Novel and Selective Fluorescent σ₂‐Receptor Ligand with a 3,4‐Dihydroisoquinolin‐1‐one Scaffold: A Tool to Study σ₂ Receptors in Living Cells

Abstract

Although sigma-2 (σ2 ) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a σ2 -selective fluorescent tracer (compound 5) as a specific tool to study σ2 receptors. By using flow cytometry with 5, we performed competition binding studies on three different cell lines where we also detected the content of the σ2 receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed σ1 /σ2 fluorescent tracer (1) also allowed for the detection of σ1 receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for σ1 , and Scatchard analysis for σ2 receptors). Thus, we have produced powerful new tools for research on the σ whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of σ receptors in oncology.

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

“…The three compounds displayed very similar maximum excitation wavelength (λex = 390 nm) and maximum emission wavelength (λem ~ 520 nm), with an important difference between λex and λem (i.e., Stokes shift) in accordance to the spectra recorded for the σ 2 ligands with the same fluorescent tag [20,21]. Measurement of quantum yields (QY) demonstrated the environment sensitive properties (low fluorescence intensity in polar solvent such as EtOH, but high fluorescence in nonpolar solvents such as CHCl 3 ), proper for the 4- N,N -dimethylphthalimide fluorescent tag, as recorded for σ 2 ligands.…”

“…Both these cell lines were thoroughly and previously characterized: the former over expresses σ 2 receptors ( B max = 2.02 pmol/mg protein) [5,20] whereas σ 1 receptors' expression is very low ( B max = 0.17 pmol/mg protein) [5,29]; the latter is obtained by stable transfection of MCF7 cells with σ 1 c-DNA to obtain a good expression of σ 1 receptors ( B max = 3.45 pmol/mg protein) [21,29]. All of the experiments were performed by incubation of the two cell lines for 45 and 75 min with either compound 5 or 6 at 100 nM, as the fluorescence signal increased in a dose-dependent manner, reaching a plateau at 100 nM.…”

“…With this aim, we produced three σ 1 receptor fluorescent tracers starting from previously well characterized σ 1 receptor ligands. We adopted the same strategy that was shown to be successful for σ 2 fluorescent ligands: the appropriate σ 1 pharmacophore was linked through a spacer to a green emitting fluorescent tag [19–21]. In particular, as σ 1 pharmacophores we selected 4-methyl-1-[4-(6-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)butyl]piperidine PB190 and 4-methyl-1-[4-(6-methoxynaphthalen-1-yl)butyl]piperidine PB212 (Fig.…”

Sigma 2 receptor expression levels in blood and bladder from healthy and bladder cancer cattle

“…The tissue sample preparation was performed according to Colabufo et al with minor modifications . Bovine bladder tissue was homogenized with a Brinkman Polytron in ice‐cold‐buffer (0.32 M sucrose and 10 mM Tris, pH 7.4).…”

“…The final pellet was stored at −80 °C until use. For each membrane preparation, the protein content was determined by the Lowry method . Blood samples were treated with THF and the precipitate was filtered on frit.…”