ABSTRACT
Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.
Subject(s)
Acid Sensing Ion Channel Blockers/therapeutic use , Acid Sensing Ion Channels/metabolism , Guanidines/therapeutic use , Hydrazones/therapeutic use , Infarction, Middle Cerebral Artery/drug therapy , Neuroprotective Agents/therapeutic use , Acid Sensing Ion Channel Blockers/chemical synthesis , Acid Sensing Ion Channel Blockers/metabolism , Acid Sensing Ion Channels/chemistry , Animals , Binding Sites , CHO Cells , Chickens , Cricetulus , Drug Design , Guanidines/chemical synthesis , Guanidines/metabolism , HEK293 Cells , Humans , Hydrazones/chemical synthesis , Hydrazones/metabolism , Mice , Molecular Docking Simulation , Molecular Structure , Neurons/drug effects , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/metabolism , Protein Binding , Rats , Structure-Activity RelationshipABSTRACT
Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments.
Subject(s)
Acid Sensing Ion Channel Blockers/chemistry , Acid Sensing Ion Channels/chemistry , Arthropod Proteins/chemistry , Neurotoxins/chemistry , Peptides/chemistry , Shab Potassium Channels/chemistry , Spider Venoms/chemistry , Acid Sensing Ion Channel Blockers/chemical synthesis , Acid Sensing Ion Channel Blockers/toxicity , Acid Sensing Ion Channels/genetics , Amino Acid Sequence , Animals , Arthropod Proteins/chemical synthesis , Arthropod Proteins/toxicity , Gene Expression , Ion Channel Gating , Kinetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Sequence Data , Neurotoxins/chemical synthesis , Neurotoxins/toxicity , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Peptides/chemical synthesis , Peptides/toxicity , Protein Binding , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Shab Potassium Channels/antagonists & inhibitors , Shab Potassium Channels/genetics , Spider Venoms/chemical synthesis , Spider Venoms/toxicity , Spiders , Unilamellar Liposomes/chemistry , Xenopus laevisABSTRACT
The synthesis, structure-activity relationship (SAR), and pharmacological evaluation of analogs of the acid-sensing ion channel (ASIC) inhibitor A-317567 are reported. It was found that the compound with an acetylenic linkage was the most potent ASIC-3 channel blocker. This compound reversed mechanical hypersensitivity in the rat iodoacetate model of osteoarthritis pain, although sedation was noted. Sedation was also observed in ASIC-3 knockout mice, questioning whether sedation and antinociception are mediated via a non-ASIC-3 specific mechanism.
