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.
Highlights
Molecular imaging (MI) agents allow for the non-invasive monitoring of molecular events in intact cells and tissues
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 [32]. These results show that DBT cells express relatively high levels of TSPO and constitute a reliable cell model to determine the pharmacological characteristics of agents targeting TSPO with nanomolar affinity
This later point is important because such a change in affinity in response to cytokines has been reported and could have accounted for the increase in NIR-conPK specific binding following cytokine treatment [43]. Together these results show that the cytokine-induced increases in NIR-conPK specific binding do not correlate with changes in either TSPO expression or the affinity of PK 11195 at TSPO, reinforcing the conclusion that this novel MI agent binds to a protein distinct from TSPO
Summary
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. [11C]-PK 11195 reliably monitors increases in TSPO expression in brain tumors (including malignant astrocytomas), as well as in the activated microglia found in patients with multiple sclerosis, stroke, epilepsy, Alzheimer’s disease, Huntington’s disease and AIDS [2,3,4,5,6,7,8,9,10] Another MI agent, [11C]DAA1106, which exhibits even higher affinity for TSPO than PK 11195, is used to monitor TSPO expression in activated microglia [11,12,13,14,15]
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