A novel NAE/UAE dual inhibitor LP0040 blocks neddylation and ubiquitination leading to growth inhibition and apoptosis of cancer cells

Article abstract of DOI:10.1016/j.ejmech.2018.05.027

NEDD8 activating enzyme (NAE) plays an important role in regulating intracellular proteins with key parts in a broad array of cellular functions. On the basis of previously work, a series of 2H-chromen-2-one based NAE inhibitors were designed and synthesized. Through enzyme-based and cell-based assays, LP0040 was identified as a non-nucleoside NAE/UAE dual inhibitor. It could inhibit NAE/UAE activities and downregulated degradations of related substrates in AGS cells, promoting apoptosis in low micromole concentrations. LP0040 possessed anti-proliferation activities with IC₅₀ values of 0.76–3.29 μM against multiple human cancer lines and had synergistic effect with bortezomib. Thus LP0040 represented valuable starting points for future development of NAE/UAE dual inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.05.027

Accepted Manuscript
A novel NAE/UAE dual inhibitor LP0040 blocks neddylation and ubiquitination leading
to growth inhibition and apoptosis of cancer cells
Peng Lu, Yahui Guo, Lijuan Zhu, Yineng Xia, Yuejiao Zhong, Yubin Wang
PII:
S0223-5234(18)30437-9
10.1016/j.ejmech.2018.05.027
EJMECH 10438
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 December 2017
Revised Date: 3 May 2018
Accepted Date: 17 May 2018
Please cite this article as: P. Lu, Y. Guo, L. Zhu, Y. Xia, Y. Zhong, Y. Wang, A novel NAE/UAE dual
inhibitor LP0040 blocks neddylation and ubiquitination leading to growth inhibition and apoptosis of
cancer cells, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.027.
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ACCEPTED MANUSCRIPT

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A Novel NAE/UAE Dual Inhibitor LP0040 Blocks Neddylation and Ubiquitination Leading to Growth
Inhibition and Apoptosis of Cancer Cells
Peng Lu 1, Yahui Guo 2, Lijuan Zhu 1, Yineng Xia 1, Yuejiao Zhong 3, Yubin Wang 1*
.
1
School of Pharmaceutical Sciences, Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009,
People's Republic of China.
2
Department of pharmaceutical science, China Pharmaceutical University, No. 24 Tongjia Xiang,
Nanjing 210009, People's Republic of China.
3
The Affiliated Cancer Hospital of Nanjing Medical University, No. 42 Baiziting, Nanjing 210009,
People’s Republic of China.
Keywords: NEDD8 activating enzyme, ubiquitin activating enzyme, dual inhibitor, apoptosis, drug
discovery.
Abstract
NEDD8 activating enzyme (NAE) plays an important role in regulating intracellular proteins with
key parts in a broad array of cellular functions. On the basis of previously work, a series of
2H-chromen-2-one based NAE inhibitors were designed and synthesized. Through enzyme-based and
cell-based assays, LP0040 was identified as a non-nucleoside NAE/UAE dual inhibitor. It could inhibit
NAE/UAE activities and downregulated degradations of related substrates in AGS cells, promoting
apoptosis in low micromole concentrations. LP0040 possessed anti-proliferation activities with IC50
values of 0.76 – 3.29 μM against multiple human cancer lines and had synergistic effect with
bortezomib. Thus LP0040 represented valuable starting points for future development of NAE/UAE
dual inhibitors.
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1. Introduction
The ubiquitin-proteasome system (UPS) is responsible for regulated degradation of intracellular
proteins with important roles in a broad array of cellular functions [1]. In UPS, proteins labelled by
ubiquitin (Ub) will be recognized and degraded by proteasome. Proteasome inhibitor bortezomib
(Velcade) which targets the proteasome is approved for the treatment of multiple myeloma and
mantle cell lymphoma [2, 3]. The successful clinical application of bortezomib raises the question
whether inhibitors of other enzymes that modulate UPS activity can lead to the development of new
anti-cancer drugs. Ubiquitin-like protein (Ubl) conjugation pathways, including NEDD8 and SUMO,
have also been identified as key players in the protein degradation process. Targeting Ubl conjugation
pathways has been considered to be more advantageous for therapeutic intervention, as Ubl
pathways have fewer downstream conjugation targets and a smaller subset of proteins will be
affected.
Ub and Ubls typically modulate protein function following covalent attachment to substrate
proteins by E1, E2 and E3 cascade catalyzations. The Ub pathway is representative of those processes
and is shown in Fig. 1. In the first step, Ubiquitin-activating enzyme (UAE, E1) consumes ATP to form
Ub-AMP, which is a rate-limiting step. Then, Ub is passed to ubiquitin conjugating enzymes (E2)
through a transthiolation reaction. The Ub-E2 then binds an ubiquitin ligase (E3), and Ub is finally
transferred to a lysine residue on its substrate protein. Ubl pathways are similar to the ubiquitin
pathway. Ubls are transferred to their substrates through cascade catalyzations which are catalyzed by
corresponding E1, E2 and E3.
