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ISSN: 1079-9893 (print), 1532-4281 (electronic)
J Recept Signal Transduct Res, Early Online: 16
! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2015.1024852
RESEARCH ARTICLE
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Zhejiang Pharmaceutical College, Ningbo, China and 2State Key Laboratory on Lead Compound Research, WuXi AppTec Co. Ltd, Shanghai, China
Abstract
Keywords
TGF-b plays a critical role in the initiation and progression of fibrosis in various organ systems
such as kidney, heart, lung and liver. TGF-b and its receptors (ALK5 and TbR II) are able to
control the cellular growth and promote several biological responses. To date, many
pharmaceutical companies have employed virtual screening to identify potent inhibitors
against ALK5. Nevertheless, none of these studies had involved the in silico ADMET evaluation
and Raccoon filtering. In our experiment, all 57423 molecules were downloaded from TCM
database and were filtered and converted to PDBQT formats by Raccoon software. Then 24 189
structures were run through AutoDock Vina in PyRx 0.8, 164 molecules were selected and
further evaluated by ADMET Predictor 6.5, and 56 structures were selected and docked by Glide
6.2. Finally, the top 10 hits were identified as promising oral ALK5 inhibitors according to their
Glide scores. The Glide scores of the best two compounds, 40686 and 33534, were 10.75 and
10.30 kcal/mol, respectively. This research provides a set of combined and detailed virtual
screening protocol and is helpful for explaining the mechanism of receptorligand interactions.
Introduction
The transforming growth factor-b (TGF-b) is the most potent
immunosuppressive cytokine. TGF-b transduces signals
through two distinct serine/threonine kinase receptors, the
type I and type II receptors (1). Following the binding of
TGF-b to the constitutively active type II receptor, the type I
receptor, also called as activin receptor-like kinase 5 (ALK5),
is phosphorylated, which further phosphorylates SMAD2/
SMAD3 proteins. Phosphorylated SMAD2/SMAD3 proteins
form a heteromeric complex with SMAD4 to translocate into
the nuclei, where they regulate the target gene transcription
involved in cell differentiation, proliferation, apoptosis,
migration and extracellular matrix production (2). TGF-b
plays a critical role in the initiation and progression of fibrosis
in various organ systems such as kidney (3), heart (4), lung (5)
and liver (6). Deregulation of TGF-b signaling has been also
implicated in various human diseases including cancer (7),
pancreatic diseases (8) and hematological malignancies (9).
ALK5 inhibitors specifically inhibit SMAD pathway by
occupying the ATP binding site of ALK5 kinase domain,
which phosphorylates SMAD2 and SMAD3.
It has been reported that several small molecule ALK5
inhibitors such as SB505154 (10), GW6604 (11), SD208
(12) and LY2157299 (13) inhibited autophosphorylation of
History
Received 28 December 2014
Revised 17 February 2015
Accepted 19 February 2015
Published online 4 June 2015
A. Lin et al.
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Library design
Natural compounds have been successfully employed in the
clinical treatment. Most of the anticancer compounds are
isolated from plants, fungi and microorganisms (19). As a
major part of natural products, traditional Chinese medicine
(TCM) has a long and successful history. TCM
Database@Taiwan is currently the worlds largest noncommercial TCM database. Both CDX (2D) and MOL2
(3D) formats of each pure compound in the database are
available for download and virtual screening (20). All 57423
MOL2 files of small molecular ligands were downloaded
from http://tcm.cmu.edu.tw/zh-tw/.
Ligands preparation and filtering
MGL Tools is a software developed at the Molecular Graphics
Laboratory (MGL) of The Scripps Research Institute for
visualization and analysis of molecular structures. Raccoon is
a graphical interface for processing ligand libraries in
different formats (PDB, multi-structure MOL2 and
PDBQT), multiple receptor conformations and flexible residues, to generate all the files required to run an AutoDock
virtual screening (21). Raccoon v1.0 software was downloaded from http://autodock.scripps.edu/resources/raccoon/.
Defaults of Raccoon were set as the following: add
Gasteiger charges, both bones and hydrogen repairs, both
merge non-polar H and delete lone pairs, all yes for special
rotatable bonds and keep largest fragment for fragmented
structures. The Lipinski rules (except for the ClogP) of filter
presets were chosen from Raccoon menu. All MOL2 ligand
files were converted to PDBQT formats automatically.
Receptor preparation
The co-crystallized structure of human ALK5 with
) was
GW857175 (PDB code: 3GXL, resolution: 1.80 A
selected for docking study. The ligand GW857175 was
extracted from the complex. Using MGL 1.5.6, the crystal
structure of receptor was analyzed and preprocessed by
adding polar hydrogen atoms and deleting water molecules.
Virtual screening by AutoDock Vina
First docking calculations were carried out using AutoDock
Vina in PyRx 0.8, freely downloaded from http://pyrx.
sourceforge.net/downloads. AutoDock Vina, a new program
for molecular docking and virtual screening, was reported to
achieve an approximately two orders of magnitude speed-up
compared to Autodock 4, while also significantly improving
the accuracy of the binding mode predictions (22). AutoDock
Vina had been used for molecular docking recently (23). In
our study, we employed Lamarckian genetic algorithm and
DOI: 10.3109/10799893.2015.1024852
Rank
1
TCM ID
Structure
40686
OH
OH
33534
OH
Glide score
(kcal/mol)
Vina score
(kcal/mol)
Key HB by Glide
10.75
12.5
10.30
12.0
9.68
11.7
His283, Lys232
9.55
12.2
His283
9.45
13.1
Asp281
9.35
12.1
His283, Asp281
9.30
11.7
His283
9.29
11.5
Lys232
9.21
13.3
No
9.06
11.9
Lys232
HN
H
N
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O
O
34815
NH
O
OH O
OH O
O
tcm03_001709
HO
O
NH
42417
OH
29110
OH
O
OH
N
N
O
tcm03_006201
O
OH
OH
44538
OH
HO
HO
OH
O
O
10
37514
OH
46707
O
O
O
O
OH
O
O
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A. Lin et al.
40686
33534
ADMET_Risk
S + logP
S + Sw (mg/ml)
S + Peff (cm/s 104)
S + Vd (l/kg)
RuleOf5
CYP_1A2_Substr
MET_UGT1A1
TOX_hERG_Filter
TOX_BRM_Rat (mg/kg/day)
TOX_AlkPhos
TOX_GGT
TOX_LDH
TOX_MUT_Risk
1.02849
3.11362
0.04205
4.75336
3.17533
0
No
Yes
No
205.402
Normal
Normal
Normal
2
5
3.95241
3.34233
1.06138
0.40079
0
Yes
Yes
No
23.7054
Elevated
Normal
Elevated
2
Figure 2. 3D receptorligand interactions of compound 40686 and 33534: (a) 40686 and (b) 33534.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Nyu Medical Center on 06/07/15
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DOI: 10.3109/10799893.2015.1024852
Figure 3. 2D receptorligand interactions of compound 40686 and 33534: (a) 40686 and (b) 33534.
Conclusion
The approach utilized in this study was successful in finding
potential inhibitors against ALK5 from TCM database. Ten
hits with good oral pharmacokinetic profiles and computational scores were identified as promising ALK5 inhibitors.
They may be chosen as drug candidates or lead compounds of
ALK5 inhibitors, and evaluated for their biological activities
experimentally in the future. This research provides a set
Declaration of interest
The authors are grateful to the Visiting Engineer Project of Zhejiang
Province Educational Department of China for financial support. The
authors declare that they have no conflicts of interest to this work.
References
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A. Lin et al.