Supplementary Information A gene expression signature-based approach reveals the mechanisms of action of the Chinese herbal medicine berberine Kuen-Haur Lee, Hsiang-Ling Lo, Wan-Chun Tang, Heidi Hao-yun Hsiao and Pei-Ming Yang* The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan *Correspondence: Dr. Pei-Ming Yang The Ph.D. Program for Cancer Biology and Drug Discovery College of Medical Science and Technology Taipei Medical University 250, Wu-Hsing Street Taipei 11031, Taiwan Tel: +886-2-27361661 ext. 7629 Fax: +886-2-26558562 E-mail:
[email protected]
Supplementary Information includes: Supplementary Methods Supplementary Figures S1-S2 Supplementary Tables S1-S4
Supplementary Methods Homology modeling Automated homology modeling was performed using SWISS-MODEL.1 The 3D model of the human HDAC6 catalytic site (with a residue range of 477~836) was built using HDAC7 (PDB ID: 3C0Z; chain A) as a template. The sequence identity between these two residues was 43.896%. Molecular docking Docking simulations were performed with iGEMDock.2 Structures of HDAC2 (4LXZ), HDAC4 (PDB ID: 2VQM), HDAC7 (PDB ID: 3C0Z), and HDAC8 (1T69) were obtained from the Protein Data Bank. The binding site of the target was prepared, and energy-minimized compounds were imported. The docking protocol consisted of 80 generations per ligand and a population size of 300 random individuals. All docking conformations were performed 10 times using a genetic evolutionary algorithm, and the fitness of the docked structures was calculated. The binding site of the target was identified at a distance of 8Å. The empirical scoring function of iGEMDOCK was estimated as: Fitness (kcal/mol) = VDW + H bond, where the VDW term is van der Waal energy and H bond term is hydrogen bonding energy. References for supplementary Methods 1. Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: An automated protein homology-modeling server. Nucleic acids research 2003; 31: 3381-5. 2. Hsu KC, Chen YF, Lin SR, Yang JM. iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis. BMC bioinformatics 2011; 12 Suppl 1: S33.
Legends to Supplementary Figures Figure S1. Chemical-protein network prediction by STITCH. CMAP compounds (Table 1) that was positively correlated with BBR were queried with STITCH 4.0 (http://stitch.embl.de/). The parameters were set as follows: Active prediction methods = Experiments, Databases, Textmining, and Predictions; Required confidence (score) = highest confidence (0.900); Interaction shown: no more than 20 interactors. The score between each node was shown in supplementary Table S4. Figure S2. Molecular docking analysis of BBR and SAHA to HDAC isoforms. The chemical structures of BBR and SAHA and the best docking models of BBR or SAHA to each HDAC isoform are shown in this figure. Magenta sphere, zinc atom; cyan, mapped BBR; purple, mapped SAHA.
Supplementary Table S1. Differentially expressed genes induced by BBR (40 M for 4 h) in HepG2 cells. This dataset (GSE47786) was obtained from NCBI GEO. Gene symbol CYR61 GDF15 PPP1R15A C8orf4 CTGF RASD1 EGR1 ANKRD1 CCL20 IER3 PIM1 DUSP1 ACTA1 WEE1 THBS1 ERRFI1 CBX4 JAG1 N4BP2L2 JUNB NEDD9 HAMP HES1 SLC30A1 PIM3 FOXQ1 KLF6 ZFP36
Gene title cysteine-rich, angiogenic inducer, 61 growth differentiation factor 15 protein phosphatase 1, regulatory (inhibitor) subunit 15A chromosome 8 open reading frame 4 connective tissue growth factor RAS, dexamethasone-induced 1 early growth response 1 ankyrin repeat domain 1 (cardiac muscle) chemokine (C-C motif) ligand 20 immediate early response 3 pim-1 oncogene dual specificity phosphatase 1 actin, alpha 1, skeletal muscle WEE1 homolog (S. pombe) thrombospondin 1 ERBB receptor feedback inhibitor 1 chromobox homolog 4 (Pc class homolog, Drosophila) jagged 1 (Alagille syndrome) NEDD4 binding protein 2-like 2 jun B proto-oncogene neural precursor cell expressed, developmentally down-regulated 9 hepcidin antimicrobial peptide hairy and enhancer of split 1, (Drosophila) solute carrier family 30 (zinc transporter), member 1 pim-3 oncogene forkhead box Q1 Kruppel-like factor 6 zinc finger protein 36, C3H type, homolog (mouse)