The covalent attachment of NEDD8, termed neddylation, is a reversible and multi-step process
analogous to ubiquitination. Neddylation plays a crucial role in the assembly and function of the
largest family of E3 ubiquitin ligases, the cullin ring ligases (CRL). Modification of the cullin-RING
ubiquitin E3 ligases (CRLs) by NEDD8 is known to be essential for the CRL-mediated ubiquitination of
downstream targets in the UPS, which is critically involved in protein homeostasis [4-6]. Other
substrates of neddylation include the mouse double minute 2 (MDM2) oncogene product [7], the p53
tumor suppressor protein [7, 8], the von Hippel-Lindau protein (pVHL) [9], the breast cancer
associated protein 3 (BCA3) [10] , and the estrogen receptor-R. (ERR) [11]. As the NEDD8 pathway
controls the degradation of a specific subset of proteins regulated by CRLs, it has been considered as a
more specific target compared to the UPS protein degradation pathway. Because NEDD8 activating
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enzyme (NAE) is involved in the rate-limiting step of neddylation, inhibition of NAE can modulate the
rate of ubiquitination and subsequent degradation of proteins which are regulated by CRLs. NAE has
recently emerged as a new target for the treatment of cancer [12-14].
MLN4924 (1, shown in Fig. 2) is the first reported NAE inhibitor. Treating cancer cells with
MLN4924 led to varieties of biological effects such as checkpoint activation, DNA re-replication and
apoptosis in multiple cancer cell lines [12, 15]. MLN4924, whose Phase 1 clinical trials is completed,
has been shown to be effective for the treatment of both solid (colon, lung) and hematological
(myeloma, lymphoma) human cancers. However, treatment-emergent heterozygous mutations of NAE
and resistance to MLN4924 have already been identified in preclinical studies [16-18]. In recent years,
some moderate NAE inhibitors has been reported, such as natural product-like compounds 2–3, metal
complex 4 and ene-rhodanine analogue 5 (Fig. 2), but while there still were some shortages about
potency and druglike physical properties in those compounds [19-22]. Besides that, ABP A3 which was
a nucleoside analogue (6, shown in Fig. 2) was reported as an NAE and UAE dual inhibitor [23]. ABP A3
would form Ub-ABP A3 and NEDD8-ABP A3 catalyzed by UAE and NAE respectively and subsequently
inhibited NAE and UAE activities. Besides that, some other NAE inhibitors were also reported
recently[24, 25]. In our previous work, M22 (7, shown in Fig. 2) was identified as a novel reversible
inhibitor of NAE through high-throughput virtual screening from a compound library containing 50000
small molecular entities [26]. M22 is reversible for NAE, inhibits multiple cancer cell lines with IC50
values in low micromolar range, induces apoptosis in A549 cells, and produces tumor inhibition in AGS
xenografts in nude mice. In this article, LP0040 is a non-nucleoside NAE and UAE dual inhibitor that
was identified through rational drug design on the basis of M22. By verification, LP0040 exhibited
proliferation inhibition against multiple cell lines and induced apoptosis in AGS cells (gastric
adenocarcinoma cells). Furthermore, synergistic effect on anti-proliferation between LP0040 and
bortezomib was also observed in this study.
2. Results and discussion
2.1. Molecular modeling and drug design
APPBP1-UBA3-NEDD8-ATP complex (PDB ID: 1R4N) was used for molecular modeling in our
previous work. M22 was docked into the ATP binding pocket, and molecular modeling results showed
that the 2, 4-dichloro-1-phenethyl group of M22 was predicted to map onto the phosphate groups of
ATP, and that benzyl group was forecasted to be located in the pocket where the adenine of ATP
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occupied. M22 was estimated to form one H-bond between the carboxyl group of Gly176 and the
secondary amine of M22. Additionally, 2, 4-dichlorophenyl group was estimated to have cation-π
interactions with Arg90 and Arg15. Herein, the replacement of benzyl group of M22 by adenine
analogous structure was considered to improve NAE inhibitory activity. Novobiocin was a DNA gyrase
and DNA topoisomerase IV dual inhibitor, whose 2H-chromen-2-one structure mapped onto the
adenine of ATP according to co-crystals of novobiocin with those two enzymes (PDB ID: 4URO,
1S14)[27, 28]. Therefore, 7-(4-(phenethylamino)piperidin-1-yl)-2H-chromen-2-one (8, shown in Fig. 3)
was designed, with benzyl group of M22 replaced by 2H-chromen-2-one. According to docking results
(Fig. 3), phenenyl group was estimated to have cation-π interactions with Arg90 and one H-bond was
formed between the carboxyl group of Gly176 and the secondary amine. More importantly, a new
H-bond was formed between the carbonyl in 2H-chromen-2-one and the backbone of Ile127.
The docking model was investigated carefully, and a hydrophobic region (in UBA3 subunit) which
was formed by Ile127, Ile149, Ala150 and Trp153was found behind the 4-position of
2H-chromen-2-one. In the co-crystal of NAE-NEDD8-MLN4924 (PDB ID: 3GZN),
2,3-dihydro-1H-inden-1-yl group was located in the hydrophobic region mentioned above. Therefore,
hydrophobic substituents were introduced to 4-position of 2H-chromen-2-one to occupy the
hydrophobic region.
