RESUMEN
To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50â pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560â nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.
Asunto(s)
Fotones , Pez Cebra , Animales , Rayos Infrarrojos , LigandosRESUMEN
Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of Gâ protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1'-(2-tolyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M1 -selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M1 positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy.
Asunto(s)
Bencimidazoles/farmacología , Carbacol/análogos & derivados , Carbacol/farmacología , Piperidinas/farmacología , Quinolinas/farmacología , Receptor Muscarínico M1/agonistas , Bencimidazoles/agonistas , Bencimidazoles/síntesis química , Bencimidazoles/metabolismo , Carbacol/agonistas , Carbacol/metabolismo , Agonismo Parcial de Drogas , Células HEK293 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Agonistas Muscarínicos/síntesis química , Agonistas Muscarínicos/metabolismo , Agonistas Muscarínicos/farmacología , Piperidinas/agonistas , Piperidinas/síntesis química , Piperidinas/metabolismo , Quinolinas/agonistas , Quinolinas/síntesis química , Quinolinas/metabolismo , Receptor Muscarínico M1/metabolismoRESUMEN
Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination, and offers new opportunities for intravital imaging and control of cardiac function.
Asunto(s)
Agonistas Muscarínicos/farmacología , Sistema Nervioso Parasimpático/efectos de los fármacos , Receptor Muscarínico M2/agonistas , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Rayos Infrarrojos , Simulación del Acoplamiento Molecular , Estructura Molecular , Agonistas Muscarínicos/síntesis química , Agonistas Muscarínicos/química , Procesos Fotoquímicos , Ratas , Ratas Wistar , Estereoisomerismo , Relación Estructura-Actividad , XenopusRESUMEN
Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra- ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra- ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo. We investigated the impact of the substitution pattern on receptor activity and evaluated the different binding modes. Compounds 9b and 15b show excellent photochemical properties and biological activity as fluorination of the azobenzene core alters not only the photochromic behavior but also the pharmacological profile at the muscarinic M1 receptor. These findings demonstrate that incorporation of fluorinated azobenzenes not just may alter photophysical properties but can exhibit a considerably different biological profile that has to be carefully investigated.
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Flúor/química , Isoxazoles/química , Isoxazoles/farmacología , Luz , Agonistas Muscarínicos/química , Agonistas Muscarínicos/farmacología , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/farmacología , Compuestos Azo/química , Células HEK293 , Halogenación , Humanos , Receptor Muscarínico M1/efectos de los fármacosRESUMEN
The investigation of the mode and time course of the activation of G-protein-coupled receptors (GPCRs), in particular muscarinic acetylcholine (mACh or M) receptors, is still in its infancy despite the tremendous therapeutic relevance of Mâ receptors and GPCRs in general. We herein made use of a dualsteric ligand that can concomitantly interact with the orthosteric, that is, the neurotransmitter, binding site and an allosteric one. We synthetically incorporated a photoswitchable (photochromic) azobenzene moiety. We characterized the photophysical properties of this ligand called BQCAAI and investigated its applicability as a pharmacological tool compound with a set of FRET techniques at the M1 receptor. BQCAAI proved to be an unprecedented molecular tool; it is the first photoswitchable dualsteric ligand, and its activity can be regulated by light. We also applied BQCCAI to investigate the time course of several receptor activation processes.