Development of sigma-1 (σ1) receptor fluorescent ligands as versatile tools to study σ1 receptors

Abstract

Despite their controversial physiology, sigma-1 (σ1) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ1 receptors, we developed three σ1 fluorescent tracers by functionalizing three well characterized σ1 ligands with a fluorescent tag. A good compromise between σ1 binding affinity and fluorescent properties was reached, and the σ1 specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ1 fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ1 receptors related studies.

Keywords: Fluorescent ligand; Sigma receptors.

Fig. 1

σ₁ Receptor lead compounds exploited for the development of σ₁ fluorescent ligands.

Fig. 2. Labeling of ARPE19 Cells with compound 9

Signal is localized to intracellular membranes. Lower intensity of labeling is detected when cells were prelabeled with PB190 100 µM. Scale bar = 20 µm.

Fig. 3

Flow Cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to compound 5 or 6 and treated with compound 10 (10 µM) to mask σ₂ receptors, or (+)-pentazocine (10 µM) to mask σ₁ receptors, or both 10 (10 µM) with (+)-pentazocine (10 µM). A) Displacement of 5 (100 nM, yellow curve): black curve: control; red curve: 10 µM 10; green curve: 10 µM (+)-pentazocine; violet curve: 10 µM 10 with (+)-pentazocine 10 µM. B) Displacement of 6 (100 nM, yellow curve): black curve: control; red curve: 10 µM 10; green curve: 10 µM (+)-pentazocine; violet curve: 10 µM 10 with (+)-pentazocine 10 µM.

Fig. 4

Flow Cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to 5 and treated with 10 (10 µM) to mask σ₂ receptors. A) Displacement of 5 (100 nM, yellow curve) with increasing concentrations of PB190: black curve: control; orange curve: 1 nM PB190; red curve: 10 nM PB190; green curve: 100 nM PB190; blue curve: 1 µM PB190; violet curve: 10 µM PB190. B) Displacement of 5 (100 nM, yellow curve) with increasing concentrations of PB212: black curve: control; orange curve: 1 nM PB212; red curve: 10 nM PB212; green curve: 100 nM PB212; blue curve: 1 µM PB212; violet curve: 10 µM PB212.

Fig. 5

Flow cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to 6 and treated with 10 (10 µM) to mask σ₂ receptors. A) Displacement of 6 (100 nM, yellow curve) with increasing concentrations of PB190: black curve: control; orange curve: 1 nM PB190; red curve: 10 nM PB190; green curve: 100 nM PB190; blue curve: 1 µM PB190; violet curve: 10 µM PB190. B) Displacement of 6 (100 nM, yellow curve) with increasing concentrations of PB212: black curve: control; orange curve: 1 nM PB212; red curve: 10 nM PB212; green curve: 100 nM PB212; blue curve: 1 µM PB212; violet curve: 10 µM PB212.

Fig. 6

Flow cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to 5 or 6 and treated with 10 (10 µM) to mask σ₂ receptors. A) Displacement of 5 (100 nM, yellow curve) with increasing concentrations of (+)-pentazocine: black curve: control; orange curve: 1 nM (+)-pentazocine; red curve: 10 nM (+)-pentazocine; green curve: 100 nM (+)-pentazocine; blue curve: 1 µM (+)-pentazocine; violet curve: 10 µM (+)-pentazocine. B) Displacement of 6 (100 nM, yellow curve) with increasing concentrations of (+)-pentazocine: black curve: control; orange curve: 1 nM (+)-pentazocine; red curve: 10 nM (+)-pentazocine; green curve: 100 nM (+)-pentazocine; blue curve: 1 µM (+)-pentazocine; violet curve: 10 µM (+)-pentazocine.

Scheme 1

Reagents and Conditions: (a) BBr₃, CH₂Cl₂, from −78 °C to room temperature, overnight; (b) K₂CO₃, DMF, 150 °C, overnight.