Discovery of candesartan cilexetic as a novel neddylation inhibitor for suppressing tumor growth

Article abstract of DOI:10.1016/j.ejmech.2019.111848

Protein neddylation is a posttranslational modification of conjugating the neuronal precursor cell-expressed developmentally down-regulated protein 8 (Nedd8) to substrates. Our previous work revealed that neddylation pathway is overactivated in various hu

Full text of DOI:10.1016/j.ejmech.2019.111848

Journal Pre-proof
Discovery of candesartan cilexetic as a novel neddylation inhibitor for suppressing
tumor growth
Shuaishuai Ni, Xin Chen, Qing Yu, Yixiang Xu, Zhiguo Hu, Junqian Zhang, Wenjuan
Zhang, Baoli Li, Xi Yang, Fei Mao, Jing Huang, Yi Sun, Jian Li, Lijun Jia
PII:
S0223-5234(19)31000-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.111848
EJMECH 111848
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 September 2019
Revised Date: 28 October 2019
Accepted Date: 3 November 2019
Please cite this article as: S. Ni, X. Chen, Q. Yu, Y. Xu, Z. Hu, J. Zhang, W. Zhang, B. Li, X. Yang,
F. Mao, J. Huang, Y. Sun, J. Li, L. Jia, Discovery of candesartan cilexetic as a novel neddylation
inhibitor for suppressing tumor growth, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111848.
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Discovery of candesartan cilexetic as a novel neddylation inhibitor for
suppressing tumor growth
1,
2,
3,
†
2
4
1
†
†
Shuaishuai Ni , Xin Chen , Qing Yu , Yixiang Xu , Zhiguo Hu , Junqian Zhang ,
Wenjuan Zhang1, Baoli Li2, Xi Yang1, Fei Mao2, Jing Huang5, Yi Sun3, Jian Li2, *,
Lijun Jia1, *
ADDRESS:
1Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese
Medicine, Shanghai, 200032, China.
2State Key Laboratory of Bioreactor Engineering, East China University of Science
and Technology, Shanghai, 200237, China
3Institute of Translational Medicine, Zhejiang University School of Medicine,
Hangzhou, Zhejiang, 30029, China
4State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular
Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of
Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 201210,
China
5Shanghai Institute of Precision Medicine, Ninth People’s Hospital, Shanghai Jiao
Tong University School of Medicine, Shanghai, 200025, China
Corresponding author: Lijun Jia, Email: ljjia@shutcm.edu.cn; Jian Li, Email:
jianli@ecust.edu.cn
1

Abstract
Protein neddylation is a posttranslational modification of conjugating the neuronal
precursor cell-expressed developmentally down-regulated protein 8 (Nedd8) to
substrates. Our previous work revealed that neddylation pathway is overactivated in
various human lung cancers and correlates with the disease progression, whereas
pharmacologically targeting this pathway has emerged as an attractive therapeutic
strategy. As a follow-up research, 1331 approved drugs were investigated the
inhibitory activities of cullin1 neddylation for screening the hit compounds via an
improved enzyme-based assay. An antihypertensive agent, candesartan cilexetic
(CDC), was identified as a novel neddylation inhibitor that ATP-competitively
suppressing Nedd8-activating enzyme (NAE, E1) in mechanism, which inhibited the
cullins neddylation superior than two representative non-covalent NAE inhibitors,
M22 and mitoxantrone. Following with the findings such as apoptotic induction and
tumor growth suppression in human lung cancer A549 in vitro and in vivo, CDC
represents a potential anticancer lead compound with promising neddylation
inhibitory activity.
Keyword: Neddylation, drug repurposing, candesartan cilexetic, NAE inhibitor.
2

