Molecular Determinants of Human Voltage-Gated Sodium Channels Blockade by Lubeluzole

Abstract

Lubeluzole displays neuroprotective activity in vitro and in vivo. Blockade of sodium channels (NaCh) was proposed as a main mechanism for preclinical and clinical efficacy. We studied the molecular determinants for lubeluzole action on whole-cell sodium currents in HEK293 cells expressing hNav1.4 NaChs, using patch clamp technique. Lubeluzole and derivatives were synthesized in our laboratories. Effect of racemic lubeluzole and enantiomers on NaChs was dose- and use-dependent, with IC50 values of 30 μM at 0.1 Hz stimulation frequency and 2 μM at 10 Hz using an holding potential (hp) of −120 mV. These are ∼8 and ∼18 times lower than the respective IC50 values for the well-known NaCh blocker mexiletine. The affinity of lubeluzole for the closed (KR) and inactivated channel (KI) were 840 and 0.03 μM, compared to 800 and 2 μM for mexiletine. Use-dependent block by lubeluzole was inhibited only partially by F1586C mutation at the local anesthetic molecular receptor, suggesting that lubeluzole may bind at a different but overlapping receptor. Indeed KR and KI values for lubeluzole binding to F1586C channel were 700 and 0.7 μM. To go further in details, we synthesized two lubeluzole derivatives, each containing about one half of the parent compound. The aryloxypropanolamine moiety recalls the structure of clenbuterol, while the benzothiazole moiety is similar to riluzole, both known NaCh blockers. However, both derivatives displayed very poor use-dependent block, with IC50 values greater than 800 μM at the hp of −120 mV. In conclusion, lubeluzole is a very potent blocker of inactivated sodium channels, which explains its huge use-dependent action. Lubeluzole probably utilizes binding interactions distinct from those of local anesthetic-like drugs, which may open the way for the development of new compounds with peculiar activity on sodium channels (Supported by Telethon-Italy).