Fold change (Log2) 3.95 2.84 2.54 2.45 2.41 2.36 2.31 2.07 2.02 1.97 1.90 1.79 1.73 1.66 1.64 1.59 1.47 1.46 1.46 1.45 1.44 1.42 1.40 1.39 1.39 1.36 1.34 1.34
SLC25A25 JUN TUFT1 SPRY4 TAGLN PDLDA DDIT3 AKR1C2
MYC FST SERTAD2 IGFBP1 CHMP1B GADD45B MIXL1 DUSP5 CDKN1A TAGLN MT2A NR0B2 ACTG1 TRIB1 ZFP36L1 BRWD1 NUAK2 IL8 TRIM25
solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 25 jun oncogene tuftelin 1 sprouty homolog 4 (Drosophila) transgelin pleckstrin homology-like domain, family A, member 1 DNA-damage-inducible transcript 3 aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III) v-myc myelocytomatosis viral oncogene homolog (avian) follistatin SERTA domain containing 2 insulin-like growth factor binding protein 1 chromatin modifying protein 1B growth arrest and DNA-damageinducible, beta Mix1 homeobox-like 1 (Xenopus laevis) dual specificity phosphatase 5 cyclin-dependent kinase inhibitor 1A (p21, Cip1) transgelin metallothionein 2A nuclear receptor subfamily 0, group B, member 2 actin, gamma 1 tribbles homolog 1 (Drosophila)
1.33
zinc finger protein 36, C3H type-like 1 bromodomain and WD repeat domain containing 1 NUAK family, SNF1-like kinase, 2 interleukin 8 tripartite motif-containing 25
1.03 1.03
1.28 1.28 1.27 1.25 1.24 1.24 1.24
1.24 1.22 1.20 1.18 1.15 1.13 1.13 1.13 1.11 1.10 1.10 1.04 1.03 1.03
1.02 1.01 -1.00
GPAM C20orf177 EIF5 GPER ZC3HAV1 TXNIP
glycerol-3-phosphate acyltransferase, mitochondrial chromosome 20 open reading frame 177 eukaryotic translation initiation factor 5 G protein-coupled estrogen receptor 1 zinc finger CCCH-type, antiviral 1 thioredoxin interacting protein
-1.01 -1.04 -1.09 -1.42 -1.51 -1.63
Supplementary Table S2. Interaction profile of HDACs to BBR or SAHA by iGEMDOCK analyses. Class
HDAC HDAC2
I HDAC8
HDAC4 IIa HDAC7
IIb
HDAC6
Drug
Localization
Total fitness energy
VDW
H bond
-113.54
-80.91
-32.62
-100.16
-85.57
-14.59
-114.64
-101.02
-13.62
BBR
-104.71
-87.25
-17.45
SAHA
-108.57
-73.68
-34.89
SAHA BBR SAHA
Nucleus
BBR
Nucleus &
-91.57
-72.23
-19.34
SAHA
cytoplasm
-97.75
-77.01
-20.74
-101.88
-84.73
-17.16
BBR SAHA
Nucleus &
-107.06
-74.65
-32.42
BBR
cytoplasm
-97.83
-78.37
-19.47
Supplementary Table S3. Target predictions for BBR through the ChEMBL website. This table displays ChEMBL single-protein targets which were predicted to interact with BBR (CHEMBL295124). Cut-off points at 1 and 10 μM were applied to ChEMBL bioactivity data used to generate the respective models, and yellow highlighted rows correspond to genuine predictions, i.e., targets not included in the original training set for this compound. Targets with a score of > 0.8 are shown. Cut-off
1 M
10 M
Target name
Organism
Score
Acetylcholinesterase Cholinesterase Butyrylcholinesterase Tubulin alpha chain Alpha-2b adrenergic receptor Tubulin beta chain Alpha-2c adrenergic receptor
Electrophorus electricus Equus caballus Equus caballus Sus scrofa Homo sapiens Bos taurus Homo sapiens
1.000 1.000 1.000 0.999 0.992 0.964 0.819
Butyrylcholinesterase Acetylcholinesterase Cholinesterase Coagulation factor III Tubulin alpha chain Tubulin beta chain
Equus caballus Electrophorus electricus Equus caballus Homo sapiens Sus scrofa Bos taurus
1.000 1.000 1.000 1.000 0.999 0.995
Alpha-1b adrenergic receptor Phosphodiesterase 11A
Rattus norvegicus Homo sapiens
0.869 0.855
Supplementary Table S4. The score between each node in STITCH analysis.
