Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: Receptor binding assays of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) in SCL60 cells (a human embryonic kidney 293 cell line stably expressing rat gonadotropin-releasing hormone (GnRH) receptor). (A) Saturation binding assay . SCL60 cells were incubated with various concentrations (0, 0.1–100 nM) of sCy 5 -D-Lys 6 -GnRH for 4 h at 4 °C. Following incubation, unbound ligands were removed by washing the cells with ice-cold Dulbecco's phosphate-buffered saline (DPBS), and relative fluorescence intensity/units (RFU) were measured at emission 680 nm (Em680) under excitation of 640 nm (Ex640). Total binding (TB) was assessed in the absence of unlabeled GnRH I, while non-specific binding (NSB) was determined in the presence of a 100-fold excess of unlabeled GnRH I. Specific binding (SB) was calculated by subtracting NSB from TB. (B) Inhibition binding assay. SCL60 cells were incubated with 10 nM sCy 5 -D-Lys 6 -GnRH and increasing concentrations of unlabeled GnRH I (0–10 μM) for 4 h at 4 °C. After incubation, unbound ligands were washed away with ice-cold DPBS, followed by measurement of RFU. Representative results from three independent experiments, each performed in quadruplicate, are shown. Data are presented as mean ± standard deviations (SD).

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Stable Transfection, Expressing, Saturation Assay, Incubation, Saline, Fluorescence, Inhibition

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: Phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2) induced by sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) , gonadotropin-releasing hormone (GnRH) I, and sCy 5 -kisspeptin 10 (sCy 5 -KP10) in human embryonic kidney 293 (HEK293) cell lines stably expressing rat GnRH receptor (SCL60) or human GnRH receptor (hGnRHR). (A) Time-dependent p-ERK1/2. SCL60 cells were treated with 10 nM sCy 5 -D-Lys 6 -GnRH or GnRH I for the indicated times. (B) Dose-dependent p-ERK1/2. SCL60 cells were treated with vehicle (–∞) or increasing concentrations of sCy 5 -D-Lys 6 -GnRH or GnRH I for 5 min. (C) Ligand-induced ERK1/2 phosphorylation and its inhibition by GnRH antagonist Cetrorelix. HEK 293 cells stably expressing hGnRHR were treated with 10 nM of sCy 5 -D-Lys 6 -GnRH, GnRH I, or 1 μM of sCy 5 -KP10, in the absence or presence of 1 μM of Cetrorelix, for 5 min. β-actin was used as a loading control. Representative blots from three independent experiments are presented. ∗∗ P < 0.01, ∗∗∗ P < 0.001 compared with vehicle-treated controls.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Inhibition, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: Protein expression levels of N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor (N-secNluc-hGnRHR) and its mutants. (A) The protein expression levels of N-secNluc-hGnRHR-K191E and N-secNluc-hGnRHR-K191 deletion (K191Δ) mutants relative to the N-secNluc-hGnRHR were determined by the measurement of relative luminescence intensity/units (RLU) in human embryonic kidney 293 (HEK293) cells transiently expressing N-secNluc-hGnRHRs. (B) The cell surface receptor expression levels of N-secNLuc-hGnRHR and N-secNluc-hGnRHR-K191Δ, transiently expressed in African green monkey kidney fibroblast (COS-7) cells, were measured by relative fluorescence intensity/units (RFU) at emission 680 (Em680) nm under excitation of 640 (Ex640) nm through receptor binding assays using 10, 50, and 100 nM, respectively, of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH). The N-secNluc-hGnRHR-K191Δ (solid/black bars) construct gave a 4.5 to 5-fold higher receptor expression than that of N-secNluc-hGnRHR (open/white bars) on the cell membrane ( ∗∗∗ P < 0.001, n = 3).

