Synthesis and characterization of a specific I-125 labeled TRPC5 radioligand

193 Objectives: Transient receptor potential channels subfamily member 5 (TRPC5) is a Ca2+ permeable cation channel that belongs to the transient receptor potential (TRP) superfamily and it is widely expressed in the brain. Compelling evidence indicates that TRPC5 plays crucial roles in depression and other mental disorders. Nevertheless, the precise functions and mechanisms of the TRPC5 channel in relevant neurological diseases and psychiatric disorders remain inadequately understood. To investigate the role of TRPC5 in pathology of the brain disorders, we radiosynthesized [125I]TZ66127 and performed initial in vitro study to determine if [125I]TZ66127 could be a radioligand that will be used for screening other new TRPC5 ligands and for studying the functions of TRPC5 in the brain. Methods: The iodine containing compound TZ66127 was synthesized by introducing a iodine atom into the benzene ring at its para-position of HC608, a TRPC5 specific inhibitor. The tin precursor TZ66128 was synthesized using conventional reaction conditions starting from commercial available 3-methylxanthine. The radiotracer [125I]TZ66127 was accomplished through an iodode/ stannylation reaction of the tributyltin precursor TZ66128 with [125I]NaI in the presence of HOAc and H2O2 for 15 min at room temperature. The crude product was purified on semi-preparation reversed phase HPLC column combined with solid phase extraction (SPE). In vitro autoradiography was conducted on 20 µm SD rat brain frozen section. Rat brain section were incubated with [125I]TZ66127 (0.03 µCi/slide in 500 μl of binding buffer) at room temperature for 60 min. For the block study, slides were incubated with [125I]TZ66127 and 1 µM of HC070, a potent TRPC5 inhibitor with IC50 values of 9.3 nM for TRPC5. Following the incubation, the brain slides were washed to remove unbound molecules, and then exposed to the Storage Phosphor Screen in an imaging cassette overnight. The ARG signal of rat brain sections was visualized by phosphor-imaging of FLA7000 scanner (GE). Intracellular live cell Ca2+ imaging experiments were performed in human TRPC5 stable transfected cell line HEK293-hTRPC5 to determining the inhibition effect of TZ66127. Fluorescence excitation wavelengths (at 340 and 380 nm) were recorded on an inverted Nikon Ti-E microscope equipped with 340 and 380 nm excitation filter wheels using NIS Elements imaging software (Nikon). Fura-2 ratios (F340/F380) were used to reflect changes in intracellular Ca2+ upon stimulation. Results: [125I]TZ66127 was successfully achieved with good radiochemical yields of 46-50% and high radiochemical purity of > 98%. In vitro autoradiography showed the brain uptake of [125I]TZ66127 is significantly reduced with the presence of HC070. Ca2+ imaging study revealed TZ66127 at 500 nM completely suppressed riluzole-induced Ca2+ responses in human TRPC5 stable transfected cell line HEK293-hTRPC5. In comparison, the inhibitory effects of HC608 and HC070 were weaker than TZ66127 in the same concentration. Moreover, TZ66127 showed dose dependent inhibitory effect on riluzole-envoked Ca2+ responses. Conclusions: Our preliminary in vitro autoradiography results suggested that [125I]TZ66127 specifically binds to TRPC5 channels in rat brain sections. Ca2+ imaging study demonstrated TZ66127 has stronger inhibition on TRPC5 channels compared to HC070 or HC608 does. Together, [125I]TZ66127 has great potential to be a radiotracer for determining the biological activity of compound binding toward TRPC5 and investigating TRPC5 function in the brain. This promising results of our initial data of [125I]TZ66127 will provide useful information for developing PET radiotracer and therapeutic drugs targeting on TRPC5.Research support: USA NIH/NINDS #NS103988, NS075527, and NS103957. References: 1) J. Biol. Chem., 2017, 292, 8158-8173. 2) PloS one, 2018, 13, e0191225.