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Development and applications of photoswitchable small molecules and peptides to control protein-protein interactions and GPCR activity

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Universitat de Barcelona. Facultat de Química

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[eng] Photopharmacology is an emerging field that relies on the development of photosensitive compounds to enable precise spatiotemporal control over endogenous proteins. Photochromic ligands· are designed to respond to specific wavelengths of light with a reversible change of structure that enhances or diminishes their activity or affinity towards the desired target. Applications are expected to lead to safer treatments in medicine and to innovative tools to investigate complex signalling networks in biology. Thus, in this thesis we aimed at further expanding the spectrum of protein-protein interactions (PPis) and G protein-coupled receptors (GPCRs), that can be addressed by means of photopharmacology. To do so, we focused on resolving different shortcomings that we identified as critical for the actual performance and widespread application of photochromic compounds. For example, Traffic Light 2 (TL2) is a photoswitchable cell-permeable peptide that can inhibit clathrin-mediated endocytosis (CME) in a light regulated manner. Despite proving effective in mammalian cells, the influence of CME over several other cellular processes limited further applications in this biological system. Thus, we validated its activity in a simpler eukaryotic system, Saccharomyces cerevisiae. Using kymography and spheroplast expressing GFP-tagged Sla-1, a coat-associated endocytic protein, we characterized the effect ofTL2 and light over CME dynamics. Studying homologies between mammalian and yeast endocytic proteins we also proposed a mechanism of action for this peptide. The same strategy used to develop Tls was then applied to design new photoswitchable peptides targeting PPis involved in developmental processes. In particular, we focused on Wnt/β-catenin signalling and synthesised a library of light-regulated peptides to reversibly inhibit or activate this pathway with light. The photophysical behaviour of the peptides was characterized and the initial design was validated in vitro by means of circular dichroism and fluorescent-polarization binding assay. The activity was then verified in mammalian cells using a dual-luciferase reporter gene assay. Finally, using Schmidtea mediterranea planarians we set up a model of tissue regeneration to probe in vivo photoregulation of Wnt/β-catenin signalling. The design of photoswitchable peptides was then broadened from PPIs to signalling peptides that bind to and activate receptors. Orexins are neuroexcitatory peptides that modulate through GPCRs neural circuits involved in regulating sleep and arousal. Analysing reported crystal structures of the orexin receptors and NMR solution structures of the peptide we identified a strategy to develop a photochromic analogue of orexins. The derivative was synthesised by standard solid-phase peptide synthesis inserting a non-natural photoswitchable amino acid into the backbone of the peptide. The photochromic behaviour of the analogue was fully characterised, and its activity was assessed by means of different techniques that allow to monitor transient increases in intracellular calcium. Using circular dichroism and molecular modelling simulations we provide a structural model to explain the difference in activity observed between the trans and cis isomers. Finally, in sight of their possible therapeutic relevance, we rationally designed a small library of photochromic ligands to target the adrenergic system. In particular, we used a non-conventional approach, based on azoheteroarenes, to render photoswitchable a class of a-adrenergic ligands. We named these compounds adrenoswitches and characterised their activity in in vitro and in vivo. Affinity towards α-adrenoceptors was assessed through a radioligand binding assay in pre-frontal cortex membranes obtained post-mortem from human brains. In addition, the potency of the compounds was screened in a model of vascular reactivity using ex vivo rat aortic rings. Finally, the most promising compound of the series was used in proof-of-concept applications to manipulate with light locomotor activity in zebrafish and pupillary responses in mice.

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[spa] La fotofarmacología es una técnica muy prometedora que permite controlar con luz y de manera reversible Ia actividad de compuestos activos biológicamente. En esta tesis doctoral hemos desarrollado péptidos y ligandos fotoconmutables para regular las interacciones entre proteínas y Ia actividad de receptores acoplados a proteínas G (GPCRs). En primer Iugar, validamos Ia actividad de TL2, un péptido fotoconmutable que inhibe Ia endocitosis mediada por clatrina, en un modelo simple de célula eucariota, Ia levadura Saccharomyces cerevisiae. Mediante Ia tecnica de "kimografía" en esferoplastos que expresan el marcador fluorescente GFP-Sia1, caracterizamos el efecto de TL2 en este proceso celular y su posible mecanismo de acción. Luego aplicamos Ia estrategia de diseño de TL2 para crear una librería de péptidos que inhiben o activan con luz Ia vía de señalización mediada por Wnt/β-catenina. Tras su caracterización química y fotocrómica, analizamos el efecto de los ciclos de luz sobre las estructuras secundarias de los péptidos y su union a Ia β-catenina. Para evaluar su actividad, empleamos un ensayo de luminiscencia que permite cuantificar Ia expresión génica y ponemos a punto un modelo in vivo de regeneración de tejidos en planarias Schmidtea mediterranea. A continuación, ampliamos las aplicaciones de los péptidos fotoconmutables desde Ia inhibición de las interacciones proteína-proteína descrita anteriormente, a Ia activación de receptores de neuropéptidos como las orexinas. En particular, creamos un análogo fotoconmutable de Ia orexina insertando un aminoácido con un grupo azobenceno en Ia secuencia lineal del péptido. Validamos este nuevo diseño mediante estudios estructurales de dicroísmo circular y modelado molecular, y su actividad mediante Ia medida de señales intracelulares como el calcio. Por ultimo,diseñamos y sintetizamos varios derivados fotoconmutables de ligandos α-adrenergicos insertando azoheteroarenos en sus estructuras. Mediante ensayos de unión evaluamos Ia afinidad de los compuestos hacia los adrenoreceptores de tipo α,y comprobamos su activad en un modelo de reactividad vascular en aorta de rata. Uno de los compuestos permitió manipular Ia motilidad de peces cebra y el tamaño pupilar en ratones, demostrando por primera vez el control de los adrenoreceptores con luz in vivo.

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Development and applications of photoswitchable small molecules and peptides to control protein-protein interactions and GPCR activity