title: Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.
abstract: Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na+channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na+channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Nav1.4 currents (IC50=60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Nav1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na+channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources.
authors.affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, 14 Sur 6301, CU, San Manuel, Puebla, México. Electronic address: bertin.paiz@gmail.com.
authors.forename: Bertin
authors.initials: B
authors.lastname: Paiz-Candia
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico; Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, 14 Sur 6301, CU, San Manuel, Puebla, México. Electronic address: angelislas@gmail.com.
authors.forename: Angel A
authors.initials: AA
authors.lastname: Islas
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Sánchez-Solano
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico. Electronic address: claudia.mancilla@gmail.com.
authors.forename: Claudia
authors.initials: C
authors.lastname: Mancilla-Simbro
authors.affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, 14 Sur 6301, CU, San Manuel, Puebla, México. Electronic address: tscior@gmail.com.
authors.forename: Thomas
authors.initials: T
authors.lastname: Scior
authors.affiliation: Centro de Química, Instituto de Ciencias, Universidad Autónoma de Puebla, 14 Sur 6301, CU, San Manuel, Puebla, México. Electronic address: lourdes.millan@correo.buap.mx.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Millan-PerezPeña
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico. Electronic address: eduardo.salinas@correo.buap.mx.
authors.forename: Eduardo M
authors.initials: EM
authors.lastname: Salinas-Stefanon
mesh_terms: D000779:Anesthetics, Local / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D055724:Electrophysiological Phenomena / Q000187:drug effects; D008012:Lidocaine / Q000378:metabolism / Q000494:pharmacology; D015767:Mefloquine / Q000378:metabolism* / Q000494:pharmacology*; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D062553:NAV1.4 Voltage-Gated Sodium Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D061567:Voltage-Gated Sodium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D000779:Anesthetics, Local; D062553:NAV1.4 Voltage-Gated Sodium Channel; D061567:Voltage-Gated Sodium Channel Blockers; D008012:Lidocaine; D015767:Mefloquine
keywords: Docking; Local anaesthetic; Mefloquine; Oocytes; Selectivity filter