RESUMO
CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.
Assuntos
Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Células CHO , Canais de Cloro CLC-2 , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Hipocampo/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeRESUMO
Select Biosciences' Fourth Annual Ion Channel Targets conference brought together scientists from industry and academia who are interested in the discovery of therapeutics targeted to various ion channels implicated in human disease. Topics addressed included methodological aspects of screening for ion channel drugs, the discovery of novel inhibitors and activators of ion channels that are drug candidates, and suggestions of potential new ion channel targets.