Allele-specific suppression in Caenorhabditis elegans reveals details of EMS mutagenesis and a possible moonlighting interaction between the vesicular acetylcholine transporter and ERD2 receptors.

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Synthesis, resolution, and in vitro evaluation of three vesicular acetylcholine transporter ligands and evaluation of the lead fluorine-18 radioligand in a nonhuman primate.

The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing cholinergic dysfunction associated with dementia. We recently reported three new potent and selective carbon-11 labeled VAChT radiotracers. Herein, we report the resolution with a Chiralcel OD column of three additional fluorine containing VAChT ligands in which a fluoroethoxy or fluoroethylamino moiety was substituted for the methoxy group. An in vitro competitive binding assay showed that (-)-7 had high potency for VAChT (Ki-VAChT = 0.31 ± 0.03 nM) and excellent selectivity for VAChT versus σ receptors (Ki-σ1 = 1870 ± 250 nM, Ki-σ2 = 5480 ± 140 nM). Three different radiolabeling approaches were explored; the radiosynthesis of (-)-[18F]7 was successfully accomplished via a stepwise two-pot, three-step method with moderate yield (11 ± 2%) and high radiochemical purity (>98%). PET imaging studies in a nonhuman primate indicated that (-)-[18F]7 rapidly entered the brain and accumulated in the VAChT-enriched striatum. The uptake of (-)-[18F]7 in the target striatal area peaked at 10 min and displayed improved clearance kinetics compared to the VAChT tracer [18F]VAT, which has been approved by the Food and Drug Administration (FDA) for first-in-man studies. These studies justify further investigation of (-)-[18F]7 and exploration of the structure-activity relationships of these fluoroethoxy and fluoroethylamino analogs.

18F-Labeled indole-based analogs as highly selective radioligands for imaging sigma-2 receptors in the brain.

We have designed and synthesized a series of indole-based σ2 receptor ligands containing 5,6-dimethoxyisoindoline or 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline as pharmacophore. In vitro competition binding assays showed that all ten ligands possessed low nanomolar affinity (Ki=1.79-5.23nM) for σ2 receptors and high subtype selectivity (Ki (σ2)/Ki (σ1)=56-708). Moreover, they showed high selectivity for σ2 receptor over the vesicular acetylcholine transporter (>1000-fold). The corresponding radiotracers [18F]16 and [18F]21 were prepared by an efficient one-pot, two-step reaction sequence with a home-made automated synthesis module, with 10-15% radiochemical yield and radiochemical purity of >99%. Both radiotracers showed high brain uptake and σ2 receptor binding specificity in mice.

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

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.

Regulation of cholinergic activity by the vesicular acetylcholine transporter.

Acetylcholine, the first chemical to be identified as a neurotransmitter, is packed in synaptic vesicles by the activity of VAChT (vesicular acetylcholine transporter). A decrease in VAChT expression has been reported in a number of diseases, and this has consequences for the amount of acetylcholine loaded in synaptic vesicles as well as for neurotransmitter release. Several genetically modified mice targeting the VAChT gene have been generated, providing novel models to understand how changes in VAChT affect transmitter release. A surprising finding is that most cholinergic neurons in the brain also can express a second type of vesicular neurotransmitter transporter that allows these neurons to secrete two distinct neurotransmitters. Thus a given neuron can use two neurotransmitters to regulate different physiological functions. In addition, recent data indicate that non-neuronal cells can also express the machinery used to synthesize and release acetylcholine. Some of these cells rely on VAChT to secrete acetylcholine with potential physiological consequences in the periphery. Hence novel functions for the oldest neurotransmitter known are emerging with the potential to provide new targets for the treatment of several pathological conditions.

Pyrrolovesamicols--synthesis, structure and VAChT binding of two 4-fluorobenzoyl regioisomers.

This Letter describes the synthesis of two regioisomers of a new class of vesamicol analogs as possible ligands for imaging the vesicular acetylcholine transporter in future PET studies. The two pyrrolovesamicols (±)-6a and (±)-6b were synthesized by nucleophilic ring opening reaction of a tetrahydroindole epoxide precursor with 4-phenylpiperidine. The reaction mechanism of the synthesis was studied by HPLC and the molecular structures were determined by X-ray structure analysis. Unexpected low binding affinities to VAChT (K(i)=312±73 nM for (±)-6a and K(i)=7320±1840 nM for (±)-6b) were determined by competitive binding analysis using a cell line stably transfected with ratVAChT and (-)-[(3)H]vesamicol.

3D QSAR study, synthesis, and in vitro evaluation of (+)-5-FBVM as potential PET radioligand for the vesicular acetylcholine transporter (VAChT).

Located in presynaptic cholinergic nerve terminals, the vesicular acetylcholine transporter (VAChT) represents a potential target for quantitative visualization of early degeneration of cholinergic neurons in Alzheimer's disease using PET. Benzovesamicol derivatives are proposed as radioligands for this purpose. We report QSAR studies of vesamicol and benzovesamicol derivatives taking into account the stereoselectivity of the VAChT binding site. Use of different data sets and different models in this study revealed that both enantiomers of 5-fluoro-3-(4-phenyl-piperidin-1-yl)-1,2,3,4-tetrahydro-naphthalen-2-ol (5-FBVM) are promising candidates, with predicted VAChT affinities between 6.1 and 0.05 nM. The synthesis of enantiopure (R,R)- and (S,S)-5-FBVM and their corresponding triazene precursors for future radiofluorination is reported. Both enantiomers exhibited high in vitro affinity for VAChT [(+)-5-FBVM: K(i)=6.95 nM and (-)-5-FBVM: K(i)=3.68 nM] and were selective for σ(2) receptors (∼70-fold), only (+)-5-FBVM is selective for σ(1) receptors (∼fivefold). These initial results suggest that (+)-(S,S)-5-FBVM warrants further investigation as a potential radioligand for in vivo PET imaging of cholinergic nerve terminals.

