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Supporting Information
The Anthelmintic Drug Niclosamide and Its Analogues
Activate the Parkinson’s Disease Associated Protein Kinase
PINK1
Erica Barini,[a] Ageo Miccoli,[b] Federico Tinarelli,[c] Katie Mulholland,[a] Hachemi Kadri,[b]
Farhat Khanim,[d] Laste Stojanovski,[e] Kevin D. Read,[e] Kerry Burness,[f] Julian J. Blow,[c]
Youcef Mehellou,*[b] and Miratul M. K. Muqit*[a, g]
cbic_201700500_sm_miscellaneous_information.pdf
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The Anthelmintic Drug Niclosamide and its Analogues Activate the Parkinson’s Disease
Associated Protein Kinase PINK1
Erica Barini,1 Ageo Miccoli,2 Federico Tinarelli,3 Katie Mulholland,1 Hachemi Kadri,2 Farhat
Khanim,4 Laste Stojanovski,5 Kevin D. Read,5 Kerry Burness,6 Julian Blow,3 Youcef
Mehellou,2,* Miratul M. K. Muqit1,7,*
1MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University
of Dundee, Dow Street, Dundee DD1 5EH, UK.
2School of Pharmacy and Pharmaceutical Sciences, College of Biomedical and Life Sciences,
Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK.
3Gene Regulation and expression department, School of Life Sciences, University of Dundee,
Dow Street, Dundee DD1 5EH, UK.
4School of Biosciences, College of Life and Environmental Sciences, University of
Birmingham, Edgbaston, Birmingham B15 2TT, UK.
5Drug Discovery Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee
DD1 5EH, UK.
6Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee,
Dow Street, Dundee DD1 5EH, UK.
7School of Medicine, University of Dundee, Dundee DD1 9SY, UK.
CONTENT
I. Antibodies and reagents (page 1)
II. Cell culture (page 2)
III. Synthesis of AM85-AM87 compounds (page 2)
IV. Supplementary figure S1 (page 3)
V. Supplementary figure S2 (page 4)
VI. Supplementary figure S3 (page 5)
I. Antibodies and reagents
The following primary antibodies were used: mouse monoclonal antibodies against Parkin
(Santa Cruz), βI-tubulin (Sigma), β-actin (Sigma), GAPDH (Santa Cruz), PSD95 (Cell
Signaling), synaptophysin (cell signalling), CISD1 (Proteintech). Horseradish-peroxidase
(HRP)-conjugated secondary antibodies (Sigma) were used. Anti-Parkin phospho-Ser65 rabbit
monoclonal antibody was raised by Epitomics in collaboration with the Michael J Fox
Foundation for Research.
Stock solutions of niclosamide (Sigma), Antimycin A (Sigma) Oligomycin A (Sigma) were
used for experiments in vitro. Unless otherwise specified, general reagents and chemicals were
from Sigma and cell culture reagents were from Invitrogen.
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II. Cell culture
HeLa cells stably expressing untagged Parkin WT and PINK 1 KO, were cultured using
DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS, 2 mM L-
glutamine, 1×penicillin/streptomycin. To uncouple mitochondria, cells were treated with 10
µM Antimycin (Sigma) and 1 µM Oligomycin dissolved in DMSO for 3 h. To express Parkin,
cell transfections were performed using polyethylenimine (Polysciences) according to the
manufacturer's instruction.
Primary cortical neurons were isolated from the brain of WT embryos of either sex at E16.5.
Embryonic cortices were collected in HBSS, and cells were dissociated by incubation with
trypsin (GIBCO) at 37°C. Cells were then diluted in Neurobasal medium containing B27
supplement, Glutamax, penicillin/streptomycin and plated at a density of 6.0 × 106 cells/well
on 6-well plates coated with 0.1 mg/ml poly-L-lysine (PLL; Sigma). Neurons were cultured at
37 °C in a humidified incubator with 5% CO2. Every 7 days, the medium was replaced with
fresh medium containing B27. Neurons were treated with 30 µM niclosamide and analogues
for 1h at 37 °C.
III. Synthesis of AM85-AM87 compounds
To a stirring solution of the relevant salicylic acid (1 eq.) in dry THF, PyBop (1.2 eq.) and
triethylamine (3 eq.) were added. After 10 min, the relevant aniline (1.5 eq.) was added to the
cloudy white suspension and stirred at room temperature for 12 h. The reaction mixture was
then concentrated under reduced pressure to afford a yellow oil, which was subsequently
dissolved in water and extracted in ethyl acetate. The combined organic layers were dried
(MgSO4), filtered and concentrated under reduced pressure to form a viscous orange oil. Flash
column chromatography employing ethyl acetate:hexane (1:1) was used to yield the desired
AM compounds as off-white solids.
5-Bromo-N-(4-bromophenyl)-2-hydroxybenzamide (AM85). Yield 10%. 1H NMR (500 MHz,
DMSO): δ 11.73 (s, 1H, OH), 10.49 (s, 1H, NH), 8.03 (d, J = 2.5 Hz, 1H), 7.69 (d, J = 8.9 Hz,
2H), 7.57 (m, 3H), 6.97 (d, J = 8.8 Hz, 1H). 13C NMR (126 MHz, DMSO): δ 165.36, 157.53,
137.98, 136.31, 132.39, 131.77, 123.10, 120.92, 119.97, 116.46, 110.59. HRMS m/z [M+H]+
calcd. for C13H10NO2Br2: 369.9078, found: 369.9067. Anal. Calcd for C13H9NO2Br2: C, 42.08;
H, 2.44; N, 3.77. Found: C, 41.93; H, 2.61; N, 3.93.
