Abstract
Botulinumneurotoksin (BoNT) er et potent og potentielt dødbringende bakterietoksin, som binder til vært motoriske neuroner, der er internaliseret i cellen, og spalter intracellulære proteiner, der er afgørende for neurotransmitter release. BoNT består af en tung kæde (HC), der medierer værtscelle binding og internalisering, og en let kæde (LC), der spalter intracellulære værtsproteiner er essentielle for frigivelse af acetylcholin. Mens behandlinger, der hæmmer toksinbinding / internalisering har et lille tidsvindue administration, forbindelser, der er målrettet intracellulær LC aktivitet har en meget større tidsvindue af forvaltninger, særligt relevante i betragtning af den ekstremt lang halveringstid af toksinet. I de seneste år, har små molekyler blevet stærkt analyseret som potentielle LC inhibitorer baseret på deres forøget cellulær permeabilitet i forhold til større terapeutika (peptider, aptamers, osv.). Bly identifikation involverer ofte high-throughput screening (HTS), hvorstore biblioteker af små molekyler screenes baseret på deres evne til at modulere terapeutisk target funktion. Her beskriver vi en FRET-baserede assay med en kommerciel BoNT / A LC substrat og rekombinant LC, der kan automatiseres for HTS af potentielle Bont hæmmere. Desuden beskriver vi en manuel teknik, der kan anvendes til opfølgning sekundær screening eller til sammenligning af styrken af flere kandidat-forbindelser.
Materials
Name | Company | Catalog Number | Comments |
HEPES | Teknova | H1021 | |
Tween-20 | Fisher Scientific | BP337-100 | |
Methanol (HPLC-grade) | Sigma-Aldrich | 34860 | |
Isopropanol (HPLC-grade) | Sigma-Aldrich | 650447 | |
96-well Black assay plate | Costar | 3915 | |
384-well Low-volume black assay plate | Greiner | 788076 | |
SNAPtide FITC/Dabcyl substrate | List Biological Laboratories | 521 | FRET-based BoNT/A LC substrate |
Pin cleaning solution | V&P Scientific | VP 110 | |
Lint-free blotting paper | V&P Scientific | VP 540DB | |
Biomek Seal and Sample Aluminum foil lids | Beckman Coulter | 538619 |
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