Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2

Abstract

Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G proteincoupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (1), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9a) as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and highthroughput manner. Further, we show that 9a can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small-molecule-based fluorescent CXCR2 ligand 9a represents a promising tool for future studies of CXCR2 pharmacology.

Development and Initial Characterization of the First 18F-CXCR2-Targeting Radiotracer for PET Imaging of Neutrophils

“…Hence, our data cannot be directly compared with that in Figure . We used transfected human embryonic kidney cells (HEK293T) and a squaramide-based fluorescent CXCR2 ligand for the NanoBRET assay . Navarixin ( 8 ) and compound 10 were used as positive controls.…”

“…We used transfected human embryonic kidney cells (HEK293T) and a squaramide-based fluorescent CXCR2 ligand for the NanoBRET assay. 50 Navarixin (8) and compound 10 were used as positive controls. A 3-fold decrease in activity of compound 14c compared to reference compound 10 was observed and considered insufficient (Scheme 1).…”

“…The constructs for the Nluc-labeled CCR2, CCR9, and CXCR2 were prepared as previously published. 50,56,57 Live Cell NanoBRET. HEK293T cells were transfected with the CXCR2_Nluc plasmid (solution A: 5.5 μg DNA in 250 μL PBS) using PEI (solution B: 7.5 μg in 250 μL PBS) as transfection reagent.…”

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1

“…For selectivity studies of 14 with CCR2, CCR9, and CXCR2, we incubated membrane preparations from HEK293T cells expressing the published CCR2-GSSG-Nluc (4 μg/well), CCR9-Nluc (2 μg/well), and CXCR2-Nluc (2 μg/well) constructs, respectively, with 5 μM 14 in the presence and absence of 10 μM NT(8–13). Nonspecific binding was determined in the presence of 10 μM CCR2-RA, ,, 10 μM vercirnon, and 1 μM cmpd24 as established intracellular ligands for CCR2, CCR9, and CXCR2, respectively.…”

“…Their complex pharmacology and probe dependence often require labor-intensive testing in various functional assays, which complicate high-throughput screening. Moreover, well-established target engagement methodologies such as radioligand binding may not be available, e.g., because of the lack of high-affinity probes. , Here, the emerging fluorescence-based binding assays represent a valuable alternative, as they do not require extremely high affinities and can be performed in a mix-and-measure setup. , Bioluminescence resonance energy transfer (BRET) ligand binding assays based on fluorescent probes and the optimized nanoluciferase (Nluc) have been established for structurally diverse GPCRs. − Especially, the combination of a receptor tagged to Nluc at its C-terminus and a TAMRA- or bodipy-labeled ligand was shown to be suitable for nanoBRET target engagement assays at the intracellular binding pockets of the chemokine receptor family. − …”