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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.

SUBMITTER: Liu H

PROVIDER: S-EPMC4369186 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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In vitro and ex vivo characterization of (-)-TZ659 as a ligand for imaging the vesicular acetylcholine transporter.

Liu Hui H Jin Hongjun H Li Junfeng J Zhang Xiang X Kaneshige Kota K Parsons Stanley M SM Perlmutter Joel S JS Tu Zhude Z

European journal of pharmacology 20150209

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 charac ...[more]

PMID: 25678250

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Project description:Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC 2.A.1.2.13) [Vardy, E., et al. (2004) Protein Sci. 13, 1832-1840]. In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12(A123.7) cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with a pK of 6.5 for binding of vesamicol and with a pK of approximately 5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [(3)H]vesamicol binding centered at pH 7.4, mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH >or=9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far from D398 and the ACh binding site in three-dimensional space. Also, a probable network of interactions involving E309 and an unknown residue having a pK of 10 has been revealed.

Dataset Information

In vitro and ex vivo characterization of (-)-TZ659 as a ligand for imaging the vesicular acetylcholine transporter.

Publications

In vitro and ex vivo characterization of (-)-TZ659 as a ligand for imaging the vesicular acetylcholine transporter.

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