Discovery of lacosamide affinity bait agents that exhibit potent voltage-gated sodium channel blocking properties

Lacosamide1 ((R)-1) is a first-in-class antiepileptic drug (AED) that has been marketed for adjuvant treatment of partial-onset seizures in adults.2 Whole animal pharmacological studies showed that the (R)-1 anticonvulsant profile is unique and prevents seizure spread by mechanisms different from other AEDs.3 Radioligand displacement assays using more than 100 potential receptors did not reveal high-affinity binding targets.4 These findings suggested that (R)-1 function is unique or that binding is weak or both. Recent patch-clamp electrophysiology studies demonstrated that (R)-1 modulated voltage-gated Na+ channel (VGSC) function by likely increasing and stabilizing the slow inactivation process without affecting fast inactivation.5−8 The experimental findings were also consistent with a mechanism in which (R)-1 blocks fast inactivated channels with very slow kinetics.5,6 No other anticonvulsant is reported to preferentially modulate Na+ channel slow inactivation. The molecular pathway for this interaction has not been determined; thus, it is not known whether (R)-1 exerts its activity by directly interacting with the Na+ channel or through an intermediary protein. Affinity baits are chemical reactive units that lead to irreversible, covalent modification of the receptor when they are appended to a ligand (drug) (Figure ​(Figure1,1, 2 + 3 ⇌ 4 → 5).9−21 This technology has been used to identify binding partners and their approximate binding location when ligand binding is modest. Different electrophilic and photoactivable affinity bait units have been advanced.9−14 Among the electrophilic affinity baits are isothiocyanate and acrylamide groups.14−16,20−30 Isothiocyanates and acrylamides display an excellent balance between stability and reactivity; they do not readily react with hydroxylic solvents (thus permitting their dissolution in aqueous solutions) but do react with biological nucleophiles (e.g., thiols, amines) with minimal nonspecific background labeling of cellular constituents.9,28−30 Figure 1 Affinity bait (AB) and strategy for target receptor modification. Proposed pathway for receptor modification by a ligand that binds with modest affinity to its cognate receptor. Ligand 2 reversibly binds to receptor 3 to provide complex 4. Modification ... We have shown that the lacosamide 4′-isothiocyanate affinity bait agent (R)-6 given orally to rats exhibited anticonvulsant activity comparable to (R)-1 in the maximal electroshock seizure (MES) test31 (ED50 (mg/kg): (R)-1, 3.9; (R)-6, 4.2).32 We also demonstrated that (R)-6, like (R)-1, likely promoted Na+ channel slow inactivation in catecholamine A differentiated (CAD) cells (IC50 (μM): (R)-1, 85; (R)-6, 8.1).7 Finally, we found that (R)-6 increased this Na+ channel inactivation far more effectively than (S)-6 (IC50 (μM): (R)-6, 8.1; (S)-6, 68), a finding consistent with their activities in the MES test in rats (ED50 (mg/kg): (R)-6, 4.2; (S)-6, >180).32 When we incubated (R)-6 (five times the calculated slow inactivation IC50 value) with CAD cells (37 °C, 10 min), it retained only a modest increase (∼10%) in the levels of inactivation after (R)-6 was removed from the reaction solution (cellular wash), indicating a low efficiency of covalent adduction by the affinity bait reagent.7 Recently, we reported that the N-(biphenyl-4′-yl)methyl amide derivative (R)-7,33 which differed from (R)-1 by an incorporated 4′-aryl unit (see box in (R)-7), had greater Na+ channel inactivation in CAD cells than (R)-1 (calculated slow inactivation IC50 (μM): (R)-1, 85; (R)-7, 2.9).34 This finding suggested that lacosamide-like affinity bait compounds containing a N-(biphenyl-4′-yl)methyl amide unit would lead to a comparable increase in slow inactivation, and with CAD cell incubation an increased percentage of the Na+ channels irreversibly converted to the slow-inactivated state. Here we report the synthesis of a series of (R)-1 affinity bait agents ((R)-8–(R)-10) designed to interact with the receptor(s) responsible for (R)-1-mediated slow inactivation. We show that the isothiocyanate analogues (R)-8 and (R)-9 potently modulated Na+ channel inactivation similar to (R)-1 and that (R)-8 function was stereospecific. We further show that (R)-9 incubation with CAD cells led to appreciable levels of inactivated Na+ channels consistent with slow inactivation after cellular wash and that the extent of slow inactivation only modestly decreased with further heating and cellular wash. The implications of these findings are discussed.