Synthesis and in vitro characterization of a specific I-125 labeled sphingosine-1-phosphate 2 receptor radioligand

409 Objectives: Sphingosine 1-phosphate (S1P) receptor is a G protein-coupled receptor that has five subtypes, S1PR1, 2, 3, 4, and 5. Out of these five subtypes, S1PR2 plays a critical role in mediating different cellular pathologies in cardiovascular, nervous, and immune systems. However, the functions of S1PR2 in diseases are not clearly illustrated and its therapeutic applications remain to be explored. Taking the advantage of relatively long half-life (t1/2 = 60 days), higher specific activity and counting rates than tritium,1 [125I]-labeled specific radiotracer can serve as a convenient tool to screen the compounds’ binding activity toward the target protein using radioactive competitive assay. Also, I-125 is a highly favorable isotope for autoradiography to check the disease pathologies.2 Therefore, [125I]-labeled S1PR2 tracer may facilitate the ligand development of S1PR2 and discover suitable PET radiotracers for assessing the changes of S1PR2 expression in living animals. Herein, we reported our effort on developing and characterizing a highly potent and selective [125I]-labeled S1PR2 tracer, [125I]TZ6544. Our competitive binding data suggest [125I]TZ6544 can be used to determine the binding potencies of other S1PR2 compounds. Methods: The aryl pinacol borate precursor TZ6568 and standard compound TZ6544 were synthesized using conventional reaction conditions. The radiosynthesis of [125I]TZ6544 was accomplished by tetrakis(pyridine)copper(II) triflate-catalyzed iodination of borate precursor TZ6568 with [125I]NaI as radioactive source under room temperature for 10 min. The radiolabeled product was purified on semi-preparation reversed phase HPLC column combined with solid phase extraction (SPE). Human recombinant S1PR2 lysophospholipid receptor membrane was applied to characterize the binding properties of [125I]TZ6544. The binding potencies of known S1PR2 compounds were determined using [125I]TZ6544 competitive binding assay. Results: [125I]TZ6544 was successfully achieved with good radiochemical yields of 45-50% and high radiochemical purity of > 99%. The estimated specific activity of [125I]TZ6544 was ~2.2 Ci/µmol because the carrier-free [125I]NaI was used for I-125 labeling and the effective separation of [125I]TZ6544 from its precursor on HPLC tomography. The saturation binding assay revealed that [125I]TZ6544 specifically binds to the human S1PR2 with high affinity (Kd = 4.31 ± 0.39 nM) and the specific binding data fit a straight line on a Scatchard plot, indicating that one-site binding model is a good model for [125I]TZ6544 binding to human S1PR2 (Figure 1). The competition assay using [125I]TZ6544 as screen radioligand determined the binding potencies (IC50) for known S1PR2 compound JTE-013, S1P, and TZ34125 were 29.4, 5.81, and 8.63 nM, respectively; for the cold compound TZ6544, the IC50 value was 5.06 nM. The result is consistent with the literature reported using [32P]S1P as competitive radioligand (Table 13, 4 ). Conclusions: The radiosynthesis of [125I]TZ6544 was achieved with good radiochemical yield (45-50%) and high purity (> 99%). The in vitro characterization of [125I]TZ6544 including saturation and competition binding demonstrated that [125I]TZ6544 specifically bound to human S1PR2 and showed comparable results as using [32P]S1P. [125I]TZ6544 could be utilized as a more suitable radioligand for screening new S1PR2 compounds by virtue of high selectivity and longer half-life than [32P]S1P (t1/2 = 14 days). Further in vitro autoradiography studies using [125I]TZ6544 to investigate the S1PR2 expression changes in disease model are ongoing. Research support: NMSS: #RG150705331; NINDS: #NS075527 and NS103988; NIBIB: #EB025815. References: (1) Practical Methods in Cardiovascular Research. Springer Berlin Heidelberg; 2005:723. (2) Eur. J. Pharmacol. 1990, 186, 373. (3) Org. Biomol. Chem. 2015, 13, 7928. (4) Bioorg. Med. Chem. Lett. 2018, 28, 488.