A novel 2-aminobenzimidazole-based compound Jzu 17 exhibits anti-angiogenesis effects by targeting VEGFR-2 signalling

Article abstract of DOI:10.1111/bph.14813

Background and Purpose: Recent development in drug discovery have shown benzimidazole to be an important pharmacophore,. Benzimidazole derivatives exhibit broad-spectrum pharmacological properties including anti-microbial, anti-diabetic and anti-tumour activity. However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its underlying mechanisms remain incompletely understood. In this study, we aim to characterize the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17, in an effort to develop novel angiogenesis inhibitor. Experimental Approach: Effects of Jzu 17 on endothelial cell proliferation, migration, invasion, and activation of signalling molecules induced by VEGF-A, were analysed by immunoblotting, MTT, BrdU, migration, and invasion assays. We performed tube formation assay, aorta ring sprouting assay, matrigel plug assay, and a mouse model of metastasis to evaluate ex vivo and in vivo anti-angiogenic effects of Jzu 17. Key Results: Jzu 17 inhibited VEGF-A-induced cell proliferation, migration, invasion, and endothelial tube formation of HUVECs. Jzu 17 suppressed VEGF-A-induced microvessel sprouting ex vivo and attenuated VEGF-A- or tumour cell-induced neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition, Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signalling molecules in VEGF-A-stimulated HUVECs. Results from computer modelling further showed that Jzu 17 binds to VEGFR-2 with high affinity. Conclusions and Implications: Jzu 17 may inhibit endothelial remodelling and suppress angiogenesis through targeting VEGF-A-VEGFR-2 signalling. These results also suggest Jzu 17 as a potential lead compound and warrant the clinical development of similar agents in the treatment of cancer and angiogenesis-related diseases.

Full text of DOI:10.1111/bph.14813

Chung Chi Li (Orcid ID: 0000-0003-0656-976X)
Hsu Ming-Jen (Orcid ID: 0000-0001-9688-8494)
Anovel 2-aminobenzimidazole-based compound Jzu 17 exhibits anti- angiogenesis effects
via targeting VEGFR-2 signaling
Jin-Cherng Lien1,2#, Chi-Li Chung3,4#, Tur-Fu Huang , Tsung-Chia Chang , Kuan-Chung Chen ,
5
1
1
Ging-Yan Gao , Ming-Jen Hsu 6,7*, Shiu-Wen Huang 6, 8*
1
1
2
School of Pharmacy, China Medical University, Taichung, Taiwan; Department of Medical
3
Research, Hospital of China Medical University, Taichung, Taiwan; Division of Pulmonary
Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei,
4
Taiwan; Division of Thoracic Medicine, Department of Internal Medicine, School of Medicine
and School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei,
5
Taiwan; Graduate Institute of Pharmacology, College of Medicine, National Taiwan
6
University, Taipei, Taiwan; Department of Pharmacology, School of Medicine, College of
7
Medicine, Taipei Medical University, Taipei, Taiwan; Cell Physiology and Molecular Image
8
Research Center, Wan Fang Hospital, Taipei Medical University; Department of Medical
Research, Taipei Medical University Hospital, Taipei, Taiwan.
Running head: Jzu 17 antagonizes VEGFR-2 and suppresses angiogenesis
#These authors contributed equally to this work
*Correspondence should be addressed to:
Dr. Ming-Jen Hsu, Department of Pharmacology, School of Medicine, Taipei Medical
University, No.250, Wu-hsing St., Taipei 11031, Taiwan; Tel: +886-2-27361661 ext. 3198; E-
mail: aspirin@tmu.edu.tw
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/bph.14813
This article is protected by copyright. All rights reserved.

Dr. Shiu-Wen Huang, Department of Medical Research, Taipei Medical University Hospital,
Taipei, Taiwan. No.252, Wu-hsing St., Taipei 11031, Taiwan; Tel: +886-2-27372181 ext. 3783;
E-mail: shiuwen@tmu.edu.tw
(Shiu-Wen Huang will communicate with the editorial office and, if necessary, the production
office.)
Abbreviations
ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FBS, fetal bovine
serum; HUVEC, human umbilical vascular endothelial cell; MTT, 3-[4, 5-dimethylthiazol-2-
yl]-2, 5-diphenyltetrazolium bromide; RTK, receptor tyrosine kinase; VEGF, vascular
endothelial growth factor;
Enzymes: (Alexander et al., 2017a)
VEGFR-2
FAK
Src
Akt
ERK
Ligands(Alexander et al., 2017a)
VEGFs
Other protein targets: (Alexander et al., 2017b)
tubulins
Alexander SP, Fabbro D, Kelly E, Marrion NV, Peters JA, Faccenda E, et al. (2017a). THE CONCISE
GUIDE TO PHARMACOLOGY 2017/18: Enzymes. Br J Pharmacol 174 Suppl 1: S272-S359.
Alexander SP, Kelly E, Marrion NV, Peters JA, Faccenda E, Harding SD, et al. (2017b). THE
CONCISE GUIDE TO PHARMACOLOGY 2017/18: Overview. Br J Pharmacol 174 Suppl 1: S1-S16.
This article is protected by copyright. All rights reserved.

