Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

Abstract

Background and purpose: Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889.

Experimental approach: UCB1244283 was characterized in vitro using radioligand binding assays with [(3)H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice.

Key results: Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [(3)H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [(3)H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions.

Conclusions and implications: These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies.

Figure 1

Chemical structures of (A) UCB1244283, (B) levetiracetam and (C) UCB30889.

Figure 2

Effect of UCB1244283 and LEV on [³H]UCB30889 binding at 4°C on rat cortex and human (Hs) and rat (Rn) recombinant SV2A. Increasing concentrations of LEV or UCB1244283 were incubated with 5.25 nM of [³H]UCB30889 for 120 min at 4°C as described in materials and methods. Data were normalized to the binding of [³H]UCB30889 in the absence of any compound (B0), n = 3 to 5.

Figure 3

Effect of UCB1244283 on the binding characteristics of [³H]UCB30889. Saturation binding curves of [³H]UCB30889 (A) on human recombinant SV2A expressed in HEK cells and (B) on rat cortex. Membranes were incubated with increasing concentrations of [³H]UCB30889 for 60 min at 37°C with (open symbols) or without (filled symbols) 10 μM of UCB1244283. Non-specific binding (NSB) was determined as the residual binding observed in the presence of 1 mM levetiracetam. The insets are the Scatschard plots from the transformed data. Specific binding = total binding – NSB. Results are representative of three independent experiments.

Figure 4

Effect of UCB1244283 on the competition profile of UCB30889 and levetiracetam against [³H]UCB30889 on rat cortex. A concentration range of both compounds was incubated with 2.6 nM of [³H]UCB30889 in the presence (open symbols) or absence (filled symbols) of 10 μM of UCB1244283 during 120 min at 4°C. B0 is the binding of [³H]UCB30889 in the absence of any competing compound. Data represent the mean ± SD of three to six independent experiments. Analysis was performed using a nonlinear regression model with variable slope as described in materials and methods.

Figure 5

Effect of UCB1244283 on the binding kinetics of [³H]UCB30889 on rat cortex at 25°C. UCB1244283 was added to the reaction mixture at different time points as indicated in the figure legend and the concentration of [³H]UCB30889 was 5.25 nM. The experiments were performed as described in materials and methods. Non-specific binding (NSB) was determined as the residual binding observed in the presence of 100 μM UCB30889. Arrows indicate the onset of dissociation by addition of 100 μM unlabeled UCB30889. (B) Close-up of the association curves presented in (A). All curves were shifted to the origin in order to allow better visual comparison of the curves. Data were fit according to a biphasic association model as described in materials and methods. (C) Close-up of the dissociation curves as presented in (A). All curves were again shifted to the origin and data were fit with a two phase (continuous line) and three phase (dotted line) dissociation model as described. Data are representative of at least three independent experiments.