Photoswitchable allosteric ligands to modulate metabotropic glutamate receptors

Abstract

Photopharmacology has the main purpose to allow the control of protein activity with light. The most exploited strategy used to achieve this objective is the freely diffusible photopharmacology and it is based in the use of photosensitive ligands. These ligands are small bioactive molecules, which include a part of their structure (i.e. photoswitch) that can experience molecular changes upon illumination with a determined wavelength of light. These ligands can freely diffuse and they can be applied with systems expressing native proteins.

Azobenzene is the most common photoswitch used in photopharmacology and it can switch with near UV light from the flat and long trans isomer to a shorter bent cis configuration. The reverse photoisomerization can be achieved either with visible light or thermally with light. Thus, if we include azobenzene in the molecular scaffold of a ligand by means of a replacement of a particular moiety (azologization), we can obtain new azo compounds that will resemble to the original ligand, but their structural shape will dramatically changes upon illumination (photoisomerization). Therefore, the two possible isomers will have distinct binding modes to the target protein and will lead to different protein activities under different light conditions, which is known as photoswitching.

Metabotropic Glutamate Receptors (mGluRs) belong to the class C/Glutamate family of G Protein-Coupled Receptors and control many neuronal and glial functions. mGlu receptors are endogenously activated by glutamate, which is the major excitatory neurotransmitter in the central nervous system (CNS), but they can also be activated or inactivated by allosteric modulators. They are usually considered better drug candidates than the orthosteric ligands because usually highly specific for a receptor and able to modulate the activity of a given receptor without blocking endogenous ligand binding.

First of all, we designed and synthesized three families of compounds, using an azo-replacement strategy, to obtain photoswitchable allosteric modulators with possible NAM activity in mGlu5 in the cis isomers, while in the trans form they are inactive. This behavior is easily controlled by illuminating with different wavelengths and it is reversible in vitro. All the three families were inactive as NAMs, but some results suggest that the compounds could act as mGlu5 PAMs in trans form. Studies are continuing in this direction (Chapter 1).

Next, we carry out the design and synthesis of compounds to improve PAM activity at the mGlu4 receptor and increase selectivity over the other group III mGluRs of at least one azo benzene candidate with a structure similar to Optogluram, the first photoswitchable positive allosteric modulator for the mGlu4 receptor. We obtained Optogluram-2 with good pharmacological potency and improved the photoisomerization properties. Under 380 nm light, the potency of Optogluram-2 is significantly reduced. The change in photoinduced potency observed is greater in Optogluram-2 than in Optogluram. Optogluram-2 has similar potency to Optogluram but is more selective for mGlu4 both on the receptors of the same group III as on the other mGluRs. All this indicates that Optogluram-2 can induce an improved activated/deactivated profile change as well as have an optimal selectivity for more complex assays, such as in vivo assays (Chapter 2).

Additionally, we synthesized two series of compounds to find the first photoswitchable compound to selectively enable optical control of the endogenous mGlu1 receptor. Photoglurax-1 arose as a PAM of mGlu1

with micromolar potency in the trans isomer. Under 380nm light, the potency is significantly reduced. Photoglurax-1 turned out to be an equipotent mGlu4 PAM and therefore its general profile is not suitable for in vivo translation as a possible mGlu1 PAM tool compound. However, a dual mGlu1/mGlu4 PAM activity could be intriguing for an antipsychotic agent, since mGlu4 PAM activity can alleviate catalepsy, a major adverse event with standard antipsychotic drug treatment. In contrast, Photoglurax-2 acts as a mGlu1 PAM and does not show any observable allosteric effect on mGlu4 or activity on mGlu5, and therefore Photoglurax-2 represents a potential in vivo photoswitchable PAM mGlu1 tool compound. Reversible monitoring of mGlu1 activity obtained with light can be very advantageous in studying the pharmacological and physiological implications of mGlu1 in many diseases with unprecedented precision (Chapter 3).

Finally, we designed and synthesized a family of novel photoswitchable azoheteroarenes as mGlu1 NAMs with an active trans isomer and an inactive cis isomer to reversibly inactivate the function of the mGlu1 receptor. The potencies of the trans configurations of some compounds of the family are in the micromolar range . Unfortunately, after 400 nm illumination the results were inconclusive due to artifacts that could originate from a possible toxicity of cis azo compounds. More experiments should be done with cells that do not express mGlu1 and also changing the light system to corroborate eventual toxicity (Chapter 4). Likewise, we use some of these compounds in their trans form, therefore without applying light, as tools to expand the knowledge about the nature of the intermediate states induced by mGlu receptor agonists in studies of fluorescence conformational dynamics. Analysis of the effect of mGlu1 NAMs on receptor conformational changes is reported in Chapter 4.

