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

Abstract

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.