Synthesis and anti-cancer activity of ND-646 and its derivatives as acetyl-CoA carboxylase 1 inhibitors

Article abstract of DOI:10.1016/j.ejps.2019.105010

Acetyl-coA carboxylase 1 (ACC1) is the first and rate-limiting enzyme in the de novo fatty acid synthesis (FASyn) pathway. In this study, through public database analysis and clinic sample test, we for the first time verified that ACC1 mRNA is overexpressed in non-small-cell lung cancer (NSCLC), which is accompanied by reduced DNA methylation at CpG island S shore of ACC1. Our study further demonstrated that higher ACC1 levels are associated with poor prognosis in NSCLC patients. Besides, we developed a novel synthetic route for preparation of a known ACC inhibitor ND-646, synthesized a series of its derivatives and evaluated their activity against the enzyme ACC1 and the A549 cell. As results, most of the tested compounds showed potent ACC1 inhibitory activity with IC₅₀ values 3–10 nM. Among them, compounds A2, A7 and A9 displayed strong cancer inhibitory activity with IC₅₀ values 9–17 nM by impairing cell growth and inducing cell death. Preliminary SAR analysis clearly suggested that (R)-configuration and amide group were vital to ACC1 and A549 inhibition, since compound (S)-A1 (the enantiomer of ND-646) had poor activity of ACC1 inhibition and the carboxylic acid ND-630 almost lost anticancer effect on A549 cells. Collectively, these findings indicate that ACC1 is a potential biomarker and target for non-small-cell lung cancer, and ND-646 and its derivatives as ACC1 inhibitors deserve further study for treatment of NSCLC.

Full text of DOI:10.1016/j.ejps.2019.105010

EuropeanJournalofPharmaceuticalSciences137(2019)105010
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Synthesis and anti-cancer activity of ND-646 and its derivatives as acetyl-
CoA carboxylase 1 inhibitors
En-Qin Lia,1, Wei Zhaob,c,1, Chenxi Zhangd,1, Lu-Zhe Qina, Sheng-Jie Liua, Zhi-Qi Fenga,
Xiaoan Wena,⁎, Cai-Ping Chena,⁎
a Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Xiang,
Nanjing 210009, China
b Department of Clinical Biochemistry, School of Laboratory Medicine, Chengdu Medical College, Chengdu 610050, China
c Department of Respiratory Medicine, The First Affiliated Hospital of Chengdu Medical College, Chengdu 610050, China
d Central Laboratory, Nanjing Chest Hospital, Medical School of Southeast University, Nanjing, Jiangsu Province 210029, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Acetyl-coA carboxylase 1 (ACC1) is the first and rate-limiting enzyme in the de novo fatty acid synthesis (FASyn)
pathway. In this study, through public database analysis and clinic sample test, we for the first time verified that
ACC1 mRNA is overexpressed in non-small-cell lung cancer (NSCLC), which is accompanied by reduced DNA
methylation at CpG island S shore of ACC1. Our study further demonstrated that higher ACC1 levels are asso-
ciated with poor prognosis in NSCLC patients. Besides, we developed a novel synthetic route for preparation of a
known ACC inhibitor ND-646, synthesized a series of its derivatives and evaluated their activity against the
enzyme ACC1 and the A549 cell. As results, most of the tested compounds showed potent ACC1 inhibitory
activity with IC50 values 3–10 nM. Among them, compounds A2, A7 and A9 displayed strong cancer inhibitory
activity with IC50 values 9–17 nM by impairing cell growth and inducing cell death. Preliminary SAR analysis
clearly suggested that (R)-configuration and amide group were vital to ACC1 and A549 inhibition, since com-
pound (S)-A1 (the enantiomer of ND-646) had poor activity of ACC1 inhibition and the carboxylic acid ND-630
almost lost anticancer effect on A549 cells. Collectively, these findings indicate that ACC1 is a potential bio-
marker and target for non-small-cell lung cancer, and ND-646 and its derivatives as ACC1 inhibitors deserve
further study for treatment of NSCLC.
Acetyl-CoA carboxylase
De novo fatty acid synthesis
Lung cancer
1. Introduction
isoforms of ACC with distinct subcellular distribution and physiological
roles have been identified, of which the cytosolic isoform ACC1 is
Cancer cells showed an extraordinary need of biological macro-
molecules including nucleic acids, proteins, and lipids to support con-
tinuous proliferation (Vander Heiden et al., 2009). Therefore, cancer
cells tend to reprogram their metabolism to meet such need, resulting in
alterations in glucose and glutamine metabolism (Martinez-Outschoorn
et al., 2017; Cairns and Mak, 2016). In addition, a number of cancers
show an increase in de novo fatty acid synthesis (FASyn) (Medes et al.,
1953; Currie et al., 2013; Menendez and Lupu, 2007; Zadra et al.,
2019). It has been demonstrated that decrease of FASyn by genetic
approaches or chemical compounds inhibited tumor growth (Chajes
et al., 2006; Svensson and Shaw, 2016). Acetyl-CoA carboxylase (ACC)
mediates the first step of FASyn by carboxylation of acetyl-CoA to form
malonyl-CoA and functions as a rate-limiting enzyme in FASyn. Two
predominant in control of the fatty acid synthesis, while the mi-
tochondrial isoform ACC2 mainly regulates the fatty acid oxidation
through inhibition of carnitine palmitoyltransferase I by localized
malonyl-CoA production (McGarry et al., 1978). With regard to cancer
progression, current research focus is on ACC1 but not ACC2, though
the both two isoforms are involved in lipid metabolism (Braig, 2018;
Wang et al., 2015). Moreover, it was reported that ACC1 was the
dominant isoform in several tested human lung cancer cell lines, while
ACC2 was nearly undetectable (Svensson et al., 2016). Upregulation of
ACC1 mRNA or protein was observed in a number of cancers, including
breast, liver and prostate cancers (Chin et al., 2006; Swinnen et al.,
2000). ACC1 silencing or deletion in cancer cells led to a loss of FASyn
and cell growth inhibition, which were rescued by addition of
⁎ Corresponding authors.