Abstract
Background and purpose: Recent development in drug discovery has highlighted the
important pharmacophore, benzimidazole. Benzimidazole derivatives exhibit broad-spectrum
pharmacological properties including anti-microbial, anti-diabetic and anti-tumor activity.
However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its
underlying mechanisms remain incompletely understood. In this study, we aim to characterize
the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17,
in an effort to develop novel angiogenesis inhibitor. Experimental approach: Jzu 17’s effects
on vascular endothelial growth factor (VEGF)-A-induced endothelial cell proliferation,
migration, invasion, as well as signaling molecules activation were analyzed by
immunoblotting, MTT, BrdU, migration and invasion assays. We performed tube formation
assay, aorta ring sprouting assay, matrigel plug assay and a metastasis mouse model to evaluate
Jzu 17’s ex vivo and in vivo anti-angiogenic effects. Key results: Jzu 17 inhibited VEGF-A-
induced cell proliferation, migration, invasion and endothelial tube formation of human
umbilical vascular endothelial cells (HUVECs). Jzu 17 suppressed VEGF-A-induced
microvessel sprouting ex vivo and attenuated VEGF-A- or tumor cells-induced
neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition,
Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signaling molecules in
VEGF-A-stimulated HUVECs. Results from computer modeling further showed that Jzu 17
This article is protected by copyright. All rights reserved.

binds to VEGFR-2 with high affinity. Conclusions and Implications: Jzu 17 may inhibit
endothelial remodeling and suppress angiogenesis through targeting VEGF-A-VEGFR-2
signaling. These results also support the role of Jzu 17 as a potential lead compound and
warrant the clinical development of agents in the treatment of cancer and angiogenesis-related
diseases
Keywords: angiogenesis; benzimidazole; human umbilical vascular endothelial cells
(HUVECs); vascular endothelial growth factor (VEGF)
What is already known about this subject
•
VEGF-A-VEGFR-2 signaling represents an attractive therapeutic target for intervention of
angiogenesis
•
Benzimidazole-base compounds may exhibit pharmacological activities capable of clinical
application
What this study adds
•
Jzu 17, a novel 2-aminobenzimidazole derivative, suppresses endothelial remodeling and
angiogenesis through targeting VEGF-A-VEGFR-2 signaling.
•
Jzu 17 suppresses VEGF-A- or tumor cells-induced neovascularization in in vivo models.
Clinical significance
•
Jzu 17 is a potential lead in developing novel anti-angiogenesis agents for future oncologic
therapy.
This article is protected by copyright. All rights reserved.

Introduction
Angiogenesis is a complicated process of forming new capillary blood vessels from pre-
existing vascular network. It primarily occurs in embryogenesis, wound healing and
reproduction (Ucuzian, Gassman, East & Greisler, 2010). However, it also participates in
various pathological states such as diabetic retinopathy, psoriasis, arthritis and cancer (Prager,
Poettler, Unseld & Zielinski, 2012). Angiogenesis is mandatory for tumor progression since
avascular tumors are restricted in their growth due to lacking blood supply. Angiogenesis is
also the most common route for metastatic spread of tumor cells, the main cause of cancer
morbidity and mortality (Bielenberg & Zetter, 2015). Therefore, modulating tumor
angiogenesis represents a rational and promising strategy for developing anti-cancer agents
(Weis & Cheresh, 2011). The balance between pro-angiogenic and anti-angiogenic stimuli
regulates angiogenesis. Numerous pro-angiogenic factors including vascular endothelial
growth factor (VEGF) (Ferrara, 2002), basic fibroblast growth factor (bFGF), epidermal
growth factor (EGF) (Javerzat, Auguste & Bikfalvi, 2002; Wang et al., 2012), as well as
angiopoietin (Holopainen et al., 2012) have been implicated in tumor angiogenesis. Of these,
VEGF-A, the member of the VEGF family, is the most critical pro-angiogenic factor
(Carmeliet, 2005). VEGF-A is expressed in various types of cancers and its overexpression
in tumors is associated with poor prognosis in cancer patients (Goel & Mercurio, 2013).
VEGF-A augments most steps of angiogenesis through binding to the receptor tyrosine
This article is protected by copyright. All rights reserved.

kinase VEGF receptor-2 (VEGFR-2, also known as Flk-1) on the endothelial cells
Yancopoulos, Davis, Gale, Rudge, Wiegand & Holash, 2000). VEGFR-2 is a receptor tyrosine
(
kinase (RTK), which undergoes ligand-induced dimerization and autophosphorylation upon
VEGF-A binding. Phosphorylated VEGFR-2 initiates downstream signaling cascades
including focal adhesion kinase (FAK), Src, Akt and extracellular signal-regulated kinase
(ERK) (Olsson, Dimberg, Kreuger & Claesson-Welsh, 2006). These signaling cascades
contribute to the regulation of endothelial cell survival, migration, proliferation and tube
formation (Brunton & Frame, 2008). Therefore, VEGF-A-VEGFR-2 signaling represents an
attractive therapeutic target for intervention of angiogenesis-associated diseases including
cancer (Ferrara & Kerbel, 2005). A variety of strategy to interfere with VEGF-A-VEGFR
signaling have been assessed in clinical trials (Jain, Duda, Clark & Loeffler, 2006). These
include soluble decoy receptors (VEGF-Trap) that sequester VEGF-A (Papadopoulos et al.,
2
012), antisense oligonucleotides that target VEGF (Levine et al., 2006) and neutralizing
monoclonal antibodies against VEGF-A or VEGFR (Ferrara, 2004). Inhibition of VEGFR-2
kinase activity using small molecule inhibitors represents another approach to suppress VEGF-
A signaling (Noble, Endicott & Johnson, 2004). To date, monoclonal antibodies bevacizumab
(Avastin®) (Summers, Cohen, Keegan & Pazdur, 2010) and ramucirumab (Cyramzar®)
(Calvetti et al., 2015) and small molecule inhibitors such as sorafenib (Nexavar®), sunitinib
(Sutent®) or pazopanib (Votrient®) have been already approved by the U.S. Food and Drug
This article is protected by copyright. All rights reserved.

