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TRANSCRIPT
Organic arsenicals target thioredoxin reductase followed by
oxidative stress and mitochondrial dysfunction resulting in
apoptosis
Xiao-Yang Fana, Yu-Jiao Liua, Kai Chend, Feng-Lei Jianga, Yan-Jun Hub, Dan Liud, Yi
Liua,b,c*, Yu-Shu Ged*
aState Key Laboratory of Virology, Key Laboratory of Analytical Chemistry for Biology
and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan
University, Wuhan 430072, P. R. China
bCollege of Chemistry and Chemical Engineering, Hubei Normal University, Huangshi
435002, P. R. China
cCollege of Chemistry and Chemical Engineering, Wuhan University of Science and
Technology, Wuhan 430081, P. R. China
dCollaborative Innovation Center of Chemistry for Life Sciences , School of Life
Sciences, University of Sciences and Technology of China, Hefei 230027, P. R. China
Figure S1. 1H and 13C NMR spectra of PDT-PAO, PIM-PAO-PDT and PAM-PAO-PDT.
Figure S2. Homology modeling of human TrxR.
Figure S3. The protective effect of DTT on cell viability.
Figure S4. The protective effect of LA on cell viability.
Figure S5. The collapse of mitochondrial membrane potential.
Figure S6. The protective effect of RR, CsA and EGTA on mitochondria swelling induced by organic
arsenicals.
Figure S7. The variation of intracellular Ca2+ level.
Table S1. Molecular docking result of two organic arsenicals with homology modeling human TrxR
protein.
A
B
C
Figure S1. 1H and 13C NMR spectra of PDT-PAO (A), PIM-PAO-PDT (B) and PAM-PAO-PDT (C).
Figure S2. Homology modeling of human TrxR. Secondary structure of modeled structure (A) and
alignment with the template structure 3EAN (B).3D structure of modeled TrxR was displayed in NEW
Cartoon colored according to secondary structure (A); Aligned structures of both protein were
displayed in Ribbons, colored with red for modeled structure and Blue for 3EAN (B). (C) Quality
evaluation of modeled structures by Verify 3D [1]. (D) Quality evaluation of modeled structures by
SOLVX [2]. (E) Quality evaluation of modeled structures by ANOLEA [3]. List of amino acids with
high energy: 12; 19; 22-25; 48; 54; 67; 77-85; 94-96; 98; 119-125; 131-136; 138; 157-166; 176-178;
190-193; 199-200; 242-251; 257-261; 264-266; 279; 336; 340-344; 346-351; 395-396; 434-435; 445-
446; 455-460; 464-468; 478-485. Total amino acids with high energy = 111. Percentage = 22.79. Total
number of amino acids = 487. Total number of atoms = 3707, Total number of non-local atomic
interactions = 64260. Total non-local energy of the protein (E/kT units) = -2160. Non-local normalized
energy Z-score = 2.12.
Figure S3. The protective effect of DTT on cell viability. HL-60 cells with 1 µM and 1.5 µM PIM-
PAO-PDT (left) or 1 µM and 1.5 µM PAM-PAO-PDT (right) and DTT (0.25 mM, 0.5 mM and 1 mM)
were incubated for 24 h, then the cell viability was obtained using trypan blue staining. The data are
expressed as the mean ± SD of three independent samples.
Figure S4. The protective effect of LA on cell viability. HL-60 cells with 1 µM and 1.5 µM PIM-
PAO-PDT (left) or 1 µM and 1.5 µM PAM-PAO-PDT (right) and LA (0.05 mM, 0.075 mM and 0.1
mM) were incubated for 24 h, then the cell viability was obtained using trypan blue staining. The data
are expressed as the mean ± SD of three independent samples.
Figure S5. The collapse of mitochondrial membrane potential. HL-60 cells were incubated with
PIM-PAO-PDT (0.75 µM and 1 µM) or PAM-PAO-PDT (0.75 µM and 1 µM) for 24 h, followed by
JC-1 staining. Data are obtained by the use of flow cytometry. The corresponding quantification data
are shown in the way of column figure and expressed as the mean ± SD of three independent samples.
**P ˂ 0.05 and ***P ˂ 0.01 vs the control group.
Figure S6. The protective effect of RR, CsA and EGTA on mitochondria swelling induced by
organic arsenicals. The mitochondria were incubated with 20 µM PIM-PAO-PDT or 20 µM PAM-
PAO-PDT and 10 µM RR or 10 µM CsA or 500 µM EGTA for 60 min, and the absorbance at 540 nm
during 60 min was recorded by the multimode plate reader.
Figure S7. The variation of intracellular Ca2+ level. HL-60 cells were incubated with PIM-PAO-
PDT for 4 h (A) or 24 h (B) or PAM-PAO-PDT for 4 h (C) or 24 h (D). Concentration of compounds:
0 (black), 0.75 µM (blue) and 1.5 µM (red). Data are obtained by the use of flow cytometry.
Table S1. Molecular docking result of two organic arsenicals with homology modeling human TrxR
protein.
Molecule PIM-PAO-PDT PAM-PAO-PDT
Site I
Size 13 12
Intermolecular H-bond O9-Leu431@N none
As-CYS@SG distance
(Å)
CYS 83: 6.36
CYS 88: 7.03
CYS 519: 5.45
SeCYS 520: 9.75
CYS 83: 6.08
CYS 88: 6.49
CYS 519: 5.70
SeCYS 520: 10.59
Binding energy
(kcals/mol)-7.8 -7.7
Site II
Size 12 14
Intermolecular H-bond noneO9-Cys519@N
O11-His132@NE2
As-CYS@SG distance
(Å)
CYS 83: 11.71
CYS 88: 10.52
CYS 519: 8.85
SeCYS 520: 11.74
CYS 83: 6.78
CYS 88: 4.80
CYS 519: 11.21
SeCYS 520: 12.28
Binding energy
(kcals/mol)-6.9 -7.6
*Size: number of residues within 5 Å of the compound.
Reference
[1] R. Luthy, J.U. Bowie, D. Eisenberg, Assessment of protein models with three-dimensional profiles,
Nature, 356 (1992) 83.
[2] L. Holm, C. Sander, Evaluation of protein models by atomic solvation preference, J Mol Biol, 225
(1992) 93-105.
[3] F. Melo, E. Feytmans, Assessing protein structures with a non-local atomic interaction energy, J
Mol Biol, 277 (1998) 1141-1152.