E-mail addresses: wxagj@126.com (X. Wen), caiping.chen@cpu.edu.cn (C.-P. Chen).
1 These authors contributed equally.
https://doi.org/10.1016/j.ejps.2019.105010
Received 22 March 2019; Received in revised form 9 July 2019; Accepted 15 July 2019
Availableonline17July2019
0928-0987/©2019ElsevierB.V.Allrightsreserved.

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
2.2. RNA collection, reverse transcription, and quantitative RT-PCR (qRT-
PCR)
RNA was extracted using PureLink™ RNA Mini Kit (Cat no.
12183025, Thermo Fisher Scientific, Carlsbad, CA, USA) following
manual and then reverse-transcribed by random primers using TaKaRa
reverse transcription kit (Dalian, China). Real-time PCR (qRT-PCR) was
carried out, using SYBR Green in an ABI 7500 StepOne Plus Real Time
PCR instrument (Applied Biosystems, USA). The relative expression
level of each target gene was normalized to β-actin in one sample.
Primers used in our study were as follows: AAC1: forward primer: 5′-
ATG TCT GGC TTG CAC CTA GTA-3′ and reverse primer: 5′-CCC CAA
AGC GAG TAA CAA ATT CT-3′; and β-actin: forward primer: 5′-CAT
GTA CGT TGC TAT CCA GGC-3′ and reverse primer: 5′-CTC CTT AAT
GTC ACG CAC GA-3′.
Fig. 1. Structures of ND-630, ND-646 and ND-654.
exogenous palmitate (Chajes et al., 2006; Svensson et al., 2016;
Brusselmans et al., 2005).
2.3. hACC1 enzyme inhibition assays
A couple of ACC inhibitors such as 5-(tetradecyloxy)-2-furoic acid
(TOFA), soraphen A and BAY ACC002 have been reported to show anti-
tumor activity (Petrova et al., 2017; Beckers et al., 2007). Very recently,
Harriman et al. identified a series of potent and highly specific allosteric
ACC dimerization inhibitors, e.g. ND-630, ND-646 and ND-654 (Fig. 1)
(Svensson et al., 2016; Harriman et al., 2016; Lally et al., 2019). These
dimerization inhibitors bind to the key residues Arg172 (ACC1) and
Arg277 (ACC2) that the AMPK-phosphorylated serine interacts with,
thus mimicking the physiological inhibition of ACC dimerization and
enzymatic activity by AMPK (Svensson and Shaw, 2016; Harriman
et al., 2016). Both ND-630 and ND-654 were shown to be liver specific.
The former could reduce hepatic steatosis, improve insulin sensitivity
and modulate dyslipidemia, and is currently in clinical trial phase II for
treatment of nonalcoholic fat liver disease (Harriman et al., 2016). The
latter was able to suppress lipogenesis and hepatocellular carcinoma
(Lally et al., 2019). However, ND-646, the amide derivative of ND-630,
was shown to be broadly distributed, significantly inhibit fatty acid
synthesis in lung tumors and strikingly suppress lung tumor growth
both in vitro and in mouse models (Svensson et al., 2016). ND-646 was
well tolerated in mice. Chronic ND-646 treatment of tumor-bearing
mice at the oral dose of 25 mg/kg twice daily or 50 mg/kg once daily
for 31 days did not cause body weight lost, and at the higher dose of
50 mg/kg twice daily or 100 mg/kg twice daily for 6 weeks only re-
duced 10% body weights (Svensson et al., 2016).
IC50 values of compounds inhibition on hACC1 were assessed by
WuXi App Tec (Shanghai) Co., Ltd. using a luminescent ADP detection
assay (ADP-Glo™ Kinase Assay Kit; Promega) according to previous
reported by Harriman et al. (Harriman et al., 2016), using recombinant
hACC1 (BPS Biosciences Catalog #50200), as the source of enzyme.
2.4. Cell lines and culture
A549 and H1975 Cells were purchased from the Cell Bank of
Shanghai Institute of Cell Biology, Chinese Academy of Sciences. A549
cells and H1975 cells were maintained in DMEM medium and modified
RPMI-1640 medium respectively, supplemented with 10% fetal bovine
serum (FBS), 100 μg/mL penicillin and 100 U/mL streptomycin in a 5%
CO2 humidified incubator at 37 °C.