Administration (FDA) or the European Medicines Agency (EMEA) for the treatment of certain
types of cancer (Meadows & Hurwitz, 2012).
Benzimidazole, a key pharmacophore with diverse biological and pharmacological properties,
has attracted considerable attention in the field of drug discovery. Therefore, synthesis of novel
benzimidazole derivatives remains a major focus for synthetic chemistry communities. Most
studies have focused on the synthesis of 2-arylbenzimidazoles and its biological activities. In
contrast, there are few studies reporting the synthesis of 2-aminobenzimidazole derivatives. A
variety of benzimidazole-based compounds have shown their potential use as antidiabetic, anti-
ulcer, anti-convulsant (Shingalapur, Hosamani, Keri & Hugar, 2010), anti-infectious (Ozkay,
Tunali, Karaca & Isikdag, 2011) or anti-inflammatory (Bukhari et al., 2016) agents. Recent
studies demonstrated that benzimidazole derivatives also exhibit anti-angiogenesis (Huang,
Lien, Kuo & Huang, 2012) and anti-tumor (Chu et al., 2015) properties. Its underlying
mechanisms, however, remain incompletely understood. These observations suggest that
additional benzimidazole-base compounds may exhibit pharmacological activities capable of
clinical application. Given their potential as lead compound for developing novel anti-
angiogenesis agents, we synthesized a series of small molecules with the core structure of 2-
aminobenzimidazole, the Jzu compounds (Supplement Figure. S1A) and evaluated their anti-
angiogenesis properties. Of these compounds, Jzu 17 was selected for its potent inhibitory
activities to reduce cell viability in VEGF-A-stimulated human umbilical endothelial cells
This article is protected by copyright. All rights reserved.

(HUVECs) (Supplement Fig. S1B). The aim of this study was to characterize the underlying
mechanisms by which Jzu 17 suppresses angiogenesis in endothelial cells.
This article is protected by copyright. All rights reserved.

Materials and methods
Reagents
3
- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) was from Sigma-
Aldrich (St Louis, MO, U.S.A). Medium 199 (M199), DMEM, TrypLE™, Fetal bovine serum
FBS) and all cell culture reagents were purchased from Invitrogen (Carlsbad, CA, U.S.A.).
(
Recombinant VEGF-A was purchased from PeproTech (Rocky Hill, NJ, USA). Antibodies
against VEGFR2 (Cell Signaling Technology Cat# 2479, RRID:AB_2212507), VEGFR2
phosphorylated at tyrosine 1175 (Y1175) (Cell Signaling Technology Cat# 3770,
RRID:AB_1642326), FAK (Cell Signaling Technology Cat# 3285, RRID:AB_2269034), FAK
phosphorylated at tyrosine 397 (Y397) (Cell Signaling Technology Cat# 3283,
RRID:AB_2173659), Src (Cell Signaling Technology Cat# 2108, RRID:AB_331137), Src
phosphorylated at tyrosine 416 (Y416) (Cell Signaling Technology Cat# 6943,
RRID:AB_10013641), Akt (Cell Signaling Technology Cat# 9272, RRID:AB_329827), Akt
phosphorylated at serine 473 (S473) (Cell Signaling Technology Cat# 9271,
RRID:AB_329825), ERK1/2 (Cell Signaling Technology Cat# 4695, RRID:AB_390779) and
ERK1/2 phosphorylated at threonine 202/tyrosine 204 (T202/Y204) (Cell Signaling
Technology Cat# 4370, RRID:AB_2315112) were purchased from Cell Signaling (Danvers,
MA, USA). Anti-mouse and anti-rabbit IgG conjugated horseradish peroxidase antibodies, as
well as antibody against α-tubulin (GeneTex Cat# GTX628802, RRID:AB_2716636) were
This article is protected by copyright. All rights reserved.

obtained from GeneTex Inc (Irvine, CA, U.S.A.). Cell Proliferation ELISA, BrdU assay kit
was from Roche (Indianapolis, IN, USA). All materials for immunoblotting were from Bio-
Rad (Hercules, CA, U.S.A.). The enhanced chemiluminescence detection kit was from
Millipore (Billerica, MA, U.S.A.). All other chemicals were obtained from Sigma-Aldrich (St
Louis, MO, U.S.A.).
Synthesis of Jzu 17
2
aminobenzimidazole-based Jzu compounds were synthesized as described in the Supplement
Information.
Cell culture
MDA-MB-231 (BCRC Cat# 60425, RRID:CVCL_0062) breast cancer and B16F10 (BCRC
Cat# 60031, RRID:CVCL_0159) melanoma cell lines and Human umbilical vascular
endothelial cells (HUVECs) were obtained from the Bioresource Collection and Research
Center (Hsinchu, Taiwan). HUVECs were maintained in M199 medium containing vascular
endothelial cell growth supplement (ECGS) (Millipore, Billerica, MA, U.S.A.), 10% FBS, 5 U
-1
-1
-1
ml heparin, 20 mM HEPES, 100 U ml of penicillin G, and 100 μg ml streptomycin in a
humidified 37 °C incubator. MDA-MB-231 and B16F10 cells were maintained in DMEM
-1
-1
medium containing 10 % FCS, 100 μg ml streptomycin and 100 U ml of penicillin G in a
This article is protected by copyright. All rights reserved.

humidified 37 °C incubator.
MTT assay
Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay as described previously (Huang, Lien, Kuo & Huang, 2012).
Lactate dehydrogenase (LDH) release assay
The CytoTox96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI, U.S.A.) was
employed to measure LDH leakage to quantify cytotoxicity as described previously (Huang,
Lien, Kuo & Huang, 2014).
Cell proliferation assay
4
HUVECs (210 per well) seeded in 96-well tissue culture plates were incubated for 24 h. Cells
were starved in M199 medium containing 2 % FBS in the absence of endothelial cell growth
supplements for another 18 h. After starvation, cells were treated with Jzu17 at different
-1
concentrations for 30 min, followed by the stimulation with VEGF-A (25 ng ml ) for another
2
4 h. Cell proliferation was determined using a BrdU Cell Proliferation kit (Millipore, Billerica,
MA, U.S.A.) based on the colorimetric detection of the incorporation of BrdU, following the
manufacturer’s instructions.
This article is protected by copyright. All rights reserved.

