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Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging

  • Yukiko Miyazaki
  • Takeshi Ishikawa
  • Yuji O. Kamatari
  • Takehiro Nakagaki
  • Hanae Takatsuki
  • Daisuke Ishibashi
  • Kazuo Kuwata
  • Noriyuki Nishida
  • Ryuichiro Atarashi
Article

Abstract

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a β-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Keywords

Prion diseases Surface plasmon resonance imaging Alprenolol hydrochloride Docking simulation 

Notes

Acknowledgments

We thank Atsuko Matsuo for the technical assistance. This work was supported by JSPS KAKENHI Grant Number JP15H04269 and a grant form Takeda Science Foundation.

We thank Kate Fox, DPhil, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Author Contributions

Y.M., N.N., and R.A. designed the entire project. Y.M., T.I., Y.O.K, T.N., H.T., D.I., and K.K. performed the experiments and analyzed the data. N.N. and R.A. supervised and discussed the data. All authors reviewed the manuscript.

Compliance with Ethical Standards

All of these experiments were approved by the Committee on the Animal Care and Use Committees of Nagasaki University. The mice were cared for according to the Guidelines for Animal Experimentation of Nagasaki University

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12035_2018_1088_Fig5_ESM.gif (114 kb)
Supplementary Fig. 1

Effects of the candidate compounds on PrPSc accumulation in N2a-FK cells. PrPSc accumulation in N2a-FK cells after treatment with the SPRi hit compounds. These 27 compounds were not effective in PrPSc accumulation. After the cells had been incubated in medium mixed with each concentration of sample compound for 48 hours, the collected cell lysates were digested with proteinase K. Western blotting was then performed for quantification of the PrPSc level. (GIF 113 kb)

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High Resolution Image (TIF 1112 kb)
12035_2018_1088_Fig6_ESM.gif (331 kb)
Supplementary Fig. 2

Histological analysis of the mouse brain at the terminal stage. (a) Survival curves in the Fukuoka-1-infected mice administered Alp. The control mice (n = 7) and Alp-treated mice (250 mg/L: n = 9; 50 mg/L: n = 10) were compared. At the terminal stage, mice from each group were euthanized for brain homogenates and histopathological analysis (Control: n = 4; 250 mg/L: n = 4; 50 mg/L: n = 4). (b) Western blotting of the brain homogenates and (c) hematoxylin and eosin staining of the brain slices were performed. Scale bars: 100 μm. (GIF 330 kb)

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High Resolution Image (TIF 4748 kb)
12035_2018_1088_Fig7_ESM.gif (47 kb)
Supplementary Fig. 3

Binding activity of the candidate compounds to rMoPrP23–231. Binding affinity of rMoPrP23–231 with each candidate compound was examined using the Biacore T200. (a) Sensorgrams and (b) affinity curves of alprenolol hydrochloride, bisoprolol fumarate, ampicillin and quinacrine for rMoPrP23–231. The concentrations of alprenolol hydrochloride, bisoprolol fumarate and ampicillin were 625, 313, 156, 78, 39, 20 and 0 μM and those of quinacrine were 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9 and 0 μM from top to bottom in the sensorgrams. (GIF 46 kb)

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High Resolution Image (TIF 265 kb)
12035_2018_1088_Fig8_ESM.gif (47 kb)
Supplementary Fig. 4

Binding activity of the candidate compounds to rHuPrP23–231. Binding affinity between rHuPrP23–231 and each candidate compound was examined using the Biacore T200. (a) Sensorgrams and (b) affinity curves of alprenolol hydrochloride, bisoprolol fumarate, ampicillin, and quinacrine for rHuPrP23–231. The concentrations of alprenolol hydrochloride, bisoprolol fumarate, and ampicillin were 625, 313, 156, 78, 39, 20, and 0 μM and those of quinacrine were 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, and 0 μM from top to bottom in the sensorgrams. (GIF 47 kb)

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High Resolution Image (TIF 266 kb)
12035_2018_1088_Fig9_ESM.gif (65 kb)
Supplementary Fig. 5

NMR analysis to evaluate alprenolol HCl binding. 1H-15N HSQC spectra of the 15N-labeled recombinant mouse PrP 121–231 (200 μM), with (red) or without (blue) Alp (4 mM) at pH 4.8 and 25.0 °C. (GIF 65 kb)

12035_2018_1088_MOESM5_ESM.tif (549 kb)
High Resolution Image (TIF 548 kb)

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Copyright information

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Authors and Affiliations

  1. 1.Department of Molecular Microbiology and ImmunologyGraduate School of Biomedical Sciences, Nagasaki UniversityNagasakiJapan
  2. 2.Program for Nurturing Global Leaders in Tropical and Emerging Infectious Diseases, Graduate School of Biomedical SciencesNagasaki UniversityNagasakiJapan
  3. 3.Molecular Epidemiology, Graduate School of Biomedical SciencesNagasaki UniversityNagasakiJapan
  4. 4.Life Science Research CenterGifu UniversityGifuJapan
  5. 5.Division of Microbiology, Department of Infectious Diseases, Faculty of MedicineUniversity of MiyazakiMiyazakiJapan
  6. 6.United Graduate School of Drug Discovery and Medical Information SciencesGifu UniversityGifuJapan

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