2.5. Cell viability assay
Cells were seeded at about 500 cells/well (100 μL) in 96-well plates
in medium containing regular 10% FBS overnight for attachment. After
being washed once with PBS, cells were switched into medium con-
taining 20% delipidated FBS (Gemini #900–123) with concentrations
of compounds. After incubating for another 7 days, cell viability were
determined by MTT assay as previously described (Huang et al., 2018;
Song et al., 2016; Chen et al., 2019). Briefly, 20 μL of MTT solution (3-
(4,5-dimethyl thiazol −2-yl)-2,5-di phenyl tetrazolium bromide) were
added into each well and incubated for 4 h. The formed insoluble for-
mazan was dissolved with 150 μL of DMSO and then measured color-
imetrically at 490 nm by Enspire (PekinElmer, Waltham, MA, USA). The
viability of control group was set as 100%, and the values of other
groups were represented as percentages of the control group. The IC50
values were calculated through nonlinear regression analysis by using
GraphPad Prism 5.0 software.
In the present study, by public database analysis and clinic sample
test, we found that ACC1 mRNA is overexpressed in non-small-cell lung
cancer (NSCLC) and higher expression of ACC1 is significantly corre-
lated with shorter progression free survival (PFS) in NSCLC patients. It
suggests that ACC1 might be a promising predictive biomarker as well
as a potential therapeutic target for NSCLC. Thus, we synthesized ND-
646 and a series of its derivatives, and evaluated their ACC1 inhibitory
activity and anti-cancer activity in NSCLC.
2. Materials and methods
2.6. Proliferation/cytotoxicity assays
2.1. Clinical specimen collection
Cells were seeded at 300 cells/well in 96-well plates and treated
with ACC1 inhibitors in delipidated FBS as mentioned above. At the
indicated time points after treatment, cell numbers were determined
using an Opera Phenix High Content Screening System (Perkin-Elmer,
Waltham, MA, USA) through digital phase contrast imaging or DAPI
staining toward the end treatment. Afterwards, cells were subjected to
cell death analysis by annexin V/PI staining. Briefly, cells in 96-well
plate were washed with PBS twice and stained with annexin V/PI
(A211–02, Vazyme, Nanjing, China) according to the manufacture's
instruction. The fluorescence and brightfield were visualized with the
Opera Phenix High Content Screening System (Perkin-Elmer, Waltham,
MA, USA).
Sixty-three patients who received surgery at the department of re-
spiratory medicine, the first affiliated hospital of Chengdu Medical
College during 2015 to 2017 were included in this study. Lung speci-
mens from cancer tissues and normal tissues (with > 5 cm distance
from the tumor edge) were immediately frozen in liquid nitrogen and
then stored at −80 °C until use. None of the patients received any
chemotherapy or radiotherapy before surgery. The Research Ethics
Committee of Chengdu Medical College approved this protocol and
informed written consent was obtained from all the participants.
For regular medium, cells were seeded at 2000 cells/well in 6-well
2

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
plates in medium containing regular 10% FBS and were treated with
ACC1 inhibitors (1 μM) or DMSO vehicle for control the next day.
Medium containing compounds were refreshed at days 6 post treat-
ment, before cell death became obvious. Then, cells were incubated for
another 6 days. During the period of culture, wet condition was always
ensured.
composed of N shore and S shore, are recently coined regions with
relatively lower CpG density, located < 2 kb flanking CpG islands
(Portela and Esteller, 2010). The methylation of CpG island shores is
reported to be closely associated with genes transcriptional inactivation
(Portela and Esteller, 2010). The DNA methylation rates of other re-
gions, including the promoter, CpG islands, 5’UTR, 3’UTR, enhancer
and N shore of ACC1 are not significantly different between lung can-
cers and normal lungs (data not shown). Therefore, the increased ACC1
mRNA abundance in lung cancers might result from the reduced DNA
methylation at CpG island S shore.
To identify whether the mRNA level of ACC1 is clinically relevant in
lung cancer, we examined correlations between ACC1 expression and
survival of lung cancer patients using an online tool (http://www.
kmplot.com) (Gyorffy et al., 2013). As results shown, individuals with
high ACC1 levels exhibited shorter progression-free survival (PFS)
compared to those with low levels (Fig. 1D). Collectively, ACC1 might
be a potential prognostic biomarker for NSCLC.
2.7. Western blot assay
To assess the effects of ACC inhibitors on ACC phosphorylation,
A549 cells were seeded at a 3 × 105/well in a 6-well plate. After 24 h,
cells were treated with compounds (1 μM) or vehicle control for 24 h.
After washed with ice-cold PBS, cells were lysed by RIPA buffer con-
taining protease and phosphatase inhibitors (Millipore). Equal amounts
of protein were subjected to Western blot assay as previously described
(Chen et al., 2019; Chen et al., 2015). Primary antibodies against ACC
(#3676S) and p-ACC (#3661S) were purchased from Cell Signaling
Technology.
3.2. Synthesis of ND-646 and its derivatives
2.8. Establishment of Osi-resistant NCI-H1975 cells
We ever attempted to synthesize ND-646 according to the patent
published procedure (Harriman et al., 2013), but encountered a lot of
difficulties. Thus, we designed a novel 15-step route as depicted in
Scheme 1. The intermediate 6 was prepared from the starting material 1
via 5 steps same as described in the patent(Harriman et al., 2013).