3,5-Dibromo-N-(4-bromophenyl)-2-hydroxybenzamide (AM86). Yield 9%. 1H NMR (500
MHz, DMSO): δ 12.75 (s, 1H, OH), 10.71 (s, 1H, NH), 8.26 (s, 1H), 8.01 (s, 1H), 7.67 (d, J =
8.6 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H). 13C NMR (126 MHz, DMSO): δ 167.09, 156.71, 139.09,
137.27, 132.07, 130.26, 124.06, 119.10, 117.41, 112.93, 110.39. HRMS m/z [M+H]+ calcd. for
C13H9NO2Br3: 447.8183, found: 447.8184. HPLC (8 min), tR = 5.21 min, purity: 97%.
3,5-Dibromo-2-hydroxy-N-phenylbenzamide (AM87). Yield 15%. 1H NMR (500 MHz,
DMSO): δ 13.01 (s, 1H, OH), 10.65 (s, 1H, NH), 8.31 (d, J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz,
1H), 7.68 (d, J = 7.7 Hz, 2H), 7.41 (t, J = 7.9 Hz, 2H), 7.21 (t, J = 7.4 Hz, 1H). 13C NMR (126
MHz, DMSO): δ 167.18, 156.98, 138.91, 137.87, 130.26, 129.21, 125.40, 122.23, 118.88,
113.14, 109.71. HRMS m/z [M+H]+ calcd. for C13H10NO2Br2: 369.9078, found: 369.9087.
Anal. Calcd for C13H9NO2Br2: C, 42.08; H, 2.44; N, 3.77. Found: C, 41.91; H, 2.37; N, 3.71.
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IV. Supplementary figure 1
Supplementary 1. Niclosamide activates PINK1 after 20 minutes treatment. A)
Niclosamide-induced Parkin activation is PINK1 dependent and can be detected after 20 min
of in vitro stimulation. HeLa cells transfected with Parkin were stimulated with either a
combination of antimycin A and oligomycin A (A/O) for 3 h or with 10 µM of niclosamide
(Niclo) for 5, 10, 20, 40 min. Detection of Parkin S65 phosphorylation (pS65Parkin), Parkin,
Full length (F/L) and cleaved OPA1, CISD1 ubiquitylation (CISD1-Ub) can be detected in
niclosamide stimulated HeLa cells at different time points. GAPDH and HSP60 were used as
loading controls. B) Quantitative analysis of Parkin S65 phosphorylation after niclosamide
treatment at different time points (5, 10, 20, 40 min). Bars represent the average ratio ± SEM
of two independent experiments and the value is a ratio between pS65 Parkin and total Parkin.
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V. Supplementary figure 2
Supplementary 2. AM85 activates PINK1 and uncouples mitochondria after 40 minutes
of treatment. A) CISD1 ubiquitylation after niclosamide and AM85 treatment is PINK1
dependent. Wildtype (WT) and PINK1 knockout (Pink1 KO) HeLa cells transfected with
Parkin were stimulated with a combination of 10 µM antimycin A and 1 µM oligomycin A
(A/O) for 3 h, 10 µM niclosamide (Niclo) and 10 µM AM85 for 40 min. CISD1 ubiquitylation
(CISD1-Ub) was detected by western blotting. B) Time course of AM85 in HeLa cells.
Detection of Parkin S65 phosphorylation (pS65 Parkin), Parkin, Full length and cleaved OPA1
and CISD1 ubiquitylation (CISD1-Ub) upon different time points of AM85 treatment (5, 10,
20, 40 min). C) Quantitative analysis of Serine 65 phosphorylation in response to AM85
treatment at different time points. Bars represent the average ratio ± SEM of 2 independent
experiments and the value is a ratio between pSer65 Parkin and total Parkin. D) Niclosamide
and AM85 uncouple mitochondria in HeLa cells. Exemplary FACS graph of CMXRos
fluorescence intensity in Hela cells treated on site. Hela cells were treated on site with
antimycin A and oligomycin A (A/O, green), niclosamide (Niclo, red), AM85 (blue) and the
vehicle, DMSO (black).
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VI. Supplementary figure 3
Supplementary 3. Mitochondrial membrane potential is restored upon wash out of AM85
and niclosamide. A) Exemplary FACS curve of CMXRos fluorescence intensity in Hela cells
subjected to 30 min washout treatment. Hela cells treated with Antimycin A/ Oligomycin A
(A/O, green curve), niclosamide (Niclo, red curve), AM85 (blue curve), normalized to the
vehicle, DMSO (black curve). B) Niclosamide and AM85 do not induce cell death. Percentage
of alive (bottom percentage) and dead cells (upper percentage) after niclosamide (bottom left
square) and AM85 (bottom right square) treatment compared to DMSO (upper left square) and
A/O (upper right square) treated cells. FSC-A (forward scatter area).