Development of Fluorescent 4-[4-(3H-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, σ 1 and σ 2 , are targets of wide pharmaceutical interest. The σ 2 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 σ 2 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 (σ 2 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 σ 2 receptor. To the best of our knowledge, these are the first red-emitting fluorescent σ 2 ligands, validated as powerful tools for the study of σ 2 receptors via fluorescence-based techniques.

Sigma-2 Receptor Ligand Binding Modulates Association between TSPO and TMEM97

“…In MCF7 cells, TMEM97 levels were reduced by shRNA, and the amount of residual TMEM97 was measured by saturating [ 3 H]DTG ( Table 1 , B max = 0.891 pmol/mg protein in shMCF7TMEM97 cells vs. B max = 1.795 pmol/mg protein in WT cells; Figure S2H,D , respectively). As recently reported, a corresponding significant reduction in the binding of two structurally different green-emitting S2R-specific fluorescent ligands (i.e., F412 and NO1) was detected [ 43 ].…”

“…The co-immunoprecipitation data in MCF7 cells were consistent with confocal microscopy results in the same cells, where colocalization was observed between a fluorescent S2R ligand and TSPO ( Figure S3A ) but not with PGRMC1, which exhibited a nuclear localization ( Figure S3B ). TSPO and PGRMC1 were immunostained by specific antibodies against each protein, while S2R (as a surrogate for TMEM97) was stained via a recently reported CY5-labeled S2R-specific ligand [ 43 ].…”