Compound 6 was reacted with o-methoxyacetophenone bromide, fol-
lowed by hydrolysis to give the intermediate 8. The carboxylic group of
8 was protected with TBDPS, and the carbonyl group was reduced by
NaBH4 and brominated through the Apple Reaction. The resulting
bromide 11 was treated with tetrahydropyranol and MgO/Mg2SO4 to
give the ether 12, which took place a Stille coupling reaction with 2-
(tripropylstannml)-1,3-oxazole in the presence of PPh3 and was subse-
quently treated by TBAF for removal of TBDPS to achieve the key ra-
cemic acid 14. The racemate 14 was successfully separated by chiral
preparative HPLC to offer a pair of enantiomers ND-630 and (S)-14
with ee > 98%. Finally, ND-630 was converted to ND-646 by ammo-
nium hydroxide under T3P.
Reagents and conditions: (f) 2-bromo-2′-methoxyacetophenone,
K2CO3, DMF, rt., overnight, 75%; (g) TFA, DCM, r.t., 1 h; (h) TBDPSCl,
Imidazole, THF, rt., overnight, 83%; (i) NaBH4, THF, rt., 5.5 h, 66%; (j)
CBr4, PPh3, THF, 0 °C-r.t., overnight, 37%; (k) MgO, MgSO4, tetrahydro-
4- pyranol, 80 °C, 10 h, 46%; (l) 2-(tripropylstannml)-1,3-oxazole, PPh3,
toluene, 110 °C, 17 h, 41%; (m) TBAF, THF, rt., 1 h, 79%; (n) chiral
preparative HPLC, > 98% ee; (o) NH3·H2O, T3P, DCM, 10 h, 40%.
Treatment of 14 and (S)-14 by diverse amines or alcohols under the
coupling reagents HOBT/EDCI or T3P offered a series of racemic deri-
vatives A1~A12 and an optically pure amide (S)-A1, that is, the en-
antiomer of ND-646 (see Supporting Information).
Osimertinib resistance H1975 cells were established by exposing
H1975 cells to gradually increasing concentrations of osimertinib from
0.05 μM to 5 μM. Cells became resistant to Osi after approximately
6 months, reflected by appearing a normal exponential growth rate at
5 μM osimertinib. The cell line was routinely cultured in the presence of
5 μM osimertinib. The newly established OSI-resistant cells were termed
as H1975/OsiR cells. During the establishment of H1975/OsiR cells, the
parental H1975 cells were always kept in drug-free medium in parallel
for control.
2.9. Chemistry
1H NMR spectra was recorded on an ACF* 300Q Bruker or ACF*
500Q Bruker spectrometer. Low- and high-resolution mass spectra
(LRMS and HRMS) were recorded in electron impact mode. The mass
analyzer type used for the HRMS measurements was TOF. Reactions
were monitored by TLC on silica gel 60 F254 plates (Qingdao Ocean
Chemical Company, China). Column chromatography was carried out
on silica gel (200–300 mesh, Qingdao Ocean Chemical Company,
China). Data for 1H NMR are recorded as follows: chemical shift (δ,
ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet
or unre solved, br = broad singlet, coupling constant (s) in Hz, in-
tegration). Commercially available reagents and solvents were used
without further purification.
3. Results
3.1. ACC1 expression is upregulated in non-small-cell lung cancer
3.3. ACC1 inhibitory activity of ND-646 and its derivatives
ACC1 has been reported to be upregulated in breast, liver and
prostate cancers (Chin et al., 2006; Swinnen et al., 2000). However, so
far as we know, there was no report on mRNA levels of ACC1 in lung
cancer tissues, despite the phosphorylation status of ACC has been
analyzed previously (Rios Garcia et al., 2017; Carretero et al., 2007). In
this study, by using the Oncomine™ Platform (Okayama et al., 2012;
Hou et al., 2010), we found that ACC1 mRNA expression is significantly
increased in both lung adenocarcinoma (LUAD) and lung squamous cell
carcinoma (LUSC) compared to normal lung tissues (Fig. 2A). Con-
sistently, we observed higher levels of ACC1 in NSCLC tissues than that
in normal lung tissues by qRT-PCR analysis of clinic samples (Fig. 2B).
According to a previous study on chicken, alterated expression of ACC1
might be associated with the epigenetic modification (Liu et al., 2016).
In the present study, by using the MethHC database (Huang et al.,
2015), we observed that DNA methylation at CpG island S shore of
ACC1 reduced in both LUAD and LUSC (Fig. 2C). CpG island shores,
The ACC1 inhibitory activity was tested according to the previously
reported method (Harriman et al., 2016). As shown in Table 1, ND-646
and its precursor ND-630 displayed very strong potency of hACC1 in-
hibition, respectively with their IC50 values of 2.89 nM and 2.39 nM,
13
identical to the literature data 3.5 nM
and 2.1 nM (Harriman et al.,
2016). The racemate of ND-646, namely compound A1, also exhibited
pretty good potency (IC50 = 5.89 nM), while the enantiomer of ND-
646,
namely
compound
(S)-A1,
showed
poor
activity
(IC50 = 1156 nM). The other compounds also exhibited quite strong
inhibitory activity (IC50 < 23 nM), among which A6 was the best
(IC50 = 3.21 nM).
