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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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Identification of ALK5 inhibitor via structure-based virtual screening


and ADMET prediction
Aibin Lin1, Zhongqi Cai1, Guoping Hu2, and Qiang Li2
1

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.

ADMET, ALK5, virtual screening, TGF-b

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

Address for correspondence: A. Lin, Zhejiang Pharmaceutical College,


Ningbo, China. E-mail: aibinlin@126.com

History
Received 28 December 2014
Revised 17 February 2015
Accepted 19 February 2015
Published online 4 June 2015

ALK5- and TGF-b-induced transcription of matrix genes. The


clinical candidate LY2157299 was proven to be safe and well
tolerated in cancer patients in a phase I clinical trial and
entered phase II clinical trials against hepatocellular carcinoma, glioma, glioblastoma and pancreatic cancer (14). The
loss of cell polarity, acquisition of motile properties and a
mesenchymal phenotype during epithelialmesenchymal transition (EMT) were considered crucial intrinsic changes of the
tumor cells (15). Preliminary results from a phase II clinical
trial of LY2157299 had shown improved clinical outcome and
also changes consistent with a reduction of EMT (16).
Currently, structure-based virtual screening is an equivalent computational process that can reduce the time and cost
of discovering new drugs. To date, many pharmaceutical
companies have employed virtual screening to identify potent
inhibitors against ALK5. GlaxoSmithKline screened their
internal compound collection for inhibitors of ALK5s ability
to phosphorylate SMAD3. This screen identified SKF104365, a cylco-fused imidazole compound that had a
2-pyridyl group (17). Lilly Research Laboratories discovered
several active compounds. Subsequent testing of these hits
against a constitutively active domain of ALK5 identified a
very potent inhibitor, LY364947 (18).
Nevertheless, none of these studies had involved the
in silico ADMET evaluation and Raccoon filtering. Here we
report the identification of ALK5 inhibitor via structure-based
virtual screening including in silico ADMET prediction and
Raccoon filtering. This research provides a set of combined

A. Lin et al.

and detailed virtual screening protocol and is helpful for


explaining the mechanism of receptorligand interactions.
Ten hits with good oral pharmacokinetic profiles and
computational scores are 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.

Materials and methods

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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.

J Recept Signal Transduct Res, Early Online: 16

250 000 maximum number of energy evaluations. The grid for


docking calculations was centered on GW857175 active
binding domain of ALK5, and the box size was set as
(X  Y  Z).
28.3  25.6  30.0 A
ADMET prediction
During virtual library screening in early drug discovery,
ADMET Predictor 6.5 can be used for estimating crucial
physicochemical and biological properties for large numbers
of candidate drug compounds (24). Its predictive models are
grouped into metabolism, physicochemical and biopharmaceutical, toxicity and simulation modules. ADMET predictor
has been consistently ranked as the most accurate, quick and
useful tool to predict physicochemical and biological properties of drug-like chemicals (25). An ADMET risk filter
enables accumulation of all the ADMET ranking criteria in
one column for easy sorting. For most drugs, the
ADMET_Risk scores are less than or equal to 5.
Precise docking by Glide
The filtered molecules were chosen for further Glide docking
calculations, which had been widely used for virtual screening
in recent years (26). The Glide 6.2 software can perform
ligand database screening and high-accuracy docking (27). A
grid size was generated around the co-crystallized
10 A
structure of 3GXL. During the grid generation, four hydrogen
bond constraints were set up including Asp281 (HBA),
His283 (HBA and HBD) and Lys232 (HBD). All the docking
calculations were performed by the Standard Precision mode,
RMSD cut-off value.
OPLS_2005 force field and 0.3 A

Results and discussion


The flowchart of virtual screening and ADMET prediction is
shown in Figure 1. Among all 57 423 MOL2 files, 24 189
ligand structures conformed to the Lipinski rules (except for

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

Figure 1. Flowchart of virtual screening and ADMET prediction.

Structure-based virtual screening and ADMET prediction

DOI: 10.3109/10799893.2015.1024852

Table 1. Structures and features of top 10 hits by virtual screening.

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

His283, Tyr249, Asp351, Ala230

10.30

12.0

His283, Lys232, Asp281

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

HB means hydrogen bond.

OH

O
O

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A. Lin et al.

