Design, synthesis and biological evaluation of photoswitchable molecules targeting G protein-coupled receptors in retina
[eng] G protein-coupled-receptors (GPCRs) are the largest family of membrane proteins and mediate important cellular responses. Metabotropic glutamate receptors (mGlu) belong to the Glutamate family/Class C GPCR. These receptors are widely expressed in the nervous system and, thus, considered promis...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/202961 |
| Acceso en línea: | https://hdl.handle.net/2445/202961 http://hdl.handle.net/10803/689160 |
| Access Level: | acceso abierto |
| Palabra clave: | Farmacologia Models moleculars Compostos azoics Pharmacology Molecular models Azo compounds |
| Sumario: | [eng] G protein-coupled-receptors (GPCRs) are the largest family of membrane proteins and mediate important cellular responses. Metabotropic glutamate receptors (mGlu) belong to the Glutamate family/Class C GPCR. These receptors are widely expressed in the nervous system and, thus, considered promising targets for the treatment of several neurologic disorders. The activity of mGlu is modulated by agonists such as glutamate but it can also be positively/negatively tuned by allosteric modulators (PAMs and NAMs, respectively). Allosteric modulators have several advantages over agonists as for example higher receptor selectivity since allosteric pocket is less conserved than the orthosteric site. Allosteric modulators targeting receptors such as mGlu5 or mGlu4 have the therapeutic potential to treat Parkinson’s disease, among others. However, mGlu6 has been barely explored due to its restricted localisation in retina and thus there is a lack of information on allosteric modulators targeting this receptor. Nevertheless, mGlu6 is known to be essential for retina function and therefore could be targeted to treat vision disorders. In the recent years, photoswitchable allosteric ligands have been developed to dynamically control mGlu within the Photopharmacology field. Photopharmacology is an emerging field of research that is based on conferring photosensitivity to proteins without the need for genetic manipulation. This approach uses light-regulated drugs to control the functional activity of proteins by means in a very precise manner. To that aim, these ligands contain a photoswitch moiety such as an azo bond (i.e. azobenzenes) which is light-sensitive. The main objective of the present thesis is to design and synthesise azobenzene-containing photoisomerisable compounds acting as allosteric modulators that would enable the light-control of mGlu6. Then, to use these pharmacological tools in the development of novel treatments for vision disorders. In the first chapter, we report the design and synthesis of two different families of a published mGlu4/6 PAM derivatives. We also describe the development of in vitro (mGlu4/6) pharmacological assays and molecular modelling (mGlu4/6) methodologies to evaluate the activity and binding of light-regulated ligands synthesised. None of the photoswitchable derivatives conserve the activity of original molecule towards mGlu6. These experimental results are in line with molecular modelling studies performed, including molecular dynamics. However, compound 75 presents a slight cis-on PAM activity. This compound is further tested in mGlu4, and we observe a similar pharmacological profile. Overall, this new compound can serve as a new scaffold for the design of cis-on allosteric modulators for the optimal control of mGlu4/6 receptors. Next, we participate in a multidisciplinary project named Drug4Sight which aims at developing trans-on ago PAMs targeting mGlu6 that show fast-thermal relaxation and red-shifted profile to treat visual disorders (Chapter 2 and 3). The starting point of the project is a slow-relaxing ago-PAM (40) showing activity towards mGlu4/6. First, we attempt to improve the selectivity of compound 40 by designing derivatives bearing a covalent warhead to target a non-conserved cysteine in mGlu6 (Chapter 2). We base our design on docking studies involving 40 and a model of mGlu6. The compounds are synthesised, photocharacterised and their reactivity towards thiol nucleophilic compounds is assessed. We found reactivity towards these nucleophiles in some derivatives and the molecular dynamics suggest that the interaction between electrophilic moiety and cysteine can take place. However, we could not test these compounds as covalent ligands in vitro since the experimental methodology required cellular assay optimizations that are beyond the scope of this thesis. In Chapter 3, we design and synthesise three different families of mGlu6 reversible allosteric ligands based on reported 40 with the aim of improving activity/selectivity, optimal wavelength and thermal-relaxation, respectively. With the first family we initially study PAM 40 in the mGlu6 binding pocket and then synthesise a family of derivatives to corroborate the molecular modelling analysis and to improve our molecular design. Compound 97 presents a better activity and photoisomerization properties than 40 as mGlu6 trans-on PAM. In contrast, compound 125 displays mGlu6 cis-on PAM activity. Both compounds are used to dynamically modulate mGlu6 cellular activity in vitro using 380 nm irradiation. The mGlu6 binding mode of the trans-on compound 97 is studied by docking and molecular dynamics. However, the cis-isomer thermal relaxation of compound 97 is too slow and not optimal for the project. In the second family, we obtained several red-shifted photoisomerization derivatives of the parent mGlu6 PAM 40. The red-shifted azobenzene 210 is more active than 40 and we apply molecular modelling to understand the structural reasons for these differences. However, we could not experimentally use 550 nm light to significantly modulate the mGlu6 cell activity of 210 in vitro. Finally, we design and synthesise a third family of fast-relaxing photoisomerisable molecules. As a result, two compounds (238 and 240) display mGlu6 trans-on PAM activity similar to that of 40 but with improved photochemical properties. Compounds 238 and 240 are tested in blind zebrafish and found to improve the visual acuity in this model of animals. In addition, these two compounds are tested in retina damaged Opn4-/- mice through conditioned place preference (CPP) experiments. The experiments result in an improvement in mice vision after topical treatment with mGlu6 trans-on PAMs 238 and 240, yielding better results in vivo than the parent azobenzene 40. In conclusion, the present thesis describes original mGlu6 trans-on PAM compounds, which are obtained using new synthetic, pharmacological and computational methodologies developed to study the mGlu6 receptor. The pharmacological tools have been applied to the development of proof-of-concept in vivo studies with encouraging results for future potential treatments of visual disorders. |
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