Design, synthesis and characterisation of photoswitchable allosteric modulators of metabotropic glutamate receptors

Abstract

Metabotropic glutamate receptors (mGlu receptors) are class C GPCRs widely distributed through the CNS and considered pharmacological targets for neurologic disorders, such as neuropathic pain and Parkinson’s disease, between others. Classical allosteric modulators receptors might be useful to treat these disorders, but they the control over of the compound, once it is administered, is lost. Photoswitchable allosteric modulators have an azo-­‐moiety in their chemical scaffold and might offer a powerful alternative to these classical drugs, since their pharmacological activity can be “switched on and off”, depending on the wavelength of illumination.

First, we designed and synthesised alloswitch-­‐1 using an azo-­‐replacement strategy, using previously described mGlu4 PAM with two diarylamides. We replaced one of these amides with an azo-­‐bond to obtain the first GPCR photoswitchable allosteric modulator with activity in-­‐vivo. Alloswitch-­‐1 selectively performed NAM activity in mGlu5 in the trans-­‐isomer, whether in the cis disposition it was inactive. This behaviour was easily controlled by illumination with 380 nm and 500 nm of wavelength and was consistent and reversible in transfected cells and native cultures. Moreover, it allowed the possibility to control the natatorial motility of native Zebrafish larvae or Xenopus Tropicalis with light, and it was also effective in the reduction of pain-­‐like behaviour in rodents, showing a clear light-­‐ dependent activity.

We next designed and synthesised new related phenylazopyiridnes not only to study the structure -­‐ pharmacological activity relationship, but also the efficiency of photoisomerisation from trans to cis isomers and the rate of thermal relaxing from cis to trans isomers. Thus, we obtained many compounds with a very similar potency to alloswitch-­‐1, but other with an enhanced potency from 4-­‐ to 10-­‐fold shift, improved photoswitching properties or other ones that requires a wider wavelength for photoisomerisating, which is less harmful for in-­‐vivo tissues. Furthermore, we demonstrated that if we adjust the wavelength of illumination, we are able to control the rate of activation of the receptor, something that only can be afforded with dose-­‐adjustment with conventional pharmacology.

We also synthesised optogluram with the previous azo-­‐replacement approach used with alloswitch-­‐1, but replacing a second amide with an azo-­‐bond. It resulted in a photoswitchable positive allosteric modulator (PAM) of mGlu4, with similar photoswitching properties to alloswitch-­‐1 and also effective in the reduction of pain-­‐like behaviour in rodents. Additionally we also designed and synthesised two non-­‐photoswitchable bioisosters of alloswitch-­‐1 and optogluram, which did not have an azo-­‐moiety in their scaffold but conserved the previous pharmacological properties as mGlu ligands.

We also synthesised a series of 12 compounds, six of them photoisomerisable, to study the relationships between mGlu4 PAMs and mGlu5 NAMs. Among them, we found two compounds having both pharmacological activities in the low-­‐micromolar range of potency. Concerning the mGlu5 NAMs of this family, we discovered the chemical particularities that make them inverse agonists or partial antagonists.

Finally, we tried to design and synthesise photoswitchable mGlu4 PAMs and mGlu5 NAMs with a trans isomer inactive and a cis isomer active (cis-­‐on). To do that, we designed new modulators and computationally verified the suitability of the cis-­‐on with molecular dockings. Despite the efforts made, we were not successful to find some cis-­‐on without unspecific effects.

Overall, we found the molecular characteristics that relate mGlu4 PAMs and mGlu5 NAMs, and how to obtain a molecular switch between both pharmacological activities. We make a success of azo-­‐ replacing approaches of previous active compounds for obtaining photoswitchable compounds. We additionally proved that optopharmacology might be more advantageous than optogenetics or conventional pharmacology, because of the possibility to perform assays with native tissues or in-­‐ vivo with no genetic modification and adjust the activation of the receptor with light with a single dose of compound, instead of changing doses to find the optimal one.

Els receptors de glutamat metabotròpics (mGlu) són GPCRs distribuïts a través del CNS i es consideren dianes farmacològiques per a trastorns neurològics, com ara el dolor neuropàtic i la malaltia de Parkinson, entre d'altres.

En primer lloc, vam dissenyar i sintetitzar l’alloswitch-­‐1 utilitzant una estratègia de azo-­‐ reemplaçament, per obtenir el primer modulador al·∙lostèric de GPCR fotocommutable amb activitat in vivo. L’alloswitch-­‐1 té activitat de NAM en mGlu5 en l'isòmer trans, mentre que en la disposició cis és inactiu. Aquest comportament es controla fàcilment amb il·∙luminació amb 380 nm i 500 nm i és reversible in-­‐vitro. D'altra banda, va permtre la possibilitat de controlar la motilitat de larves de peix zebra o capgrossos amb llum. També vam poder controlar la sensació de dolor en rosegadors depenent de la llum.

Seguidament, vam realitzar el disseny i síntesi de noves feniliazopiridines, no només per l'estudi de la relació SAR, sinó també per l'eficiència de fotoisomerizació. Vam obtenir compostos amb bona potència farmacològica i vam millorar les propietats de fotoisomerització. També vam sintetitzar l’optogluram amb l’estratègia azo-­‐reemplaçament anterior. El resultat va ser un modulador al·∙lostèric positiu (PAM) de mGlu4 fotoisomerable, amb propietats òptiques similars a l’alloswitch-­‐1 i també és eficaç en el control de la sensació de dolor en rosegadors. A més també vam dissenyar i sintetitzar dos bioisòsters no fotisomeritzables que conservaven les propietats farmacològiques anteriors.

Vam sintetitzar una sèrie per trobar compostos amb activitat dual com a PAM de mGlu4 i NAM de mGlu5. Dos dels compostos ho va arribar a ser i amb els que van resultar NAMs de mGlu5, en vam descobrir les particularitats químiques que els fan agonistes inversos o antagonistes parcials. Finalment, vam intentar de dissenyar i sintetitzar PAMs de mGlu4 i NAMs de mGlu5 amb un isòmer trans inactiu i un isòmer cis actiu, però sense èxit.

En resum, vam trobar les característiques moleculars que relacionen PAMS de mGlu4 i NAMS mGlu5, i com obtenir un interruptor molecular entre ambdues activitats farmacològiques. A més, vam demostrar que l’optofarmacologia presenta avantatges respecte la farmacologia convencional, ja que permet ajustar l'activació del receptor amb llum.