Biaryl structures, with rotatable bond, suitable rigidity, volume and shape, are fit for binding with
proteins and are considered as privileged structures in anti-tumor drugs[29]. For the reason above,
hydrophilic and hydrophobic aryl rings were introduced on the 4 position of 2H-chromen-2-one (9-14,
shown in Fig. 4A). As shown in Table 1, 2H-chromen-2-one derivatives with phenyl substituents (9-12)
increased anti-proliferation activities against A549 (lung cancer), K562 (chronic myelogenous
leukemia), HCT-116 (colorectal carcinoma), SKOV-3 (ovarian cancer), BxPC-3 (pancreatic cancer) and
AGS (gastric cancer) cells, comparing with M22. Cancer cells used in this study were neddylation
up-regulated (A549) or MLN4924 sensitive [2, 12-14, 30]. Furthermore, compound 11 could inhibit
proliferation of AGS cells with the lowest IC50 value of 0.76 μM. However, uracil (13) and
1,2,3-1H-triazole (14) substituted derivatives could not inhibit proliferation of tested cells even at the
concentration of 30 μM. Those results suggested that 4-hydrophobic substituents would favor
inhibition activities, but 4- hydrophilic substituents would not.
In this study, ethynyl group was introduced as a linker connecting 2H-chromen-2-one with rings,
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owing to its hydrophobicity and linear structure. So compounds 15-18 (shown in Fig. 4A) were
designed, synthesized and evaluated. As shown in Table 1, those 4-ethynyl substituted derivatives
exhibited increased anti-proliferation activities, indicating that introducing ethynyl groups on
4-position of 2H-chromen-2-one would also contribute to the increasing of anti-proliferation activity.
One of the drawbacks of 2H-chromen-2-ones described earlier is the red blood cell hemolysis.
The hemolytic activities of 9, 10, 11, 12, 15, 16, 17 and 18 on rabbit red blood cells were assessed to
gain further insight into the potential toxic effects of the tested compounds. As shown in Fig. 4B, there
was no apparent sign of toxicity (<10% at 100 μM) to erythrocytes for 11, 16, and 17 at the
concentration of 100 μM, compared to MLN4924 (5.80% at 100 μM) In addition, Log P values of
compound 9, 10, 11, 12, 15, 16, 17 and 18 were also measured, and Log P value of each compound
was between 1 and 5 (shown in Fig. 4C).
Then, 11 was chose to investigate its toxicity against normal cells. In this study, HEK293T, HUVEC
and L-02 cells were used and LC50 values of 11 were respectively 12.93 μM, 10.69 μM and 13.54 μM
(shown in Table 2), which were about 14-18 times than IC50 values against AGS cells. However,
bortezomib showed non-selectivity against cancer cells and normal cells. Considering that NAE was
widely expressed in non-cancer cells at a low level, NAE inhibitors would have some effect on
non-cancer cells at high concentrations. The difference between the anti-proliferation activities of 11
against AGS cells and tested normal cells suggested that proliferation inhibition of 11 against AGS cells
might be induced by NAE inhibition rather than general toxicity. Furthermore, those results indicated
that NAE inhibitor might target cancer cells more selectively over normal cells.
With the lowest IC50 value against AGS cells and moderate selectivity to normal cells, 11 (LP0040) was
selected for further validation.
2.2. LP0040 inhibited NAE activity in enzyme-based assay
A dose-response experiment was performed using an enzymatic assay which measured the level
of NAE-mediated formation of the Ubc12-NEDD8 (Ubc12 is one of E2s for NEDD8 conjugation)
thioester product in a cell-free system. Decreased level of Ubc12-NEDD8 conjugation indicates the
inhibition of NAE. Recombinant human NAE was incubated with NEDD8, Ubc12 and ATP in the
presence of LP0040 for 1h. Then levels of Ubc12-NEDD8 thioester product were detected by western
blot. A concentration-dependent reduction of the intensity of Ubc12-NEDD8 bands was observed and
formation of Ubc12-NEDD8 was evidently inhibited at 0.37 μM (Fig. 5A and Supplementary Fig. S1A),
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indicating that compound LP0040 was an effective NAE inhibitor.
2.3. LP0040 inhibited NAE activity in cell-based assay
To validate whether LP0040 could inhibit NAE in AGS cells, AGS cells which were treated with
LP0040 under 0 – 90 μM for 24 h were lysed and protein extracts were analyzed by western blot. The
NEDD8 combined Uba3 was noticeably decreased in a concentration-dependent manner, as well as
the decrease of Ubc12-NEDD8 and Cullins-NEDD8 (Fig. 5B and Supplementary Fig. S1B), indicating
that NAE activity was inhibited and that neddylation was suppressed by LP0040. Inhibition of NAE
activity results in the inactivation of CRLs, leading to a decreased degradation of proteins which are
mediated by CRLs. As shown in Fig. 5B and Fig. S1C, treatment of AGS cells with LP0040 for 24 h
resulted in a reciprocal increase in the abundance of known CRL substrates CDT1, p27 and NRF2,
suggesting that LP0040 could stabilize CRL substrates through suppressing neddylation. However, 13
which was selected as a negative control in this assay was not able to inhibit NAE activity or block
neddylation of CRLs (Fig. 5D and Supplementary Fig. S1D). From those data, LP0040 was considered to
suppress NAE activity and to downregulate neddylation in vitro.
2.4. Selectivity of LP0040
As NAE was homologous to Ub and Ubl E1s, the selectivity profile of LP0040 for NAE was also
investigated in cell-based assays evaluating levels of E2-UBL thioester products by western blot. As
shown in Fig. 5C, formation of those Ubc9-SUMO and USE1-Ub (Ubc9 and Use1 are E2 enzymes that
participate in the conjugation cascade for SUMO and FAT10 labeling, respectively) were almost not
suppressed at different concentrations of LP0040, while neddylation was blocked by LP0040 at those
concentrations, which illustrated that LP0040 had no inhibition on SAE and Uba6. However, LP0040
showed a degree of inhibitory activity on UAE, so that LP0040 might be an NAE/UAE dual inhibitor.