Los receptores metabotrópicos de glutamato (mGlu) son GPCRs distribuidos a través del CNS y se consideran dianas farmacológicas para trastornos neurológicos, tales como el dolor neuropático y la enfermedad de Parkison, entre otras.

En primar lugar, diseñamos y sintetizamos tres familias de compuestos, utilizando una estrategia de azo- reemplazo, para obtener moduladores alostéricos de GPCR fotoconmutable con posible actividad NAM en mGlu5 en los isomeros cis, mientras que en la disposición trans son inactivos. Este comportamiento se controla fácilmente con iluminación con diferentes longitudes de onda y es reversible in vitro. Ninguna familia resultò activa como NAMs, pero algunos resultados sugieren que los compuestos podrían actuar como PAMs mGlu5 en forma trans. La investigación continúa siguiendo esta dirección (Capítulo 1).

Seguidamente, realizamos el diseño y sintesis de compuestos para mejorar la actividad de PAM en el receptor mGlu4 y aumentar la selectividad sobre los otros mGluR del grupo III de al menos un candidato a azobenceno con estructura similar a Optogluram, el primer modulador alostérico positivo fotoconmutable para el receptor mGlu4. Obtuvimos Optogluram-2 con buena potencia farmacologica y mejoramos las propriedades de fotoisomerizacion. Bajo una luz de 380 nm, la potencia de Optogluram-2 se reduce significativamente. El cambio de potencia fotoinducido observado es mayor en Optogluram-2 que en Optogluram.Optogluram-2 tiene potencia parecida a Optogluram pero es màs selectivo para mGlu4 tanto sobre los receptores del mismo grupo III como sobre los demas. Todo esto indica que Optogluram-2 puede inducir un cambio de perfil

activado/desactivado mejorado asì como tener una selectividad optimal para ensayos más complejos, como los ensayos in vivo (Capítulo 2).

Sintetizamos dos series para encontrar el primer compuesto fotoconmutable para habilitar selectivamente el control óptico del receptor mGlu1 endógeno. Photoglurax-1 surgió como un PAM de mGlu1 con potencia micromolar en el isómero trans. Bajo una luz de 380 nm, la potencia se reduce significativamente. Photoglurax- 1 resultó ser un mGlu4 PAM equipotente y por eso su perfil general no es apropiado para una traducción in vivo como una posible herramienta molecular mGlu1 PAM. Sin embargo, una actividad dual mGlu1/mGlu4 PAM podría ser intrigante para un agente antipsicótico,ya que la actividad mGlu4 PAM puede aliviar la catalepsia, un evento adverso importante con el tratamiento estándar con fármacos antipsicóticos. En cambio, Photoglurax-2 actúa como un PAM mGlu1 y no muestra ningún efecto alostérico observable en mGlu4 ni actividad en mGlu5 y por lo tanto Photoglurax-2 representa una potencial herramienta molecular PAM mGlu1 fotoconmutable in vivo. El control reversible de la actividad de mGlu1 obtenido con luz puede ser muy ventajoso para estudiar las implicaciones farmacológicas y fisiológicas de mGlu1 en muchas enfermedades con una precisión sin precedentes (Capítulo 3).

Finalmente, intentamos diseñar y sintetizar una familia de novedosos azoheteroarenos fotoconmutables como NAMs de mGlu1 con un isomero trans activo y un isomero cis inactivo para inactivar reversiblemente la función del receptor mGlu1. Las potencias de las configuraciones trans de algunos compuestos de la familia estan en el rango de micromolaridad. Desafortunadamente, tras una iluminación de 400 nm los resultados fueron no concluyentes debido a artefactos que podrían originarse a partir de una posible toxicidad de los compuestos cis azo. Se deben realizar más experimentos con células que no expresen mGlu1 y cambiando tambien el sistema de luz para comprobar si se trata de toxicidad (Capítulo 4). Asimismo, utilizamos algunos de estos compuestos en su forma trans, por lo tanto sin aplicar luz, como herramientas para ampliar el conocimiento sobre la naturaleza de los estados intermedios inducidos por agonistas de los receptores mGlu en estudios de dinámica conformacional de fluorescencia. El análisis del efecto de los NAMs de mGlu1 sobre los cambios conformacionales del receptor están reportados en el Capítulo 4.

En resumen, encontramos como obtener un interruptor molecular entre varias actividades farmacologicas. Ademàs, demostramos que la fotofarmacologia presenta ventajas respecto a la farmacologia convencional, ya que permite ajustar la activacion del receptor con luz.