title: 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-[(125)I]iodobenzyl)-2'-deoxyguanosine ([(125)I]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 [(125)I]IBdG in 70-85% isolated radiochemical yield. [(125)I]IBdG bound specifically to pure AGT with an IC(50) of 7.1 microM. From paired-label assays, [(125)I]IBdG showed a 2- to 3-fold higher cellular uptake than [(131)I]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 [(125)I]IBdG and [(131)I]IBG in DAOY cells more efficiently (IC(50)<1 microM vs >10 microM for BG). There was no significant change in cell-bound activity of [(125)I]IBdG and [(131)I]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 [(125)I]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 [(125)I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [(125)I]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.
authors.affiliation: Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710, USA.
authors.forename: Sriram
authors.initials: S
authors.lastname: Shankar
authors.forename: Michael R
authors.initials: MR
authors.lastname: Zalutsky
authors.forename: Ganesan
authors.initials: G
authors.lastname: Vaidyanathan
mesh_terms: D045744:Cell Line, Tumor; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis*; D019275:Radiopharmaceuticals / Q000138:chemical synthesis* / Q000493:pharmacokinetics
chemical_list: D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals; C088636:O(6)-benzyl-2'-deoxyguanosine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003849:Deoxyguanosine