Since 2H-chromen-2-one are known as promiscuous binders to ATP binding pocket, LP0040 was
also submitted to a selectivity screen against a panel of 13 kinases. As shown in Table 3, LP0040
showed less than 15 % inhibition against tested kinases at 30 μM, indicating that LP0040 might have
little effect to other kinases.
2.5. NAE and UAE Dual Inhibition by LP0040
To test whether LP0040 was an UAE inhibitor, an enzyme based assay was also conducted. Similar
to NAE inhibition assay, UAE was incubated with Ub, Ubc10 and ATP in the presence of LP0040 for 1h.
Levels of Ubc10-Ub thioester product were detected by western blot. As shown in Fig. 5A and
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Supplementary Fig. S1A, LP0040 also suppressed Ubc10-Ub formation, suggesting that LP0040 was an
NAE/UAE dual inhibitor.
2.6. Apoptosis Induced by LP0040 In AGS Cells
Flow cytometic analysis showed that apoptotic cells appeared with a dose-dependent pattern in
LP0040-treated AGS cells when the cells were double labelled with annexin-V-FITC and PI (Fig. 6).
After treatment with LP0040, the apoptotic rates (Annexin V+) of AGS cells were increased to 83.44%
of total cells (10 μМ, 12 h) whereas only few cells were observed as apoptotic cells in the control
group. Besides that, necrotic rates (PI+/Annexin V-) of AGS cells were always below 10%. Caspase-3 is
a cytosolic protein that is activated by proteolytic cleavage. It is one of the key factors in the apoptotic
pathway. The inactive 32 kDa proenzyme can be cleaved into 20 kDa (p20) and 11 kDa (p11) active
subunits, which can lead to cell apoptosis. In this study, we examined the promotion effects of LP0040
on the activation of caspase-3 in AGS cells after 24 h treatment. As shown in Fig. 5B immunoblot
analysis indicated that LP0040 treatment increased the expression of cleaved caspase 3. Taken
together, these results suggested that treatment of LP0040 promoted cell apoptosis in AGS through
activating caspase 3.
2.7. G1 Arrest and Apoptosis Induced by LP0040
To further explore whether growth inhibition and apoptosis induced by LP0040 was associated
with regulation of the cell cycle, DNA content of cell nuclei was detected by flow cytometry. The
effects of LP0040 on AGS cell cycle distribution were shown in Fig. 7A-7B. With the percentage of G1
phase increased from 22.82% to 48.88%, AGS cell cycle was arrested in G1, treated with 4 μM LP0040.
Furthermore, treating AGS cells with 4 μM LP0040, sub-G1 cells were detected at 24 h, indicating that
LP0040 could induce apoptosis.
2.8. Synergistic Effect of LP0040 with Bortezomib
Because NEDD8 pathway locates on the upstream of UPS, LP0040 may have synergistic effects
with bortezomib which is a selective inhibitor of 26S proteasome on the downstream of UPS. To verify
the effect of combination of LP0040 and bortezomib on AGS cells, Jin equation (the modified Burgi
formula, shown below) whose concept was universally accepted in pharmacology was employed [31,
32]. In Jin equation, the q values presenting < 0.85, 0.85–1.25, 1.25–2 and ˃ 2 suggest antagonism,
simple addition, synergism and apparent synergism respectively. Inhibition efficiency of drug alone
and drug combination on AGS cells’ growth at 48 h after drug introduction were measured and
q
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values for every drug combination at different concentrations were calculated. Results are shown in
Fig. 7C and Table 4. Synergistic effect on anti-proliferation was observed in those drug combinations
as follows: 10 nM BTZ with 1 μM LP0040, 10 nM BTZ with 1.25 μM LP0040 and 10 nM BTZ with 1.5 μM
LP0040. In addition, apparent synergism was noticed in drug combinations, such as 1.25 μM LP0040
with 5 nM or 7 nM BTZ.
ELP0040+BTZ
ꢀ =
ELP0040 + EBTZ − ELP0040 × EBTZ
Where ELP0040, EBTZ and ELP0040+BTZ are the inhibition rates of LP0040, bortezomib, and combination
of LP0040 and bortezomib, respectively.
Proteasome inhibitors bortezomib and carfilzomib are clinically used as first-in-line treatments
for multiple myeloma now, and show clear clinical efficacy. However, these agents have not found use
as therapeutic agents to treat solid tumors. In some cases, proteasome inhibitors also have shown
limited clinical efficacy in treating multiple myeloma [33-35]. These limited responses are in part due
to the alternative degradation of misfolded proteins via the aggresomal pathway. Therefore, new
approaches to UPS are urgently needed. Inhibitors of UAE and NAE activating enzyme are potential
therapeutic agents. Pharmacological dual inhibition of UAE and NAE inhibits ubiquitin conjugation,
resulting in protein degradation suppression. This paper reported the discovery of LP0040, a
non-nucleoside NAE/UAE dual inhibitor, which inhibited both NEDD8-independent and
NEDD8-dependent ubiquitin conjugation and protein degradation. Then LP0040 was used to validate
the therapeutic potential of the UPS to treat solid tumor through using AGS cells as a model system.
