Optimized Translocator Protein Ligand for Optical Molecular Imaging and Screening.
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, Kd= 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 and3H-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.
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Jun
J
Li
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Jarrod A
JA
Smith
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Eric S
ES
Dawson
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Allie
A
Fu
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Michael L
ML
Nickels
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Michael L
ML
Schulte
Interdisciplinary Materials Science Program, ∥Vanderbilt University Center for Structural Biology, and ■Department of Biomedical Engineering, Vanderbilt University , Nashville, Tennessee 37232, United States.|Vanderbilt University Institute of Imaging Science (VUIIS), §Center for Molecular Probes, ⊥Department of Radiology and Radiological Sciences, #Department of Biochemistry, ¶Vanderbilt-Ingram Cancer Center (VICC), and ▽Department of Neurosurgery, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
H Charles
0000-0002-5057-4518
HC
Manning
D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D008958:Models, Molecular; D057054:Molecular Imaging; D061848:Optical Imaging; D011485:Protein Binding; D051381:Rats; D018079:Receptors, GABA / Q000032:analysis* / Q000378:metabolism
D008024:Ligands; D018079:Receptors, GABA; C499229:TSPO protein, human