Search for the acetylcholine and vesamicol binding sites in vesicular acetylcholine transporter: the region around the lumenal end of the transport channel.

Vesicular acetylcholine transporter (VAChT; TC 2.A.1.2.13) mediates storage of acetylcholine (ACh) by synaptic vesicles. A three-dimensional homology model of VAChT is available, but the binding sites for ACh and the allosteric inhibitor (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) are unknown. In previous work, mutations of invariant W331 in the lumenal beginning of transmembrane helix VIII (TM VIII) of rat VAChT led to as much as ninefold loss in equilibrium affinity for ACh and no loss in affinity for vesamicol. The current work investigates the effects of additional mutations in and around W331 and the nearby lumenal end of the substrate transport channel. Mutants of human VAChT were expressed in the PC12(A123.7) cell line and characterized using radiolabeled ligands and filtration assays for binding and transport. Properties of a new and a repeat mutation in W331 are consistent with the original observations. Of 16 additional mutations in 13 other residues (Y60 in the beginning of lumenal Loop I/II, F231 in the lumenal end of TM V, W315, M316, K317, in the lumenal end of TM VII, M320, A321, W325, A330 in lumenal Loop VII/VIII, A334 in the lumenal beginning of TM VIII, and C388, C391, F392 in the lumenal beginning of TM X), only A334F impairs binding. This mutation decreases ACh and vesamicol equilibrium binding affinities by 14- and 4-fold, respectively. The current results, combined with previous results, demonstrate existence of a spatial cluster of residues close to vesicular lumen that decreases affinity for ACh and/or vesamicol when the cluster is mutated. The cluster is composed of invariant W331, highly conserved A334, and invariant F335 in TM VIII and invariant C391 in TM X. Different models for the locations of the ACh and vesamicol binding sites relative to this cluster are discussed.

Multiple protonation states of vesicular acetylcholine transporter detected by binding of [3H]vesamicol.

Vesicular acetylcholine transporter (VAChT) is inhibited by (-)-vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol], which binds tightly to an allosteric site. The tertiary alkylamine center in (-)-vesamicol is protonated and positively charged at acidic and neutral pH and unprotonated and uncharged at alkaline pH. Deprotonation of the amine has been taken to explain loss of (-)-vesamicol binding at alkaline pH. However, binding data deviate from a stereotypical bell shape, and more binding occurs than expected at alkaline pH. The current study characterizes the binding of (-)-vesamicol from pH 5 to pH 10 using filter assays, (-)-[3H]vesamicol (hereafter called [3H]vesamicol), and human VAChT expressed in PC12(A123.7) cells. At acidic pH, protons and [3H]vesamicol compete for binding to VAChT. Preexposure or long-term exposure of VAChT to high pH does not affect binding, thus eliminating potential denaturation of VAChT and failure of the filter assay. The dissociation constant for the complex between protonated [3H]vesamicol and VAChT decreases from 12 nM at neutral pH to 2.1 nM at pH 10. The simplest model of VAChT that explains the behavior requires a proton at site 1 to dissociate with pK1 = 6.5 +/- 0.1, a proton at site A to dissociate with pKA = 7.6 +/- 0.2, and a proton at site B to dissociate with pKB = 10.0 +/- 0.1. Deprotonation of the site 1 proton is obligatory for [3H]vesamicol binding. Deprotonation of site A decreases affinity (2.2 +/- 0.5)-fold, and deprotonation of site B increases affinity (18 +/- 4)-fold. Time-dependent dissociation of bound [3H]vesamicol is biphasic, but equilibrium saturation curves are not. The contrasting phasicity suggests that the pathway to and from the [3H]vesamicol binding site exists in open and at least partially closed states. The potential significance of the findings to development of PET and SPECT ligands based on (-)-vesamicol for human diagnostics also is discussed.

Mutational and bioinformatics analysis of proline- and glycine-rich motifs in vesicular acetylcholine transporter.

The vesicular acetylcholine transporter (VAChT) contains six conserved sequence motifs that are rich in proline and glycine. Because these residues can have special roles in the conformation of polypeptide backbone, the motifs might have special roles in conformational changes during transport. Using published bioinformatics insights, the amino acid sequences of the 12 putative, helical, transmembrane segments of wild-type and mutant VAChTs were analyzed for propensity to form non-alpha-helical conformations and molecular notches. Many instances were found. In particular, high propensity for kinks and notches are robustly predicted for motifs D2, C and C'. Mutations in these motifs either increase or decrease Vmax for transport, but they rarely affect the equilibrium dissociation constants for ACh and the allosteric inhibitor, vesamicol. The near absence of equilibrium effects implies that the mutations do not alter the backbone conformation. In contrast, the Vmax effects demonstrate that the mutations alter the difficulty of a major conformational change in transport. Interestingly, mutation of an alanine to a glycine residue in motif C significantly increases the rates for reorientation across the membrane. These latter rates are deduced from the kinetics model of the transport cycle. This mutation is also predicted to produce a more flexible kink and tighter tandem notches than are present in wild-type. For the full set of mutations, faster reorientation rates correlate with greater predicted propensity for kinks and notches. The results of the study argue that conserved motifs mediate conformational changes in the VAChT backbone during transport.