On the basis of M22 which was a new class of NAE inhibitor, a series of 4-substituted
2H-chromen-2-one derivatives were designed and synthesized. In this study, the modification strategy
used was similar to a previous work reported by J.L. Lukkarila [30]. However, rigid groups instead of
flexible groups were introduced to occupy the back hydrophobic pocket, which might improve
selectivity. Through anti-proliferation assay, 4-hydrophobic substituents was found favor activities of
2H-chromen-2-one derivatives, and that 4- hydrophilic substituents was not. LP0040 could inhibit
proliferation of AGS cells with evidently inhibition against NAE. Meanwhile, compound 13 whose
4-position of 2H-chromen-2-one was substituted by a hydrophilic group almost have no effect on NAE
activity or AGS proliferation. Those results indicated necessity of hydrophobic substituent on the
4-position of 2H-chromen-2-one for NAE inhibition. Investigating the docking model carefully, a
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hydrophobic region was found locating behind the 4-position of 2H-chromen-2-one. The docking
model coincided with experiment results, suggesting that the docking model could be used as a
prediction model for molecular modification．
LP0040 showed the lowest IC50 value against AGS cells. Treatment of AGS cells with LP0040 for 24
h induced a dose-dependent decrease of NEDD8 and Ub conjugations, resulting in a reciprocal
increase in the abundance of the known CRL substrates CDT1, NRF2 and p27. When the concentration
of LP0040 was 8 μM, NEDD8 conjugation was almost completely inhibited. On contrast, CRL substrates
were accumulated noticeably and cleaved caspase 3 was observed at 8 μM of LP0040.
Correspondingly, Anexin V-positive cells were detected at 8 μM of LP0040. Therefore, proliferation
inhibition and apoptosis were induced by UAE/NAE dual suppression of LP0040, but not cytotoxicity.
Although expected selectivity was not achieved, an UAE/NAE dual inhibitor was identified.
Furthermore, LP0040 was proved to inhibit proliferation of multiple cell lines and have synergistic
action on anti-proliferation activity with bortezomib. Thus LP0040 might improve the therapeutic
efficacy of bortezomib in solid tumors in the future.
3. Conclusion
The identification of LP0040 and related 2H-chromen-2-ones as UAE/NAE inhibitors represents
an important step forward in developing leads for preclinical evaluation of inhibitors of UAE/NAE in
cancer and potentially other diseases. LP0040 increases the level of a cell cycle inhibitor, activates
caspase 3, and induces cell apoptosis. All of these are desirable effects for a cancer therapeutic.
However, inhibitors of UAE/NAE are considered to induce undesirable side effects on protein dynamics
as proteasome inhibitors. In this paper, a simple structure-activity relationship (SAR) study was
conducted, and a more detailed SAR investigation was needed to develop leads for preclinical
evaluation of inhibitors of UAE/NAE in cancer and potentially other diseases. Further investigations of
the pharmacological properties of LP0040 in vitro and in vivo will also be reported in the near future.4.
4. Experiment sections
4.1 Chemistry
4.1.1 General procedures for the synthesis of target compounds
The synthetic strategy used to prepare the target compounds 9-18 is outlined in Scheme 1-2. The
key intermediate 28 was synthesized from phenethylamine (19) and 4-bromo-2-hydroxyacetophenone
(23) as start materials. Intermediate 20 was made from 19 and N-benzyl-4-piperidone through Borch
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reduction. Compound 21 was obtained by protection of 20’s secondary amine, which was followed by
Pd/C catalytic hydrogenation to provide intermediate 22. In present of NaH, start material 23 reacted
with dimethyl carbonate to provide compound 24 by one-pot synthesis. Intermediate 25 was obtained
by protection of hydroxyl group in compound 24. Through Buchwald–Hartwig coupling reaction,
compound 26 was synthesized from intermediate 22 and 25. With Pd/C catalytic hydrogenation of
compound 26, compound 27 was obtained. Then compound 27 reacted with trifluoromethanesulfonic
anhydride to get key intermediate 28. From intermediate 28, Target compounds 9-13 could be
achieved through Suzuki-Miyaura coupling reaction and deprotection of Boc, while compounds 15-18
would be provided through Sonogashira coupling reaction and deprotection of Boc. Target compound
14 was synthesized from intermediate 28 through Sonogashira coupling with Trimethylsilylacetylene,
addition reaction with trimethylsilylacetylene, and deprotection of Trimethylsilyl group and Boc group.
4.1.2 Synthetic method
Synthetic method is detailedly described in supplementary methods.
4.2. Tested compounds
Bortizomib (B48966) was provided by Bepharm (Shanghai, China). Tested 2H-chromen-2-one
derivatives were synthesized according to Scheme 1–2. All synthesized target compounds were
characterized by 1H NMR (Supplementary Fig. S2–Fig. S21), 13C NMR (Supplementary Fig. S22–Fig. S31)
and HPLC (Supplementary Fig. S32–Fig. S41). Purity of all tested compounds was all over 95%.
4.3. Cell Lines and Culture Conditions
The tumor cell lines used in this study were obtained from the American Type Culture Collection
(ATCC), unless specified otherwise. K562 (provided by professor Hai Qian, China Pharmaceutical
University), BxPC-3 (CRL-1687), and A549 (CCL-185) cells were grown in RPMI 1640 (Wisent)/10% (v/v)
fetal bovine serum (FBS, Wisent). AGS (CL-0022, from Procell) cells were cultured in Ham’s F12
medium (Wisent)/10% (v/v) FBS. HCT-116 (CCL-247) and SKOV-3 (HTB-77) were cultured in DMEM
medium (Wisent)/10% (v/v) FBS. All cells were cultured at 37 ˚C with 5% CO2. All cell lines were
obtained between 2010 and 2016 and kept in culture up to 15 to 20 passages, but not longer than 6
months. All cell lines were authenticated by the suppliers using short tandem repeat (STR) analysis.
