Determinants of interactions of a novel next-generation gabapentinoid NVA1309 and mirogabalin with the Cavα2δ-1 subunit

Abstract

NVA1309 is a non-brain penetrant next-generation gabapentinoid shown to bind Cavα2δ at R243 within a triple Arginine motif forming the binding site for gabapentin and pregabalin. In this study we have compared the effects of NVA1309 with Mirogabalin, a gabapentinoid drug with higher affinity for the voltage-gated calcium channel subunit Cavα2δ-1 than pregabalin which is approved for post-herpetic neuralgia in Japan, Korea and Taiwan. Both NVA1309 and mirogabalin inhibit Cav2.2 currents in vitro and decrease Cav2.2 plasma membrane expression with higher efficacy than pregabalin. Mutagenesis of the classical binding residue arginine R243 and the newly identified binding residue lysine K615 reverse the effect of mirogabalin on Cav2.2 current, but not that of NVA1309.

Keywords: Calcium channel; Cavα2δ; Gabapentinoids; Mirogabalin; Neuropathic pain.

Fig. 1

Effect of mirogabalin on Cav2.2 function and trafficking. a Current density–voltage (I/V) relationships of cells co-expressing Cav2.2 with wild-type Cavα2δ-1 and treated with 100 μM NVA1307 for 48 h. The inset shows maximum conductance (**p = 0.0035, Mann–Whitney test). b Current density–voltage (I/V) relationships of cells expressing Cav2.2 with R243A Cavα2δ-1 and treated with 100 μM mirogabalin for 48 h. The inset shows maximum conductance. c Current density–voltage (I/V) relationships of cells co-expressing Cav2.2 with K615A Cavα2δ-1 and treated with 100 μM mirogabalin for 48 h. The inset shows maximum conductance. d Current density–voltage (I/V) relationships of cells expressing Cav2.2 with R243A + K615A Cavα2δ-1 and treated with 100 μM mirogabalin for 48 h. The inset shows maximum conductance. e Average peak current density from cells treated with 100 µM of NVA1309 (data from Ref. (7)), mirogabalin, PGB or GPB for 48 h. (**p = 0.0028 for NVA1309 and **p = 0.0098 for mirogabalin compared to DMSO, Kruskal–Wallis test followed by Dunn’s multiple comparisons test). f Effect of mirogabalin on membrane expression of Cav2.2HA after 48 h. g Effect of PGB on membrane expression of Cav2.2HA after 48 h. Fluorescence intensity was quantified using Image J software. Scale bar: 10 µm. h Overlapping of the docked NVA1309 (red) and mirogabalin (blue) within the full-length structure of Cavα2δ-1. i Predicted 2D NVA1309 interactions in the Cavα2δ-1 binding pocket. j Predicted 2D mirogabalin interactions in the Cavα2δ-1 binding pocket. k Predicted 3D NVA1309 interactions with Cavα2δ-1. l Predicted 3D mirogabalin interactions with Cavα2δ-1. Interactions are represented by green (conventional hydrogen bonding), light green (van der Waals bond), pink (π-alkyl interactions), orange (alkyl interactions), and red (unfavorable donor-donor)