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: Hui Liu, Hongjun Jin, Junfeng Li, Xiang Zhang, Kota Kaneshige, Stanley M Parsons, Joel S Perlmutter, Zhude Tu
• Journal: European journal of pharmacology (Eur J Pharmacol) Vol. 752 Pg. 18-25 (Apr 05 2015) ISSN: 1879-0712 [Electronic] Netherlands
• PMID: 25678250 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical References

• Aniline Compounds
• Ligands
• Piperidines
• TZ659 compound
• Vesicular Acetylcholine Transport Proteins

Topics

• Aniline Compounds (metabolism)
• Animals
• Brain (metabolism)
• Humans
• Isotope Labeling
• Ligands
• Male
• Molecular Docking Simulation
• Molecular Imaging
• PC12 Cells
• Piperidines (metabolism)
• Protein Conformation
• Protein Transport
• Rats
• Substrate Specificity
• Vesicular Acetylcholine Transport Proteins (chemistry, metabolism)