1. Introduction
Protein neddylation is an important posttranslational modification that conjugates
Nedd8 to substrates via a three-step enzymatic cascade reaction by E1 (NAE), E2s
(Ubc12/UBE2M or UBE2F) and Nedd8 ligases (E3s, RBX1/ROC1, RBX2/ROC2,
etc.) [1, 2]. In the process of neddylation, mature Nedd8 is first adenylated and then
activated via binding NAE in an Mg2+/ATP dependent manner to form NAE-Nedd8
[3]. Subsequently, activated Nedd8 is transferred to one of two E2s through a
transthiolation reaction and forms the E2-Nedd8 [4]. Nedd8 is ultimately transferred
from E2-Nedd8 to the specific substrates in the presence of E3s (Figure 1A). The
best-known substrates of neddylation are members of the cullin family, which are
involved in the assembly of cullin-RING E3 ubiquitin ligases (CRLs). CRLs act as a
large family of ubiquitin ligases that target cellular protein substrates for proteasomal
degradation [5, 6]. The inhibition of the neddylation pathway inactivates CRLs and
decreases the protein levels of ubiquitination and subsequent degradation of substrates
which are regulated by CRLs substrates, such as Wee1 [7], p27 [8], and Nrf2 [9],
thereby leading to the suppression of the tumor progression. Besides CRL substrates,
neddylation also regulates several non-cullin substrates, such as MDM2, p53, and
VHL [10] (Fig 1A). In recent years, overactivated neddylation was observed in
various types of human cancers and positively correlated with poor prognosis [11-15].
Thus, the inhibition of neddylation pathway has been developed as an attractive
anticancer strategy recently [16-18].
NAE has emerged as a novel target in the neddylation pathway for drug discovery.
3

MLN4924 is a neddylation inhibitor that suppresses neddylation modification by
covalently binding the adenosine triphosphate (ATP) binding pocket of NAE, which
has been advanced into phase II/III clinical trials (Fig S1) [19, 20]. However, recent
preclinical studies identified the resistance to MLN4924 for the heterozygous
mutations nearby its covalently binding site with NAE, highlighting the necessity to
develop other neddylation inhibitors [21, 22]. Therefore, Chung-Hang Leung and
other researchers discovered a series of noncovalent NAE inhibitors by utilizing the
structure-based virtual screening of compound libraries, including natural product 6,
6’’-biapigenin, metal complex 1, M22, mitoxantron, 7g, LP0040 and deoxyvasicinone
derivatives (Fig S1) [23-30]. Notably, the core skeleton of 7g, mitoxantron, LP0040
and deoxyvasicinone derivatives are similar to MLN4924, suggesting that these
benzohetercyclic may be an essential group for maintaining the neddylation inhibitory
effects of these inhibitors. Furthermore, long and flexible side links with amide or
amino groups increased the affinities of compounds and NAE. In addition to NAE
inhibitors, several E3 inhibitors were obtained by the high throughput screening (HTS)
[31-35]. However, a majority of currently reported neddylation inhibitors lack enough
druggability-related evaluations or structural potentiality, and accompany with seldom
exploring the inhibitory effects of cullins neddylation comprehensively.
Utilizing new approaches and conceptions to discover novel therapeutic regimes
and potential lead compounds has gradually become a trend in the field of drug
research and development (R&D) [24, 36-38]. Drug repurposing is the re-exploration
of clinically approved drugs and compounds for one indication being applied to new
4

and different diseases, which can significantly reduce cost and save time during the
R&D of new drugs [39-41]. In this study, drug repurposing was performed for
screening potential neddylation inhibitors, while the cullin1 neddylation level as the
important phenotype was explored by an improved enzymatic assay in this screen.
With a lab in-house “Old drug bank”, 1331 approved drugs were firstly screened in
vitro with this assay, and investigated whether some agents could suppress the growth
of human tumor cell with the neddylation inhibition. An antihypertensive agent
candesartan cilexetil (CDC) presented superior inhibitory activity on cullin1
neddylation and structurally modificational potentiality than other hit compounds
after two rounds of screening. Further experiments revealed that CDC was able to
block cullins neddylation by inhibiting the NAE activity in enzymatic and cellular
assays, which also displayed potent anticancer activity in vitro and in vivo. These
evidences indicated that CDC is suitable for further structural modification as a lead
compound. In addition, CDC as a benzimidazole-derived neddylation inhibitor is also
similar to some reported inhibitors in the chemical structure [19-22]. This finding may
afford us more rational design about novel derivatives in the following-up studies
2. Materials and Methods
2.1. Cell culture and reagents
Human lung cancer cell lines A549, EKVX, H1299, Human liver cancer cell lines
HepG2, Huh7, Human breast cancer cell lines T-47D, MCF-7, Human gastric cancer
cell lines MKN45, MGC803, PLC were obtained from the American Type Culture
Collection (Manassas, VA) and passaged five to six times before use. Cells were
5

cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT), containing
10% fetal bovine serum (Biochrom AG, Berlin, Germany) and 100 units ml-1
penicillin/streptomycin solution and maintained in a humidified atmosphere of 5%
CO2 at 37°C (standard culture conditions). MLN4924, CDC and other drugs were
dissolved in dimethyl sulfoxide (DMSO) and kept at -20°C for in vitro studies.
MLN4924
and
CDC
were
dissolved
respectively
in
5%
2-hydroxypropyl-β-cyclodextrin and 5% castor oil (Macklin Reagent, Shanghai,
China) in vivo study. The solution of either MLN4924 or CDC was freshly made
when was used.
2.2. Enzyme-based neddylation activity assay
The protein Nedd8, NAE, Ubc12, Rbx1/Cullin1CTD were prepared and gifted by
Prof. Jin Huang, Shanghai Jiao Tong University School of Medicine, China. The
similar protocol was described [42] and applied by Prof. Yi Sun, Zhejiang University
School of Medicine, China. In respect of drug screen, 1µL UBA3-NAE (final
concentration, 0.025µM), 1µL Nedd8 (final concentration, 10 µM), 1µL Ubc12 (final
concentration, 1µM), 1 µL RBX1/cullin1CTD (final concentration, 1µM), 1µL Tris-HCl
(1M, pH=7.4; final concentration, 50 mM), 1µL MgCl2 (0.1M), 1µL DTT (10mM),
0.2µL 0.1mg/mL BSA, 2.8µL ddH2O, were added into in each well of 96-well plates,
followed with 2.0µL water solution of MLN4924 (final concentration, 10µM) or test
drugs (final concentration, 50µM). 4.0µL (final concentration, 20µM) ATP was added
to reaction after the mixture was incubated for 10min at room temperature. The
mixture was incubated at 37°C for 30min. The reaction was quenched by 10% loading
6

buffer, and heated for 5 min at 95°C by a real-time PCR. Protein samples were
electrophoresed under non-reducing conditions on a 10~15% SDS-PAGE gel, and
cullin1-Nedd8 levels were determined by immunoblotting analysis. To determine the
neddylation inhibitory activities of agent CDC, various concentration CDC groups
(final concentration, 6.125µM, 12.5µM, 25µM, 50µM, and 100µM) were used. For
the ATP-competitive assay, CDC (final concentration, 50µM) and ATP (final
concentration, 0µM, 10µM, 100µM, 500µM, and 1000µM) were used, which all other
conditions remained the same.
2.3. Cell-based neddylation activity assay
A549 cells were exposed to the indicated concentrations of MLN4924 (10µM) or
CDC (25µM, 50µM, 75µM, 100µM, or 50µM, 60µM, 70µM, 80µM, 90µM, 100µM,
or 50µM, 75µM, 100µM, 125µM) or 0.1% (v/v) DMSO for 6h or 12h, respectively.
Cells were washed three times with ice-cold PBS, resuspended in RIPA lysis buffer,
and incubated on ice for 30min. Cell debris was removed by centrifugation at
15,000rpm for 10min at 4°C. The protein concentration of the supernatant was
determined with Thermo Fisher protein assay dye reagent (Thermo Fisher). Equal
protein amounts were electrophoresed under non-reducing (cullin1-Nedd8,
cullin2-Nedd8, cullin3-Nedd8, cullin4a-Nedd8, and cullin5-Nedd8) or reducing
(Wee1, p27 or Clever-Parp) conditions on SDS-PAGEs and subjected to western blot
analysis.
2.4. Cell proliferation and cell clonogenic assays
For evaluating the proliferation of cultured cells, cells seeded (A549, EKVX,
7

