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Introduction: Development of a selective and specific high affinity PET tracer, [(11)C]UCB-A, for the in vivo study of SV2A expression in humans. Radiochemistry and preclinical studies in rats and pigs including development of a tracer kinetic model to determine VT. A method for the measurement of percent intact tracer in plasma was developed and the radiation dosimetry was determined in rats., Results: 3-5GBq of [(11)C]UCB-A could be produced with radiochemical purity exceeding 98% with a specific radioactivity of around 65GBq/μmol. In vitro binding showed high selective binding towards SV2A. [(11)C]UCB-A displayed a dose-dependent and reversible binding to SV2A as measured with PET in rats and pigs and the VT could be determined by Logan analysis. The dosimetry was favorable and low enough to allow multiple administrations of [(11)C]UCB-A to healthy volunteers, and the metabolite analysis showed no sign of labeled metabolites in brain., Conclusions: We have developed the novel PET tracer, [(11)C]UCB-A, that can be used to measure SV2A expression in vivo. The dosimetry allows up to 5 administrations of 400MBq of [(11)C]UCB-A in humans. Apart from measuring drug occupancy, as we have shown, the tracer can potentially be used to compare SV2A expression between individuals because of the rather narrow range of baseline VT values. This will have to be further validated in human studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.

Levtiracetam (Lev), an inhibitor of SV2A (synaptic vesicle protein A2), affected the ATP-dependent priming of Ca(2+)-regulated exocytosis in antral mucous cells of guinea pig. In antral mucous cells, the Ca(2+)-regulated exocytosis, which is activated by acetylcholine (ACh), consists of an initial peak that declines rapidly (initial phase) followed by a second slower decline (late phase). Dinitrophenol (DNP), which depletes ATP, inhibits the ATP-dependent priming. DNP abolished the initial phase by reducing the number of primed granules, Lev decreased the frequency of initial phase, but not in the presence of DNP. Moreover, 8-bromoguanosine 3'5'-cyclic monophosphate (8BrcGMP) accelerates the ATP-dependent priming. 8BrcGMP enhances the frequency of initial phase by increasing the number of primed granule. Lev added prior to 8BrcGMP addition decreased the frequency of initial phase, but Lev added after 8BrcGMP addition did not. Thus, Lev affected the granules in the process of priming, but it did not affect the granules already primed. Lev did not affect [Ca(2+)]i in unstimulated or ACh-stimulated antral mucous cells. Immunohistochemistry and western blotting demonstrated that SV2A exists in antral mucous cells. The results suggest that SV2A plays an essential role in maintaining the process of ATP-dependent priming in antral mucous cells. In conclusion, Lev decreases the frequency of Ca(2+)-regulated exocytosis the number of primed granules by inhibiting SV2A functions, leading to a decrease in antral mucous cells., (© 2013 Elsevier B.V. All rights reserved.)

Levetiracetam (LEV) is a prominent antiepileptic drug that binds to neuronal synaptic vesicle glycoprotein 2A protein and has reported effects on ion channels, but with a poorly defined mechanism of action. We investigated inhibition of voltage-dependent Ca(2+) (Ca(V)) channels as a potential mechanism through which LEV exerts effects on neuronal activity. We used electrophysiological methods to investigate the effects of LEV on cholinergic synaptic transmission and Ca(V) channel activity in superior cervical ganglion neurons (SCGNs). In parallel, we investigated the effects of the inactive LEV R-enantiomer, (R)-α-ethyl-2-oxo-1-pyrrolidine acetamide (UCB L060). LEV but not UCB L060 (each at 100 μM) inhibited synaptic transmission between SCGNs in long-term culture in a time-dependent manner, significantly reducing excitatory postsynaptic potentials after a ≥30-min application. In isolated SCGNs, LEV pretreatment (≥1 h) but not short-term application (5 min) significantly inhibited whole-cell Ba(2+) current (I(Ba)) amplitude. In current-clamp recordings, LEV reduced the amplitude of the afterhyperpolarizing potential in a Ca(2+)-dependent manner but also increased the action potential latency in a Ca(2+)-independent manner, which suggests additional mechanisms associated with reduced excitability. Intracellular LEV application (4-5 min) caused rapid inhibition of I(Ba) amplitude, to an extent comparable to that seen with extracellular LEV pretreatment (≥1 h). Neither pretreatment nor intracellular application of UCB L060 produced any inhibitory effects on I(Ba) amplitude. These results identify a stereospecific intracellular pathway through which LEV inhibits presynaptic Ca(V) channels; resultant reductions in neuronal excitability are proposed to contribute to the anticonvulsant effects of LEV.

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861-9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [3H]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetam binding site/SV2A in rat brain [Gillard, M., Fuks, B., Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1-9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [3H]ucb 30889 binding in human brain and to SV2A was saturable and reversible. At 4 degrees C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to Kd values for [3H]ucb 30889 of 53+/-7, 55+/-9, 70+/-11 and 75+/-33 nM in human cerebral cortex, hippocampus, cerebellum and CHO cells expressing SV2A respectively. Bmax values around 3-4 pmol/mg protein were calculated in all brain regions. Some of the saturation curves displayed curvilinear Scatchard plots indicating the presence of high and low affinity binding sites. When this was the case, Kd values from 25 to 30 nM for the high affinity sites (24% to 34% of total sites) and from 200 to 275 nM for the low affinity sites were calculated. This was observed in all brain regions and in CHO cell membranes expressing the SV2A protein. It cannot be explained by putative binding of [3H]ucb 30889 to SV2B or C isoforms but may reflect different patterns of SV2A glycosylation or the formation of SV2A oligomers. Competition experiments were performed to determine the affinities for SV2A of a variety of compounds including levetiracetam, some of its analogues and other molecules known to interact with levetiracetam binding sites in rat brain such as bemegride, pentylenetetrazol and chlordiazepoxide. We found an excellent correlation between the affinities of these compounds measured in human brain, rat brain and CHO cells expressing human SV2A. In conclusion, we report for the first time that the binding characteristics of native levetiracetam binding sites/SV2A in human brain and rat brain share very similar properties with human recombinant SV2A expressed in CHO cells.

The novel antiepileptic drug levetiracetam ((2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA possesses a specific binding site in brain, which has very recently been identified as the synaptic vesicle protein SV 2 A. The aim of this study was to evaluate the presence of a levetiracetam binding site in the spinal cord and compare its properties to that in rat brain. We used [(3)H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide), a levetiracetam analogue, to perform binding assays, photoaffinity labelling and autoradiography experiments, and revealed the presence of SV 2 A by Western-blot analysis. [(3)H]ucb 30889 binding kinetics at 4 degrees C were biphasic and saturation binding curves were compatible with the labelling of a homogenous population of binding sites with a K(d) similar to that in brain. Competition curves with ligands known to interact with levetiracetam binding sites and photolabelling experiments indicated that [(3)H]ucb 30889 labels the same 90 kDa protein in both spinal cord and brain. Levetiracetam binding site was localised in the grey matter of the spinal cord and its expression was not modified in a model of neuropathic pain. This study demonstrates the presence of a specific levetiracetam binding site in the rat spinal cord, which is similar to that found in rat brain.

Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of approximately 90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.