RESUMO
Determining permeability of a given compound through human skin is a principal challenge owing to the highly complex nature of dermal tissue. We describe the application of an ambient mass spectrometry imaging method for visualizing skin penetration of sodium channel modulators, including novel synthetic analogs of natural neurotoxic alkaloids, topically applied ex vivo to human skin. Our simple and label-free approach enables successful mapping of the transverse and lateral diffusion of small molecules having different physicochemical properties without the need for extensive sample preparation.
Assuntos
Espectrometria de Massas/métodos , Absorção Cutânea , Pele/metabolismo , Bloqueadores dos Canais de Sódio/farmacocinética , Administração Tópica , Alcaloides/administração & dosagem , Alcaloides/química , Alcaloides/farmacocinética , Animais , Humanos , Permeabilidade , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/administração & dosagem , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/metabolismoRESUMO
The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.
Assuntos
Venenos , Canais de Sódio Disparados por Voltagem , Batraquiotoxinas/metabolismo , Sítios de Ligação , Conformação Molecular , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
The steroidal neurotoxin (-)-batrachotoxin functions as a potent agonist of voltage-gated sodium ion channels (NaVs). Here we report concise asymmetric syntheses of the natural (-) and non-natural (+) antipodes of batrachotoxin, as well both enantiomers of a C-20 benzoate-modified derivative. Electrophysiological characterization of these molecules against NaV subtypes establishes the non-natural toxin enantiomer as a reversible antagonist of channel function, markedly different in activity from (-)-batrachotoxin. Protein mutagenesis experiments implicate a shared binding side for the enantiomers in the inner pore cavity of NaV These findings motivate and enable subsequent studies aimed at revealing how small molecules that target the channel inner pore modulate NaV dynamics.
Assuntos
Batraquiotoxinas/síntese química , Batraquiotoxinas/farmacologia , Proteínas Musculares/antagonistas & inibidores , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Sítios de Ligação , Proteínas Musculares/química , Proteínas Musculares/genética , Mutação Puntual , Estrutura Secundária de Proteína , Ratos , Canais de Sódio/química , Canais de Sódio/genética , Bloqueadores do Canal de Sódio Disparado por Voltagem/químicaRESUMO
A novel family of small molecule inhibitors of voltage-gated sodium channels (NaVs) based on the structure of batrachotoxin (BTX), a well-known channel agonist, is described. Protein mutagenesis and electrophysiology experiments reveal the binding site as the inner pore region of the channel, analogous to BTX, alkaloid toxins, and local anesthetics. Homology modeling of the eukaryotic channel based on recent crystallographic analyses of bacterial NaVs suggests a mechanism of action for ion conduction block.