H1299, HepG2, Huh7, T-47D, MCF-7, MKN45, MGC803, and PLC) in 96-well
plates with 2500 cells per well, in triplicate, and cultured overnight were treated with
MLN4924 or CDC for 48 hours, followed by cell counting kit-8 (CCK-8) assay. For
clonogenic assays, A549 cell were seeded in six-well plates (150 cells per well) in
triplicate, and were treated with MLN4924 (0.1µM, 0.33µM, and 1µM) or CDC (5µM,
10µM, 15µM, and 20µM) and cultured for 10 days. Colonies were considered as 50
cells or more and counted. The representative results of three independent
experiments with similar trend were presented.
2.5. Cell cycle and cell apoptosis assays
Cells treated with DMSO or various concentrations of agent CDC (25µM, 50µM,
75µM) were harvested and fixed in 90% ethanol at - 20°C for 24h, then centrifuged at
15,00rpm for 10min at 4°C in tubes. The residue in each tube was washed three times
with ice-cold PBS, and stained with 0.5mL propidium iodide solution (36µg/mL;
Sigma, St. Louis, MO) contained RNase A (10µg/mL; Sigma) at 37 °C for 30 minutes,
and analyzed for cell-cycle profile with Flowjo. Representative results of three
independent experiments with similar trends are presented.
Apoptosis was evaluated using FITC Annexin V apoptosis detection kit. A549 cells
were seeded at 2.5×105 cells per well in 6-well plates and allowed to attach overnight.
Cells were treated with increasing concentrations of CDC (25µM, 50µM, 75µM) for
48h. Cells were dissociated using trypsin, washed three times with ice-cold PBS, and
resuspended in 100µL 1×binding buffer. The addition of 5µL FITC Annexin V
staining solution and 5µL PI staining solution were added. After incubation at room
8

temperature in the dark for 15min, another 400µL 1×binding buffer was added.
Stained cells were analyzed immediately by fluorescence-activated cell sorting
(FACS).
2.6. Molecular modeling
The X-ray structure of NAE was downloaded from the Protein Data Bank (PDB ID:
3GZN). Crystallographic water, ligands and alternate conformations were removing
by using the protein preparation protocol in Autodock 4.0. The docking procedure was
performed by employing DOCK program in Autodock 4.0, and the structural image
was obtained using PyMOL software.
2.7. Tumor xenograft growth inhibition
Female nude mice were subcutaneously injected with 2×106 A549 cells in 100 µL
PBS according to protocols of tumor transplant research. After tumor induction (about
7 days), nude mice were divided into five groups randomly, including mock group
(10% 2-hydroxypropyl-β-cyclodextrin/water solution), positive group (MLN4924-60
mg/kg), and the other three were for various treatments of CDC, respectively.
Compounds were administrated by intraperitoneal injection (60 mg/kg vs 30 mg/kg).
All groups were treated with one dose per day. Body weights and tumor volumes were
measured every 2 days. On the 32th day after inoculation, all the mice were sacrificed.
Tumor volumes and mass were measured for each group.
3. Results and Discussion
3.1. Discovery of optimal neddylation inhibitor CDC
Cullin1-regulated CRL (CRL1) emerges as the most studied cullins-associated
9

complex in neddylation [43-45]. In order to rationally evaluate the neddylation
inhibitory activities of the approved drugs, an enzyme-based assay that simulated the
process of cullin1 neddylation in vivo was set up in this study. The assay was operated
with ATP under the cascade reaction of protein Nedd8-E1 (NAE)-E2 (Ubc12)-E3
(RBX1)-cullin1 pathway (Figure 1B). Taking advantage of the assay, we primarily
screened 1331 FDA-approved drugs and explored their neddylation inhibitory effects
(Figure 1C). Theoretically, Nedd8 in control groups with ATP could conjugate the
substrate cullin1, thereby forming the cullin1-Nedd8 adduction, whereas the groups
without ATP failed to form cullin1-Nedd8 adduction (Figure S2). Similar to the
groups without ATP, MLN4924 groups competitively suppressed the formation of
cullin1 neddylation in the presence of ATP (Figure S2, WB-01). If the cullin1-Nedd8
conjugation of the drug-treated groups decreased similar to that of MLN4924, these
data would suggest these drugs are neddylation inhibitors. In this assay, 106 drugs that
presented either partially accumulating of cullin1 or deceasing of cullin1-Nedd8
adduction were identified as hit compounds and selected for reinspection. (Figure S3).
In the secondary hit compounds screening, five hit drugs displayed superior
neddylation inhibitory activities than the others, including CDC (01, MT04D0301),
Trifluoperazine (02, MT05C1901), Bismuth Potassium Citrate (03, MT08A1201),
Cetylpyridinium Chloride (04, MT20B0301), and Procyanidin (05, MT24A0201)
(Figure 1D). In this study, we first investigated the cullin1-Nedd8 adduction inhibition
of those five hit compounds by a cell-based assay for removing the pan-assay
interference compounds (PAINS) in the enzymatic assay. In general, CDC presented
10