3.4. Anticancer activity of ND-646 and its derivatives against A549 cells
ND-646 and its derivatives were evaluated by cell viability assay for
3

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
Fig. 2. ACC1 expression is upregulated in lung cancers and higher ACC1 levels predict a poor outcome. (A) Analysis of human ACC1 expression in LUAD (left) and
LUSC (right) as well as control normal lung samples, using Oncomine database (www.oncomine.org). ***P < 0.001 (Student's t-test). (B) Box whisker plot pre-
senting the mean (min to max) value of ACC1 mRNA expression levels in 46 human NSCLC tumors and 24 adjacent normal lung tissues by qRT-PCR. **P < 0.01
(Mann Whitney test). (C) The methylation rate at S Shore of ACC1 is significantly reduced in both LUAD and LUSC compared with normal lung tissues according to
the MethHC database (http://methhc.mbc.nctu.edu.tw/php/diffMeth.php).**P < 0.005.(D) Kaplan-Meyer plots showing progression free survival (PFS) in lung
cancers. Patients with higher ACC1 expression associated with shorter PSF compared with those with low ACC1 expression (P = 0.03). Cox's proportional hazards
model was used to calculate hazard ratio (HR). HR > 1 indicates that patients with high ACC1 expression exhibited a poor prognosis.
their anticancer activity against A549 cells, which expressed the highest
ACC1 levels among the three NSCLC cell lines that we detected (Fig.
S1).
To best reflect the FASyn inhibition on tumor cell viability, cells
were cultured in delipidated fetal bovine serum (FBS) (Svensson et al.,
2016). The IC50 values are summarized in Table 1. Six compounds
showed strong proliferation inhibitory activity in A549 cells
(IC50 < 100 nM). Among them, the cyclohexyl amide A7 displayed the
of A2, A3 and A4 gradually decreased. Strangely, when it was a ring, a
large ring seemed favourable, since the activity of A5, A6 and A7
gradually increased. Besides, introduction of hydroxy group had effects
on the potency of anticancer activity. The alcohols A9 and A10 were
much more active than the corresponding non-alcohols A3, A4 and A7,
while the 4-hydroxypiperidine A11 was less active than the piperidine
A7.
best activity (IC50 = 9.4
3.2 nM). The methyl amide A2
the hydroxyethyl amide A9
(IC50 = 16.8
(IC50 = 23
3.8 nM)
and
3.5. Effect of ND-646 and its derivatives on ACC phosphorylation
4.6 nM) showed potency comparable to ND-646
10.6 nM). However, the enantiomer of ND-646,
(IC50 = 16.2
Crystallographic study has demonstrated that ND-646 binds to the
same residues as the AMPK-phosphorylated serines in ACC1 and ACC2
interact with, mimicking the physilogical dimerization inhibition by
AMPK. Reduction in AMPK-phosphorylated serines of ACC (p-ACC) has
been used to reflect ACC engagement of ACC inhibitors (Svensson et al.,
2016; Lally et al., 2019). Thus, we explored effects of compounds A2,
A7 and A9 on ACC phosphorylation. It was found that similar with ND-
646, these 3 compounds could eliminate p-ACC levels (Fig. 3), con-
firming their mechanism of action.
namely
(S)-A1,
showed
almost
no
anticancer
activity
(IC50 > 50,000 nM), consistent with its low hACC1 inhibitory activity.
Surprisingly, ND-630 also displayed disappointed anticancer activity
(IC50 > 20,000 nM) in spite of its hACC1 inhibitory activity strong as
ND-646, maybe due to poor uptake in A549 cells, since ND-630 was
reported highly liver specific (Harriman et al., 2016).
Prelimilary SAR analysis revealed that when the R group was a
chain, a long chain seemed unfavourable, since the anticancer activity
4

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
Scheme 1. Synthesis of ND-646.
3.6. ACC1 inhibitors impaired cell growth and caused cell death
FBS (Fig. 4D).
To further understand the tumor inhibitory effects of the synthe-
sized ACC1 inhibitors, we examined their effects on cell proliferation
and cell death. Compounds A2, A7 and A9 time-dependently decreased
the cell number of A549 cells, similar to ND-646 (Fig. 4A and B).
Moreover, annexin V-FITC/PI staining revealed that dying cells with
annexin V-positive/PI- positive or Annexin V-negative/PI-positive, re-
presenting late apoptosis and necrosis respectively, were extensively
seen after ACC1 inhibitors treatment for 7 days (Fig. 4C).
3.7. Sensitivity to ACC1 inhibitors reduced in osimertinib-resistant H1975
cells
As is known, patients harboring mutations in epidermal growth
factor receptor (EGFR), even if they initially could benefit from tyrosine
kinase inhibitors (TKIs), later likely become resistant to such drugs
including the third generation of TKI Osimertinib (Osi, also known as
AZD9291) (Jiang et al., 2018; Romaniello et al., 2018). Treatment of
TKI-resistant patients remains tremendously challenging. In the present
study, we sought to verify whether ACC inhibitors have anticancer
activity in Osi-resistant lung cancer cells. Hence, we established Osi-
Notably, ACC1 inhibitors treatment of A549 cells grown in regular
(not delipidated FBS) also inhibited cell growth and caused cell death,
though it required longer time compared to that of cells in delipidated
5

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
altered FASyn metabolism or multi-drug resistance, which needs to be
further studied.
