A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Mitsuharu Ueda, Masamitsu Okada, Mineyuki Mizuguchi, Barbara Kluve-Beckerman, Kyosuke Kanenawa, Aito Isoguchi, Yohei Misumi, Masayoshi Tasaki, Akihiko Ueda, Akinori Kanai, Ryoko Sasaki, Teruaki Masuda, Yasuteru Inoue, Toshiya Nomura, Satoru Shinriki, X. Tsuyoshi Shuto, Hirofumi Kai, Taro Yamashita, Hirotaka Matsui, Merrill BensonYukio Ando

Research output: Contribution to journalArticle

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 ofTTRex vivo in patient plasma.Weconclude 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.

Original languageEnglish (US)
Pages (from-to)11259-11275
Number of pages17
JournalJournal of Biological Chemistry
Volume294
Issue number29
DOIs
StatePublished - Jan 1 2019

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High-Throughput Screening Assays
Prealbumin
Amyloid
Screening
Throughput
Apomorphine
Amyloidosis
Pharmaceutical Preparations
Drug Repositioning
Amyloidogenic Proteins
Tissue
Patents
Amyloid Plaques
Neuroglia
Gene expression
Cultured Cells
Deposits
Gene Expression
Cells
Apoptosis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ueda, M., Okada, M., Mizuguchi, M., Kluve-Beckerman, B., Kanenawa, K., Isoguchi, A., ... Ando, Y. (2019). A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH. Journal of Biological Chemistry, 294(29), 11259-11275. https://doi.org/10.1074/jbc.RA119.007851

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH. / Ueda, Mitsuharu; Okada, Masamitsu; Mizuguchi, Mineyuki; Kluve-Beckerman, Barbara; Kanenawa, Kyosuke; Isoguchi, Aito; Misumi, Yohei; Tasaki, Masayoshi; Ueda, Akihiko; Kanai, Akinori; Sasaki, Ryoko; Masuda, Teruaki; Inoue, Yasuteru; Nomura, Toshiya; Shinriki, Satoru; Shuto, X. Tsuyoshi; Kai, Hirofumi; Yamashita, Taro; Matsui, Hirotaka; Benson, Merrill; Ando, Yukio.

In: Journal of Biological Chemistry, Vol. 294, No. 29, 01.01.2019, p. 11259-11275.

Research output: Contribution to journalArticle

Ueda, M, Okada, M, Mizuguchi, M, Kluve-Beckerman, B, Kanenawa, K, Isoguchi, A, Misumi, Y, Tasaki, M, Ueda, A, Kanai, A, Sasaki, R, Masuda, T, Inoue, Y, Nomura, T, Shinriki, S, Shuto, XT, Kai, H, Yamashita, T, Matsui, H, Benson, M & Ando, Y 2019, 'A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH', Journal of Biological Chemistry, vol. 294, no. 29, pp. 11259-11275. https://doi.org/10.1074/jbc.RA119.007851
Ueda, Mitsuharu ; Okada, Masamitsu ; Mizuguchi, Mineyuki ; Kluve-Beckerman, Barbara ; Kanenawa, Kyosuke ; Isoguchi, Aito ; Misumi, Yohei ; Tasaki, Masayoshi ; Ueda, Akihiko ; Kanai, Akinori ; Sasaki, Ryoko ; Masuda, Teruaki ; Inoue, Yasuteru ; Nomura, Toshiya ; Shinriki, Satoru ; Shuto, X. Tsuyoshi ; Kai, Hirofumi ; Yamashita, Taro ; Matsui, Hirotaka ; Benson, Merrill ; Ando, Yukio. / A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 29. pp. 11259-11275.
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AU - Kluve-Beckerman, Barbara

AU - Kanenawa, Kyosuke

AU - Isoguchi, Aito

AU - Misumi, Yohei

AU - Tasaki, Masayoshi

AU - Ueda, Akihiko

AU - Kanai, Akinori

AU - Sasaki, Ryoko

AU - Masuda, Teruaki

AU - Inoue, Yasuteru

AU - Nomura, Toshiya

AU - Shinriki, Satoru

AU - Shuto, X. Tsuyoshi

AU - Kai, Hirofumi

AU - Yamashita, Taro

AU - Matsui, Hirotaka

AU - Benson, Merrill

AU - Ando, Yukio

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N2 - 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 ofTTRex vivo in patient plasma.Weconclude 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.

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