significant neddylation inhibitory activity at the concentration of 100µM comparable
to MLN4924, whereas hit compounds 02, 03, and 04 failed to display efficacy (Figure
S4). Meanwhile, 05 not only showed inferior neddylation inhibitory activities than
CDC, but seriously violated the Lipinski’s rule of five in the respect of structure as
well [46, 47]. Thus, drug CDC was selected as the best lead compound to explore the
mechanism of neddylation inhibition.
3.2. CDC suppresses neddylation pathway via inhibiting NAE
To further explore the efficacy and interactive mechanism of CDC, a
dose-dependent experiment with various concentration of CDC was firstly performed
for measuring the level of cullin1-Nedd8 adduction. Meanwhile, since NAE activates
Nedd8 and transfers it to Ubc12 in the process of neddylation, the level of
Ubc12-Nedd8 conjugation was assessed in parallel for preliminary determining target
enzyme of CDC. The results showed that the formation of cullin1-Nedd8 adduction
was significantly inhibited by CDC at the concentration of 25~50µM, while the
formation of Ubc12-Nedd8 adduction was completely inhibited at 50µM as well
(Figure 2A), suggesting that CDC inhibits the enzyme in the upstream of neddylation
(NAE or Ubc12). ATP-binding pocket of NAE emerged as an excellent drug target
with distinct mechanism in neddylation has been utilized for the discovery of
neddylation inhibitors, such as MLN4924, M22, mitoxantrone. Thus, an ATP
concentration-dependent assay was conducted to determine whether CDC was an
ATP-competitive inhibitor. The results showed that the inhibitory activities of CDC on
cullin1-Nedd8 and Ubc12-Nedd8 were reduced with increasing concentrations of ATP
11

(Figure 2B).
Furthermore, in order to obtain enzyme inhibition properties of CDC, a kinetic
analysis on the inhibition was performed using Lineweaver-Burk representation
analysis. The Lineweaver-Burk plot showed that the Vmax values were regardless of
concentration of CDC while the Km values were elevated with increasing
concentrations of CDC (Figure S5), demonstrating that CDC represents an
ATP-competitive inhibitor to suppress the neddylation. Meanwhile, an additional
kinetics binding assay was addressed for exploring the binding affinity of CDC with
NAE by the application of surface plasmon resonance (SPR) technology. The SPR
results showed CDC displayed moderate binding effect (KD=195µM) with NAE
(Figure S6). Consequently, these results suggested that CDC acts as an
ATP-competitive neddylation inhibitor by targeting the enzyme NAE.
We have previously shown the overactivated neddylation-CRLs axis amplified the
lung cancer development [13]. Thus, we chose human A549 lung cancer cells to
explore the efficacy against the neddylation modification, and then identified the level
of global protein neddylation using a specific Nedd8 antibody to investigate the
efficacy of CDC against the neddylation pathway (Figure 2C). In contrast to the
control group, the treatment groups could diminish the level of global protein
neddylation, even comparable to MLN4924. Cullin family members act as the
best-known substrates of neddylation that involved in the assembly of CRLs are
modulated by Ubc12 and UBE2F. In cullin family members, individual cullin1, 2, 3,
4a or 4b is catalyzed by the Ubc12 neddylation, while cullin5 is catalyzed by the
12