Table 1
The hACC1 and A549 cell proliferation inhibitory activities of ND646 and its
derivatives.
4. Discussion
A number of cancers exhibit an increase in de novo fatty acid
synthesis (FASyn). The elevated FASyn in cancers is reflected by the
upregulation of lipogenic enzymes, including ACC1, fatty acid synthase
(FASN) and ATP citrate lyase (ACLY). In the present study, we found
the mRNA levels of ACC1 are increased in NSCLC patients, consistent
with the previous findings in breast, liver and prostate cancers (Chin
et al., 2006; Swinnen et al., 2000). Moreover, by using public database,
we revealed that higher ACC1 levels are associated with shorter PFS in
NSCLC patients, suggesting that ACC1 might be a potential prognostic
biomarker for NSCLC.
ND-646 is an allosteric inhibitor of ACC, with a unique mechanism
of action. It binds to the BC domain of ACC, where the AMPK phos-
phorylated serine of ACC interacts to prevent dimerization and acti-
vation of ACC (Svensson and Shaw, 2016; Svensson et al., 2016),
leading to constitutive dephosphorylation of ACC. Therefore, the
phosphorylation status of ACC was used as a biomarker to evaluate how
ND-646 acted. Our synthesized derivatives of ND-646 seemed to act
with the same mechanism as ND-646, since compounds A2, A7 and A9
diminished the ACC phosphorylation as ND-646 did.
Compd
R
IC50 (nM) hACC1
inhibition
IC50 (nM)a against A549
cells
A1
A2
5.89
6.69
117
18
16.8
3.8
Blockage of cancer cell FASyn by genetical or pharmacological
targeting of lipogenic enzymes caused a marked decrease of lipogenesis
and consequently cell growth arrest and apoptosis or autophagy de-
pending on cancer cell types (Chajes et al., 2006; Svensson and Shaw,
2016). Our synthesized ACC1 inhibitors, A2, A7 and A9 as well as ND-
646, indeed caused A549 NSCLC growth inhibition and cell death.
However, the anti-cancer mechanism of ACC1 inhibitors has not been
fully elucidated. Fatty acids not only supply with building blocks for
synthesis of membranes during cell division by conversion into phos-
pholipids (Stoiber et al., 2018), but also function as signaling molecules
that trigger physiological responses directly or through lipidation of
proteins, e.g., WNT and Hedgehog (Petrova et al., 2017). Therefore,
ACC1 inhibition might cause comprehensive influence to cancer cells,
including effects on cellular membranes and numerous signaling
pathways that link to proliferation and survival.
A3
A4
8.90
7.86
1029
1967
488
1227
A5
A6
A7
A8
8.90
3.21
434
63.4
9.4
21
11.8
3.2
6.87
22.87
7231
1902
A9
6.57
7.56
23.0
395
4.6
A10
214
In conclusion, we for the first time verified that ACC1 mRNA was
upregulated in NSCLC and higher ACC1 level was correlated with poor
outcome in lung cancer patients, indicating ACC1 might be as a prog-
nostic index for NSCLC patients. Moreover, we identified a series of
ACC1 inhibitors with IC50 values from 2 nM to 25 nM. Compounds A2,
A7 and A9 displayed strong cancer inhibitory activity in A549 cells by
impairing cell growth and inducing cell death. These compounds are
worth further evaluation for NSCLC treatment. Evaluation of a new
concept of combination therapy with them as well as their safety pro-
files will be conducted in the coming future and results will be pub-
lished elsewhere.
A11
A12
17.23
10.83
89.7
31.4
719
1300
(S)-A1
(R)-A1
1156
2.79
> 50,000
16.2
10.6
ND-646
ND-630
2.39
> 20,000
a
The values were obtained from at least two independent experimental re-
standard deviation (SD).
sults and are given as mean
Credit author statement
C.P. Chen and X. Wen contribute to conceptualization, project ad-
ministration, funding acquisition, manuscript writing and editing; C.P.
Chen, E.Q. Li, W. Zhao, C. Zhang, L.Z. Qin, S.J. Liu, and Z.Q. Feng
contribute to data curation, formal analysis, investigation, methodology
and writing review; E.Q. Li and C.P. Chen wrote the original draft; W.
Zhao and C. Zhang contribute to resources.
Fig. 3. ACC phosphorylation eliminated by ND646, A2, A7 and A9.
Acknowledgements
resistant H1975 cells (H1975/OsiR) (Fig. 5A) and tested the cytotoxic
activity of ND-646, A2, A7 and A9 on H1975/OsiR by MTT assay.
