A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH
| Autor: | Barbara Kluve-Beckerman, Mineyuki Mizuguchi, Masayoshi Tasaki, Akinori Kanai, Toshiya Nomura, Kyosuke Kanenawa, Satoru Shinriki, Mitsuharu Ueda, Ryoko Sasaki, Yukio Ando, Hirofumi Kai, Teruaki Masuda, Tsuyoshi Shuto, Hirotaka Matsui, Masamitsu Okada, Yohei Misumi, Merrill D. Benson, Akihiko Ueda, Aito Isoguchi, Taro Yamashita, Yasuteru Inoue |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
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 |
| Zdroj: | J Biol Chem |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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