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

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 synthesized by conjugating sCy5 on the substituted D-Lys6 of the native GnRH I. Substitution of Gly6 of GnRH I with sCy5-D-Lys6 stabilizes 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 (IL6) secretory signal peptide (secNluc) to the N-terminus of the hGnRHR and deleting Lys191 (K191Δ) within the 2nd extracellular loop. This modification, N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (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.