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Electronic Supplementary Information (ESI)

Celastrol binds to its target protein via specific noncovalent interactions and

reversible covalent bond

Duo Zhang,†a Ziwen Chen,†a Chaochao Hu,a Siwei Yan,a Zhuoer Li,a Baohuan Lian,a Yang Xu,a Rong Ding,a Zhiping Zeng,a Xiaokun Zhang*a,b and Ying Su*a,b aSchool of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, China bCancer center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA *Corresponding authors: Xiaokun Zhang, Ph.D. Ying Su, Ph.D. Phone: 858-646-3100 Fax: 858-646-3195 E-mail: xzhang@sbpdiscovery.org or ysu@sbpdiscovery.org

Table of Contents:

I. Supplementary Fig. S1-S8……………………………………………………..S2-S9

II. Compound experimental data and NMR Spectra……………………………..S10-S15

III. Experimental Methods……………………………………………………….S16-S19

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2018

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I. Supplementary Figures

A B

Figure S1. (A) HPLC spectra of Nur77-LBD and celastrol-bound Nur77-LBD. Nur77-LBD protein has a main peak (protein peak) at the retention time of 7.63 min. A second peak appears at the retention time of 8.04 min after incubating Nur77-LBD with celastrol. (B) HPLC spectra of Nur77-LBD and Nur77-LBD incubated with XS0419. A second peak is not observed for the Nur77-LBD incubated with XS0419.

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Figure S2. Crystal structure analysis shows six cysteines in the Nur77-LBD: C475, C505 and C534 are buried, C465 and C566 are partially solvent-exposed, and C551 is highly exposed.

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Figure S3. (A) CD spectra showed mutations didn’t affect the overall secondary conformation of Nur77-LBD. (B) Mutations did not impact the ability of Nur77 to form heterodimer with RXRα.

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Figure S4. (A) Addition reaction of celastrol with glutathione (GSH). (B) Absorption spectrum of celastrol. Celastrol in PBS (1‰ DMSO) was treated with or without the indicated concentration of GSH, after which UV-visible absorption spectra were recorded (left panel); then the reaction of celastrol and GSH was diluted 10-fold with PBS, and the absorption spectra were recorded again (right panel). Celastrol was released slowly when adduct was diluted in PBS. (C) The absorption spectra of celastrol (100 μM in PBS) in the presence of increasing concentrations of GSH (2.0 μM–10.0 mM, PBS). Equilibrium dissociation constants determined by titrating GSH to the Michael acceptor of celastrol. (D) Addition reaction of celastrol with beta-mercaptoethanol (βME). (E) Celastrol in PBS (1‰ DMSO) was treated with or without the indicated concentration of βME, after which UV-visible absorption spectra were recorded (left panel); then the reaction of celastrol and βME was diluted 10-fold with PBS and the absorption spectra were recorded again (right panel). Celastrol was released slowly when adduct was diluted in PBS. (F) The absorption spectra of celastrol (100 μM in PBS) in the presence of increasing concentrations of βME (0.15–25 mM, PBS). Equilibrium dissociation constants determined by titrating βME to the Michael acceptor of celastrol.

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Figure S5. 1H-NMR spectra showing reversible addition of βME to celastrol. Celastrol (22 mM, top spectrum) treated with 25 mM βME in DMSO-d6 resulting in a 1:33.3 mixture (ratio of peak area) that favored the βME adduct (middle spectrum). Upon ten-fold dilution, the equilibrium shifts to the left (bottom spectrum), ratio 1:16.7. Red and blue asterisks indicate protons used to determine the population ratios.

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Figure S6. Enzymolysis assays and the Orbitrap HCD tandem mass spectrometry (MS/MS) spectrum of Celastrol (A), peptide (B) and Celastrol-peptide complex (C) (Mass analyzer: FTMS ; Match tolerance: 0.02 Da). The results showed that only peptide EHVAAVAGEPQPASCLSR which contained C551 could be labelled by celastrol, however, celastrol didn’t bind to the peptide or C551 directly, indicating that the covalent binding of celastrol to Nur77 was reversible.

