Development of Fluorescent 4-[4-(3 H-Spiro[isobenzofuran-1,4'-piperidin]-1'-yl)butyl]indolyl Derivatives as High-Affinity Probes to Enable the Study of σ Receptors via Fluorescence-Based Techniques

Abstract

Sigma (σ) receptor subtypes, σ₁ and σ₂, are targets of wide pharmaceutical interest. The σ₂ receptor holds promise for the development of diagnostics and therapeutics against cancer and Alzheimer's disease. Nevertheless, little is known about the mechanisms activated by the σ₂ receptor. To contribute to the exploitation of its therapeutic potential, we developed novel specific fluorescent ligands. Indole derivatives bearing the N-butyl-3H-spiro[isobenzofuran-1,4'-piperidine] portion were functionalized with fluorescent tags. Nanomolar-affinity fluorescent σ ligands, spanning from green to red to near-infrared emission, were obtained. Compounds 19 (σ pan affinity) and 29 (σ₂ selective), which displayed the best compromise between pharmacodynamic and photophysical properties, were investigated in flow cytometry, confocal, and live cell microscopy, demonstrating their specificity for the σ₂ receptor. To the best of our knowledge, these are the first red-emitting fluorescent σ₂ ligands, validated as powerful tools for the study of σ₂ receptors via fluorescence-based techniques.

Figure 1

σ₂ receptor reference compounds and fluorescent ligands.

Scheme 1. Synthetic Pathways for the Synthesis of Final Compounds 16–20

Reagents: (a) 3H-spiro[isobenzofuran-1,4′-piperidine], CDI, dry THF, RT, overnight; (b) 3-bromopropanenitrile, KOH, K₂CO₃, CH₃CN, MW, 150 °C, 1 h; (c) BH₃·DMS, dry MeOH, reflux, 4 h; (d) 1,6-dibromohexane, TBAB, KOH, dry DMF, RT, 2 h; (e) NaN₃, dry DMF, 60 °C, 20 h; (f) PPh₃, dry MeOH, 80 °C, 1 h; (g) 4-(dimethylamino)phthalic acid, CDI, dry DMF, RT, overnight; (h) BODIPY-TR-COOH, HATU, DIPEA, CH₂Cl₂, 30 °C, O/N; (i) Cy-5-COOH, HATU, DIPEA, CH₂Cl₂, 30 °C, overnight; (j) Cy-7-COOH, HATU, DIPEA, CH₂Cl₂, 30 °C, overnight.

Scheme 2. Synthetic Pathways for the Synthesis of Final Compounds 23 and 28–30

Reagents: (a) 4-(dimethylamino)phthalic acid, CDI dry DMF, RT, overnight; (b) 1-bromo-4-chloro-butane, t-BuOK, dry DMF, RT, 1 h; (c) 3H-spiro[isobenzofuran-1,4′-piperidine], K₂CO₃, CH₃CN, reflux, overnight; (d) 2,5-hexanedione, dry toluene, reflux, 6 h; (e) 1-bromo-4-chloro-butane, TBAB, KOH, dry DMF, RT, 2 h; (f) NH₂OH·HCl, 2:1 EtOH/H₂O, 30 °C, overnight; (g) BODIPY-TR-COOH, HATU, DIPEA, CH₂Cl₂, 30 °C, overnight; (h) Cy-5-COOH HATU, DIPEA, CH₂Cl₂, 30 °C, overnight; (i) Cy-7-COOH, HATU, DIPEA, CH₂Cl₂, 30 °C, overnight.

Figure 2

Top-scoring docking poses of (A) siramesine within the binding pocket of σ₁ (PDB entry 6DK1), (B) siramesine within the binding pocket of σ₂ (PDB entry 7M95), (C) 8 within the binding pocket of σ₁ (PDB entry 6DK1), and (D) 8 within the binding pocket of σ₂ (PDB entry 7M95). For the sake of clarity, only polar hydrogen atoms are shown. Important residues are rendered as sticks, while the proteins are represented as cartoon. Salt-bridge and cation−π interactions are depicted as red and green lines, respectively.

Figure 3

Top-scoring docking poses of (A) 17 within the binding pocket of σ₁ (PDB entry 6DK1), (B) 17 within the binding pocket of σ₂ (PDB entry 7M95), (C) 23 within the binding pocket of σ₁ (PDB entry 6DK1), and (D) 23 within the binding pocket of σ₂ (PDB entry 7M95). For the sake of clarity, only polar hydrogen atoms are shown. Important residues are rendered as sticks, while the proteins are represented as a cartoon. Salt-bridge and cation−π interactions are depicted as red and green lines, respectively.

Figure 4

Top-scoring docking poses of (A) 29 within the binding pocket of σ₁ (PDB entry 6DK1), (B) 29 within the binding pocket of σ₂ (PDB entry 7M95), (C) 30 within the binding pocket of σ₁ (PDB entry 6DK1), and (D) 30 within the binding pocket of σ₂ (PDB entry 7M95). For the sake of clarity, only polar hydrogen atoms are shown. Important residues are rendered as sticks, while the proteins are represented as a cartoon. Salt-bridge and cation−π interactions are depicted as red and green lines, respectively.

Figure 5

(A) Saturation binding assay on MCF7 and MCF7KO (silenced in TMEM97/σ₂) cells. (B) Flow cytometry. Dose-dependent increase in the mean fluorescence intensity (MFI) in MCF7 and MCF7KO (one representative image of three repetitions) cells upon administration of compound 19, 29, or 3 at the indicated concentrations.

Figure 6

Representative confocal microscopy images showing the co-localization of 19 and 29 with σ₂ receptor expression in MCF7 cells. Cells were preincubated with 10 μM selective σ₁ receptor agonist (+)-pentazocine for 2 h to mask residual σ₁ receptor expression. This was followed by incubation for 1 h with the indicated fluorescent ligands at 5 μM. Then cells were fixed with paraformaldehyde, stained for σ₂ receptor expression (TMEM97 ab) as well as nuclei (by DAPI), and analyzed by confocal microscopy. The ligands are colored red (scale bar of 20 μm).

Figure 7

Representative spinning disk images showing the localization of ligand 19 as well as 29 with the mitochondria as well as reduction of the Cy-5 signal of the ligands in the presence of the nonfluorescent σ₂ ligand DTG in living MCF7 cells. Cells were preincubated with 10 μM 31 to mask residual σ₁ receptor expression with or without 20 μM DTG for 75 min. This was followed by administration of 0.05 μM 19 or 29 for 30 min (red stain in the Cy-5 channel). In addition, mitochondria were stained with 250 nM MitoTracker-Bodipy FL (green stain in the GFP channel) and the nuclei were counterstained with Hoechst 33342 (blue stain in the DAPI channel). The depicted scale bar indicates 20 μm.

Figure 8

Representative microscopically live cell images showing time-dependent accumulation of ligand 19 as well as 29 in MCF7 cells from 0 to 20 min. Cells were preincubated with 10 μM selective σ₁ receptor agonist 31 for 2 h to mask residual σ₁ receptor expression. This was followed by administration of 19 and 29. For time-lapse experiments, images were collected every 5 min for a period of 4 h. The bright field (BF) and the fluorescence channel Cy-5 for the ligands (red) are shown (scale bar of 40 μm).