4.4. Antiproliferation assay
Cell counting kit-8 (CCK-8, Dojindo) was used to evaluate the proliferation of cultured cells [36,
37]. Cancer cells (K562, BxPC-3, A549, HCT-116, SKOV-3 and AGS) were prepared and diluted to a
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concentration of 6×104 cells/mL. 100 µL of the cell suspension was seeded in each well of 96-well
plates, totaling 6×103 cells/well. After 12 h incubation at 37 ˚C in 5% CO2, the medium in the plates
was replaced by 150 µL medium containing serial dilutions of compounds. Bortizomib was used as a
positive control. Following 48 h culture, 10 μL CCK-8 was added to each well and cells were incubated
for another 4 h. The light absorption value (OD) of each well was measured at 450 nm. The
experiment was repeated 3 times. IC50 values were calculated from the inhibition curves by
normalized nonlinear regression analysis using GraphPad Prism 5 software.
4.5. Enzyme based NAE activity assay
This assay was performed using the NEDD8 conjugation initiation kit (K-800, R&D) according to
the instructions of the manufacturer. 2 μL NAE (2.5 μM), 2 μL NEDD8 (250 μM), 2 μL Ubc12 (50 μM,
Ubc12 is the E2 enzyme that participate in the conjuation cascade for Nedd8 labeling), 2 μL reaction
buffer and 10 μL water solution of LP0040 (0 μM, 0.12μM, 3.7 μM, 1.11 μM, 3.33 μM, 10 μM) were
added into 100 μL reaction tubes in order. Reaction solutions were incubated at 37 ˚C for 10 min. The
reaction was initiated by the addition of 2 μL Mg2+/ATP solution (10 mM), and the mixture was
incubated at 37 ˚C for 60 min. In negative control group, 2 μL ddH2O was added instead of ATP
solution. The reaction was quenched by the addition of 2 μL EDTA (1 M), and protein samples were
electrophoresed under non-reducing conditions on a 15% SDS-PAGE gel. Ubc12-NEDD8 levels were
determined by western blot analysis.
4.6. Enzyme based UAE activity assay
This assay was performed using the ubiquitin conjugation initiation kit (K-995, R&D) according to
the instructions of the manufacturer. 2 μL UAE (2.5 μM), 2 μL Ub (250 μM), 2 μL Ubc10 (50 μM, Ubc10
is the E2 enzyme that participate in the conjuation cascade for ubiquitin labeling), 2 μL reaction buffer
and 10 μL water solution of LP0040 (0 μM, 0.12μM, 3.7 μM, 1.11 μM, 3.33 μM, 10 μM) were added
into 100 μL reaction tubes in order. Reaction solutions were incubated at 37 ˚C for 10 min. The
reaction was initiated by the addition of 2 μL Mg2+/ATP solution (10 mM), and the mixture was
incubated at 37 ˚C for 60 min. In negative control group, 2 μL ddH2O was added instead of ATP
solution. The reaction was quenched by the addition of 2 μL EDTA (1 M), and protein samples were
electrophoresed under non-reducing conditions on a 15% SDS-PAGE gel. Ubc10-Ub levels were
determined by western blot analysis.
4.7. Cell based NAE activity assay
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AGS cells were exposed to the indicated concentrations of LP0040 (2 μM, 4 μM, 6 μM, 8 μM, 10
μM) or 0.1% (v/v) DMSO for 24 h. Cells were washed three times with ice-cold PBS, resuspended in
RIPA lysis buffer (P0013B, Beyotime), and incubated on ice for 30 min. Cell debris was removed by
centrifugation at 13,000 rpm for 30 min at 4 ˚C. The protein concentration of each supernatant sample
was determined with Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher). Equal protein amounts
were electrophoresed under non-reducing (Uba3-NEDD8, Ubc12-NEDD8, Cullins-Nedd8, Ubc9-SUMO,
Ubc10-Ub and USE1-Ub) or reducing (p27, NRF2, CDT1 and cleaved caspase 3) conditions on
SDS-PAGEs and subjected to western blot analysis. All experiment was repeated 3 times.
4.8. Western blot analysis
Protein samples were transferred to a PVDF membrane. The membrane was blocked with 5%
(w/v) milk for 1 h at room temperature, and probed with primary antibody diluted (1:1000, v/v ) in 5%
(w/v) milk overnight at 4 ˚C. The membrane was washed with TBS/0.1% (v/v) Tween 20 (TBST) and
incubated with horseradish peroxide-conjugated secondary antibody diluted (1:4000, v/v) in 5% (w/v)
milk for 1.5 h at room temperature. Protein bands were detected using SignalFire™ ECL Reagent (6883,
Cell Signaling Technology, CST).
In this assay anti-NEDD8 antibody (2745, CST) was used to measure Uba3-NEDD8, Ubc12-NEDD8
and Cullins-NEDD8. Anti-Ubc9 (4918, CST), anti-Ubc10 (A-650, R&D), anti-USE1 (Sc-102150, Santa
Cruz), anti-p27 (KF446, Nanjing Jiancheng Bioengineering Institute), anti-CDT1 (8064, CST), anti-NRF2
(12721, CST) and anti-cleaved caspase 3 (9661, CST) antibodies were utilized to recognize Ubc9-Ub,
Ubc10-SUMO, USE1-Ub, p27, CDT1, NRF2 and cleaved caspase 3 respectively. Densitometry analysis of
the Western blot was conducted by using Image J. All experiment was repeated 3 times.
