RESUMO
Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.
Assuntos
Quimiocina CXCL12 , Receptores CXCR4 , Humanos , Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL11/metabolismo , Transdução de Sinais , Ligantes , Ligação CompetitivaRESUMO
Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits ß-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of ß-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of ß-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both ß-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for ß-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that ß-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for ß-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential ß-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when ß-arrestin recruitment is impaired or in the absence of ß-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced ß-arrestin recruitment, ACKR3 trafficking and internalization.
Assuntos
Endocitose , Receptores CXCR/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo , Técnicas Biossensoriais , Quimiocina CXCL12/farmacologia , Transferência Ressonante de Energia de Fluorescência , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Células HEK293 , Humanos , Cinética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptores CXCR/agonistas , Receptores CXCR/genética , beta-Arrestina 1/genética , beta-Arrestina 2/genéticaRESUMO
G protein-coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer-based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development. SIGNIFICANCE STATEMENT: The CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor-G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.
Assuntos
Quimiocina CXCL12/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Cinética , Ligação Proteica , Multimerização Proteica , Transdução de SinaisRESUMO
Dysregulation of the chemokine system is implicated in a number of autoimmune and inflammatory diseases, as well as cancer. Modulation of chemokine receptor function is a very promising approach for therapeutic intervention. Despite interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAbs) and antibody-based molecules have been successfully applied in the clinical therapy of cancer and represent a potential new class of therapeutics targeting chemokine receptors belonging to the class of G protein-coupled receptors (GPCRs). Besides conventional mAbs, single-domain antibodies and antibody scaffolds are also gaining attention as promising therapeutics. In this review, we provide an extensive overview of mAbs, single-domain antibodies, and other antibody fragments targeting CXCR4 and ACKR3, formerly referred to as CXCR7. We discuss their unique properties and advantages over small-molecule compounds, and also refer to the molecules in preclinical and clinical development. We focus on single-domain antibodies and scaffolds and their utilization in GPCR research. Additionally, structural analysis of antibody binding to CXCR4 is discussed. SIGNIFICANCE STATEMENT: Modulating the function of GPCRs, and particularly chemokine receptors, draws high interest. A comprehensive review is provided for monoclonal antibodies, antibody fragments, and variants directed at CXCR4 and ACKR3. Their advantageous functional properties, versatile applications as research tools, and use in the clinic are discussed.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/metabolismo , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Receptores CXCR/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidoresRESUMO
Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of both solid and hematological malignancies, rendering this receptor an attractive therapeutic target. Besides the only FDA-approved CXCR4 antagonist Plerixafor (AMD3100), multiple other classes of CXCR4-targeting molecules are under (pre-)clinical development. Nanobodies (Nb), small single variable domains of heavy-chain only antibodies from Camelids, have appeared to be ideal antibody-fragments for targeting a broad range of epitopes and cavities within GPCRs such as CXCR4. Compared to conventional antibodies, monovalent nanobodies show fast blood clearance and no effector functions. In order to further increase their binding affinities and to restore antibody-mediated effector functions, we have constructed three different bivalent nanobody Fc-fusion molecules (Nb-Fc), targeting distinct epitopes on CXCR4, via fusion of Nbs to a Fc domain of a human IgG1 antibody. Most Nb-Fc constructs show increased binding affinity and enhanced potency in CXCL12 displacement, inhibition of CXCL12-induced signaling and CXCR4-mediated HIV entry, when compared to their monovalent Nb counterparts. Moreover, Nb-Fc induced ADCC- and CDC-mediated cell-death of CXCR4-overexpressing CCRF-CEM leukemia cells and did not affect cells expressing low levels or no CXCR4. These highly potent CXCR4 Nb-Fc constructs with Fc-mediated effector functions are attractive molecules to therapeutically target CXCR4-overexpressing tumors.