title: Kinetic assay for high-throughput screening of in vitro transthyretin amyloid fibrillogenesis inhibitors.
abstract: Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.
authors.affiliation: Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, E-08017 Barcelona, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Dolado
authors.forename: Joan
authors.initials: J
authors.lastname: Nieto
authors.forename: Maria João M
authors.initials: MJ
authors.lastname: Saraiva
authors.forename: Gemma
authors.initials: G
authors.lastname: Arsequell
authors.forename: Gregori
authors.initials: G
authors.lastname: Valencia
authors.forename: Antoni
authors.initials: A
authors.lastname: Planas
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D000686:Amyloidosis / Q000378:metabolism*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D011228:Prealbumin / Q000037:antagonists & inhibitors* / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D013963:Thyroid Hormones / Q000494:pharmacology; D014260:Triclosan / Q000494:pharmacology
chemical_list: D000682:Amyloid; D000894:Anti-Inflammatory Agents, Non-Steroidal; D011228:Prealbumin; D011994:Recombinant Proteins; D013963:Thyroid Hormones; D014260:Triclosan