Cell migration assay
HUVECs were allowed to grow to confluence in 12-well tissue culture plates coated with 0.1
%
gelatin (Sigma-Aldrich St Louis, MO, U.S.A.). Cells were starved with M199 medium
,
containing 2% FBS for 18 h. After starvation, monolayer HUVECs were wounded by
scratching with pipette tips. Cells were washed with PBS, followed by the treatment with Jzu17
at different concentrations in the presence or absence of 25 ng/mL VEGF-A for another 24 h.
After treatment, cells were fixed with cold 4 % paraformaldehyde. Cells were stained with 0.5
%
toluidine blue. Microscope images were taken at 40× magnification by an OLYMPUS
Biological Microscope digital camera (Yuan Li Instrument Co., Taipei, Taiwan). The rate of
cell migration was determined by comparing the sizes of scratch area as a percentage of the
values obtained with their respective controls at the beginning of experiments (time 0) using
an Image J program (http://rsbweb.nih.gov/ij/index.html) (ImageJ, RRID:SCR_003070).
Transwell invasion assay
The cell invasion assays were performed using transwell plate (Corning, NY, U.S.A.). The
bottom face of the filter in the transwell plate was coated with 0.2 % gelatin. The bottom
chambers were filled with M199 medium containing 2 % FBS in the presence of VEGF-A (25
-1
4
ng ml ). HUVECs (10 cells per well) in 200 µLM199 medium (with 2 % FBS) with or without
Jzu17 at different concentrations were seeded in the top chambers. Cells were allowed to invade
This article is protected by copyright. All rights reserved.

for 18 h. Non-invaded cells (on the top side of filter) were scraped with a cotton swab, and
invaded cells were fixed and stained with 0.5% toluidine blue in 4% paraformaldehyde. The
cells were photographed under an inverted contrast phase light microscope (×40, Nikon, Japan).
Stained HUVECs that invaded through the membrane were quantified by counting in three
random fields.
Matrigel tube formation assay
The tube formation assay was performed as described previously (Huang, Lien, Kuo & Huang,
2
014). Matrigel, a basement membrane matrix (Becton Dickinson, Mountain View, CA, USA),
was polymerized at 37 °C for 30 min. HUVECs suspended in M199 medium containing 2 %
-1
FBS in the presence or absence of VEGF-A (25 ng ml ) were seeded onto the Matrigel. They
were then treated with vehicle or Jzu17 at indicated concentrations. After 18 h, cells were
photographed using phase-contrast microscopy. The cells were photographed under an inverted
contrast phase light microscope (×40, Nikon, Japan).
Immunoblotting
Cells were harvested in an extraction buffer containing 140 mM NaCl, 10 mM Tris (pH 7.0),
0
.5 % NP-40, 0.2 mM leupeptin, 0.05 mM pepstatin A and 2 mM PMSF. Equal amounts of
protein samples were subjected to SDS-PAGE and transferred onto a NC membrane (Pall
This article is protected by copyright. All rights reserved.

Corporation, Washington, NY, U.S.A.). After blocking in a 5 % non-fat milk-containing
blocking buffer for 1 h, proteins were recognized using specific primary antibodies followed
by horseradish peroxidase-conjugated secondary antibodies. To detect immunoreactivity,
enhanced chemiluminescence was employed as per the manufacturer’s instructions.
Quantitative data was obtained using a computing densitometer with a scientific imaging
system (Biospectrum AC System, UVP).
Randomization and blinding
In each experiment, the same cell was used to evaluate the effects of Jzu17 versus the related
control. Formal randomization was therefore not employed. Mice used in this study were
randomly allocated to cages by vivarium staff and randomized into vehicle-treated or Jzu17-
treated group before the treatment. For blinding, we have different people conducting
experiments (operator) and analyzing data (analyst).
Ethic statement
Animal studies are reported in accordance with the ARRIVE guidelines (Kilkenny, Browne,
Cuthill, Emerson & Altmans, 2010; McGrath & Lilley, 2015). The Taipei Medical University
Laboratory Animal Care and Use Committee approved the protocols described below (Permit
Number: LAC-2017-0241). We performed this study in strict accordance with the
This article is protected by copyright. All rights reserved.

recommendations in the Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health (NIH publication No 85-23, revised 1996).
Aortic ring sprouting assay
Aorta ring sprouting assay was performed as described previously (Huang, Lien, Kuo & Huang,
2
014). 8- to 10-week old male Sprague-Dawley rats were obtained from National Laboratory
Animal Center (Taipei, Taiwan) and used for the experiment presented in Fig. 2C. Rats were
scarified using CO asphyxiation to dissected aortic arch. After removing the surrounding
2
fibro-adipose tissues and thoroughly rinsing with M199 culture medium, the aorta was cut into
about 1 mm ring segments. The aortic rings were immersed in Matrigel in the wells of 48-well
-1
plate. VEGF-A(25 ng ml ) with or without Jzu17 was added to the wells. The aortic rings were
cultured in 37 °C with 5% CO and the cultured medium was changed every 3 days. Growing
2
sprouts of endothelial cells were observed and photographed on day 7. The images were
photographed under microscope, and sprouting area was determined on the computer-digitized
images with Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) (Image-
Pro Plus, RRID:SCR_007369). An observer who was unaware to the treatment group assessed
the sprouting area. The study conforms to the Guide for the Care and Use of Laboratory
Animals (NIH publication No 85-23, revised 1996) and was approved by the Taipei Medical
University Animal Care and Use Committee.
This article is protected by copyright. All rights reserved.

