O6-3-[ 125I]iodobenzyl-2′-deoxyguanosine ([ 125I]IBdG): synthesis and evaluation of its usefulness as an agent for quantification of alkylguanine-DNA alkyltransferase (AGT)

Abstract

The development of O 6-(3-[ 125I]iodobenzyl)-2′-deoxyguanosine ([ 125I]IBdG), the glycosylated analogue of the O 6-3-iodobenzylguanine (IBG), as an agent for the in vivo mapping of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) is described. Synthesis of its tin precursor, O 6-3-trimethylstannylbenzyl-2′-deoxyguanosine (TBdG) was achieved in four steps from deoxyguanosine. Radioiodination of TBdG in a single step gave [ 125I]IBdG in 70–85% isolated radiochemical yield. [ 125I]IBdG bound specifically to pure AGT with an IC 50 of 7.1 μM. From paired-label assays, [ 125I]IBdG showed a 2- to 3-fold higher cellular uptake than [ 131I]IBG in DAOY medulloblastoma, TE-671 rhabdomyosarcoma, SK-Mel-28 melanoma, and HT-29 colon carcinoma human cell lines. Uptake of both labeled compounds in these cell lines decreased with increasing concentrations of unlabeled O 6-benzylguanine (BG) when BG was present in the medium during incubation with the labeled compounds. Compared to BG, unlabeled IBdG diminished the uptake of [ 125I]IBdG and [ 131I]IBG in DAOY cells more efficiently (IC 50 < 1 μM vs >10 μM for BG). There was no significant change in cell-bound activity of [ 125I]IBdG and [ 131I]IBG when BG was removed from the incubation medium before incubating cells with the tracers, suggesting that only a very small portion of radioactivity taken up by the cells is AGT bound. This was corroborated by gel-electrophoresis performed on extracts from cells treated with varying amounts of BG and then incubated with [ 125I]IBdG in the presence of BG. No radiolabeled AGT band was discernable by phosphor-imaging, signifying low cellular AGT binding of the radiotracer. In contrast, when cell extracts were prepared from BG pre-treated cells and aliquots were incubated with [ 125I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [ 125I]IBdG that binds to AGT does so in a concentration dependent manner. These data suggest that IBdG is transported across the cell membrane to a higher degree than IBG. However, to be a practical tracer for quantifying cellular AGT, considerable localization of such derivatives need to occur within the cell nucleus where AGT is present predominantly.