UBE2F neddylation [48, 49]. In the present study, those characteristics were utilized
for evaluating the neddylation inhibitory ability and identifying target enzyme of
CDC by comprehensively exploring the levels of cullins-Nedd8 adduction (Figure
2D). In general, CDC represented an efficient neddylation inhibitor for suppressing
the whole cullins neddylation significantly at 50µM. On the other hand, the
neddylation inhibition resulted in a corresponding decrease in the abundance of
cullins-Nedd8 with the dosage increase of CDC, indicating that the whole neddylation
pathway was blocked by CDC. Meanwhile, the degradations of two representative
CRLs substrates Wee1 and p27 were decreased after incubating with CDC in this
assay (Figure 2E), validating that CDC stabilized CRLs substrates by suppressing
neddylation-CRLs pathway.
Nedd8 activating enzyme NAE in neddylation pathway is closely related to some
enzymes in ubiquitination or sumoylation [19], suggesting that CDC may also block
these pathways. Herein, Sumo-conjugating enzyme Ubc9, Ub-conjugating enzyme
UbcH10, Sumo1 and ubiquitin antibodies were introduced for investigating the effects
of CDC on ubiquitination and sumoylation by immunoblotting (Figure S7). The
results showed that the formation of either Ubc9-Sumo or Ubc10-Ub adduct were
almost not suppressed at different concentrations of CDC. Furthermore, the levels of
Sumo1- and Ub-modified proteins also had no significant decrease in the presence of
CDC, suggesting that CDC selectively blocks the neddylation pathway. Small
molecule M22 as a known noncovalent NAE inhibitor that displays the significant
antitumor activities in vitro and in vivo was suitable for comparing the inhibition of
13

CDC [25]. In this study, we synthesized compound M22 (Figure S8) and compared its
cullin1-Nedd8 and global Nedd8 modification inhibitory effects with those of CDC.
Despite M22 had significant inhibitory effect against Nedd8-associated conjugation at
the range of 60~80µM in correspondence to literature reports, the cullin1-Nedd8 and
global neddylation inhibition of M22 was inferior to those of CDC (Figure S9A).
Similarly, another known NAE inhibitor mitoxantrone (an antitumor drug) was also
used in our study to compare with CDC [26]. The results showed that mitoxantrone
had no effect on cullin1 neddylation at the concentration of 50µM but partly inhibited
the formation of cullin1-Nedd8 at 75µM, which was inferior to CDC as well (Figure
S9B).
3.3. The effect of neddylation inhibitor CDC on anticancer activity in vitro
Considering that blocking the neddylation pathway can effectively suppress tumor
progression [11-15], we examined the antiproliferation activities of CDC against
A549 cell by CCK-8 cell viability assay (Figure 3A). The results displayed that CDC
could inhibit A549 cell proliferation in the micromolar range (IC50=63.93±4.18µM).
To further investigate the anti-cell survival effects of CDC, colony formation assay
was performed as a side-by-side assay (Figure 3B, C). Similarly, the results
demonstrated CDC inhibited the clonogenic survival in a dose-dependent manner.
To further explore whether growth inhibition suppressed by CDC was associated
with cell cycle, DNA content of cell nuclei was detected by flow cytometry, as shown
in Figure 3D. In general, phases G1, S, G2 had no significant difference in various
concentrations of CDC-treated groups in 24h, whereas the cell population in Sub-G1
14

phase increased 0.89% to 30.45%, suggesting that CDC induced cancer cell apoptosis.
Moreover, Annexin-V/PI double staining assay was further performed for evaluating
the capacity of CDC to induce apoptosis (Figure 3E). After treatment with various
concentrations (25, 50, or 75µM, 24h) of CDC, the total numbers of early and late
apoptotic cells were 9.6%, 25.2%, and 50.9%, respectively. In addition, we found that
cleaved-PARP (Poly ADP ribose polymerase, cleaved by caspase-3), as another
classical apoptosis marker, was accumulated after treatment with the increasing
concentration of CDC (Figure 3F). Taken together, these results validated that the
treatment of CDC induced cell death through apoptosis in the A549 cells.
To evaluate the capacity of CDC against other cell lines, we tested additional nine
cancer cell lines using the CCK-8 assay. These cell lines included EKVX (lung
cancer), H1299 (lung cancer), HepG2 (liver cancer), Huh7 (liver cancer), T-47D
(breast cancer), MCF-7 (breast cancer), MKN45 (gastric cancer), MGC803 (gastric
cancer), and PLC (gastric cancer), as shown in Table 1. We observed that CDC
decreased the proliferation of various types of cell lines in the micromolar range. In
addition, two normal cell lines BEAS-2B (Normal lung epithelial cell) and 16HBE
(Normal bronchial epithelial cell) were utilized for exploring the selectivity of CDC
(Table 1). In general, neddylation inhibitor CDC presented minor proliferation
inhibitory activities against these two normal cell lines (IC50 = 89.94 ± 5.16µM and
IC50 > 100µM, respectively), along with the overactivation of target neddylation
pathway in various cancer tissues in vivo as described above [11, 12], suggesting that
CDC has a certain selective effect in suppressing cancer cell lines.
15

