Optimized Translocator Protein Ligand for Optical Molecular Imaging and Screening

Abstract

Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (29, K_d = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an n-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand-protein interference. Probe 29 was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe 29 quantified by fluorescence and ³H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand 29 for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties.

Figure 1.

Compound structures of TSPO ligands cited in the text.

Figure 2.

Best score modeling structure of probe 29 interaction with human TSPO (see all poses in Supporting Information).

Figure 3.

Fluorescent probe 29 aqueous spectroscopy (A) and molar extinction coefficient curve (B).

Figure 4.

Saturation curve of fluorescent probe 29 binding to C6 glioma cell lysate (A black curve, and B) and displacement experiments (A red curve). C6 glioma cell lysates were coincubated with 0.50−100 nM fluorescent probe and 100 nM of its parent ligand.

Figure 5.

Confocal microscopy images of C6 rat glioma cells incubated with optical probe 29. (A,D) fluorescent images of optical probe; (B,E) fluorescent images of MitoTracker Red; (C,F) merged images of optical probe and MitoTracker Red. (D,E,F) fluorescent images of the chosen area in (A); (G,H,I) and (J,K,L) images of displacement experiment. C6 cells were coincubated with 0.1 μM optical probe 29 and 10 μM of its parent ligand (G,H,I) or PK 11195 (J,K,L); (M,N,O) images of C6 glioma cells dosed with MitoTracker Red only.

Scheme 1.

Synthesis of TSPO Fluorescent Ligands 23−29