Drug binding assays do not reveal specific binding of lacosamide to collapsin response mediator protein 2 (CRMP-2)

Abstract

Aims: Lacosamide (LCM; SPM 927, Vimpat®) is an antiepileptic drug (AED) used as adjunctive treatment for adults with partial-onset seizures. LCM has a different mode of action from traditional sodium channel blocking AEDs in that it selectively enhances slow inactivation of sodium channels without affecting fast inactivation. Initial investigations suggested that LCM might have an additional mode of action by binding to the collapsin response mediator protein 2 (CRMP-2), which is further investigated here.

Methods: LCM binding to native and cloned human CRMP-2 was determined using radioligand binding experiments and surface plasmon resonance measurements.

Results: No specific binding of [(3) H]LCM (free concentration 100-1450 nM) to isolated or membrane bound human CRMP-2 expressed in mammalian cell systems and bacteria was observed. Surface plasmon resonance analysis also showed that LCM, over a concentration range of 0.39-100 μM, does not specifically bind to human CRMP-2.

Conclusion: The diverse drug binding methods employed here are well suited to detect specific binding of LCM to CRMP-2 in the micromolar range, yet the results obtained were all negative. Results of this study suggest that LCM does not specifically bind to CRMP-2.

Figure 1

[³H]LCM shows no specific binding to rat brain membranes. Binding of [³H]LCM (300 nM) to rat brain membranes (200 μg/assay) at 4°C and 25°C was determined by manual filtration in a final volume of 50 μL using a 50‐mM Tris‐HCl buffer (pH 7.4). NSB was determined in the presence of 1 mM unlabeled LCM. Data are presented as mean ± SEM (n = 3).

Figure 2

[³H]LCM does not specifically bind to CRMP‐2 expressed in oocyte membrane fractions. Binding of [³H]LCM (969 nM) to oocyte membrane fractions (100 μg/assay) expressing human CRMP‐2. Total binding (gray bars) and NSB (open bars) were determined on membrane fractions from CRMP‐2 and control (–CRMP‐2) injected oocytes. Binding experiments were performed in a final volume of 30μL in either Tris buffer (Tris 10 mM, NaCl 150 mM, pH 7.4) or a buffer composition mimicking the intracellular environment (HEPES 15 mM, KCl 135 mM, CaCl₂ 1 mM, NaCl 4 mM, MgCl₂ 2 mM: pH 7.2). NSB was determined in the presence of 1mM unlabeled LCM. Data are presented as mean ± SEM (n = 3–5). Insert: Western blot analysis of CRMP‐2 expression in oocyte fractions: pellet (P), supernatant (S) and membranes (M) (20 μg/sample).

Figure 3

(A) Western blot analysis of CRMP‐2 expression in COS cells: Analysis of soluble and membrane fractions, respectively (20 μg/lane), of untransfected cells (1,2), GST‐tagged CRMP‐2 (3,4) and His‐tagged CRMP‐2 (5,6). (B) Binding of [³H]LCM to His‐ or GST‐tagged CRMP‐2 proteins using a scintillation proximity assay: CRMP‐2 proteins expressed in cytosolic or membrane fractions of COS‐7 cells were coupled to Copper‐PVT beads and YSi beads, respectively and incubated for 60 min (25°C) in the presence of 1200 nM [³H]LCM. Radioactivity coupled to the beads was measured by luminescence. Data represent total binding (gray bars) and NSB (open bars) in the presence of 1 mM unlabeled LCM (mean ± SD; n = 4). Ct‐His_CRMP‐2 and Nt‐GST_CRMP‐2 are the C‐ and N‐terminal tagged versions, respectively. (C) [³H]LCM does not bind to purified CRMP‐2: His(C_t) and GST(N_t) CRMP‐2 were expressed in E. Coli and coupled to SPA beads. Binding experiments were performed at 25°C during 1 h in the presence of [³H]LCM (969 nM) and in a final volume of 30 μL in Tris buffer (Tris 10 mM, NaCl 150 mM: pH 7.4). NSB was determined in the presence of 1 mM unlabeled LCM. Data are presented as mean ± SEM (n = 10). Total binding (gray bars), NSB (open bars).

Figure 4

Analysis of LCM binding to immobilized CRMP‐2 or HSA using Biacore. Surface plasmon resonance report point data (n = 3, ±SD) for the binding of LCM (0.39–100μM) to immobilized CRMP‐2 (black diamonds) or HSA (open squares). CRMP‐2 and HSA were immobilized at binding response levels of ∼18700 RU and ∼10800 RU, respectively. LCM was titrated over the immobilized protein surfaces. Report points (n = 3, ± SD) for the binding of LCM or HSA to CRMP‐2 were recorded 5 seconds before the end of the injection (see insert). The predicted R_max binding levels of LCM to CRMP‐2 and HSA based on 1:1 stoichiometry (horizontal dotted lines) are 75 RU and 40.5 RU, respectively. Insert: SPR overlaid sensorgrams depicting the interaction of LCM (0.39–100 μM) to the immobilized human CRMP‐2.