4.9. Apoptosis/necrosis assay
Apoptosis/necrosis was evaluated using FITC annexin V apoptosis detection kit (556547, BD
Biosciences). AGS cells were seeded at 3×105 cells per well in 6-well plates and allowed to attach in 12
h. Cells were treated with different concentrations of LP0040 (0 μM, 4 μM, 6 μM, 8 μM, and 10 μM)
for 36 h. Cells were dissociated using trypsin. Dissociated cells were washed twice with cold PBS and
resuspended in 100 μL 1× binding buffer, followed by the addition of 5 μL FITC annexin V staining
solution and 5 μL PI staining solution. After incubation at room temperature in the dark for 15 min,
another 400 μL 1×binding buffer was added. Stained cells were analyzed immediately by flow
cytometry (FACSCalibur, BD Biosciences). In each sample 10000 cells were examined, and fluorescence
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was measured at an excitation wave length of 488 nm through FL-1 (530 nm) and FL-3 (630 nm) filters.
4.10. Cell cycle assay
AGS cells were seeded at a density of 1×105 cells per well in a final volume of 2 mL/well in a
six-well plate and left to attach for 24 h at 37 ˚C in a 5% CO2 incubator. Cells treated with 4 μM LP0040
for different time were harvested and fixed in 70% ethanol at −20 °C overnight. They were then
incubated with PI/RNase staining buffer (550825, BD Biosciences) in the dark for 15–20 min and
analyzed by flow cytometry (FACSCalibur, BD Biosciences). In each sample 20000 cells were examined,
and fluorescence was measured at an excitation wave length of 488 nm through FL-3 (630 nm) filters.
The experiment was repeated 3 times.
4.11. Synergistic effect of LP0040 with bortezomib
AGS cells (6×103/well) were plated in 96-well plates. After 12 h for attachment, AGS cells were
treated with indicated concentrations of LP0040, bortezomib and combination, respectively. Following
48 h incubation, 5 μL CCK-8 was added to each well and cells were incubated for 4 h. The light
absorption value (OD) of each well was measured at 450 nm. Inhibition rates were calculated, and the
q value was calculated according to the equation:
ELP0040+BTZ
ꢀ =
ELP0040 + EBTZ − ELP0040 × EBTZ
Where ELP0040, EBTZ and ELP0040+BTZ are the inhibition rates of LP0040, bortezomib, and combination
of LP0040 and bortezomib, respectively.
The experiment was repeated 3 times.
4.12. Hemolysis assay
Hemolysis assay was conducted as the method previously reported[38]. Rabbit erythrocytes were
harvested by centrifugation for 10 min at 3000 rpm, washed two times with PBS and finally suspended
in PBS for immediate use. Of that suspension, 150 μL was added in 1.5 mL centrifuge tube containing
150 μL of 200 μM tested compounds in the same buffer. 0 and 100% hemolysis was respectively
achieved with PBS and 1% Triton X-100. Centrifuge tubes were incubated for 1 h at 37 ˚C and
suspensions were centrifuged at 3000 rpm for 5 min. Supernatant were transferred to 96-well plates
and hemoglobin release was measured by microplate reader at 540 nm. Hemolytic ratio was
calculated according to the equation:
ꢁ
ꢅꢁ
ꢆꢇꢈꢂꢉꢇꢊ
ꢂꢃꢄꢂ
Hemolysis% = ꢁ
× 100%
ꢅꢁ
ꢆꢇꢈꢂꢉꢇꢊ
ꢋꢉꢌꢂꢇꢈꢍꢎꢏꢐꢐ
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4.13. Log P measurement
Log P values of 9, 10, 11, 12, 15, 16, 17 and 18 were measured using shake-flask method. Each
compound was dissolved in 1 mL water (water was saturated with 1-octanol), and then an equal
volume of 1-octanol (1-octanol was saturated with water) was added. The tube was shaken for 24 h at
37 °C to allow the compound to partition to each phase. After being centrifugated at 5000 rpm ,
compound in each phase was determined by HPLC (LC-20A, SHIMADZU) at 254 nM. All the
experiments were carried out in triplicate. The partition coefficient was calculated using the equation
below.
ꢓꢔꢕ
ꢓꢔꢕ
ꢖꢗꢘꢙꢚꢖꢛ
ꢜꢙꢘꢝꢞ
Log P=log
ꢑꢒ
Supporting information. Supplementary data related to this article can be found at
https://doi.org/ XXXXXXXXX.
Author information
Corresponding author
* Yubin Wang, School of Pharmaceutical Sciences, Nanjing Tech University, No. 5 Xinmofan Road,
Nanjing 210009, People's Republic of China. Tel. +86 25 83172108; Fax: +86 25 83172105; E-mail:
wyb5393@njtech.edu.cn.
Conflicts of interest
None of the authors of the above manuscript has declared any conflict of interest which may
arise from being named as an author on the manuscript.
Acknowledgements
The authors thank Professor Hai Qian (China Pharmaceutical University, Nanjing, China) for
providing the K562 cells. This work was supported by the Natural Science Foundation for Colleges and
Universities in Jiangsu Province (No. 11KJB350002), the Natural Science Foundation of Jiangsu
Province (No.BK2012422, BK20141015) and the National Natural Science Foundation of China (No.