Regrettably, compared to H1975 cells, H1975/OsiR cells exhibited less
sensitivity to the tested ACC inhibitors (Fig. 5B). This might due to
This work was supported by grants from the National Natural
Science Foundation of China (grants 31501182, 81773584);
Fundamental Research Funds for the Central Universities
(2632017ZD05) and the scientific research fund for new teacher of
6

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
Fig. 4. Effects of ACC1 inhibitors on A549 cell growth and cell death. (A-B) Growth assay of A549 cells that were grown in delipidated FBS with 1 μM of ACC
inhibitors for 7 days. Cell numbers were measured using the Opera Phenix system through digital phase contrast imaging (A) or DAPI staining toward the end
treatment (B). Experiments were performed in sextuplicate. Statistical differences of cell numbers were calculated toward the end treatment. ***P < 0.001
(Student's t-test). (C) Cell death were analyzed by annexin V/PI staining after ACC1 inhibitors treatment for 7 days. Annexin V, PI and cell morphology were
visualized with FITC (green), Alexa 568 (red) and brightfield respectively. Scale bars = 50 μm (D) Representative microphotographs of A549 cells cultured in regular
FBS after receiving with ACC1 inhibitors (1 μM) for 12 days. Extensive cell death were seen after ACC1 inhibitors treatment. Scale bars = 75 μm. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Osi-resistant H1975 cells were less sensitive to ACC inhibitors. (A) Establishment of Osi-resistant H1975 cells. (B) Inhibitory effects of ND-646, A2, A7 and A9
in H1975 cells and H1975/OsiR cells.
7

E.-Q. Li, et al.
EuropeanJournalofPharmaceuticalSciences137(2019)105010
China Pharmaceutical University.
Nucleic Acids Res. 43, D856–D861.
Huang, Y., Sun, G., Wang, P., Shi, R., Zhang, Y., Wen, X., et al., 2018. Synthesis and
biological evaluation of complex I inhibitor R419 and its derivatives as anticancer
agents in HepG2 cells. Bioorg. Med. Chem. Lett. 28, 2957–2960.
Jiang, W., Cai, G., Hu, P.C., Wang, Y., 2018. Personalized medicine in non-small cell lung
cancer: a review from a pharmacogenomics perspective. Acta Pharm. Sin. B 8,
530–538.
Lally, J.S.V., Ghoshal, S., DePeralta, D.K., Moaven, O., Wei, L., Masia, R., et al., 2019.
Inhibition of acetyl-CoA carboxylase by phosphorylation or the inhibitor ND-654
suppresses lipogenesis and hepatocellular carcinoma. Cell Metab. 29 (1), 174–182.
Liu, Z., Li, Q., Liu, R., Zhao, G., Zhang, Y., Zheng, M., et al., 2016. Expression and me-
thylation of microsomal triglyceride transfer protein and acetyl-CoA carboxylase are
associated with fatty liver syndrome in chicken. Poult. Sci. 95, 1387–1395.
Martinez-Outschoorn, U.E., Peiris-Pages, M., Pestell, R.G., Sotgia, F., Lisanti, M.P., 2017.
Cancer metabolism: a therapeutic perspective. Nat. Rev. Clin. Oncol. 14, 113.
McGarry, J.D., Leatherman, G.F., Foster, D.W., 1978. Carnitine palmitoyltransferase I. the
site of inhibition of hepatic fatty acid oxidation by malonyl-CoA. J. Biol. Chem. 253,
4128–4136.
Medes, G., Thomas, A., Weinhouse, S., 1953. Metabolism of neoplastic tissue. IV. A study
of lipid synthesis in neoplastic tissue slices in vitro. Cancer Res. 13, 27–29.
Menendez, J.A., Lupu, R., 2007. Fatty acid synthase and the lipogenic phenotype in
cancer pathogenesis. Nat. Rev. Cancer 7, 763–777.
Okayama, H., Kohno, T., Ishii, Y., Shimada, Y., Shiraishi, K., Iwakawa, R., et al., 2012.
Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative
lung adenocarcinomas. Cancer Res. 72, 100–111.
Petrova, E., Scholz, A., Paul, J., Sturz, A., Haike, K., Siegel, F., et al., 2017. Acetyl-CoA
carboxylase inhibitors attenuate WNT and hedgehog signaling and suppress pan-
creatic tumor growth. Oncotarget 8, 48660–48670.
Portela, A., Esteller, M., 2010. Epigenetic modifications and human disease. Nat.
Biotechnol. 28, 1057–1068.
Declaration of Competing Interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ejps.2019.105010.
References
Beckers, A., Organe, S., Timmermans, L., Scheys, K., Peeters, A., Brusselmans, K., et al.,
2007. Chemical inhibition of acetyl-CoA carboxylase induces growth arrest and cy-
totoxicity selectively in cancer cells. Cancer Res. 67, 8180–8187.
Braig, S., 2018. Chemical genetics in tumor lipogenesis. Biotechnol. Adv. 36, 1724–1729.
Brusselmans, K., De Schrijver, E., Verhoeven, G., Swinnen, J.V., 2005. RNA interference-
mediated silencing of the acetyl-CoA-carboxylase-alpha gene induces growth in-
hibition and apoptosis of prostate cancer cells. Cancer Res. 65, 6719–6725.
Cairns, R.A., Mak, T.W., 2016. The current state of cancer metabolism FOREWORD. Nat.
Rev. Cancer 16, 613–614.
Carretero, J., Medina, P.P., Blanco, R., Smit, L., Tang, M., Roncador, G., et al., 2007.