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Figure S7. Binding affinity of celastrol to proteins (A) Nur77-LBD (B) Nur77-LBD/C551A (C) Nur77-LBD/C566A (D) Nur77-LBD/D499L (E) Nur77-LBD/Q547W detected by Fluorescence Titration. Mutants that block covalent bond or noncovalent bond interactions all influence the binding affinity to varying degrees.

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Figure S8. (A) Binding affinity of XS0419 to the wild type Nur77-LBD detected by Fluorescence Titration. (B) Binding affinity of XS0419 to Nur77-LBD/C551A detected by Fluorescence Titration. (C) XS0419 induces Nur77 interaction with p62 at a higher concentration than celastrol. HepG2 cells transfected with Flag-p62 (2 μg) and Myc-Nur77 (2 μg) , after treatment with 2 μM celastrol or 2/4/8 μM XS0419 and 20 ng/mL TNFα for 3hr, were examined by co-IP. (D) Mutating C551 to Ala does not affect the XS0419-induced interaction of Nur77 with p62. HepG2 cells transfected with Flag-p62 (2 μg) and Myc-Nur77 or mutants C551A (2 μg) were treated with XS0419 (8 μM) and TNFα (20 ng/mL) for 3 hr and analyzed by co-IP.

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II. Compound information

Analyses of the purchased celastrol 1H and 13C NMR spectra were recorded in CDCl3, CD3OD or DMSO-d6 (dimethyl sulfoxide-d6) on a Bruker 600 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts were expressed in δ (ppm) units downfield from TMS. All coupling constants (J-values) were reported in Hertz (Hz). Signals were described as follow: s, single; br. s., broad signal; d, doublet; t, triplet; m, multiplet. Chemical shifts of common trace 1H-NMR impurities (ppm): H2O: 3.29-3.4 in DMSO-d6, DMSO-d6: 2.50, CHCl3: 7.27. Mass spectra were further characterized by HRMS (high resolution mass spectroscopy), using a Q-Exactive apparatus (ThermoFisher, Shanghai, China).

Celastrol was purchased from Chengdu Pufei De Biotech Co., Ltd.

1H NMR (600 MHz, CHLOROFORM-d) d 7.07 (d, J = 7.15 Hz, 1H), 6.50 (s, 1H), 6.33 (d, J = 7.15 Hz, 1H), 2.52 (d, J = 15.96 Hz, 1H), 2.27 (d, J = 13.94 Hz, 1H), 2.22 (s, 3H), 2.12 - 2.16 (m, 1H), 2.08 - 2.12 (m, 1H), 1.87 - 1.91 (m, 1H), 1.84 - 1.87 (m, 1H), 1.79 - 1.84 (m, 1H), 1.72 - 1.79 (m, 1H), 1.66 (dd, J = 4.58, 13.39 Hz, 1H), 1.59 - 1.64 (m, 1H), 1.55 - 1.59 (m, J = 7.30 Hz, 2H), 1.50 - 1.55 (m, 1H), 1.47 (dd, J = 3.94, 14.95 Hz, 1H), 1.44 (s, 3H), 1.36 (dt, J = 4.68, 13.98 Hz, 1H), 1.28 (s, 3H), 1.25 (s, 3H), 1.10 (s, 3H), 0.94 (d, J = 13.94 Hz, 1H), 0.58 (s, 3H); 13C NMR (151 MHz, CHLOROFORM-d)d 182.4, 178.3, 172.7, 165.0, 147.0, 135.4, 127.5, 120.6, 120.5, 118.3, 45.3, 44.3, 43.0, 39.9, 39.3, 38.3, 36.3, 34.5, 33.8, 32.4, 31.4, 31.0, 30.6, 29.5, 29.3, 28.7, 21.4, 18.7, 10.4; HRMS (ESI) calcd. for C29H39O4+ [M+H]+: 451.2843, found: 451.2841.

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1H-NMR (600 MHz, CHOLORIDE-d) spectrum of celastrol

13C-NMR (151 MHz, CHOLORIDE-d) spectrum of celastrol

Celastrol-C-NMR.esp

180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)

0.05

0.10

0.15

0.20

Norm

alize

d In

tens

ity

10.4

4

18.6

621

.43

28.7

129

.27

30.6

536

.33

38.3

339.2

839

.90

43.0

445

.30

76.7

877

.00

77.2

1

118.

