July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Drug discovery for the treatment of Fuchs corneal endothelial dystrophy by cell-based drug screening system
Author Affiliations & Notes
  • Risako Nakagawa
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Naoki Okumura
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Takako Onishi
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Takeshi Oshima
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Emi Ueda
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Kyoko Watanabe
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Theofilos Tourtas
    University of Erlangen-Nürnberg, Germany
  • Ursula Schlotzer-Schrehardt
    University of Erlangen-Nürnberg, Germany
  • Friedrich E Kruse
    University of Erlangen-Nürnberg, Germany
  • Noriko Koizumi
    Doshisha University, Kyotanabe, Kyoto, Japan
  • Footnotes
    Commercial Relationships   Risako Nakagawa, None; Naoki Okumura, ActualEyes, Inc. (I), Doshisha University (F), Doshisha University (P), Kowa Company Ltd. (C), Senju Pharmaceutical Co.,Ltd. (P); Takako Onishi, None; Takeshi Oshima, None; Emi Ueda, None; Kyoko Watanabe, None; Theofilos Tourtas, None; Ursula Schlotzer-Schrehardt, None; Friedrich Kruse, None; Noriko Koizumi, ActualEyes, Inc. (I), Doshisha University (F), Doshisha University (P), Japan Innovative Therapeutics, Inc. (F), Kowa Company Ltd. (F), Kowa Company Ltd. (C), M’s Science Corporation (F), M’s Science Corporation (C), Senju Pharmaceutical Co.,Ltd. (F), Senju Pharmaceutical Co.,Ltd. (C), Senju Pharmaceutical Co.,Ltd. (R), Senju Pharmaceutical Co.,Ltd. (P)
  • Footnotes
    Support  Program for the Strategic Research Foundation at Private Universities from Japanese Ministry of Education, Culture, Sports, Science and Technology, JSPS KAKENHI Grant Numbers 16K11307 and 18K09464.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3877. doi:
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      Risako Nakagawa, Naoki Okumura, Takako Onishi, Takeshi Oshima, Emi Ueda, Kyoko Watanabe, Theofilos Tourtas, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Noriko Koizumi; Drug discovery for the treatment of Fuchs corneal endothelial dystrophy by cell-based drug screening system. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3877.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We previously reported that transforming growth factor-β (TGF-β) signaling plays an important role in the pathophysiology of Fuchs endothelial corneal dystrophy (FECD) by inducing endoplasmic reticulum (ER) stress (Okumura et al., Sci Rep. 2017), and the establishment of a drug screening system for FECD via the use of a cellular model derived from FECD patients. The purpose of this study was to select candidate compounds for treating FECD from an FDA-approved drug library.

Methods : Corneal endothelial cells were isolated from Descemet’s membrane including corneal endothelium obtained from FECD patients who underwent Descemet's membrane endothelial keratoplasty. The Corneal endothelial cells were cultured and immortalized to produce an FECD cell model (iFECD), and then seeded in a 96-well plate and treated with transforming growth factor beta 2 (TGF-β2) (10 ng/mL) to induce cell death. The antiapoptotic effect of 765 FDA-approved drugs was screened by using the Caspase-Glo® 3/7 Assay kit. Hit compounds were further verified by phase-contrast microscopy and western blotting. The effective concentration and drug toxicity were evaluated by phase-contrast microscopy, Caspase-Glo® 3/7 Assay, and flow cytometry.

Results : Of the 765 FDA-approved drugs, 46 inhibited the activation of caspase 3/7 induced by TGF-β2 less than 60% in comparison to TGF-β2 treated iFECD. Phase-contrast microscopy evaluation of those 46 drugs showed that 9 (including rapamycin) had a strong inhibitory effect and that 16 had a moderate inhibitory effect on cell death, while 21 had no inhibitory effect. Further examination by western blotting showed that TGF-β2 induced cleavage of caspase 3 and poly (ADP-ribose) polymerase, yet rapamycin suppressed the cleavage of those apoptotic proteins. Caspase-Glo® 3/7 Assay showed that rapamycin significantly suppressed the TGF-β2-mediated caspase 3/7 activity at the concentration of 0.1 nM to 10 μM (p<0.01). As to the toxicity of rapamycin, phase contrast microscopy showed that rapamycin (from 0.1 nM to 10 μM) did not result in significant cell death.

Conclusions : Our cell-based screening findings suggest that rapamycin is a potential drug for FECD, and although further studies are needed to elucidate the mechanism of action, the mammalian target of rapamycin (mTOR) signaling may be a novel therapeutic target for FECD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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