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Cell Surface Receptor Assay, Fluorescence, Binding Assay, Construct, Membrane

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: Confocal microscopy image analysis of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) binding to human embryonic kidney 293 (HEK293) cells expressing gonadotropin-releasing hormone (GnRH) receptor. (A) Fluorescence of sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ). (B) Overlay of both fluorescence and brightfield/differential interference contrast (DIC) images of the cells in sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing N-secNluc-hGnRHR-K191Δ. (C) Abolishment of fluorescence of sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing N-secNluc-hGnRHR-K191Δ by co-treatment of the cells with 1 μM of GnRH I. (D) Overlay of both fluorescence and brightfield/DIC images of the HEK293 cells expressing N-secNluc-hGnRHR-K191Δ treated with both sCy 5 -D-Lys 6 -GnRH and 1 μM of GnRH I. (E) The cell membrane localisation of fluorescence of sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing GnRHR and the monomeric teal fluorescent protein-H-Ras membrane biomarker (mTFP-H-Ras). (F) Cell membrane localisation of mTFP-H-Ras membrane biomarker of the sCy 5 -D-Lys 6 -GnRH incubated HEK293 cells expressing GnRHR and mTFP-H-Ras.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Confocal Microscopy, Binding Assay, Expressing, Fluorescence, Membrane, Biomarker Discovery, Incubation

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: NanoLuciferase (Nluc) bioluminescence resonance energy transfer (NanoBRET)-based saturation and inhibition binding assays between sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) andN-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor and its K191 deletion (N-secNluc-hGnRHR and N-secNluc-hGnRHR-K191Δ) transiently expressed in human embryonic kidney 293 (HEK293) cells. For saturation binding assays, transfected HEK293 cells were incubated with various concentrations (0, 1 nM–100 nM) of sCy 5 -D-Lys 6 -GnRH in the absence of (total binding, TB) or presence (non-specific binding (NSB)) of a 100-fold excess of unlabeled gonadotropin-releasing hormone I (GnRH I) for 30 min at 37 °C, followed by the immediate addition of the substrate furimazine (1:1000) before measurement of emissions. For inhibition assays, transfected HEK293 cells were incubated with 10 nM sCy 5 -D-Lys 6 -GnRH and increasing concentrations of unlabeled GnRH I (0, 0.1 nM to 1 μM) for 4 h at 4 °C. The plates were then brought to room temperature, and furimazine (1:1000) was added immediately before measurement. (A) Saturation binding of sCy 5 -D-Lys 6 -GnRH to N-secNluc-hGnRHR. (B) Saturation binding of sCy 5 -D-Lys 6 -GnRH to N-secNluc-hGnRHR-K191Δ. (C) Inhibition binding of N-secNluc-hGnRHR by GnRH I. (D) Inhibition binding of N-secNluc-hGnRHR-K191Δ by GnRH I. Raw bioluminescence resonance energy transfer (BRET) ratios were calculated by the division of the emission at 610 nm longpass (610LP) by the emission at 460 nm with a bandpass of 80 (460BP80). Representative results from at least three independent experiments, each performed in triplicate, are shown.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Bioluminescence Resonance Energy Transfer, Inhibition, Binding Assay, Transfection, Incubation

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: Kinetics of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) binding toN-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) stably expressed in human embryonic kidney 293 (HEK293) cells were examined by measurement of bioluminescence resonance energy transfer (BRET) within a continuous time. The cells were seeded into a 96-well plate at a density of 5 × 10 5 cells per well and incubated with furimazine (1:1000 dilution) for 3 min. After the incubation, three different concentrations of sCy 5 -D-Lys 6 -GnRH, approximating the dissociation equilibrium constant ( K D ) value, were added. Emissions were simultaneously measured at 610 nm longpass (610LP) and 460 nm with a bandpass of 80 nm (460BP80) over 40 min at 37 °C. A representative result from three independent experiments, each performed in triplicate, is shown. Data are presented as mean ± standard deviations (SD).

