RESUMO
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.
Assuntos
Corantes Fluorescentes , Macrófagos , Receptor A2B de Adenosina , Humanos , Células HEK293 , Receptor A2B de Adenosina/metabolismo , Ligantes , Corantes Fluorescentes/química , Macrófagos/metabolismo , Macrófagos/imunologia , Ligação Competitiva , Antagonistas do Receptor A2 de Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologiaRESUMO
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.
Assuntos
Corantes Fluorescentes , Receptor A1 de Adenosina , Ligantes , Receptor A1 de Adenosina/metabolismo , Receptor A1 de Adenosina/química , Humanos , Corantes Fluorescentes/química , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Células HEK293 , Neurônios/metabolismoRESUMO
CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)-based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling.
Assuntos
Quimiocinas CXC , Glicosaminoglicanos , Quimiocinas CXC/metabolismo , Glicosaminoglicanos/farmacologia , Ligantes , Quimiocinas/metabolismo , Transdução de Sinais , Receptores CXCR4/genética , Quimiocina CXCL12RESUMO
The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.
Assuntos
Corantes Fluorescentes , Receptores CXCR4 , Receptores CXCR4/metabolismo , Ligantes , Corantes Fluorescentes/química , Ligação Proteica , Quimiocinas/metabolismo , Quimiocina CXCL12/metabolismoRESUMO
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.
Assuntos
Antagonistas do Receptor A1 de Adenosina/síntese química , Corantes Fluorescentes/química , Receptor A1 de Adenosina/química , Antagonistas do Receptor A1 de Adenosina/metabolismo , Compostos Bicíclicos com Pontes/química , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Octanos/química , Receptor A1 de Adenosina/metabolismo , Relação Estrutura-Atividade , Xantina/química , Xantina/metabolismoRESUMO
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.
Assuntos
Antagonistas do Receptor A2 de Adenosina/química , Corantes Fluorescentes/química , Pirimidinas/química , Receptor A2A de Adenosina/análise , Triazóis/química , Antagonistas do Receptor A2 de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Descoberta de Drogas , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Imagem Óptica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Receptor A2A de Adenosina/metabolismo , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.
Assuntos
Corantes Fluorescentes , Receptores Acoplados a Proteínas G/metabolismo , Triazinas , Triazóis , Marcadores de Afinidade , Desenho de Fármacos , Células HEK293 , Humanos , Ligantes , Receptor A2A de Adenosina/metabolismoRESUMO
A new reactivity mode of hindered lithium amides with terminal epoxides is described whereby aldehyde enamines are produced via a previously unrecognized reaction pathway. Some of these aldehyde enamines display unprecedented C-alkylation reactivity toward unactivated primary and secondary alkyl halides. For comparison, the reactivity of aldehyde enamines synthesized via a traditional condensation method was examined. C- rather than N-alkylation was the dominant reaction pathway found with a range of electrophiles, making this route to alpha-alkylated aldehydes more synthetically useful than previously reported.
Assuntos
Aldeídos/síntese química , Aldeídos/química , Alquilação , Amidas/química , Compostos de Epóxi/química , Lítio/química , Compostos Organometálicos/químicaRESUMO
The human P2Y2 receptor ( hP2Y2R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y2R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y2R antagonists, based on the known, non-nucleotide hP2Y2R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y2R (p Kd = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y2R. These properties make 98 one of the first tools for studying hP2Y2R distribution and organization.
Assuntos
Dibenzocicloeptenos/farmacologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Antagonistas do Receptor Purinérgico P2/síntese química , Antagonistas do Receptor Purinérgico P2/farmacologia , Pirimidinonas/farmacologia , Receptores Purinérgicos P2Y2/efeitos dos fármacos , Astrocitoma/metabolismo , Linhagem Celular , Dibenzocicloeptenos/química , Humanos , Ligantes , Microscopia Confocal , Sondas Moleculares , Ligação Proteica , Pirimidinonas/química , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
A novel molecular scaffold has been synthesized, and its incorporation into new analogues of biologically active molecules across multiple target classes will be discussed. In these studies, we have shown use of the tricyclic scaffold to synthesize potent inhibitors of the serine peptidase DPP-4, antagonists of the CCR5 receptor, and highly potent and selective PI3K δ isoform inhibitors. We also describe the predicted physicochemical properties of the resulting inhibitors and conclude that the tractable molecular scaffold could have potential application in future drug discovery programs.
Assuntos
Antagonistas dos Receptores CCR5/química , Antagonistas dos Receptores CCR5/farmacologia , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Dipeptidil Peptidase 4/metabolismo , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Receptores CCR5/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
[reaction: see text] Reaction of hindered lithium amides with readily available (enantiopure) terminal epoxides gives 2-ene-1,4-diols via carbenoid dimerization of the corresponding alpha-lithiated epoxides. D-Mannitol and D-iditol were synthesized using this method in three steps from (S)-tritylglycidyl ether.
Assuntos
Álcoois/síntese química , Compostos de Epóxi/química , Lítio/química , Manitol/síntese química , Álcoois Açúcares/síntese química , Amidas/química , Catálise , Éteres de Glicerila/química , Estrutura Molecular , EstereoisomerismoRESUMO
A new reactivity mode of lithium amides with epoxides leads to hindered enamines. The reaction of some of these enamines with unactivated primary and secondary alkyl halides is described, which expands the range of electrophiles that one can use in the synthesis of mono-alkylated aldehydes.