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A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH
- Source :
- J Biol Chem
- Publication Year :
- 2019
- Publisher :
- American Society for Biochemistry and Molecular Biology, 2019.
-
Abstract
- Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.
- Subjects :
- 0301 basic medicine
Amyloid
Apomorphine
Protein Conformation
Protein aggregation
Biochemistry
03 medical and health sciences
Pyrvinium Compounds
Protein structure
medicine
Humans
Prealbumin
Trypsin
Molecular Biology
Cells, Cultured
Inflammation
Neurons
Amyloid Neuropathies, Familial
030102 biochemistry & molecular biology
biology
Drug discovery
Chemistry
Amyloidosis
Methods and Resources
Drug Repositioning
nutritional and metabolic diseases
Cell Biology
Hydrogen-Ion Concentration
medicine.disease
In vitro
High-Throughput Screening Assays
Transthyretin
030104 developmental biology
Proteolysis
biology.protein
Neuroglia
Ex vivo
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- J Biol Chem
- Accession number :
- edsair.doi.dedup.....4e48c31017e98e6e58277dbb722f7f07