VEGF-A- or tumor cells-induced angiogenesis Matrigel plug assay
The angiogenesis matrigel plug assay with nudenu/nu mice as described previously (Huang, Lien,
Kuo & Huang, 2012) was used to determine Jzu 17’s in vivo anti-angiogenic effect. 3- to 5-
week old male nudenu/nu mice with body weight about 20 g were obtained from BioLasco
(Taipei, Taiwan) and used for the experiment presented in Fig. 3. All the mice were housed (3
mice per cage) in clean specific pathogen free (SPF) rooms (standard 12-hour light/12-hour
dark cycle at 22°C) in LaboratoryAnimal Center of Taipei Medical University, and maintained
on standard chow and autoclaved water. The cage floor was covered with Bed
O’Cobs animal bedding (The Andersons, Maumee, OH, USA). All mice were randomly
allocated to individually ventilated cage (IVC) by vivarium staff, upon transfer from
BioLASCO into the animal housing room. All mice purchased from BioLASCO were
acclimatized in the animal housing room for 7 days prior to starting experiments. Mice
were anesthetized with intraperitoneal pentobarbital (50 mg/kg). Once anesthesia was induced,
-1
an aliquot (500 μl) of Matrigel containing VEGF-A (200 ng ml ) with heparin (20 U) was
injected subcutaneously into the right flank of each mouse (VEGF-A-induced angiogenesis
model). In the other set of experiments, MDA-MB-231 cells were harvested and re-suspended
6
in PBS. Cells (5×10 cells) in a volume of 150 μl in the presence of heparin (20 U) were mixed
This article is protected by copyright. All rights reserved.

with Matrigel (150 μl) and injected subcutaneously into the right flank of each mouse (Tumor
cells-induced angiogenesis model). After implantation, animals were randomized to either the
vehicle-treated control group or the treatment group, which received Jzu17 at indicated
concentrations. The treatment was administrated intraperitoneally once daily for 7 (VEGF-A-
induced angiogenesis model) or 10 (Tumor cells-induced angiogenesis model) days. At the end
of treatment, animals were sacrificed using CO asphyxiation, Matrigel plugs were removed
2
and the surrounding tissues trimmed. Hemoglobin levels of the Matrigel plugs were evaluated
with Drabkin’s reagent kit (Sigma-Aldrich) according to the manufacturer’s instructions. The
concentration of hemoglobin was calculated based on a set of hemoglobin standards. The study
conforms to the Guide for the Care and Use of Laboratory Animals (NIH publication No 85-
2
3, revised 1996) and was approved by the Taipei Medical University Animal Care and Use
Committee.
Melanoma lung metastatic mouse model
The melanoma lung metastatic model with C57BL/6 mice as described previously (Lin, Chen,
Chou & Wang, 2011) was used to determine Jzu 17’s anti-metastatic effect. 6- to 8-week old
male C57BL/6 mice with body weight about 35 g were obtained from National Laboratory
Animal Center (Taipei, Taiwan) and used for the experiment presented in Fig. 4. All the mice
were housed (5 mice per conventional cage) in clean conventional animal housing rooms
This article is protected by copyright. All rights reserved.

(standard 12-hour light/12-hour dark cycle at 22°C) in Laboratory Animal Center of Taipei
Medical University and maintained on standard chow and autoclaved water. The cage floor was
covered with Bed O’Cobs animal bedding (The Andersons, Maumee, OH, USA). All mice
were randomly allocated to conventional cage by vivarium staff, upon transfer from National
Laboratory Animal Center into the animal housing room. Mice were anesthetized with
intraperitoneal pentobarbital (50 mg/kg). Once anesthesia was induced, mice were inoculated
6
at the tail vein with B16F10 melanoma cells (10 cells per mouse) suspended in 150 μl of sterile
saline. Mice were randomized to either the vehicle-treated control group or the treatment group,
-1
-1
which received Jzu 17 (10 mg kg day ). The treatment was administrated intraperitoneally
once daily for 18 days. At the end of treatment, mice were scarified using CO asphyxiation
2
and dissected. Lung tissue was lungs collected and fixed in 10% formalin. Metastatic melanoma
nodules were scored by counting. Paraffin wax-embedded sections (5 μm) of lung tissue were
stained with hematoxylin and eosin (H&E) staining and photographed under microscope to
assess metastatic nodules area. Computer-digitized images with Image-Pro Plus software
(Media Cybernetics, Inc., Rockville, MD, USA). The study conforms to the Guide for the Care
and Use of Laboratory Animals (NIH publication No 85-23, revised 1996) and was approved
by the Taipei Medical University Animal Care and Use Committee.
VEGF-A binding analysis
This article is protected by copyright. All rights reserved.

We performed VEGF-A binding analysis using human VEGF-A biotinylated Fluorokine Kit
(
R&D Systems). Briefly, HUVECs were deattached using Accutase™ cell detachment solution
BD Biosciences). Cells were washed with PBS, and incubated with biotinylated recombinant
(
VEGF-A or negative control reagent (biotinylated soybean trypsin inhibitor) in the absence or
presence of Jzu17 for 1 h. Cells were incubated subsequently with avidin-conjugated
fluorescein for another 30 min. Cells were washed with PBS and fluorescence of labelled cells
was determined using flow cytometer (FACScan; BD Biosciences, San Jose, CA, USA) and
analysed with CellQuest software (BD Biosciences) (BD CellQuest Pro, RRID:SCR_014489).
Molecular Docking Simulation
For docking simulation, the X-ray crystallography structure for VEGF-A (PDB ID: 3V2A
(Brozzo et al., 2012) and VEGFR-2 (PDB ID: 5EW3 (Bold et al., 2016) were downloaded from
RCSB Protein Data Bank. The preparation of protein was performed by Prepare Protein module
in Discovery Studio 2.5 (DS2.5) to remove crystal water in crystallography structure, insert
missing atoms in incomplete residues, protonate the structure of both protein with Chemistry
at HARvard Macromolecular Mechanics (CHARMM) force field (Brooks, Bruccoleri, Olafson,
States, Swaminathan & Karplus, 1983), and optimize side-chain conformation for residues with
inserted atoms. For VEGF-A, there are two small receptor cavities between VEGF-A and
VEGFR-2. We combine these two small receptor cavities to define the binding site with the
This article is protected by copyright. All rights reserved.

