Date Published: December 18, 2009
Publisher: Public Library of Science
Author(s): Michelle Sexton, Grace Woodruff, Eiron Cudaback, Faith R. Kreitzer, Cong Xu, Yi Hsing Lin, Thomas Möller, Mingfeng Bai, H. Charles Manning, Darryl Bornhop, Nephi Stella, Yuan Luo. http://doi.org/10.1371/journal.pone.0008271
Abstract: PK 11195 and DAA1106 bind with high-affinity to the translocator protein (TSPO, formerly known as the peripheral benzodiazepine receptor). TSPO expression in glial cells increases in response to cytokines and pathological stimuli. Accordingly, [11C]-PK 11195 and [11C]-DAA1106 are recognized molecular imaging (MI) agents capable of monitoring changes in TSPO expression occurring in vivo and in response to various neuropathologies.Here we tested the pharmacological characteristics and TSPO-monitoring potential of two novel MI agents: NIR-conPK and NIR-6T. NIR-conPK is an analogue of PK 11195 conjugated to the near-infrared (NIR) emitting fluorophore: IRDye 800CW. NIR-6T is a DAA1106 analogue also conjugated to IRDye 800CW.We found that NIR-6T competed for [3H]-PK 11195 binding in astrocytoma cell homogenates with nanomolar affinity, but did not exhibit specific binding in intact astrocytoma cells in culture, indicating that NIR-6T is unlikely to constitute a useful MI agent for monitoring TSPO expression in intact cells. Conversely, we found that NIR-conPK did not compete for [3H]-PK 11195 binding in astrocytoma cell homogenate, but exhibited specific binding in intact astrocytoma cells in culture with nanomolar affinity, suggesting that NIR-conPK binds to a protein distinct, but related to, TSPO. Accordingly, treating intact astrocytoma cells and microglia in culture with cytokines led to significant changes in the amount of NIR-conPK specific binding without corresponding change in TSPO expression. Remarkably, the cytokine-induced changes in the protein targeted by NIR-conPK in intact microglia were selective, since IFN-γ (but not TNFα and TGFβ) increased the amount of NIR-conPK specific binding in these cells.Together these results suggest that NIR-conPK binds to a protein that is related to TSPO, and expressed by astrocytomas and microglia. Our results also suggest that the expression of this protein is increased by specific cytokines, and thus allows for the monitoring of a particular subtype of microglia activation.
Partial Text: Molecular imaging (MI) agents allow for the non-invasive monitoring of molecular events in intact cells and tissues. They include high affinity receptor ligands that are labeled with radioactive isotopes or conjugated to biocompatible imaging moieties. Thus, the signal emitted by MI agents – be it radioactivity or light–fluctuates in parallel with any change in the expression of the targeted receptor.
To determine the pharmacological characteristics of the new MI agents targeting TSPO that we are studying, we required a cell line that expresses relatively high levels of TSPO. Using qPCR, we found that DBT cells, a highly malignant mouse astrocytoma cell line , express high levels of TSPO mRNA compared to two other mouse astrocytoma cell lines, D30 and D1A cells (Table 1) . By comparison, levels of TSPO mRNA in DBT cells were also higher than in healthy mouse brain, mouse microglia in primary culture and BV-2 cells (a mouse microglia cell line) (Table 1) . High TSPO expression in DBT cells was confirmed by filtration radioligand binding assays using [3H]-PK 11195 (we detected 14 pmol of TSPO per gram of protein, Fig. 1A, Table 2). Increasing concentrations of PK 11195 and DAA1106 competed for [3H]-PK 11195 binding with Ki of 2.0 and 0.2 nM, respectively (Fig. 1B, C), values that are well within the range of what has been reported . These results show that DBT cells express relatively high levels of TSPO and therefore constitute a reliable cell model to determine the pharmacological characteristics of agents targeting TSPO with nanomolar affinity.
Cancerous transformation and immune cell activation are associated with changes in the expression of hundreds to thousands of proteins , . Some of these proteins, such as TSPO, constitute valuable targets for non-invasive in vivo MI and prognosis. However, when developing new MI agents, it is essential to systematically verify several criteria: that the selectivity and affinity of the MI agent at the targeted protein remains optimal, and that this MI agent reliably monitors any change in the expression of the targeted protein. Indeed, as we show in this study, the addition of an imaging moiety, such as a large hydrophobic fluorophore, to a ligand can greatly reduce its affinity toward the targeted protein, while increasing its affinity at a related protein. Thus, it is important to systematically verify that the amount of specific binding measured for a new MI agent indeed fluctuates concomitantly to changes in the expression of the targeted protein. Another challenge is to increase our understanding of the physiopathological significance of any changes in the expression of the targeted protein. Our aim was to address these questions for two MI agents, NIR-conPK and NIR-6T, originally developed to monitor TSPO expression in intact cells.