title: In vitro and ex vivo characterization of (-)-TZ659 as a ligand for imaging the vesicular acetylcholine transporter.
abstract: The loss of cholinergic neurons and synapses relates to the severity of dementia in several neurodegenerative pathologies; and the vesicular acetylcholine transporter (VAChT) provides a reliable biomarker of cholinergic function. We recently characterized and (11)C-labeled a new VAChT inhibitor, (-)-TZ659. Here we report the in vitro and ex vivo characterization of (-)-TZ659. A stably transfected PC12(A123.7) cell line which expresses human VAChT (hVAChT) was used for the in vitro binding characterization of (-)-[(3)H]TZ659. A saturated binding curve was obtained with Kd=1.97±0.30nM and Bmax=3240±145.9fmol/mg protein. In comparison, a PC12(A123.7) cell line that expresses mutant hVAChT showed decreased binding affinity (Kd=15.94±0.28nM). Competitive binding assays using a panel of other CNS ligands showed no inhibition of (-)-[(3)H]TZ659 binding. On the other hand, binding inhibitions were observed only using VAChT inhibitors (Ki=0.20-31.35nM). An in vitro assay using rat brain homogenates showed that (-)-[(3)H]TZ659 had higher binding in striatum than in cerebellum, with a target: non-target ratio>3.46. Even higher ex vivo striatum-to-cerebellum ratios (9.56±1.11) were observed using filtered homogenates of brain tissue after rats were injected intravenously with (-)-[(11)C]TZ659. Ex vivo autoradiography of (-)-[(11)C]TZ659 confirmed high striatal uptake, with a consistently high striatum-to-cerebellum ratio (2.99±0.44). In conclusion, (-)-TZ659 demonstrated high potency and good specificity for VAChT in vitro and in vivo. These data suggest that (-)-[(11)C]TZ659 may be a promising PET tracer to image VAChT in the brain.
authors: Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors: Hui
authors: H
authors: Liu
authors: Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors: Hongjun
authors: H
authors: Jin
authors: Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors: Junfeng
authors: J
authors: Li
authors: Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors: Xiang
authors: X
authors: Zhang
authors: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
authors: Kota
authors: K
authors: Kaneshige
authors: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
authors: Stanley M
authors: SM
authors: Parsons
authors: Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors: Joel S
authors: JS
authors: Perlmutter
authors: Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: tuz@mir.wustl.edu.
authors: Zhude
authors: Z
authors: Tu
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D008297:Male; D062105:Molecular Docking Simulation; D057054:Molecular Imaging; D016716:PC12 Cells; D010880:Piperidines / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport; D051381:Rats; D013379:Substrate Specificity; D050494:Vesicular Acetylcholine Transport Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D008024:Ligands; D010880:Piperidines; C000603497:TZ659 compound; D050494:Vesicular Acetylcholine Transport Proteins
keywords: Autoradiography; Binding assay; Radioligand; Vesicular acetylcholine transporter