Adrenergic Modulation With Photochromic Ligands

© 2020 Wiley-VCH GmbH Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice

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

[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.[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

In vivo photocontrol of orexin receptors with a nanomolar light-regulated analogue of orexin-B

Orexinergic neurons are critically involved in regulating arousal, wakefulness, and appetite. Their dysfunction has been associated with sleeping disorders, and non-peptide drugs are currently being developed to treat insomnia and narcolepsy. Yet, no light-regulated agents are available to reversibly control their activity. To meet this need, a photoswitchable peptide analogue of the endogenous neuroexcitatory peptide orexin-B was designed, synthesized, and tested in vitro and in vivo. This compound (photorexin) is the first photo-reversible ligand reported for orexin receptors. It allows dynamic control of activity in vitro (including full agonism, nanomolar activity, and subtype selectivity to human OX2 receptors) and in vivo in zebrafish larvae by direct application in water. Photorexin induces dose- and light-dependent changes in locomotion and a reduction in the successive induction reflex that is associated with sleep behavior. Molecular dynamics calculations indicate that trans and cis photorexins adopt similar bent conformations and that the only discriminant between their structures and activities is the positioning of the N-terminus at the extracellular region of the orexin receptor. Thus, our approach could be extended to a broad and important family of neuropeptides that share a “message-address” mechanism when binding to their cognate receptors

Light-dependent inhibition of clathrin-mediated endocytosis in yeast unveils conserved functions of the AP2 complex

Summary: Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-β-adaptin/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the β-adaptin/β-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved

Adrenergic Modulation with Photochromic Ligands

Adrenoceptors are ubiquitous and regulate heart and respiratory rate, digestion, metabolism, and vascular tone. They can be activated or blocked with adrenergic drugs, but systemic administration causes broad adverse effects. We have developed photochromic ligands (adrenoswitches) to switch on and off adrenoceptor activity on demand at selected locations. Their pharmacology, photochromism, bioavailability and lack of toxicity allow photomodulating adrenergic signalling, as demonstrated by controlling locomotion in zebrafish and pupillary responses in blind mice