BACKGROUND AND PURPOSEThe 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 Na v 1.1-Na v 1.7. EXPERIMENTAL APPROACHEach 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.
The first total synthesis of chiriquitoxin, the most structurally complex analogue of tetrodotoxin isolated from a Costa Rican dart frog, has been accomplished from a newly designed intermediate for a variety of tetrodotoxin derivatives. The synthesis includes the third total synthesis of tetrodotoxin in this laboratory, and its intermediate was transformed into chiriquitoxin by a stereocontrolled aldol reaction with a D-camphor-derived lactone for installation of the unique side chain, and a new deprotection of methylthiomethyl (MTM) ether by using a Pummerer rearrangement.
Background GGC repeat expansions in NOTCH2NLC are associated with neuronal intranuclear inclusion disease. Very recently, asymptomatic carriers with NOTCH2NLC repeat expansions were reported. In these asymptomatic individuals, the CpG island in NOTCH2NLC is hypermethylated, suggesting that two factors repeat length and DNA methylation status should be considered to evaluate pathogenicity. Long-read sequencing can be used to simultaneously profile genomic and epigenomic alterations. We analyzed four sporadic cases with NOTCH2NLC repeat expansion and their phenotypically normal parents. The native genomic DNA that retains base modification was sequenced on a per-trio basis using both PacBio and Oxford Nanopore long-read sequencing technologies. A custom workflow was developed to evaluate DNA modifications. With these two technologies combined, long-range DNA methylation information was integrated with complete repeat DNA sequences to investigate the genetic origins of expanded GGC repeats in these sporadic cases. Results In all four families, asymptomatic fathers had longer expansions (median: 522, 390, 528 and 650 repeats) compared with their affected offspring (median: 93, 117, 162 and 140 repeats, respectively). These expansions are much longer than the disease-causing range previously reported (in general, 41–300 repeats). Repeat lengths were extremely variable in the father, suggesting somatic mosaicism. Instability is more frequent in alleles with uninterrupted pure GGCs. Single molecule epigenetic analysis revealed complex DNA methylation patterns and epigenetic heterogeneity. We identified an aberrant gain-of-methylation region (2.2 kb in size beyond the CpG island and GGC repeats) in asymptomatic fathers. This methylated region was unmethylated in the normal allele with bilateral transitional zones with both methylated and unmethylated CpG dinucleotides, which may be protected from methylation to ensure NOTCH2NLC expression. Conclusions We clearly demonstrate that the four sporadic NOTCH2NLC-related cases are derived from the paternal GGC repeat contraction associated with demethylation. The entire genetic and epigenetic landscape of the NOTCH2NLC region was uncovered using the custom workflow of long-read sequence data, demonstrating the utility of this method for revealing epigenetic/mutational changes in repetitive elements, which are difficult to characterize by conventional short-read/bisulfite sequencing methods. Our approach should be useful for biomedical research, aiding the discovery of DNA methylation abnormalities through the entire genome.
Tetrodotoxin, a toxic principle of puffer fish intoxication, is one of the most famous marine natural products owing to its complex structure and potent biological activity, which leads to fatal poisoning. Continuous synthetic studies on tetrodotoxin and its analogues to elucidate biologically interesting issues associated with tetrodotoxin have led to the development of versatile routes for a variety of tetrodotoxin derivatives. With the aim of investigating the structure-activity relationship of tetrodotoxin with voltage-gated sodium channels, this study describes the first total syntheses of 5-deoxytetrodotoxin, a natural analogue of tetrodotoxin, and 8-deoxytetrodotoxin, an unnatural analogue, from a newly designed, versatile intermediate in an efficient manner. An estimation of the biological activities of these compounds reveals the importance of the hydroxy groups at the C-5 and C-8 positions on the inhibition of voltage-gated sodium channels.
5,6,11-Trideoxytetrodotoxin, a naturally occurring analog of tetrodotoxin, was synthesized from a synthetic intermediate of 5-deoxytetrodotoxin by continuous radical deoxygenation of two hydroxy groups at the C-6 and C-11 positions.
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