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Development and applications of photoswitchable ligands for G protein-coupled receptors
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[eng] G protein-coupled receptors (GPCRs) modulate diverse cellular responses to the majority of neurotransmitters and hormones within the human body. They exhibit much structural and functional diversity and are responsive to a plethora of ligands and stimuli including both endogenous (e.g., biogenic amines, cations, lipids, peptides, and glycoproteins) and exogenous (e.g., therapeutic drugs, photons, tastants, and odorants). Due to the key roles of GPCRs in a myriad of physiological processes, signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, ranging from metabolic, immunological, and neurodegenerative disorders to cancer and infectious diseases. Approximately 40% of clinically approved drugs mediate their effects by modulating GPCR signaling pathways, which makes them attractive targets for drug screening and discovery. In this work, we focus on distinct GPCRs which are involved in vital pathways, such as the muscarinic acetylcholine receptors, subtype -1 and subtype -2 (M1 and M2 mAChRs; class A); the dopamine receptor 1 (D1R; class A), and the metabotropic glutamate 6 receptor (mGlu6 receptor; class C). Both M1 receptor (M1R) and M2 receptor (M2R) are important drug targets for several diseases that affect the central nervous system (CNS), including Alzheimer's disease, Parkinson’s disease (PD), schizophrenia, sleep disorders, and acute brain diseases. M2R also represent a target for cardiac disfunctions; the D1R for hypertension and PD, and mGlu6 receptor for retinal degenerative diseases, like retinitis pigmentosa. Traditional pharmacological approaches for the modulation of the GPCRs activity are still limited since precise spatiotemporal control of a ligand is lost as soon it is administrated. Photopharmacology proposes the use of small diffusible molecules called photochromic ligands (PCLs) (or photoswitches) to overcome this shortfall, since their activity can be reversibly controlled by light with high precision. In this thesis, we combined photochromic, cell-based (calcium fluorescence assays), and in vivo photopharmacological approaches to characterize several photoswitches either agonists, antagonists, dualsteric ligands or allosteric modulators. In Chapter 3 we introduce a novel method of photoswitch design named “cryptoazologization”, which replaces the tricyclic core of “privileged” drugs with different azobenzenes as molecular photoswitches. This strategy offers the advantage of producing photoswitchable compounds that are inactive in the trans configuration, the most thermodynamically stable. The cis isomer can be obtained upon illumination with the appropriate wavelength, mimicking the tricyclic geometry of the parent drug and its effect. The potential of the “cryptoazologization” has been demonstrated by the development of four pirenzepine derivatives, termed “cryptozepines”. We have characterized these new compounds for mAChRs through a calcium imaging assay in HEK cells overexpressing the M1R. The cryptozepine-2 that showed a stronger inhibitory activity was studied ex vivo in cardiac atria as well as in cortical network. The latter study is the focus of Chapter 4 where it is shown the antagonistic effect of cryptozepine-2 in ferret slices where it blocks muscarinic activation of slow oscillations. In addition, we found that cryptozepine-2 suppresses M1R-mediated epileptiform seizures in its cis active form. In parallel, it was also demonstrated the ability of the reported dualsteric agonist benzyl quinolone carboxylic acid-azo-iperoxo (BAI) to increase the frequency of slow oscillatory activity in cortical networks both ex vivo and in vivo in its trans configuration. Phthalimide-azo-iperoxo (PAI) is another dualsteric photoswitchable compound which is also active in its trans form (Chapter 8). Differently from BAI, PAI targets the M2Rs, being the first photoswitchable M2 mAChR agonist to be reported. This compound enables the control of cardiac activity with light in wild-type animals without genetic manipulation. However, the limitation of PAI and BAI is that both trans-to-cis isomerize under UV light. Consequently, the property of noninvasiveness of light, a key advantage of pharmacological agent, is lost. We managed to activate PAI with longer, tissue-penetrating wavelengths such as 840 nm under two- photon excitation (2PE) (Chapter 9). In this thesis, thereby, we also aimed at further expanding the photopharmacological toolset with the development of compounds or the optimization of methods that can resolve different shortcomings that we have identified as critical for in vivo applications. In principle, the tissue penetration depth limitation can be overcome by