2612

0.63

127.

52

135.

40

147.

03

165.

01

172.

70178.

3118

2.37

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13C-NMR (151 MHz, CHOLORIDE-d) 135 ℃ DEPT spectrum of celastrol

HR-MS spectrum of celastrol

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XS0419

(2R,4aS,6aS,12bS,14aS,14bR)-10,11-dihydroxy-2,4a,6a,9,12b,14a-hexamethyl-

1,2,3,4,4a,5,6,6a,8,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid.

Preparation of XS0419 (CAS#: 193957-88-9)

50 mg (0.11 mmol, 1 equiv) of celastrol were stirred to dissolve in 2 mL CD3OD in a 25-mL flask, 44 mg (1.1 mmol, 10 equiv) of NaBH4 were added. Then reaction was stirred to react at room temperature for 30 min. 0.1 mol/L HCl (3 mL) was added to quench the reaction, subsequently the aqueous phase was separated and extracted with DCM (15 mL) three times. The organic layers were combined and dried with anhydrous sodium sulfate, and then purified by removing the solvent under vacuum conditions. White solid, 50.1 mg, yield: 99%. m. p.: 178.3-180.1 ℃. [α]24

D

= -4.0 (c = 0.1, MeOH). 1H NMR (600 MHz, DMSO-d6) δ 12.05 (br. s., 1H), 8.80 (s, 1H), 7.82 (s, 1H), 6.61 (s, 1H), 5.72 (dd, J = 1.74, 6.14 Hz, 1H), 3.18 (dd, J = 6.24, 20.54 Hz, 1H), 2.91 (dd, J = 1.47, 19.99 Hz, 1H), 2.34 (d, J = 15.59 Hz, 1H), 2.04 (d, J = 13.57 Hz, 1H), 2.01 (s, 3H), 1.94 - 2.00 (m, 2H), 1.86 (dt, J = 5.00, 13.80 Hz, 1H), 1.79 (dt, J = 6.50, 13.80 Hz, 1H), 1.63 - 1.69 (m, 1H), 1.59 - 1.63 (m, 1H), 1.53 - 1.59 (m, 1H), 1.43 - 1.51 (m, 3H), 1.36 - 1.41 (m, 1H), 1.29 (dt, J = 4.40, 13.70 Hz, 1H), 1.22 (s, 3H), 1.17 (s, 3H), 1.11 (s, 3H), 1.05 (s, 3H), 0.85 (d, J = 13.94 Hz, 1H), 0.66 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δ 179.5, 149.2, 143.1, 140.6, 139.4, 123.1, 120.1, 117.7, 108.2, 43.8, 43.3, 39.4, 37.1, 36.6, 36.1, 34.4, 34.1, 34.1, 32.4, 31.4, 30.2, 30.1, 29.8, 29.5, 28.4, 27.3, 22.7, 18.1, 11.5; HRMS (ESI) calcd. for C29H40NaO4+ [M+Na]+: 475.2819, found: 475.2823.

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1H-NMR (600 MHz, DMSO-d6) spectrum of XS0419

13C-NMR (151 MHz, DMSO-d6) spectrum of XS0419

XS-0419_H.esp

9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7No

rmal

ized

Inte

nsity

3.021.043.013.173.023.471.251.143.031.270.971.101.061.081.983.131.051.050.090.081.051.091.041.051.031.07

DMSO

Water

0.64

0.67

0.87

1.061.11

1.181.23

1.491.50

1.98

1.99

2.02

2.052.33

2.36

2.902.93

3.17

3.18

3.20

3.215.72

5.72

5.73

5.73

6.62

7.82

8.81

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HR-MS spectrum of XS0419

F:\Spectrum Data\XS0419 2017/1/10 星期二 下午 3:02:19

XS0419 #39 RT: 0.38 AV: 1 NL: 8.06E6T: FTMS + p ESI Full ms [100.00-1000.00]

420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce475.2823

451.2845

727.4681505.2927441.2978

543.2696

470.3267

557.2856425.2877514.2932 689.1587491.2569 615.1392 633.1509565.2523 591.4958 707.1686 764.1782 788.6783659.2856 737.4990

