Photoswitchable ligands targeting beta-adrenoceptors for in vitro, in vivo and structural studies
[eng] G protein-coupled receptors (GPCRs) are a large family of membrane proteins responsible for signaling transduction processes. Due to their roles in modulating essential physiological functions, GPCRs are classical pharmacological targets and the focus of numerous research lines. Within this su...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2022 |
| País: | España |
| Recursos: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/189622 |
| Acesso em linha: | https://hdl.handle.net/2445/189622 http://hdl.handle.net/10803/675582 |
| Access Level: | acceso abierto |
| Palavra-chave: | Farmacologia Química clínica Adrenoceptors beta Compostos azoics Lligands (Bioquímica) Pharmacology Clinical chemistry Beta adrenoreceptors Azo compounds Ligands (Biochemistry) |
| Resumo: | [eng] G protein-coupled receptors (GPCRs) are a large family of membrane proteins responsible for signaling transduction processes. Due to their roles in modulating essential physiological functions, GPCRs are classical pharmacological targets and the focus of numerous research lines. Within this superfamily of receptors, beta-adrenoceptors (β-AR) have been widely studied. Their relevant contribution to the modulation of the cardiac output, among other physiological functions, has historically signaled these receptors as therapeutic targets. Many approved drugs modulate the activation of β-AR, which evidences the high potential of these GPCRs in clinical and research applications. On the other hand, photopharmacology has arisen as an innovative approach with therapeutic potential and many research applications. This technique uses synthetic light-regulated molecules to render light-controllable proteins without genetic manipulation. In this context, the main objective of the present thesis is the development of light-sensitive molecules that allow the modulation of beta-adrenoceptors through light application. In the first chapter, we report the development of a caged analogue of carvedilol, an approved inhibitor of β-AR. Upon illumination at 405 nm, Caged-Carvedilol photolitically releases the beta-blocker carvedilol. The caged ligand was used to explore light-dependent modulation of beta-adrenoceptors in several physiological systems, including native cardiac tissues and living zebrafish larvae. Overall, this novel caged compound provides an innovative molecular tool to precisely control the activation state of β-AR in space and time. Therefore, C-C could be used in future studies to better understand the complex role of beta-adrenoceptors in physiology. Caged ligands are valuable molecular tools but present one main limitation, their irreversible nature. Once the bioactive molecule is photolitically released, no compound deactivation is possible. To produce ligands that allow a reversible control of the target receptors with light, a series of azobenzene-based compounds were designed, synthesized, and photochemically characterized in Chapter 2. Additionally, we explored the light-dependent pharmacological properties of Photoazolols 1-3 (PZLs 1-3) in vitro against two different receptor subtypes, β1- AR and β2-AR. The pharmacological results obtained for the first series of photochromic ligands guided the design of two additional azobenzenes to modulate β1-AR selectively. Both ligands, designed with a para-substituted azobenzene (p-AB) scaffold, displayed good light-dependent properties and an excellent β1-AR selectivity profile. Additionally, in vivo assays were performed using zebrafish larvae. These experiments highlighted that light-dependent cardiac modulation was achieved when larvae were treated with p-AB 84 under different illumination conditions. Importantly, all photochromic ligands described in the second chapter of this thesis display trans→cis isomerization wavelengths within the UV range. This photochemical feature can limit the research applications for the developed photoswitches, considering that highly energetic ultraviolet light can introduce safety issues due to its phototoxicity. It could be especially problematic if the developed compounds were intended for therapeutic applications. For this reason, we developed red-shifted azobenzenes that enable modulation of β-AR with the application of visible light, reported in Chapter 3. Two series of aminoazobenzenes (aABs) were synthesized, which could be photoisomerized efficiently through the application of visible light. Photopharmacological properties of the red-shifted ligands were evaluated in vitro for both β1-AR and β2-AR subtypes. Finally, we intended to use the developed azobenzenes to perform structural studies on β-AR. The last chapter of this thesis describes the research conducted during a 6-months stay performed at the Paul Scherrer Institut (Switzerland), where a joint subproject was established with the Standfuss group. This subproject aimed to crystallize both β1-AR and β2-AR, bound to one of the trans-on compounds from our library of photochromic ligands. After crystallization, we intended to perform conventional X-Ray Crystallography experiments with the obtained crystals in the dark and after illumination with the appropriate wavelength to evaluate the light-induced structural changes in our target receptors. Firstly, receptor expression and purification protocols were optimized and established for different constructs of β1-AR and β2- AR. Finally, crystallization trials were set up using purified protein bound to PZL-1, and preliminary crystals were obtained for the ultra-stable (US) construct of β1-AR. However, we did not obtain the diffraction-quality crystals required to perform X-Ray Crystallography experiments. To sum up, the present thesis reports a variety of novel photopharmacological tools to study beta-adrenoceptors with spatiotemporal precision. We performed in vitro, in vivo, and structural studies using different molecules from the developed library to demonstrate the possibilities offered by the described compounds in therapeutic and research applications. |
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