shifting the operational wavelengths into the “biological window” (650-1100 nm). Besides the operational wavelength, another shortcoming is represented by the large excitation volume caused by the linear dependence of one-photon excitation (1PE). In contrast, the nonlinearity of multiphoton excitation at the focal point affords subcellular resolution in three dimensions. To meet both requirements of deep tissue penetration and focalized photoswitching, multiphoton pharmacology takes advantage of NIR pulsed lasers and nonlinear absorption of certain photochromic moieties. In Chapter 5, we developed a set of cis-on xanomeline (i.e., muscarinic agonist) derivatives named “xanoswitches”. The best-in-class compounds were selected based on their responses to an in vitro assay and these photoswitches were chemically modified to allow photoisomerization by two-photon excitation (2PE) using a pulsed NIR light laser. This was done using single point, bio-isosteric modifications at the azobenzene. These 2PE xanoswitches afforded photocontrol of calcium oscillation in various settings, including in vitro and, for the first time, bidirectional, reversible photomodulation of neuronal activity in vivo using NIR-light. In Chapter 9 we have presented the first proof of concept for photoactivation of a photoswitchable compound (i.e., PAI) by three-photon excitation (3PE) in vitro and in vivo. The wide application of two photon and three-photon pharmacology would be transformative for research and development in chemical biology and to progress with advanced phototherapies. Depending on the biological application, the three-dimensional micrometric spatial resolution obtained by 2PE might not be necessary or desired. Instead, 1PE possesses significant advantages to activate larger areas of tissue. Chapter 6 describes the development of 1PE xanoswitches through the introduction into the molecular structure of a tetra-ortho chlorinated azobenzene. These compounds allowed reversible modulation of calcium oscillation in vitro with orange light and photocontrol of zebrafish motility. This would also allow using standard “off-the-shelf” light emitting diodes (LEDs) which are lightweight and widely accessible, simplifying its applicability for photopharmacological therapies for the PNS and CNS. Another goal of this work was to develop the first reversibly photoswitchable allosteric modulators for class A GPCRs, described in Chapter 7. Using the prototypical M1R positive allosteric modulator benzyl quinolone carboxylic acid (BQCA), a set of Photo-BQCAs was developed. These compounds exhibit complementary photopharmacological properties, one being cis- and one being trans-on. Biological characterization demonstrated their ability to modulate receptor activation in presence of an orthosteric agonist in a light dependent manner. These photoswitches represent interesting and useful tools in the ongoing efforts to understand complex signaling mechanism of GPCRs. Moreover, this work presents the development and application of azodopa, a photopharmacological agent that is aimed at light-regulation of dopaminergic system (Chapter 10). We demonstrated that this compound activates D1-like receptors in vitro in a light-dependent manner and it enables reversibly photocontrolling zebrafish motility on a time scale of seconds. Moreover, it increases the overall neural activity in the cortex of anesthetized mice. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals, opening the way to remotely control dopaminergic neurotransmission for fundamental and therapeutic purposes. In Chapter 11, we describe the development and application of a photoswitchable compound library targeting metabotropic glutamate 6 (mGlu6) receptors and acting as agonist and positive allosteric modulators (ago-PAM) with nanomolar potency. We performed a high-throughput screening of 15 photoswitchable allosteric ligands using a microplate reader and we identified two compounds (i.e., 1492 and 1495) that and are fast relaxing, blue light switching, and allow in vivo restoration of visual acuity for the first time. Moreover, they restore light-avoidance behavior in blind mice by topical administration. The characteristics of these compounds make them excellent candidates for further preclinical studies and a potential drug-based therapy for sight restoration in humans. The presented findings broaden the availability of photopharmacological tools not only to investigate complex signaling pathways that underlie many (patho)physiological processes but also for innovative and noninvasive treatments of different pathologies with light.
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SORTINO, Rosalba. Development and applications of photoswitchable ligands for G protein-coupled receptors. [consulta: 9 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/210883]