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Stable Transfection, Bioluminescence Resonance Energy Transfer, Incubation

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: NanoLuciferase (Nluc) bioluminescence resonance energy transfer (NanoBRET)-based ligand binding and the Z′ factor of ligand displacements of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) at(N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) stably expressed in human embryonic kidney 293 (HEK293) cells. (A) Saturation binding of sCy 5 -D-Lys 6 -GnRH at HEK293 cells stably expressing N-secNluc-hGnRHR-K191Δ. The cells were incubated with the indicated concentrations of sCy 5 -D-Lys 6 -GnRH in the absence of (total binding, TB) or presence (non-specific binding (NSB)) of a 100-fold excess of unlabeled gonadotropin-releasing hormone (GnRH) I for 30 min at 37 °C. The emissions at 610 nm longpass (610LP) and 460 nm with a bandpass of 80 nm (460BP80) were measured following the addition of the substrate furimazine. (B) Competitive inhibition binding of HEK293 cells stably expressing N-secNluc-hGnRHR-K191Δ. The cells were incubated with 10 nM of sCy 5 -D-Lys 6 -GnRH and increasing concentrations of unlabeled Cetrorelix, GnRH I, or GnRH II, ranging from 0 (B 0 , the maximum binding) to 1 μM, and the emissions were then measured as above. (C) Z′ factor values were calculated for the competitive inhibition bindings of sCy 5 -D-Lys 6 -GnRH to N-secNluc-hGnRHR-K191Δ. Data are presented as mean ± standard deviations (SD) from three independent assays performed in triplicate.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Bioluminescence Resonance Energy Transfer, Ligand Binding Assay, Stable Transfection, Binding Assay, Expressing, Incubation, Inhibition

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: Validation of high-throughput screening (HTS) using a small-scale library. Human embryonic kidney 293 (HEK293) cells stably expressing N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ ) were plated at densities of 5 × 10 5 cells per well in 96-well plates and were then incubated with 10 nM of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) and increasing concentrations of various ligands, such as gonadotropin-releasing hormone (GnRH) I and II, N-lactoyl-phenylalanine, respectively, ranging from 0 (B 0 , the maximum binding) to 10 μM at 4 °C for 4 h. The plates were then brought to room temperature, and furimazine (1:2000, 5 μM) was added and incubated for 5 min. Emissions were simultaneously measured at 610 nm longpass and 460 nm with a bandpass of 80 nm. Raw bioluminescence resonance energy transfer (BRET) ratios were calculated by the division of the emission at 610 nm by the emission at 460 nm. (A) Inhibition of specific BRET signal between sCy 5 -D-Lys 6 -GnRH and N-secNluc-hGnRHR-K191Δ by endogenous GnRHs and their analogues, peptide and non-peptide antagonists. (B) Effect of various GnRH-unrelated molecules on the BRET ratio. Representative results from three independent experiments, each performed in quadruplicate, are shown.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Biomarker Discovery, High Throughput Screening Assay, Stable Transfection, Expressing, Incubation, Binding Assay, Bioluminescence Resonance Energy Transfer, Inhibition, Analogues

Journal: Journal of Pharmaceutical Analysis

Article Title: 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

Figure Lengend Snippet: NanoLuciferase (Nluc) bioluminescence resonance energy transfer (NanoBRET)-based saturation and inhibition binding of Boron-dipyrromethene 630/650-D-Lys 6 -gonadotropin-releasing hormone (BODIPY630/650-D-Lys 6 -GnRH) atN-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) stably expressed in human embryonic kidney 293 (HEK293) cells. (A) Saturation binding assay. HEK293 cells stably expressing S-N-Nluc-GnRHR-K191Δ were incubated with various concentrations of BODIPY630/650-D-Lys 6 -GnRH in the absence of (total binding, TB) or presence (non-specific binding (NSB)) of a 100-fold excess of unlabeled gonadotropin-releasing hormone (GnRH) I for 30 min at 37 °C. Emissions at 610 nm longpass (610LP) and 460 nm with a bandpass of 80 nm (460BP80) were measured simultaneously following the addition of the substrate furimazine. (B) Inhibition binding assay. HEK293 cells stably expressing S-N-Nluc-GnRHR-K191Δ were incubated with 10 nM BODIPY630/650-D-Lys 6 -GnRH and increasing concentrations of unlabeled GnRH I (0–1 μM) for 4 h at 4 °C. The plate was then brought to room temperature, and furimazine was added immediately before measurement of emissions. The raw bioluminescence resonance energy transfer (BRET) ratios were then calculated. Representative results from at least three independent experiments, each performed in triplicate, are shown.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Bioluminescence Resonance Energy Transfer, Inhibition, Binding Assay, Stable Transfection, Saturation Assay, Expressing, Incubation