volume of 341.375 Å for VEGF-A (Supplement Fig. S3A). The binding site of VEGFR-2 was
defined as volume of the co-crystallized compound in the X-ray crystallography (Supplement
Fig. S3B). The docking poses of compound was performed by Ligand Fit module in DS2.5
using a shape filter and Monte-Carlo ligand conformation generation and optionally minimized
with CHARMM force field (Brooks, Bruccoleri, Olafson, States, Swaminathan & Karplus,
1
983).
Data and Statistical analysis
The data and statistical analysis in this study comply with the recommendations on
experimental design and analysis in pharmacology (Curtis et al., 2018). The exact group size
(n) is equal for each experiment in this study. Results are expressed as mean ± standard error
of mean (SEM) (n ≥ 5), where 'n' refers to independent values, and not replicates. To control
for unwanted sources of variation and to reveal relevant trends, normalization was performed
to compare the differences after the treatment. For MTT or BrdU assay, the viability or BrdU
incorporation was expressed as fold changes over that of the vehicle-treated cells, whose
expression was set to 1 (100 %). For LDH assay, LDH release from lysis buffer-treated cells
(TL group) was considered to be 100 % and LDH release from the VEGF-A-treated cells in the
presence or absence of Jzu17 was expressed as a percentage of the control. For immunoblotting,
the levels of protein modification (e.g., VEGFR-2 or FAK phosphorylation) were normalized
This article is protected by copyright. All rights reserved.

to that of un-modified protein (e.g., VEGFR-2 or FAK). The status of protein modification was
expressed as fold changes over that of the vehicle-treated cells, whose expression was set to 1
(100 %). The SEM was normalized appropriately. The status of protein modification was
expressed by normalization that generates control values with no variance (SEM = 0) to reduce
the effect of variation from different exposure of blotting, and such data are subjected to non-
parametric statistical analysis. The group data subjected to statistical analysis have a minimum
of n = 5 independent samples per group in this study. Statistical analysis was performed using
SigmaPlot 10 (Build 10.0.0.54; Systat Software, San Jose, CA, U.S.A.) (SigmaPlot,
RRID:SCR_003210). Statistical comparisons between two groups were evaluated by the
unpaired Student's t-test for parametric analysis or Mann-Whitney test for non-parametric
analysis. Statistical comparisons among more than two groups were evaluated by one-way
analysis of variance (ANOVA) with Tukey’s post-hoc test for parametric analysis or Kruskal-
Wallis test followed by Dunn's multiple comparison for non-parametric analysis. Post hoc tests
were run only if F achieved P < 0.05 and there was no significant inhomogeneity. A P value
smaller than 0.05 was defined as statistically significant.
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
This article is protected by copyright. All rights reserved.

Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a; Alexander et al.,
2
017b).
This article is protected by copyright. All rights reserved.

Results
A novel 2-aminobenzimidazole derivative Jzu 17 inhibited VEGF-A-induced HUVEC
proliferation, migration and invasion
Vascular endothelial cell proliferation, migration, invasion, and tube formation are critical steps
in angiogenesis (Solinas et al., 2012). To investigate whether the novel 2-aminobenzimidazole-
based compounds with benzyl, methylbenzyl, fluorobenzyl, or chlorobenzyl structure, the Jzu
compounds (Jzu 01~24) (Supplement Fig. S1A), exhibit anti-angiogenic activities, we evaluate
their effects on cell proliferation in VEGF-A-stimulated HUVECs. HUVECs were
synchronized by starvation medium (2 % FBS-containing M199 medium) for 18 h. After
-1
starvation, cells were stimulated by VEGF-A (25 ng ml ) in the absence or presence of the Jzu
compounds (1, 3 or 10 μM) for another 24 h. As shown in Supplement Fig. S1B, Jzu 01 (2-
aminobenzimidazole, the core structure) and its derivatives (Jzu 04 ~ Jzu 13, Jzu 17, Jzu 20 ~
Jzu 22 and Jzu 24) significantly reduced cell viability in VEGF-A-stimulated HUVECs as
determined by MTT assay. Among these 16 effective compounds. Jzu 17 exhibited the most
prominent effects in reducing cell viability in VEGF-A-stimulated HUVECs (Supplement Fig.
S1B). Therefore, we sought to explore the underlying mechanisms by which Jzu 17 suppresses
VEGF-A-induced angiogenesis in the following experiments. Fig. 1A illustrates the chemical
structure of the novel 2-aminobenzimidazole-based compound, Jzu 17. Results from BrdU
labeling analysis demonstrated that Jzu 17 concentration-dependently suppressed VEGF-A-
This article is protected by copyright. All rights reserved.

