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.