Novel Irreversible Fluorescent Probes Targeting the 18 kDa Translocator Protein: Synthesis and Biological Characterization

Abstract

The 18 kDa translocator protein (TSPO) is a mitochondrial protein whose basal density is altered in several diseases, with the result that the evaluation of its expression levels by means of molecular imaging techniques represents a promising diagnostic approach. Experimental procedures using a labeled ligand often cause loss of the bound probe, and consequently high affinity ligands covalently binding the receptor protein are needed to overcome this problem. We have previously described a series of N,N-dialkyl-(2-phenylindol-3-yl)glyoxylamides as potent and selective TSPO ligands. Starting from these derivatives, we designed novel TSPO irreversible ligands bearing an electrophilic isothiocyanato group (7, 8), together with an irreversible NBD-fluorescent probe (18). The TSPO affinity of the new irreversible ligands was measured on rat tissue homogenates by [(3)H]Ro 5-4864 radiobinding kinetic assays, all compounds showing high affinities for the target protein. Further biological characterization of the fluorescent irreversible TSPO probe 18 was carried out by using fluorescent spectroscopy in human glioma cells.

TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance

“…The irDE-MPIGA ligand is structurally related to 2-phenylindol-3-ylglyoxylamide specific TSPO ligands developed by our group [40], and it has been previously investigated in detail, demonstrating the irreversible covalent nature of its interaction with the target protein [39]. A number of irreversible TSPO ligands have been previously employed to improve the understanding of TSPO structure and ligand binding sites [62][63][64][65][66].…”

“…The TSPO ligands irDE-MPIGA and PIGA were synthesized essentially as previously reported [39,40]. The low passage number human U87MG GBM cell line was obtained from the National Institute for Cancer Research of Genoa (Italy) and monitored for DNA profiling.…”

“…Specifically, [ 3 H]PK11195 binding assay was explored in U87MG cell membrane homogenates after 1.5 h cell treatment (time required for a covalent irDE-MPIGA-TSPO interaction [39]) and 24 h cell treatment. After cell treatments, it would be expected that [ 3 H]PK11195 did not bind to TSPO binding sites, as they should be already occupied by irDE-MPIGA.…”

“…loading and after 2 h exposure with irDE-MPIGA before Ca 2? loading, which is the time required for the formation of covalent binding [39]. Interestingly, when the irDE-MPIGA low nanomolar dose was simultaneously added to Ca 2?…”

“…For this purpose, nanomolar doses of irDE-MPIGA, a synthetic ligand that covalently binds TSPO [39] (Fig. 1a), were used at different times.…”