Dysfunctional AMPK activity, signalling through mTOR and survival in response to
energetic stress in LKB1-deficient lung cancer. Oncogene 26, 1616–1625.
Chajes, V., Cambot, M., Moreau, K., Lenoir, G.M., Joulin, V., 2006. Acetyl-CoA carbox-
ylase alpha is essential to breast cancer cell survival. Cancer Res. 66, 5287–5294.
Chen, C.P., Chen, X., Qiao, Y.N., Wang, P., He, W.Q., Zhang, C.H., et al., 2015. In vivo
roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and
T852 in bladder smooth muscle contraction. J. Physiol. 593, 681–700.
Chen, C.P., Chen, K., Feng, Z., Wen, X., Sun, H., 2019. Synergistic antitumor activity of
artesunate and HDAC inhibitors through elevating heme synthesis via synergistic
upregulation of ALAS1 expression. Acta Pharm. Sin. B. https://doi.org/10.1016/j.
apsb.2019.05.001.
Rios Garcia, M., Steinbauer, B., Srivastava, K., Singhal, M., Mattijssen, F., Maida, A., et al.,
2017. Acetyl-CoA carboxylase 1-dependent protein acetylation controls breast Cancer
metastasis and recurrence. Cell Metab. 26, 842–855 (e5).
Romaniello, D., Mazzeo, L., Mancini, M., Marrocco, I., Noronha, A., Kreitman, M., et al.,
2018. A combination of approved antibodies overcomes resistance of lung Cancer to
Osimertinib by blocking bypass pathways. Clin. Cancer Res. 24, 5610–5621.
Song, Z., Chen, C.P., Liu, J., Wen, X., Sun, H., Yuan, H., 2016. Design, synthesis, and
biological evaluation of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate deriva-
tives as anti-proliferative agents through ROS-induced cell apoptosis. Eur. J. Med.
Chem. 124, 809–819.
Chin, K., DeVries, S., Fridlyand, J., Spellman, P.T., Roydasgupta, R., Kuo, W.L., et al.,
2006. Genomic and transcriptional aberrations linked to breast cancer pathophy-
siologies. Cancer Cell 10, 529–541.
Stoiber, K., Naglo, O., Pernpeintner, C., Zhang, S., Koeberle, A., Ulrich, M., et al., 2018.
Targeting de novo lipogenesis as a novel approach in anti-cancer therapy. Br. J.
Cancer 118, 43–51.
Currie E, Schulze A, Zechner R, Walther TC, Farese RV, Jr. Cellular fatty acid metabolism
and cancer. Cell Metab. 2013;18:153–61.
Svensson, R.U., Shaw, R.J., 2016. Lipid synthesis is a metabolic liability of non-small cell
lung cancer. Cold Spring Harb. Symp. Quant. Biol. 81, 93–103.
Gyorffy, B., Surowiak, P., Budczies, J., Lanczky, A., 2013. Online survival analysis soft-
ware to assess the prognostic value of biomarkers using transcriptomic data in non-
small-cell lung cancer. PLoS One 8, e82241.
Harriman, G., Greenwood, J., Bhat, S., Huang, X., Wang, R., Paul, D., et al., 2016. Acetyl-
CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin
sensitivity, and modulates dyslipidemia in rats. Proc. Natl. Acad. Sci. U. S. A. 113,
E1796–E1805.
Harriman, G.C., Masse, C.E., Harwood, J., Bhat, S., Greewood, J.R., 2013. Acetyl-CoA
carboxylase inhibitors and uses thereof. World intellectual property organization
(WO 2013/071169 A1).
Hou, J., Aerts, J., den Hamer, B., van Ijcken, W., den Bakker, M., Riegman, P., et al., 2010.
Gene expression-based classification of non-small cell lung carcinomas and survival
prediction. PLoS One 5, e10312.
Svensson, R.U., Parker, S.J., Eichner, L.J., Kolar, M.J., Wallace, M., Brun, S.N., et al.,
2016. Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor
growth of non-small-cell lung cancer in preclinical models. Nat. Med. 22, 1108–1119.
Swinnen, J.V., Vanderhoydonc, F., Elgamal, A.A., Eelen, M., Vercaeren, I., Joniau, S.,
et al., 2000. Selective activation of the fatty acid synthesis pathway in human
prostate cancer. Int. J. Cancer 88, 176–179.
Vander Heiden, M.G., Cantley, L.C., Thompson, C.B., 2009. Understanding the Warburg
effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033.
Wang, C., Ma, J., Zhang, N., Yang, Q., Jin, Y., Wang, Y., 2015. The acetyl-CoA carboxylase
enzyme: a target for cancer therapy? Expert. Rev. Anticancer. Ther. 15, 667–676.
Zadra, G., Ribeiro, C.F., Chetta, P., Ho, Y., Cacciatore, S., Gao, X., et al., 2019. Inhibition
of de novo lipogenesis targets androgen receptor signaling in castration-resistant
prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 116, 631–640.
Huang, W.Y., Hsu, S.D., Huang, H.Y., Sun, Y.M., Chou, C.H., Weng, S.L., et al., 2015.
MethHC: a database of DNA methylation and gene expression in human cancer.
8