induced HUVEC proliferation (Fig. 1B). We explored whether Jzu 17 alters cell motility in
HUVECs after VEGF-A exposure. As shown in Fig. 1C, Jzu 17 significantly inhibited VEGF-
A-induced HUVEC migration as determined by wound-healing migration assay. We also used
transwell invasion assay to examine Jzu 17’s effects on VEGF-A-induced cell invasion. As
shown in Fig. 1D, Jzu 17 at 0.3 to 10 μM significantly reduced the number of invade cell
through the gelatin-coated transwell membrane barrier, using VEGF-A as the chemoattractant.
Jzu 17 inhibited VEGF-A-induced tube formation of HUVECs and suppressed microvessel
sprouting ex vivo
We next examined Jzu 17’s effects on tubular formation of endothelial cells, a key step in
-1
angiogenesis. HUVECs seeded on top of matrigel were stimulated by VEGF-A (25 ng ml )
with or without Jzu 17 (0.3-10 μM). As shown in Fig. 2A, HUVECs became elongated and
formed capillary-like structure after 24 h exposure to VEGF-A. However, Jzu 17 significantly
inhibited the formation of capillary-like network in a concentration-dependent manner. Results
from the LDH assay further showed that Jzu 17 at 0.3 to 10 μM did not increase LDH release
in HUVECs exposed to VEGF-A for 24 h (Fig. 2B). Together these results indicate that Jzu 17
exhibits anti-angiogenesis property through suppression of cell proliferation, migration,
invasion, as well as tube formation of endothelial cells. It also suggests that Jzu 17’s inhibitory
actions in VEGF-A-stimulated HUVECs may not attribute to its cytotoxic effects. Moreover,
This article is protected by copyright. All rights reserved.

we also performed an ex vivo rat aortic ring sprouting assay to examine the Jzu 17’s anti-
-1
angiogenesis effects. As shown in Fig. 2C, VEGF-A (25 ng ml ) induced significant increases
in sprouting microvessels to form complex network around the aortic rings. However,
treatment with Jzu 17 (0.3-10 μM) significantly reduced VEGF-A’s effects on microvessel
sprouting (Fig. 2C). It indicates that Jzu 17 is effective in suppressing VEGF-A-induced
angiogenesis ex vivo.
Jzu 17 suppressed VEGF-A- or tumor cells-induced angiogenesis in vivo
We employed murine Matrigel plug models to explore whether Jzu 17 exhibits anti-
angiogenesis effects in vivo. As shown in Fig. 3A, VEGF-A significantly induced microvessel
formation in the Matrigel plug. The pale color of the plugs removed from the Jzu 17-treated
mice indicates that VEGF-A induced less neovascularization after 7-day treatment with Jzu 17
-1
-1
(
2.5 or 5 mg kg day ) (Fig. 3A, upper panel). We also examine the hemoglobin content of the
plugs to quantify angiogenesis level. A significant reduction in neovascularization was
observed in plugs from Jzu 17-treated mice when compared with those from vehicle-treated
mice (Fig. 3A, bottom panel). Moreover, we used a tumor cells-induced angiogenesis model to
investigate whether Jzu 17 suppresses tumor angiogenesis. MDA-MB-231 breast cancer cells
mixed with Matrigel were injected into the flanks of mice. After implantation for 10 days,
Matrigel plugs were harvested. As shown in Fig. 3B, MDA-MB-231 cells markedly increased
This article is protected by copyright. All rights reserved.

neovascularization in the plug while Jzu 17 reduced this effect (Fig. 3B, upper panel). The
angiogenesis level was also quantified. As compared with the vehicle-treated control group,
Jzu 17 significantly reduced tumor cells-elicited angiogenesis in vivo (Fig. 3B, bottom panel).
We further examined whether Jzu 17 affects MDA-MB-231 cell viability. As shown in
Supplement Fig. S2, Jzu 17 slightly decreased MDA-MB-231 cell viability. Jzu 17 at 10 μM
caused about 10 % reduction in cell viability in MDA-MB-231 cells (Supplement Fig. S2).
These results suggest that Jzu 17 inhibits MDA-MB231 tumor cells-induced angiogenesis by
targeting the proliferating endothelial cells, rather than tumor cells. It also indicates that
systemic administration with Jzu 17 is capable of suppressing angiogenesis in vivo.
Jzu 17 reduced lung metastasis of B16F10 melanoma cells
We exploited the well-described lung metastasis assay to evaluate the anti-metastatic effects
of Jzu 17 in vivo. C57BL/6 mice were tail-vein inoculated with B16F10 mouse melanoma cells
exhibiting high metastasis activity from small inoculums. After inoculation for 18 days, lungs
from the vehicle-treated mice exhibited multiple metastasis nodules of different sizes (Fig.
4
A, upper panel). In contrast, the lungs from mice receiving daily intraperitoneal injections
-1
-1
of Jzu 17 (10 mg kg day ) displayed fewer and smaller melanoma nodules (Fig. 4A, upper
panel). We also quantified the level of lung metastasis by determining the number of nodules.
A significant reduction in the number of lung metastatic nodules was shown in lungs from Jzu
This article is protected by copyright. All rights reserved.

1
7-treated mice when compared with those from vehicle-treated control mice (Fig. 4A, bottom
panel). The lung tissues were also paraffin-embedded for histological analysis with H&E
staining (Fig. 4B, upper panel). Jzu 17 remarkably reduced lung metastatic nodular areas as
compared to the vehicle-treated control group (Fig. 4B, bottom panel). In addition,
-1
-1
administration with Jzu 17 at 10 mg kg day had no significant effects on mouse body weight
as compared to the control group within 18 days (Fig. 4C). These findings suggest that Jzu 17
also exhibits inhibitory effects on tumor metastasis in vivo.
Jzu 17 inhibited VEGFR-2 signaling in VEGF-A-stimulated HUVECs
VEGF-A signaling via VEGFR-2 plays a prominent role in regulating angiogenesis (Shibuya,
2
011). Upon VEGF-A exposure, VEGFR-2 becomes phosphorylated, resulting in the
activation of many downstream signaling molecules such as focal adhesion kinase (FAK)
Farhan, Azad, Touret & Murray, 2017), Src (Sun et al., 2012), Akt and ERK1/2 (Huang, Lien,
(
Kuo & Huang, 2014). VEGF-A triggers auto-phosphorylation of VEGFR-2 on several tyrosine
residues. Of these, VEGFR-2 Tyr 1175 phosphorylation is believed to be a key determinant of
angiogenic signaling in VEGF-A-stimulated HUVECs (Olsson, Dimberg, Kreuger &
Claesson-Welsh, 2006). We therefore sought to determine whether Jzu 17 affects VEGFR-2
Tyr 1175 phosphorylation in HUVECs after VEGF-A exposure. As shown in Fig. 5, Jzu 17
significantly inhibited VEGF-A-induced VEGFR-2 Tyr 1175 phosphorylation (Fig. 5B).
This article is protected by copyright. All rights reserved.