J Recept Signal Transduct Res, Early Online: 16

the ClogP) filtering of Raccoon. Before docking, the ligand


GW857175 in 3GXL was extracted from the complex and
treated as the same way by Raccoon and AutoDock Vina. The
RMSD value between the obtained conformation and original
ligand is 1.5920 indicating that the parameters for docking
simulation are good in reproducing the X-ray crystal structure. After virtual screening by AutoDock Vina in PyRx, the
ligands were sorted by the binding energy. 164 compounds
with binding energy less than or equal to 11.4 kcal/mol were
chosen for the next step. Among these 164 compounds, 56
ligands passed the screening of ADMET Predictor 6.5
(ADMET_Risk value 5). Then these 56 ligands were
docked by Glide accurately. Finally, the top 10 hits were
identified as oral ALK5 inhibitors according to their Glide
scores (Table 1). The Glide scores of the best two compounds,
40686 and 33534, were 10.75 and 10.30 kcal/mol,
respectively.
The selected ADMET properties of the best two compounds are shown in Table 2. In this table, ADMET_Risk
means the overall evaluation of ADMET Predictor 6.5.
S + logP means octanol-water partition coefficient. S + Sw
means native water solubility. S + Peff means human jejunal
effective permeability. S + Vd means pharmacokinetic
volume of distribution in human. RuleOf5 means computational filter for oral absorption in human identical to the
Lipinskis Rule of Five. In terms of ADMET Predictor
descriptors and models, the RuleOf5 model rules can be

Table 2. Selected ADMET properties of the best two compounds.


ADMET properties

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

formulated in the following way: MlogP44.15 (excessive


lipophilicity), MWt4500 (large size), HBDH45 (too many
potential hydrogen bond donors), M_NO410 (too many
potential hydrogen bond acceptors). Most commercial
drugs violate no more than one of the corresponding
rules.CYP_1A2_Substr means qualitative assessment of a
molecule being the substrate of CYP 1A2 in human.
MET_UGT1A1 means qualitative model of a glucuronidation
by the UDP glucuronosyltransferase 1A1 enzyme. TOX_
hERG_Filter means qualitative estimation of the likelihood
of the hERG potassium channel inhibition in human.
TOX_BRM_Rat is defined as the oral dose of a compound
required to induce tumors in 50 percent of a rat population
after exposure over a standard lifetime. TOX_AlkPhos means
human liver adverse effect as the likelihood of causing
elevation in the levels of Alkaline Phosphatase enzyme.
TOX_GGT means human liver adverse effect as the likelihood of causing elevation in the levels of GGT enzyme.
TOX_LDH means human liver adverse effect as the likelihood of causing elevation in the levels of LDH enzyme.
TOX_MUT_Risk means ADMET Risk for mutagenicity in
S. typhimurium
The 3D receptorligand interactions of compound 40686
and 33534 are illustrated in Figure 2. The 2D interactions are
shown in Figure 3. Both the compounds show good binding
modes and docking scores. The hydrogen bonding interaction
of ALK5 inhibitor and His283 was reported to be important
for inhibitory activity in the previous study (28). The
compound 40686 forms four hydrogen bond with ALK5,
including Tyr249, Asp351, Ala230 and His283. The compound 33534 generates three hydrogen bond with ALK5,
including Lys232, Asp281 and His283.
A growing number of studies suggest that the hepatocellular carcinoma onset and progression are not just dependent
on the tumor cells or injured hepatocyte, but on other factors
as well. Using a more holistic view, it is becoming
increasingly clear that the cross-talk between tumor cells
and host stroma plays a key role in tumor growth. In such a
context, TGF-b orchestrates the homeostasis of microenvironment components and tumor cells by balancing the activity
of inflammatory cells or fibroblast with tumor cell growth or
progression. For example, in treatment-resistant viral hepatitis, TGF-b and IL-10 secretions are increased and associated
with increased T regulatory cells; these have a local

Figure 2. 3D receptorligand interactions of compound 40686 and 33534: (a) 40686 and (b) 33534.

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DOI: 10.3109/10799893.2015.1024852

Structure-based virtual screening and ADMET prediction

Figure 3. 2D receptorligand interactions of compound 40686 and 33534: (a) 40686 and (b) 33534.

immunosuppressive effect and consequently contribute to a


persistent viral infection. With continued inflammation of the
liver, reversible and irreversible remodeling are often
observed, including fibrosis and early cirrhosis. This remodeling is characterized by accumulation of extracellular matrix
(ECM) proteins. The rich ECM deposition is driven in large
part by activation of TGF-b; if not stopped, this leads to
cirrhosis and eventually to liver failure. The accumulation of
ECM proteins leading to fibrosis is still a reversible step. This
phase would be particularly important for therapeutic interventions aimed at blocking the progression of liver disease,
thus preventing hepatocellular carcinoma occurrence.
One of the main challenges for developing an effective
therapy against hepatocellular carcinoma is related to the
underlying cirrhosis, which is not only associated with a
reduced liver function, but also provides the support for tumor
cell growth. Because of this unique microenvironment, new
drugs should not only treat the tumor cells, but also prevent
new tumoral lesions. Hence, the ideal drug should have a lowtoxicity profile, antitumor activity and chemopreventive
activity.

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

of combined and detailed virtual screening protocol, and is


helpful for explaining the mechanism of receptorligand
interactions. It may be a practical virtual screening way for
peers to refer.

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.

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