Homology Experimental Knowledge Textmining
Combined
Node1
Node2
AKT1
vorinostat
0
0
0.7
0.842
0.951
AKT1
RPS6KB1
0.883
0.846
0.9
0.967
0.985
AKT1
CCND1
0
0
0
0.954
0.954
AKT1
rapamycin
0
0
0.9
0.925
0.992
AKT2
AKT1
0.968
0.62
0.9
0.954
0.96
AKT2
MTOR
0
0
0.9
0.76
0.974
AKT2
RPTOR
0
0
0.9
0.317
0.927
AKT2
rapamycin
0
0
0.9
0.876
0.987
CCND1
rapamycin
0
0
0.9
0.882
0.987
CYP2D6
thioridazine
0
0.9
0.9
0.922
0.999
DHFR
pyrimethamine
0
0.9
0.8
0.944
0.998
EIF4E
CCND1
0
0
0
0.918
0.919
EIF4E
rapamycin
0
0
0.9
0.929
0.992
EIF4E
RHEB
0
0
0.9
0.543
0.951
EIF4EBP1
CCND1
0
0
0
0.906
0.906
EIF4EBP1
RPS6KB1
0
0.62
0
0.969
0.987
EIF4EBP1
EIF4E
0
0.999
0.9
0.969
0.999
EIF4EBP1
RHEB
0
0
0.9
0.752
0.973
EIF4EBP1
rapamycin
0
0
0.9
0.939
0.993
EIF4EBP1
AKT1
0
0.621
0.9
0.954
0.998
EIF4EBP1
RPTOR
0
0.999
0.9
0.947
0.999
FKBP1A
RPTOR
0
0.845
0
0.61
0.935
FKBP1A
rapamycin
0
0.9
0.9
0.94
0.999
FKBP1A
MTOR
0
0.999
0
0.961
0.999
FKBP3
rapamycin
0
0.9
0.9
0.339
0.992
HDAC1
trichostatin A
0
0.9
0.7
0.654
0.989
HDAC1
FKBP3
0
0.621
0.9
0
0.959
HDAC1
vorinostat
0
0.9
0.8
0.399
0.987
HDAC1
MS-275
0
0.9
0.8
0.399
0.987
HDAC1
CCND1
0
0.846
0.9
0.54
0.991
HDAC1
HIF1A
0
0.951
0
0.373
0.967
score
HDAC1
HDAC6
0.699
0
0.9
0.81
0.924
HDAC2
MS-275
0
0.897
0
0.338
0.929
HDAC2
HDAC1
0.97
0.999
0.9
0.97
0.999
HDAC2
HDAC6
0.66
0
0.9
0.748
0.924
HDAC2
vorinostat
0
0.9
0.8
0.391
0.987
HDAC2
trichostatin A
0
0.9
0.7
0.641
0.988
HDAC6
trichostatin A
0
0.9
0.7
0.397
0.98
HDAC6
vorinostat
0
0.9
0.8
0.399
0.987
HIF1A
rapamycin
0
0
0.9
0.873
0.986
HIF1A
cycloheximide
0
0
0.7
0.831
0.947
HIF1A
AKT1
0
0
0.9
0.946
0.994
HTR2A
thioridazine
0
0.9
0.8
0.765
0.995
IRS1
RPS6KB1
0
0
0.9
0.927
0.992
IRS1
AKT1
0
0.621
0
0.975
0.99
IRS1
rapamycin
0
0
0.9
0.886
0.988
MTOR
AKT1
0
0.989
0.9
0.979
0.999
MTOR
CCND1
0
0.079
0
0.943
0.944
MTOR
RHEB
0
0.998
0.9
0.974
0.999
MTOR
RPTOR
0
0.999
0.9
0.967
0.999
MTOR
RPS6KB1
0
0.999
0.9
0.979
0.999
MTOR
HIF1A
0
0
0.8
0.927
0.984
MTOR
vorinostat
0
0
0.7
0.837
0.949
MTOR
EIF4E
0
0.621
0.9
0.954
0.998
MTOR
IRS1
0
0.621
0.9
0.961
0.998
MTOR
rapamycin
0
0.9
0.9
0.966
0.999
MTOR
EIF4EBP1
0
0.999
0.9
0.979
0.999
RHEB
rapamycin
0
0
0.9
0.927
0.992
RHEB
RPS6KB1
0
0
0.9
0.967
0.996
RPS6
MTOR
0
0.614
0
0.971
0.988
RPS6
RPS6KB1
0
0.846
0.9
0.981
0.999
RPS6
rapamycin
0
0
0.9
0.938
0.993
RPS6
EIF4E
0
0.845
0.9
0.751
0.995
RPS6KB1
rapamycin
0
0
0.9
0.954
0.995
RPTOR
RHEB
0
0.834
0.9
0.752
0.995
RPTOR
IRS1
0
0.621
0.9
0.43
0.975
RPTOR
EIF4E
0
0
0.9
0.611
0.958
RPTOR
AKT1
0
0
0.9
0.566
0.953
RPTOR
RPS6KB1
0
0.989
0.9
0.947
0.999
RPTOR
rapamycin
0
0
0.9
0.939
0.993
0
0
0
0.823
0.952
vorinostat trichostatin A
