RESUMEN
The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.
Asunto(s)
Colorantes Fluorescentes , Receptor de Adenosina A1 , Ligandos , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A1/química , Humanos , Colorantes Fluorescentes/química , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Células HEK293 , Neuronas/metabolismoRESUMEN
Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.
Asunto(s)
Colorantes Fluorescentes , Macrófagos , Receptor de Adenosina A2B , Humanos , Células HEK293 , Receptor de Adenosina A2B/metabolismo , Ligandos , Colorantes Fluorescentes/química , Macrófagos/metabolismo , Macrófagos/inmunología , Unión Competitiva , Antagonistas del Receptor de Adenosina A2/farmacología , Agonistas del Receptor de Adenosina A2/farmacologíaRESUMEN
A major limitation of pulmonary delivery is that drugs can exhibit suboptimal pharmacokinetic profiles resulting from rapid elimination from the pulmonary tissue. This can lead to systemic side effects and a short duration of action. A series of dibasic dipeptides attached to the poorly lung-retentive muscarinic M3 receptor antagonist piperidin-4-yl 2-hydroxy-2,2-diphenylacetate (1) through a pH-sensitive-linking group have been evaluated. Extensive optimization resulted in 1-(((R)-2-((S)-2,6-diaminohexanamido)-3,3-dimethylbutanoyl)oxy)ethyl 4-(2-hydroxy-2,2-diphenylacetoxy)piperidine-1-carboxylate (23), which combined very good in vitro stability and very high rat lung binding. Compound 23 progressed to pharmacokinetic studies in rats, where, at 24 h post dosing in the rat lung, the total lung concentration of 23 was 31.2 µM. In addition, high levels of liberated drug 1 were still detected locally, demonstrating the benefit of this novel prodrug approach for increasing the apparent pharmacokinetic half-life of drugs in the lungs following pulmonary dosing.
Asunto(s)
Profármacos , Animales , Semivida , Pulmón , Antagonistas Muscarínicos/farmacología , Profármacos/química , RatasRESUMEN
The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.
Asunto(s)
Antagonistas del Receptor de Adenosina A1/síntesis química , Colorantes Fluorescentes/química , Receptor de Adenosina A1/química , Antagonistas del Receptor de Adenosina A1/metabolismo , Compuestos Bicíclicos con Puentes/química , Diseño de Fármacos , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Cinética , Ligandos , Octanos/química , Receptor de Adenosina A1/metabolismo , Relación Estructura-Actividad , Xantina/química , Xantina/metabolismoRESUMEN
Among class A G protein-coupled receptors (GPCR), the human adenosine A2A receptor (hA2AAR) remains an attractive drug target. However, translation of A2AAR ligands into the clinic has proved challenging and an improved understanding of A2AAR pharmacology could promote development of more efficacious therapies. Subtype-selective fluorescent probes would allow detailed real-time pharmacological investigations both in vitro and in vivo. In the present study, two families of fluorescent probes were designed around the known hA2AAR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hA2AAR, selectivity over all other adenosine receptor subtypes and allowed clear visualization of specific receptor localization through confocal imaging. Furthermore, the Alexa Fluor 647-labeled conjugate allowed measurement of ligand binding affinities of unlabeled hA2AAR antagonists using a bioluminescence resonance energy transfer (NanoBRET) assay. The fluorescent ligands developed here can therefore be applied to a range of fluorescence-based techniques to further interrogate hA2AAR pharmacology and signaling.
Asunto(s)
Antagonistas del Receptor de Adenosina A2/química , Colorantes Fluorescentes/química , Pirimidinas/química , Receptor de Adenosina A2A/análisis , Triazoles/química , Antagonistas del Receptor de Adenosina A2/metabolismo , Antagonistas del Receptor de Adenosina A2/farmacología , Descubrimiento de Drogas , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Células HEK293 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Imagen Óptica , Pirimidinas/metabolismo , Pirimidinas/farmacología , Receptor de Adenosina A2A/metabolismo , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
The therapeutic action of a drug depends on its ability to engage with its molecular target in vivo. However, current drug discovery strategies quantify drug levels within organs rather than determining the binding of drugs directly to their specific molecular targets in vivo. This is a particular problem for assessing the therapeutic potential of drugs that target malignant tumors where access and binding may be impaired by disrupted vasculature and local hypoxia. Here we have used triple-negative human breast cancer cells expressing ß2-adrenoceptors tagged with the bioluminescence protein NanoLuc to provide a bioluminescence resonance energy transfer approach to directly quantify ligand binding to a G protein-coupled receptor in vivo using a mouse model of breast cancer.