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III. Materials and Methods Cell Culture Conditions Human hepatocellular carcinoma cell line HepG2 was maintained in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (FBS). Plasmids Plasmids pcmv-myc-Nur77, pFlag-cmv-2-TRAF2, pFlag-cmv-2-p62, RFP-LC3 and pcmv-3*flag-RXRα were described previously. Plasmids pcmv-myc-Nur77/C551A, pcmv-myc-Nur77/C465A, pcmv-myc-Nur77/C566A, pET-15b-Nur77-LBD/C566A, pcmv-myc-Nur77/D499L, pcmv-myc-Nur77/Q547W, pcmv-myc-Nur77/D499L/Q547W, pET-15b-Nur77-LBD/D499L and pET-15b-Nur77-LBD/Q547W were constructed by using PCR or QuikChange mutagenesis kit. Protein Expression and Purification The human Nur77-LBD (367-598) was cloned as an N-terminal histidine-tagged fusion protein in pET15b expression vector and overproduced in Escherichia coli BL21 DE3 strain. Briefly, cells were harvested and sonicated, and the extract was incubated with the His60 Ni Superflow resin, then the His-tagged Nur77-LBD-resin complexes were washed and eluted with 300 mM NaCl in 20 mM imidazole buffer (pH 7.5). Nur77-LBD was further purified by gel filtration on a Superdex 200 Increase 10/300 column (lot 10243519, GE). For HPLC-MS/MS and Orbitrap MS/MS experiments, the buffer was replaced with 50 mM NH4HCO3 solution by ultrafiltration (3000 MWCO). For CD and UV-Vis absorption assays, the buffer was exchanged to phosphate buffer (10 mM, pH7.4). Antibodies and Reagents Anti-Myc (9E10) (Cat. ab32) antibody was from Abcam (UK); anti-β-actin (Cat. 4970S), anti-IκBα (Cat.4814S) antibodies were from Cell Signal Technology (Beverly, MA, USA); anti-Flag (Cat.F3165) antibody was from Sigma; human TNFα was from peprotech; phusion site-directed mutagenesis kit (Cat.F-541) was from Thermo. Circular Dichroism (CD) Briefly, Nur77-LBD mutant proteins (0.1mg/mL) was measured with Jasco J-810 spectropolarimeter, and the CD spectra were obtained from 190 nm to 260 nm. Nur77-LBD was used as control.

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Western Blotting (WB) Cell lysates were boiled in sodium dodecyl sulfate (SDS) sample loading buffer, resolved by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose. The membranes were blocked in 5% milk in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris– HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween 20) for 1 hr at room temperature. After washing twice with TBST, the membranes were incubated with appropriate primary antibodies in TBST for 1 hr and then washed twice, probed with horseradish peroxide-linked anti-immunoglobulin (1:5000 dilution) for 1 hr at room temperature. After three washes with TBST, immunoreactive products were visualized using enhanced chemiluminescence reagents and autoradiography. Co-immunoprecipitation (co-IP) HepG2 cells transfected with Flag-TRAF2/p62 (2 μg) and Myc-Nur77 or mutants C551A, C566A, D499L, or Q547W (2 μg) were treated with celastrol (2 μM) and TNFα (20 ng/ml) for 3 hr. Cells were harvested in lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, and 5 mM ethylenediaminetetraacetic acid, and containing protease inhibitors). Lysate was incubated with 1 µg antibody at 4℃ for 2 hr. Immunocomplexes were then precipitated with 30 µL of protein A/Gsepharose. After an extensive washing with lysis buffer, the beads were boiled in SDS sample loading buffer and assessed by Western blotting (WB). HPLC-MS/MS Celastrol was incubated with purified Nur77-LBD or mutant proteins (3.3 μM in 50 mM NH4HCO3 solution). After incubation for 5 min or more at room temperature. The complex was separated in a C8 column (3.5 μm, 2.1×100 mm from Agilent), and eluted with solvent A (0.1% formic acid) and solvent D (0.1% formic acid in acetonitrile) by DIONEX Ultimate 3000 (UHPLC from Thermo). The solvent gradient at a flow rate of 0.3 mL/min was 0-4 min 40% solvent D; 4-12 min, 40 to 90% solvent D; 12-15 min, 90% solvent D and re-equilibrate to initial condition for 5 min. Then constituents eluted analyzed by MS (Q Exactive – Orbitrap MS from Thermo). The protein MS analysis was performed in the positive ion mode: In-source CID was 30.0 ev, and capillary temperature was 200 ℃. Finally, the LC spectrum was imported by Xcalibur Qual Browser (from Thermo) and MS data was calculated by Protein Deconvolution (from Thermo).

