization of alloswitch-1 by using two-photon stimulation, with the aim of exploring the resolution limits of reversible optical switches (Chapter 4). Overall, this thesis shows for the first time the design and characterization of optical switches for the allosteric and remote control of endogenous mGlu receptors in vitro and in vivo with light. This advance broadens the availability of optical tools in research to manipulate mGlu receptors with high temporal and spatial resolution, and represents a step forward in innovative opportunities to treat neuropathologies with light.En el sistema nerviós els esdeveniments es desenvolupen en l’escala temporal dels milisegons, i els processos que tenen lloc en neurones i cèl·lules de la glia presenten compartimentalitzacions microscòpiques. Aquesta organització determina uns patrons d’activitat ben definits temporal i espacialment, els quals permeten el precís funcionament del sistema nerviós per tal de transmetre, integrar i processar la informació d’una forma rapida i especifica. Per entendre millor el modus operandi del cervell en el temps i l’espai, calen noves eines que permetin superar les limitacions espaitemporals de les tecnologies existents per l’observació passiva o l’activa manipulació del sistema nerviós. Una de les estratègies més rapides i precises per activar e inactivar proteïnes neuronals es basa en la seva fotosensibilització, per tal de poder-les controlar mitjançant la precisió espai-temporal incomparable que la llum ofereix. Aquesta tesi fa un resum de les eines òptiques disponibles per detectar (sensors) e induir (commutadors) l’activitat d’una família de proteïnes neuronals denominades receptors metabotropics de glutamat (mGlu). Estem interessats en aquests receptors per la importància que tenen com moduladors de la neurotransmissió, i el seu rol en el desenvolupament de neuropatologies. L’objectius de la tesi fou desenvolupar eines optofarmacològiques pel control òptic i reversible dels receptors mGlu amb llum, considerant els grans avantatges d’especificitat espaitemporal que ofereix el fotocontrol de proteïnes i l’escassetat de tals eines. El primer capítol descriu el disseny, la síntesi i la caracterització d’alloswitch-1, el primer fotocommutador al·lostèric capaç d’activar receptors mGlu amb llum de forma reversible. El segon capítol il·lustra la caracterització de G4optoNAM, un fotocommutador al·lostèric actiu en receptors mGlu4. El tercer capítol recull una llibreria de compostos derivats del precursor alloswitch-1 amb diverses substitucions químiques, que presenten característiques fotofísiques i optofarmacològiques variades. Al quart i últim capítol demostrem la capacitat dels alloswitches de fotoisomeritzar amb il·luminació micromètrica amb un làser multifotó. La nostra capacitat d’expandir el ventall d’eines optofarmacològiques que permeten un control farmacològic de receptors neuronals amb llum, de forma remota i no invasiva, ha aportat a la comunitat científica noves metodologies farmacològiques per a l’estudi de la fisiopatologia del sistema nerviós

Development of light-modulated allosteric ligands for remote, non-invasive control of neuronal receptors