3.4. The neddylation inhibitory activity of CDC originates from its unique structure
Molecular modeling studies were performed for investigating the binding mode
between CDC and NAE by Autodock 4.0. As shown in Figure 4A, the binding mode
of CDC in the NAE complex was similar to that of MLN4924 in general, but its
binding conformation had some difference with MLN4924 (Figure 4B, C). In
comparison with MLN4924 (PDB ID: 3GZN), CDC was estimated to form various
interactions in the binding pocket, including two H-bonds to both the side chain
guanidine group of Arg22 (3.1Å) and carboxylate group of Asp167 (1.9Å) through its
tetrazolium substituent. Meanwhile, CDC had the same H-bond to the side chain
amide group of Gln149 (2.3Å) through its tetrazolium cilexetil substituent as that of
MLN4924. Unlike MLN4924, CDC lacked the sulphamate group to form the covalent
adduct with NAE. These molecular interactions suggested that CDC had high affinity
with NAE comparable to MLN4924.
CDC is a well-known prodrug of candesartan that owns the moderate angiotensin II
type 1 receptor (A2T1R) antagonistic activity, which transfers into potent candesartan
by oral administration. In this study, we could not preclude the possibility that CDC
would suppress the neddylation through its A2T1R antagonistic activity. Thus, other
nine FDA-approved A2T1R antagonists, including losartan, irbesartan, azilsartan,
olmesartan medoxomil, eprosartan, telmisartan, valsartan, candesartan, and valsartan
were explored for neddylation inhibitory activities in A549 cell line (Figure 4D). The
results showed that formations of cullin1-Nedd8 failed to be blocked after the
treatment of these antagonists in contrast to that of CDC. Notably, candesartan, a
16

metabolic product of CDC, displayed no anti-neddylation activity, indicating that the
inhibition effect of CDC is related to its unique structure and irrelevant to A2T1R
antagonistic effect.
3.5. Neddylation inhibitor CDC suppresses the growth of A549 xenograft tumors in
mice
To evaluate the antitumor activity of CDC in vivo, we administered CDC to nude
mice bearing human tumor cell xenografts and monitored tumor growth rate. Tumors
were established after 8 days after subcutaneous inoculation of A549 lung cancer cells
(the tumor volume is at 70~100mm3), tumor bearing mice were then randomly
allocated into four groups: mock group, MLN4924-treated group as positive control
(60mg/kg qd) and CDC-treated groups (60mg/kg qd and 30mg/kg qd) with six mice
per group. The indicated agents were administrated by intraperitoneal injection. As
shown in Figure 5A, B, in contrast to mock group, all treatment groups significantly
decreased tumor volume and tumor weight after 22 days. Furthermore, CDC
displayed the dose-dependent activities through comparative analyses with those CDC
treatment groups (Figure 5A). To our surprise, although the MLN4924 group
presented superior antitumor activity in the assay, CDC treated with 60mg/kg group
had no significant difference with MLN4924 in vivo. Compared with either Mock or
MLN4924 groups, CDC-treated with 60 or 30mg/kg group did not affect the body
weight (Figure 5B). In addition, the tumor weights and sizes were measured and the
two CDC-treated groups had a decrease in tumor growth compared to the Mock group
(Figure 5C, D). In particular, CDC-treated with 60 mg/kg group decreased the tumor
17