Orbitrap Mass Mpectrometry (MS/MS) Purified Nur77-LBD protein in 50 mM NH4HCO3 buffer was incubated with Celastrol over night at 4 ℃, the complex (100 ng) was then pretreated by 10 mM dithiothreitol (37 ℃, 30 min) and then with 40 mM iodoacetamide (RT, 20 min). The sample was digested overnight in 50 mM NH4HCO3 buffer with sequencing grade trypsin using an enzyme: substrate radio of 1:50 (w/w). After desalting by ZipTipC18 column, the peptides (5 ng) were separated in Acclaim PepMap RSLC (75

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μm × 15 cm, nanoViper, C18, 2 μm, 100 A, from Thermo) and eluted with solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) by EASY-nLC 1000 (nano-LC from Thermo). Peptides were eluted with a linear gradient of solvent B (3-35% in 113 min) at a flow rate of 10 μL/min, and then detected by Q Exactive – Orbitrap MS (from Thermo). Peptide MS/MS analysis was performed in the positive ion mode using a mass range of 200-2000 m/z. The data were processed using Proteome Discover (from Thermo). UV-Visible Absorption Assay Celastrol (100 μM) in PBS (1‰ DMSO) was treated with or without 1 mM GSH or βME (2 mL of 100 μM celastrol in PBS, pH=7.4 was added with 2 μL of 1M GSH or βME, in PBS, pH=7.4), after which UV-visible absorption spectra were recorded. Each reactions (Reaction A: 100 μM celastrol, B: 100 μM celastrol with 1mM GSH or βME) were then diluted 10-fold with PBS or (1 mM) GSH/PBS (or βME/PBS), and the absorption spectra were recorded again. Determination of equilibrium dissociation constants (Kd) for thiol/Michael acceptor adducts Reactions of celastrol with GSH and bME were monitored with UV-Vis absorption spectra. Reactions were initiated by titrating tiny volumes of thiol (300 mM GSH or 1 M bME in PBS, pH 7.4) to the celastrol solution (100 μM in PBS, pH 7.4) with final concentration of the thiol at 0–10 mM (or 0-25 mM, while bME was used). Then absorption spectra were acquired (300–600 nm). Equilibration of celastrol with GSH or bME occurred within seconds or less. Formation of the thiol adduct was quantified by monitoring the disappearance of the absorbance peak (about 450 nm max) relative to the no-thiol control sample. Data were fit using Origin 2016 to obtain equilibrium dissociation constants. Fluorescence Titration Wild-type Nur77-LBD protein or mutant protein (1 μM in 3mL phosphate buffer) was measured with Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer, and the fluorescence spectra were obtained from 300 nm to 500 nm. Celastrol or XS0419 (from 100 nM to 1 μM with an increment of 100 nM, from 1 μM to 2 μM with an increment of 250 nM, from 2 μM to 5 μM with an increment of 500 nM) were added to the protein. After incubation for 30 s at RT, the incubation buffer was measured with the spectrophotometer. Data were processed and fitted to obtain the binding affinities using Origin. Modeling The crystal structure of Nur77-LBD in complex with DPDO (PDB ID: 4KZI) was used. The disordered loop containing 541AVAGEPQPAS550 was reconstructed using Schrodinger’s Prime

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(Default settings were used) and the built protein model was used for docking studies. Schrodinger’s Glide, a grid-based docking module, was employed to study how celastrol bound to Nur77-LBD. Center of the grid box was set at C551with a box of 15 angstrom in each direction. Standard Precision mode was used for scoring.