Moreover, Jzu 17 also reduced the phosphorylation of FAK (Fig. 5C), Src (Fig. 5D), Akt (Fig.
E) and ERK1/2 (Fig. 5F) in HUVECs exposed to VEGF-A. These results suggest that Jzu 17
5
exhibits its anti-angiogenesis activities through suppressing VEGF-A-VEGFR-2 signaling.
Jzu 17, sunitinib or sorafenib inhibited VEGFR-2 tyrosine kinase activity in vitro
We next compared the inhibitory effects of Jzu 17, sunitinib and sorafenib on VEGFR-2
phosphorylation in HUVECs exposed to VEGF-A. As shown in Supplement Fig. S3, Jzu 17
(1-10 μM) suppressed VEGF-A-induced VEGFR-2 phosphorylation with similar potency to
sunitinib (Supplement Fig. S3A) or sorafenib (Supplement Fig. S3B). The potency of
angiogenesis inhibitors might vary in cell-based systems and in cell-free systems (Kankanala,
Latham, Johnson, Homer-Vanniasinkam, Fishwick & Ponnambalam, 2012; Latham et al.,
2
012). We thus used an in vitro kinase assay to examine the effects of Jzu 17, sunitinib and
sorafenib on the intrinsic tyrosine kinase activity of VEGFR-2. Sunitinib (Latham et al., 2012)
and sorafenib (Pattarozzi et al., 2017) are multi-targeted RTK inhibitors, which also suppress
the kinase activity of bFGFR. The effects of these compounds on bFGFR1 kinase activity were
also determined. As shown in Supplement Fig. S4A, sunitinib and sorafenib at concentrations
ranging from 0.1 to 3 μM markedly inhibited purified recombinant VEGFR-2 tyrosine kinase
activity. Both drugs at 1 μM elicited approximately 90 % inhibition of VEGFR-2 kinase
activity in vitro. To our surprisingly, Jzu 17 exhibited only about 30 % inhibition of kinase
This article is protected by copyright. All rights reserved.

activity at a high concentration of 10 μM (Supplement Fig. S4A). The suppressive effects of
these compounds on bFGFR1 kinase activity were less pronounced. Sunitinib and sorafenib at
a concentration of 1 μM displayed about 50 % inhibition of bFGFR1 kinase activity. However,
Jzu 17 is a much weaker inhibitor of this receptor (Supplement Fig. S4B).
Computational modeling of the interactions between Jzu 17 and VEGF-A or VEGFR-2
We next examined whether Jzu 17 affects VEGF-A binding to VEGFR in HUVECs. As shown
in Fig. 6A, Jzu 17 suppressed VEGF-A binding to VEGFR in HUVECs as determined by flow-
cytometry. It is likely that Jzu17 may antagonize VEGF-A-VEGFR-2 signaling through
binding to VEGFR-2 or VEGF-A. We thus conducted molecular docking simulations to
analyze the possible interactions between Jzu 17 and VEGF-A or VEGFR-2. The binding site
of VEGF-A was defined by two small receptor cavities between VEGF-A and VEGFR-2
(Supplement Fig. S5A). In addition, the binding site of VEGFR-2 was defined as the volume
of the co-crystallized compound (Supplement Fig. S5B). Table 1 shows the docking score and
scoring results (Table 1). The docking pose of Jzu 17 has π-cation interactions with Lys868 and
His1026 residues of VEGFR-2 (Fig. 6B). It forms a stable docking pose with VEGFR-2, which
is similar to the docking pose of the co-crystallized compound in the X-ray crystallography.
Fig 6C further illustrated the 2D ligand-protein interaction diagram between Jzu 17 and
VEGFR-2. In contrast, there are three different possible docking poses of Jzu 17 for VEGF-A
This article is protected by copyright. All rights reserved.

(Supplement Fig. S6). The poses 1 and 3 have π-cation interactions with Lys84 and Phe36
residues of VEGF-A. Whereas, the pose 1, 2 or 3 lay in different position of binding site. It
appears that Jzu 17 does not have a stable docking pose for VEGF-A. Taken together, these
observations suggest that Jzu 17 may bind to VEGFR-2 rather than VEGF-A to interrupt
VEGF-A-VEGFR2 signaling.
Discussion
In the last decades, cancer remains the leading cause of death globally with increasing
incidence. Tumor metastasis is the primary cause of cancer mortality. It is estimated that tumor
metastasis is responsible for about 90 % of cancer-related deaths. Inhibition of angiogenesis
potentially prevents tumors from growing and metastatic spread to other organs (Fischer,
Mazzone, Jonckx & Carmeliet, 2008). It may also improve chemotherapy targeting tumors
through vascular remodeling. Therefore, angiogenesis blockade has been considered an
essential modality for normalizing the tumor-associated vasculature and serves as a
complementary therapeutic paradigm for cancer therapy (Heath & Bicknell, 2009). Clinical
efforts developing novel anti-angiogenesis agents have largely focused on targeting VEGF-A-
VEGFR-2 signaling due to its central role in angiogenesis (Welti, Loges, Dimmeler &
Carmeliet, 2013). There is increasing evidence highlighting the beneficial effects of
benzimidazole derivatives in the treatment of cancer (El Rashedy & Aboul-Enein, 2013). In the
This article is protected by copyright. All rights reserved.