[eng] In the brain events happen in the scale of milliseconds, and the fine processes of neurons and neuroglia are highly compartmentalized at a microscopic level. These exclusive features of the brain define extremely precise temporal and spatial patterns of cellular activity, which are of fundamental importance for its proper functioning, because they allow the fast processing, sorting, integration, and flow of information with high reproducibility and precision. To gain deeper understanding of how these patterns are organized in time and space, we need new tools that overcome the spatiotemporal limitations of the currently available techniques. Recently, neurobiology was revolutionized by the idea of using light to control neuronal proteins remotely with millisecond- and micrometer-precision, which led to the development of a new field of study called optogenetics. After more than a decade, the control of protein function with light has gone far beyond optogenetics and its need of genetic manipulations. Optopharmacology is now gaining importance because it is less invasive and suitable for controlling endogenous proteins with light, and each year compounds with enhanced photochemical and pharmacological properties are developed. This thesis reviews the optopharmacological tools developed so far to study a family of neuronal proteins called metabotropic glutamate (mGlu) receptors. We are interested in these proteins because they participate in neurotransmission, and are link to neuropathologies when dysfunctional. Thanks to pioneering advances in probing these receptors with light, many features of mGlu signaling have been unraveled, and it is now emerging that these receptors follow activation mechanisms different from those initially foreseen. Still, it is not clear what their exact kinetics are, or which are the functional consequences of temporal and spatial patterns of activity – which are widespread both among brain structures and across evolution. Despite the fundamental relevance of mGlu receptors to brain computing in physiology and disease, the mechanisms that govern their functioning are still partially understood, and this is mainly due to the scarcity of tools to activate mGlu receptors with spatiotemporal precision. The aim of this thesis was to expand the toolbox of optical switches to activate with light mGlu receptors, with special interest in respecting the physiological context of their activation. For that purpose, we discarded approaches based on genetic engineering of receptors, as well as irreversible uncaging of compounds. We preferred the use of optopharmacology, and specifically applied it to allosteric modulators, which display higher selectivity and more physiological activation than orthosteric ligands. This objective implied technical challenges due to the structural restrictions of mGlu allosteric binding pockets, but at the same time offered high gains to spatiotemporally control these receptors both in therapeutic and basic research applications. From these premises, we: 1. developed the first light-regulated allosteric modulators targeting metabotropic glutamate receptors. The molecular design, in vitro and in vivo characterization of alloswitch-1 and G4optoNAM are presented in Chapters 1-2. 2. expanded the knowledge about this new class of compounds through a library of compounds derived from alloswitch-1, and present the inferred data about structure-activity relationship and optimal optopharmacological characteristics for allosteric photoswitches of mGlu receptors (Chapter 3). 3. demonstrated the functional photoisomerization of alloswitch-1 by using two-photon stimulation, with the aim of exploring the resolution limits of reversible optical switches (Chapter 4). Overall, this thesis shows for the first time the design and characterization of optical switches for the allosteric and remote control of endogenous mGlu receptors in vitro and in vivo with light. This advance broadens the availability of optical tools in research to manipulate mGlu receptors with high temporal and spatial resolution, and represents a step forward in innovative opportunities to treat neuropathologies with light.[cat] En el sistema nerviós els esdeveniments es desenvolupen en l’escala temporal dels milisegons, i els processos que tenen lloc en neurones i cèl·lules de la glia presenten compartimentalitzacions microscòpiques. Aquesta organització determina uns patrons d’activitat ben definits temporal i espacialment, els quals permeten el precís funcionament del sistema nerviós per tal de transmetre, integrar i processar la informació d’una forma rapida i especifica. Per entendre millor el modus operandi del cervell en el temps i l’espai, calen noves eines que permetin superar les limitacions espaitemporals de les tecnologies existents per l’observació passiva o l’activa manipulació del sistema nerviós. Una de les estratègies més rapides i precises per activar e inactivar proteïnes neuronals es basa en la seva fotosensibilització, per tal de poder-les controlar mitjançant la precisió espai-temporal incomparable que la llum ofereix. Aquesta tesi fa un resum de les eines òptiques disponibles per detectar (sensors) e induir (commutadors) l’activitat d’una família de proteïnes neuronals denominades receptors metabotropics de glutamat (mGlu). Estem interessats en aquests receptors per la importància que tenen com moduladors de la neurotransmissió, i el seu rol en el desenvolupament de neuropatologies. L’objectius de la tesi fou desenvolupar eines optofarmacològiques pel control òptic i reversible dels receptors mGlu amb llum, considerant els grans avantatges d’especificitat espaitemporal que ofereix el fotocontrol de proteïnes i l’escassetat de tals eines. El primer capítol descriu el disseny, la síntesi i la caracterització d’alloswitch-1, el primer fotocommutador al·lostèric capaç d’activar receptors mGlu amb llum de forma reversible. El segon capítol il·lustra la caracterització de G4optoNAM, un fotocommutador al·lostèric actiu en receptors mGlu4. El tercer capítol recull una llibreria de compostos derivats del precursor alloswitch-1 amb diverses substitucions químiques, que presenten característiques fotofísiques i optofarmacològiques variades. Al quart i últim capítol demostrem la capacitat dels alloswitches de fotoisomeritzar amb il·luminació micromètrica amb un làser multifotó. La nostra capacitat d’expandir el ventall d’eines optofarmacològiques que permeten un control farmacològic de receptors neuronals amb llum, de forma remota i no invasiva, ha aportat a la comunitat científica noves metodologies farmacològiques per a l’estudi de la fisiopatologia del sistema nerviós

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. © 2016 American Chemical Society.We are grateful to C. Serra and L. Muñoz for synthetic and analytical support, F. Malhaire for technical support in cell-based pharmacological assays, and Y. Pérez for NMR support. This research has been supported by RecerCaixa foundation (2010ACUP00378 to P.G., J.G., and A.L.), the Marató de TV3 Foundation (110230 to J.G., 110231 to A.L., 110232 to C.G., and 111531 to P.G.), the Catalan government (2010 BP-A 00194 to X.R., 2012FI_B 01122 to S.P., 2012 CTP 00033 and 2012 BE1 00597 to X.G.-S., 2014SGR-1251 to P.G., and 2014SGR-00109 to A.L.), the Spanish Government (CTQ2014-57020-R and PCIN-2013-017-C03-01 to A.L., PIE14/00034, SAF2014-55700-P, and PCIN-2013-019-C03-03 to F.C., and SAF2014-58396-R to J.G.), AWT (SBO-140028) to F.C., and the ERANET Neuron LIGHTPAIN project (to A.L., F.C., J.G., and J.-P.P.); SynBio MODULIGHTOR, Human Brain Project WAVESCALES and Ramon Areces foundation grants (to P.G.); the Agence Nationale de la Recherche (ANR-16-CE16-0010 to A.L. and C.G.).Peer reviewe

Glutamete receptor photomodulators

The present invention relates to the discovery of particular aromatic compounds of formula (1), possessing activity as modulators of metabotropic glutamate receptors (mGluR) whose modulatory activity on the receptor may be controlled by irradiation with suitable light resulting in the optical control of receptor biological function, to the use of said compounds as a medicament, and to pharmaceutical compositions comprising said compounds of formula (1).Peer reviewedConsejo Superior de Investigaciones Científicas, Universitat Autónoma de Barcelona, Fundació Institut de Bioenginyeria de Catalunya (IBEC), Centre National de la Recherche Scientifique, Institució Catalana de Recerca i Estudis AvançatsA2 Solicitud de patente sin informe sobre el estado de la técnic