Development of a NanoBRET-Based Sensitive Screening Method for CXCR4 Ligands

Abstract

A critical part of the development of CXCR4 modulators is to have a simple and sensitive assay system to complement the search by screening and evaluating the binding affinity. Herein, a NanoBRET assay system was developed, and its feasibility as a high-throughput screening tool for potent CXCR4 ligands was ascertained. TAMRA-Ac-TZ14011, a fluorescent-labeled CXCR4 antagonist, was adopted as a fluorescent acceptor of bioluminescent energy from N-terminally fused NanoLuc-CXCR4 stably expressed in CHO cells. The ratio of fluorescence at 620 nm to the luminescence at 460 nm represents the interaction between test compounds and CXCR4. We have demonstrated in the present study that the NanoBRET assay system is applicable for the evaluation of CXCR4 ligands using the combination of TAMRA-Ac-TZ14011 as an acceptor and NanoLuc tagged to CXCR4 as a bioluminescent donor expressed in living cells. IC50 values of known CXCR4 ligands were determined and found to be compatible with the values obtained by other existing and sensitive methods, such as SDF-1:3.2 nM, Ac-TZ14011:15.3 nM, and FC131:4.5 nM, which confirmed the feasibility of our system (Z′ values ≥0.5). The introduction of an IL-6 secretory signaling peptide (secNluc-CXCR4) further enhanced the expression and trafficking of the tagged receptor, which, in turn, increased the dynamic range of the NanoBRET assay system. Thus, we have successfully developed a NanoBRET system in living cells, which is simple, homogeneous, and useful in multiwell plate screening of potent CXCR4 ligands.

NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells

“…1) was carried out as previously reported in five steps 28,29 . Subsequently, 6 was treated with the respective labeling reagent (13)(14)(15)Fig. 2) in the presence of triethylamine resulting in 8-10.…”

“…Furthermore, the management of radioactive waste is becoming increasingly regulated and expensive. To circumvent these issues, techniques using fluorescently labeled ligands like flow cytometry and the recently described BRET-based binding assay 10,11 , which has been adapted to several G-protein coupled receptors (GPCRs) [12][13][14][15][16][17][18][19][20][21][22][23][24] , have gained great importance.…”

Fluorescent ligands for dopamine D2/D3 receptors

“…We selected three different fluorophores, the commercially available dyes Alexa488 and Cy3B and a recently described bis-trifluoroethyl substituted rhodamine 38 for the synthesis of our fluorescent dopamine receptor probes. Alexa488 and Cy3B, but also tetramethylrhodamine dyes (TAMRA), have already been shown to be suitable for characterizing ligand binding to GPCRs 11 , 22 , 29 , 45 , and especially Cy3B-labeled ligands have proven useful for fluorescence microscopy 14 , 17 , 29 . For the synthesis of these ligands, we focused on the N -propyl substituted 2-amino-dihydro-1 H -indene scaffold.…”

“…Similar to radioligands, they can be detected in very low concentration and with high specificity. In the context of GPCR research, fluorescent ligands have been successfully employed to study receptor internalization 17 , receptor-receptor interactions within the cellular membrane 14 – 16 , 18 , and recently also ligand binding 11 , 22 – 24 , 29 , 45 , 53 , by techniques like fluorescence microscopy, fluorescence polarization and resonance energy transfer. Starting from phenylpiperazine and indanylamine scaffolds, known dopamine-isosteres with antagonistic or agonistic properties, respectively, we have designed and synthesized a small library of fluorescent ligands for D 2 -like receptors.…”

“…In contrast to Renilla luciferase, which is frequently used to study intracellular protein–protein interactions, N-terminal Nluc fusion proteins can be easily targeted to the cell surface 11 . NanoBRET between fluorescently labeled ligands and Nluc-receptor fusion proteins has already been used to study ligand binding at a number of therapeutically relevant GRCRs, including β-adrenergic 11 , adenosine 11 , histamine 20 , free fatty acid 21 , and chemokine 22 receptors. Despite the high therapeutic relevance of the D 2 -like receptor family, only a small number of fluorescent ligands targeting these receptors have been reported so far.…”