RESUMEN
A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuroâ 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7â nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1â nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .
Asunto(s)
Saxitoxina/química , Bloqueadores de los Canales de Sodio/síntesis química , Canales de Sodio Activados por Voltaje/metabolismo , Potenciales de Acción/efectos de los fármacos , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Técnicas de Placa-Clamp , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Teoría Cuántica , Saxitoxina/metabolismo , Saxitoxina/farmacología , Bloqueadores de los Canales de Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/química , Tetrodotoxina/metabolismo , Canales de Sodio Activados por Voltaje/química , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
BACKGROUND AND PURPOSE: The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7. EXPERIMENTAL APPROACH: Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings. KEY RESULTS: The inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively. CONCLUSIONS AND IMPLICATIONS: The reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.