weight comparable with MLN4924 group. Taken together, these data indicated that
CDC was efficacious in suppressing tumor growth in vivo with no observable toxicity.
4. Conclusion
One major challenge in developing neddylation inhibitors as therapeutic drugs is
lack of enough druggability studies and the costs of research and development. The
advantage of drug repurposing is cost and time effectiveness as absorption,
distribution, metabolism, excretion, and basic toxicity are already well established and
can be immediately taken to phase II/III clinical trials [39-41]. In this study, we
screened 1331 approved drugs from our in house “Old drug bank” to discover CDC as
a potential neddylation inhibitor based on a new enzyme-based assay with evaluating
the cullin1-Nedd8 inhibition. The findings from this study demonstrated the
practicability of this screening strategy for the discovery of novel neddylation
inhibitors.
The additional enzyme-based assays revealed that CDC not only significantly
inhibited neddylation pathway by evaluating the formations of cullin1-Nedd8 and
Ubc12-Nedd8 adductions, but ATP-competitively suppressed the neddylation
modification as well. As for cellular-based assays, CDC could significantly suppress
various cullins neddylation pathway in cancer cells, comparable to its performance in
enzymatic assay. Further study with investigating the inhibitions of other structurally
similar A2T1R antagonists validated that the neddylation inhibition of CDC
originated from its unique structure, not related to A2T1R antagonistic activity. A
molecular modeling verified the high affinity of CDC with NAE. In addition, CDC
18

inhibited the cullins neddylation more significant than two representative NAE
inhibitors M22 and mitoxantrone. Taken together, CDC represents a novel
neddylation inhibitor for suppressing the enzyme NAE.
Furthermore, anticancer experiments in vitro showed that CDC inhibited cancer cell
growth and survival in the micromolar range. Additional cell cycle and Annexin-V/PI
double staining assays displayed that CDC induced apoptosis in the A549 cancer cell.
Antitumor activity in vivo indicated CDC significantly inhibited the growth of A549
xenograft tumors in mice. These findings demonstrated that CDC presents the
passable efficacy on suppressing the tumor growth in vitro and in vivo. However, the
neddylation and anticancer inhibitory activities of CDC are inferior to those of clinical
agent MLN4924. Meanwhile, the defects of structurally easy hydrolysis by oral
administration and potential A2T1R-related toxicity also limit CDC to develop a
novel cancer therapeutic drug. Thus, subsequent studies will focus on the structural
modification by medicinal chemistry to overcome those weaknesses. Especially,
based on the results of docking model, we will chemically modify the hydrolyzable
cilexetil group in the structure of CDC for elevating the neddylation and anticancer
inhibitory activities and in vivo stability. In summary, CDC significantly inhibits the
neddylation modification by targeting enzyme NAE, and represents a promising lead
compound for the development of new anticancer drugs.
ASSOCIATED CONTENT
Supporting Information
Current reported neddylation inhibitors; The list of “Old Drug Bank”; Primary
19

approved drugs screen blocks the cullin1-Nedd8 pathway at the concentrate of 50µM;
Secondary hit compounds screen blocks the cullin1-Nedd8 pathway at the concentrate
of 50µM; The synthetic route of M22 and the related spectrum; The preliminary
neddylation inhibitory activities of hits compounds in human lung cancer cell A549;
Lineweaver-Burk representation of the kinetics assay; The kinetics binding assay by
the surface plasmon resonance (SPR) technology; Various concentrations of M22 and
mitoxantrone blocked the cullin1-Nedd8 pathway and Nedd8-mediated modification
in A549 cell.This material is available free of charge via the Internet.
AUTHOR INFORMATION
Corresponding Author
*For Lijun Jia: ljjia@shutcm.edu.cn; For Jian Li: jianli@ecust.edu.cn.
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interests.
Abbreviations
CRLs, cullin-RING ligases; Nedd8, neuronal precursor cell-expressed
developmentally down-regulated protein 8; NAE, Nedd8-activating enzyme E1; ATP,
adenosine triphosphate; A2T1R, Angiotensin II type 1 receptor; intraperitoneal
injection, I.P.
Ethics approval and consent to participate
All experimental protocols were approved by the Animal Experimental Ethics
20

Committee of Longhua Hospital of Shanghai University of Traditional Chinese
Medicine.
Conflicts of interest statement
The authors agree with the content of this manuscript and declare there are no
competing interests about this study.
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