Development of molecular probes for imaging sigma-2 receptors in vitro and in vivo

Abstract

The sigma-2 (sigma(2)) receptor is proving to be an important protein in the field of cancer biology. The observations that sigma(2) receptors have a 10-fold higher density in proliferating tumor cells than in quiescent tumor cells, and that sigma(2) receptor agonists are capable of killing tumor cells via apoptotic and non-apoptotic mechanisms, indicate that this receptor is an important molecular target for the development of radiotracers for imaging tumors using techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) and for the development of cancer chemotherapeutic agents. In spite of recent promising results towards achieving these goals, research in this field has been hampered by the fact that the molecular identity of the protein sequence of the sigma(2) receptor is currently not known. Consequently, most of what is known about this protein has been obtained using either radiolabeled or fluorescent probes for this receptor, or biochemical analysis of the effect of sigma(2) selective ligands on cells growing under tissue culture conditions. This article provides a review of the development and use of sigma(2) receptor ligands, and how these ligands have been used with a variety of in vitro and in vivo models to gain a greater understanding of the role this receptor plays in cancer.

Figure 1

Differences in the density of the σ₂ receptors in 66P and 66Q cells (A) and the upregulation kinetics of the σ₂ receptors in 66 cells during the Q to P transition and the downregulation kinetics during the P to Q transition (B).

Figure 2

Structures of ligands that are selective for σ₂ versus σ₁ receptors (Ki values).

Figure 3

σ₂ receptor ligands based on the granatane analog, 1 (Ki values).

Figure 4

Structures of the Conformationally-flexible benzamide analogs. Receptor affinity measurements are Ki values for the respective receptors.

Figure 5

Radiolabeled σ₂ receptor ligands for in vitro binding studies.

Figure 6

Tumor uptake and biodistribution studies of [¹⁸F]16 under no-carrier added and σ₁ blocking conditions. Data for the metabolic tracer, [¹⁸F]FDG, and the thymidine analog, [¹²⁵I]IUdR, are included for comparison.

Figure 7

MicroPET imaging studies with [¹⁸F]16 under no-carrier-added (nonselective) and σ₁ blocking (σ₂ selective) conditions. Note the higher background in normal tissues under the nonselective condition.

Figure 8

In vivo studies with ¹¹C-labeled conformationally-flexible benzamide analogs

Figure 9

Structures and in vitro binding affinities (Ki values) of the ortho-2-fluoroethoxy conformationally-flexible benzamide analogs

Figure 10

MicroPET/MicroCT imaging study of [¹⁸F]18 at 2 h post-i.v. injection of the radiotracer. Note the high uptake of the radiotracer in the EMT6 mammary adenocarcinoma and low uptake in the surrounding normal tissues.

Figure 11

Comparison of the σ₂ receptor imaging agent, [⁷⁶Br]8 (right) with the thymidine analog, [¹⁸F]FLT (left) in EMT-6 tumors. Note the high signal : noise ratio of [⁷⁶Br]8, relative to [¹⁸F]FLT.

Figure 12

Structures of ^99mTc-labeled agents having a moderate to high affinity for σ₂ receptors. Affinity measurements are Ki values.

Figure 13

Two-photon microscopy studies with SW107. These “Tracker” co-localization studies reveal that σ₂ receptors are found in the mitochondria (A), lysosomes (B), endoplasmic reticulum (C), and plasma membrane (D), but not in the nucleus.

Figure 14

A: Time-lapsed confocal microscopy studies with K05-138 showing that the σ₂ receptors are located in the cytoplasm, but not in the nucleus. B: The rapid uptake of the fluorescent probe in MDA-MB-435 cells suggested that internalization of the probe may occur by receptor-mediated endocytosis. C: Blocking studies with PAO, a known inhibitor of endocytosis, confirm that the internalization of the σ₂ receptor probe is, in part, by receptor-mediated endocytosis.