Discovery of LacosamideAffinity Bait Agents That Exhibit Potent Voltage-Gated Sodium ChannelBlocking Properties

摘要

Lacosamide ((R)->1) is a recently marketed, first-in-class, antiepileptic drug. Patch-clamp electrophysiology studies are consistent with the notion that (R)->1 modulates voltage-gated Na⁺ channel function by increasing and stabilizing the slow inactivation state without affecting fast inactivation. The molecular pathway(s) that regulate slow inactivation are poorly understood. Affinity baits are chemical reactive units, which when appended to a ligand (drug) can lead to irreversible, covalent modification of the receptor thus permitting drug binding site identification including, possibly, the site of ligand function. We describe, herein, the synthesis of four (R)->1 affinity baits, (R)-N-(4″-isothiocyanatobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((R)->8), (S)-N-(4″-isothiocyanatobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((S)->8), (R)-N-(3″-isothiocyanatobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((R)->9), and (R)-N-(3″-acrylamidobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((R)->10). The affinity bait compounds were designed to interact with the receptor(s) responsible for (R)->1-mediated slow inactivation. We show that (R)->8 and (R)->9 are potent inhibitors of Na⁺ channel function and function by a pathway similar to that observed for (R)->1. We further demonstrate that (R)->8 function is stereospecific. The calculated IC50 values determined for Na⁺ channel slow inactivation for (R)->1, (R)->8, and (R)->9 were 85.1, 0.1,and 0.2 μM, respectively. Incubating (R)->9 with the neuronal-like CAD cells led to appreciable levelsof Na⁺ channel slow inactivation after cellular wash, andthe level of slow inactivation only modestly decreased with furtherincubation and washing. Collectively, these findings have identifieda promising structural template to investigate the voltage-gated Na⁺ channel slow inactivation process.