Characterization of Sigma-2 Receptor-Specific Binding Sites Using [H-3]DTG and [I-125]RHM-4

The sigma-2 receptor/transmembrane protein 97 (sigma 2R/TMRM97) is a promising biomarker of tumor proliferation and a target for cancer therapy. [H-3]DTG has been used to evaluate sigma 2R/TMEM97 binding affinity in compound development studies. However, [H-3]DTG has equal and moderate binding affinities to both sigma 1 receptor (sigma 1R) and sigma 2R/TMEM97. Furthermore, co-administration with the sigma 1R masking compound (+)-pentazocine may cause bias in sigma 2R/TMEM97 binding affinity screening experiments. We have developed a radioiodinated ligand, [I-125]RHM-4, which has high affinity and selectivity for sigma 2R/TMEM97 versus sigma 1R. In this study, a head-to-head comparison between [H-3]DTG and [I-125]RHM-4 on the binding affinity and their effectiveness in sigma 2R/TMEM97 compound screening studies was performed. The goal of these studies was to determine if this radioiodinated ligand is a suitable replacement for [H-3]DTG for screening new sigma 2R/TMEM97 compounds. Furthermore, to delineate the binding properties of [I-125]RHM-4 to the sigma 2R/TMEM97, the structure of RHM-4 was split into two fragments. This resulted in the identification of two binding regions in the sigma 2R, the "DTG " binding site, which is responsible for binding to the sigma 2R/TMEM97, and the secondary binding site, which is responsible for high affinity and selectivity for the sigma 2R/TMEM97 versus the sigma 1R. The results of this study indicate that [I-125]RHM-4 is an improved radioligand for in vitro binding studies of the sigma 2R/TMEM97 versus [H-3]DTG.