81202393, 81600159).
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Table 1. IC50 values of tested compounds (μM, n=3)
Cell line
9
A549
AGS
K562
HCT-116
5.82 ± 2.40
6.00 ± 0.90
1.33 ± 0.13
6.11 ± 0.23
> 30
BxPC-3
8.44 ± 1.47
7.67 ± 1.56
2.80 ± 0.20
2.82 ± 0.37
> 30
SKOV-3
6.42 ± 0.48
5.94 ± 0.99
3.29 ± 1.28
3.22 ± 0.13
> 30
3.77 ± 0.67 1.89 ±0.27 3.26 ± 0.64
3.09 ± 0.99 1.39 ± 0.35 3.29 ± 0.78
2.14 ± 0.37 0.76 ± 1.26 2.45 ± 0.31
3.83 ± 0.88 1.26 ± 0.17 1.53 ± 0.39
10
11
12
> 30
> 30
> 30
> 30
> 30
> 30
13
> 30
> 30
> 30
14
2.99 ± 0.41 1.13 ± 0.32 1.17 ± 0.18
3.10 ± 1.12 1.42 ± 0.17 1.32 ± 0.32
3.35 ± 0.79 1.66 ± 0.15 3.30 ± 1.01
2.48 ± 0.27 1.71 ± 1.17 2.63 ± 0.26
2.81 ± 0.33
4.27 ± 1.01
2.69 ± 0.28
2.79 ± 1.00
4.08 ± 1.26
1.33 ± 0.24
4.33 ± 1.25
2.01 ± 0.25
4.17 ± 0.29
7.50 ± 1.66
7.70 ± 0.42
4.35 ± 0.19
9.26 ± 1.11
15
16
17
18
5.55 ± 0.17 9.92 ± 0.91 5.51 ±1.18 10.57 ± 1.84 12.40 ± 3.68
M22
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Table 2. LC50 values of LP0040, MLN4924 and bortezomib against normal cells (μM, n=3)
Cells
LP0040(11)
MLN4924
BTZ
HEK293T
12.93 ± 1.09
6.17 ± 0.44
0.016 ± 0.003
HUVEC
L-02
10.69 ± 0.83
4.79 ± 0.27
0.018 ± 0.002
13.54 ± 2.37
9.01 ± 0.98
0.398 ± 0.041
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Table 3. Percentage inhibition values of kinase activity by LP0040 (30 μM)
Kinase
PKAα
PKCα
Aur A
B-raf
CDK1
CDK2E
CHK1
CHK2
LCK
% inhibition
5.17 ± 0.49
11.49 ± 1.27
6.03 ± 0.77
1.75 ± 0.26
-1.18 ± 0.21
4.57 ± 0.39
1.01 ± 0.14
9.44 ± 1.44
-2.51 ± 0.53
5.58 ± 0.48
9.87 ± 1.21
2.77 ± 0.37
1.07 ± 0.14
PLK
AKT
EGFR
IKKb
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Table 4. q values for each combination of BTZ and LP0040
LP0040 (μM)
BTZ
(nM)
1
1.25
1.5
4.34**
3.1**
1.75*
5
0
1.15
1.05
1.35*
7.5
10
1.13
1.14
** apparent synergism；* synergism
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Figure Legends
Fig. 1 overview of the UPS
Fig. 2 Structures of reported NAE inhibitors.
Fig. 3 Scaffold replacement of M22: (A) design of compound 8; (B) low-energy binding conformations
of 8 bound to NAE heterodimer generated by AutoDock 4. APPBP1-UBA3-NEDD8-ATP complex (PDB ID:
1R4N) was used in docking study (red dashed: H-bond; yellow line: π-cation interaction); (C) merging
between top-ranked 8 pose (shown in blue) and ATP conformation (shown in red) from
APPBP1-UBA3-NEDD8-ATP complex (PDB ID:1R4N).
Fig. 4 Structures, Log P and hemolysis rate of 7-(4-(phenethylamino) piperidin-1-yl)-2H-chromen-2-
one based NAE inhibitors. (A) Structures of designed NAE inhibitors; (B) hemolysis of designed NAE
inhibitors (* hemolysis rate < 10%, n=3); (C) Log P of designed NAE inhibitors (n=3).
Fig. 5 Compound LP0040 was an NAE and UAE dual inhibitor: (A) LP0040 inhibited NAE and UAE
activities in enzyme-based assay; (B) LP0040 could inhibited neddylation and CRL substrate
degradation in AGS cells; (C) selectivity of compound LP0040 in AGS cells; (D) compound 13 could not
inhibit NAE activity in AGS cells.
Fig. 6 Apoptosis induced by LP0040 in AGS cells. The ratio of apoptotic cells (Annexin V+ cells)
increased from about 6% to more than 80% with concentration of LP0040 increasing from 0 µM to 10
µM.
Fig. 7 Cell-based activity evaluation of LP0040: (A) cell cycle analysis through flow cytometry (n=3); (B)
cell cycle statistics of AGS cells treated with LP0040; (C) synergistic effect between LP0040 and
Bortezomib against AGS cells. Inhibition efficiency of drug alone and drug combination on 6 × 103
AGS cells’ growth at 48 h after drug introduction. Results are presented as mean ± SD (error bar)
of triplicate experiments. Symbols “*” and “**” represent the grades of synergism and apparent
synergism, respectively, for evaluating effect of drug combination.
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