Development of A Novel NanoBRET High-Throughput Drug Screening Assay for Human GnRH Receptor Using Sulfo-cyanine 5 Fluorophore

Li Shen; Xiaozhe Du; Yakai Yang; Ming Su; Rong Rong; Jia Meng; Lee Wei Lim; David G. Fernig; Zhi-Liang Lu

doi:10.1016/j.jpha.2025.101532

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Li Shen, Xiaozhe Du, Yakai Yang, Ming Su, Rong Rong, Jia Meng, Lee Wei Lim, David G. Fernig, Zhi-Liang Lu. Development of A Novel NanoBRET High-Throughput Drug Screening Assay for Human GnRH Receptor Using Sulfo-cyanine 5 Fluorophore[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2025.101532

Citation:

Li Shen, Xiaozhe Du, Yakai Yang, Ming Su, Rong Rong, Jia Meng, Lee Wei Lim, David G. Fernig, Zhi-Liang Lu. Development of A Novel NanoBRET High-Throughput Drug Screening Assay for Human GnRH Receptor Using Sulfo-cyanine 5 Fluorophore[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2025.101532

Citation:

Li Shen, Xiaozhe Du, Yakai Yang, Ming Su, Rong Rong, Jia Meng, Lee Wei Lim, David G. Fernig, Zhi-Liang Lu. Development of A Novel NanoBRET High-Throughput Drug Screening Assay for Human GnRH Receptor Using Sulfo-cyanine 5 Fluorophore[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2025.101532

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Development of A Novel NanoBRET High-Throughput Drug Screening Assay for Human GnRH Receptor Using Sulfo-cyanine 5 Fluorophore

doi: 10.1016/j.jpha.2025.101532

a. Department of Biosciences and Bioinformatics, and Suzhou Municipal Key Lab of Cancer Biology and Chronic Disease, School of Science, Xi’an Jiaotong-Liverpool University, Suzhou, Jiangsu, 215123, China;
b. Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 7BE, United Kingdom

Funds:

This work was supported by the XJTLU Research Development Fund, China (Grant No.: RDF-22-01-045 and RDF-TP-003), the XJTLU Key Program Special Fund, China (Grant No.: KSF-E-33), and the National Natural Science Foundation of China (Grant No.: NSFC 81373469).

Received Date: Apr. 17, 2025
Accepted Date: Dec. 15, 2025
Rev Recd Date: Dec. 15, 2025
Available Online: Dec. 18, 2025

Abstract

Abstract

G protein-coupled receptors (GPCRs), the largest superfamily of cell surface receptors and targets for over 30% of current clinical drugs, remain crucial for future therapeutic development. This study introduces a novel NanoLuciferase (NanoLuc, Nluc) bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay, utilizing the gonadotrophin-releasing hormone (GnRH) receptor as a model system. Our study demonstrates that sulfo-cyanine 5 (sCy⁵) is an ideal fluorophore compatible with NanoBRET, enabling sensitive measurement of ligand binding on living cell membranes. A novel GnRH analogue, sCy⁵-D-Lys⁶-GnRH, was synthesised by conjugating sCy⁵ on the substituted D-Lys⁶ of the native GnRH I. Substitution of Gly⁶ of GnRH I with sCy⁵-D-Lys⁶ stabilises the βII’ turn configuration of the decapeptide that exhibits high affinity and specificity for GnRH receptors while maintaining agonist activity. To address the characteristically low expression of the human GnRH receptor (hGnRHR), we engineered a modified receptor by fusing NanoLuc with an interleukin-6 secretory signal peptide (secNluc) to the N-terminus of the hGnRHR and deleting Lys191 (K191Δ) within the 2^nd extracellular loop. This modification (N-secNluc-hGnRHR-K191Δ) significantly enhances receptor expression without altering ligand binding affinity, resulting in a robust BRET signal detection (Z' ≥ 0.5) between sCy⁵-D-Lys⁶-GnRH and the modified receptor. Our innovative approach using sCy⁵ to conjugate ligands offers several key advantages: high sensitivity and specificity, remarkably low non-specific binding (NSB), compatibility with live-cell assays, and suitability for high-throughput drug screening, which may accelerate the discovery of new therapeutics for GnRH receptor signal-selective drugs and potentially for other GPCRs.
- G protein-coupled receptors,
- high throughput drug screening,
- NanoBRET,
- fluorescent ligand,
- gonadotrophin-releasing hormone (GnRH),
- GnRH receptor

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References(50)

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