Discovery of a novel sodium taurocholate cotransporting polypeptide (NTCP) inhibitor: Design, synthesis, and anti-proliferative activities

Article abstract of DOI:10.1016/j.cclet.2020.03.017

Sodium taurocholate cotransporting polypeptide (NTCP) is identified as the functional receptor for HBV entry, which is responsible for upregulated HBV transcription in the HBV life cycle. Besides, NTCP is also implicated in the progression of HBV-induced hepatocellular carcinoma (HCC). Thereby, NTCP-targeting entry inhibitors are proposed to suppress HBV infection and replication in HBV-induced hepatoma therapy. Herein, we integrated in silico screening and chemical synthesis to obtain a small-molecule NTCP inhibitor B7, which exhibited moderate anti-proliferative activities against HepG2 cells and anti-HBV activity in vitro. Additionally, CETSA assay, molecular docking, and MD simulation validated that B7 could bind to NTCP. Furthermore, western blot analysis demonstrated that B7 induced apoptosis with an increased expression of Bax and caspase 3 cleaving as well as a decreasing expression of Bcl-2 in HepG2 cells. Taken together, our study identified B7 as a novel NTCP inhibitor with anti-proliferation activities which might provide a new opportunity for HCC therapy.

Full text of DOI:10.1016/j.cclet.2020.03.017

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Discovery of a novel sodium taurocholate cotransporting polypeptide
(NTCP) inhibitor: Design, synthesis, and anti-proliferative activities
Honggang Xiang, Yanmei Chen, Jifa Zhang, Jin Zhang, Dabo Pan,
Bo Liu, Liang Ouyang
PII:
S1001-8417(20)30136-4
https://doi.org/10.1016/j.cclet.2020.03.017
CCLET 5515
DOI:
Reference:
To appear in:
Chinese Chemical Letters
Accepted Date:
6 March 2020
Please cite this article as: Xiang H, Chen Y, Zhang J, Zhang J, Pan D, Liu B, Ouyang L,
Discovery of a novel sodium taurocholate cotransporting polypeptide (NTCP) inhibitor:
Design, synthesis, and anti-proliferative activities, Chinese Chemical Letters (2020),
doi: https://doi.org/10.1016/j.cclet.2020.03.017
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Communication
Discovery of a novel sodium taurocholate cotransporting polypeptide (NTCP)
inhibitor: Design, synthesis, and anti-proliferative activities
a,1
a,1
a,1
a
b,
a,
a,
Honggang Xiang , Yanmei Chen , Jifa Zhang , Jin Zhang , Dabo Pan *, Bo Liu *, Liang Ouyang *
a
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy,
Chengdu 610041, China
b
Institute of Traditional Chinese Medicine & Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New
Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, China
Graphical Abstract
We discovered a small-molecule NTCP inhibitor B7 that was identified to bind to NTCP and induced apoptosis in HepG2 cells.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Sodium taurocholate cotransporting polypeptide (NTCP) is identified as the functional
receptor for HBV entry, which is responsible for upregulated HBV transcription in the HBV life
cycle. Besides, NTCP is also implicated in the progression of HBV-induced hepatocellular
carcinoma (HCC). Thereby, NTCP-targeting entry inhibitors are proposed to suppress HBV
infection and replication in HBV-induced hepatoma therapy. Herein, we integrated in silico
screening and chemical synthesis to obtain a small-molecule NTCP inhibitor B7, which
exhibited moderate anti-proliferative activities against HepG2 cells and anti-HBV activity in
vitro. Additionally, CETSA assay, molecular docking, and MD simulation validated that B7
could bind to NTCP. Furthermore, western blot analysis demonstrated that B7 induced apoptosis
with an increased expression of Bax and caspase 3 cleaving as well as a decreasing expression
of Bcl-2 in HepG2 cells. Taken together, our study identified B7 as a novel NTCP inhibitor with
anti-proliferation activities which might provide a new opportunity for HCC therapy.
Available online
Keywords:
Sodium taurocholate cotransporting polypeptide
(NTCP)
Hepatocellular carcinoma (HCC)
HBV infection
NTCP inhibitor
Apoptosis
Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide as a severe global problem
of public health, with over 80% of HCC cases occurring in low- and middle-resource countries, where medical and social care resources
are often constrained [1]. As the main risk factor of HCC, chronic HBV infection accounts for most of global HCC cases of 257 million,
especially in Asia-Pacific regions [2]. According to the latest statistics, on average, the disease progression for ∼25% of untreated
individuals will finally die of HCC and/or cirrhosis complications, and this rate even increases to 50% if only men are taken into
consideration [3]. Currently, approved treatment agents for HCC are broadly classified into immunomodulatory agents [4,5] and antiviral
agents [6], the former including conventional IFNα-2b and Peg-IFNα-2a and the latter including nucleoside- or nucleotide-based reverse-
*
Correspondence authors.
E-mail addresses: pandabo05@163.com (D. Pan), liubo2400@163.com (B. Liu), ouyangliang@scu.edu.cn (L. Ouyang).
These authors contributed equally to this work.
1

transcriptase inhibitors such as nucleotide analogues (NUCs), namely, lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir
disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). The mainstay treatment for HCC in most countries is NUCs. Nevertheless,
the clinical utilization of IFNα-2b and Peg-IFNα-2a is subject to a low response rates (about 20%–30%) among patients with chronic
hepatitis B and a host of side effects, including flu-like symptoms, anemia, leucopenia, thrombocytopenia, anorexia, and depression [7].
Besides, the emergence of drug resistance during long-term NUCs-treatment is almost inevitable and represents a clinical challenge. For
instance, approximately 70% of patients resistant to lamivudine [8] and entecavir also occurs among 43% lamivudine-pretreated patients
after 4 years [9]. Accordingly, to improve the treatment of HCC and reduce drug resistance, there stands an urgency to develop novel
agents with different therapeutic targets.
In recent years, targeted drugs for various types of cancer have developed rapidly [10-13]. In particular, the researches about drug
development for HCC are increasing and attract a lot of interests [14,15]. Sodium taurocholate cotransporting polypeptide (NTCP) is an
integral membrane glycoprotein with seven predicted transmembrane domains, which in humans is encoded by the SLC10A1 (solute
carrier family 10 member 1) gene [16], and is expressed in the basolateral membranes of hepatocytes. Functionally, as a key component
of the enterohepatic recovery of bile acids, NTCP participates in the enterohepatic circulation of bile acids. It is responsible for the
basolateral uptake of bile acids from the portal blood into hepatocytes. What is more, NTCP was identified to serve as the functional
receptor for HBV and HDV [17]. NTCP can specifically interact with the receptor-binding region of the pre-S1 domain of HBV/HDV
envelope protein and thus virus gain access to cells. And further mechanism research has elucidated that NTCP can upregulate HBV
transcription via farnesoid X receptor α (FxRα)-mediated activation of the HBV EN2/core promoter at the postentry step in an NTCP-
dependent manner, and NTCP-targeting entry inhibitor manage to suppress HBV infection and replication [18]. Increasing researches
have implicated that NTCP is involved in the progress of HCC: NTCP was down-regulated in HCC tissues and was up-regulated when
HCC cell lines were arrested in the G0/G1 phase [19]. Decreased SLC10A1/NTCP expression was also reported in a recent research
about integrated proteogenomic characterization of HBV-related HCC [20]. Therefore, these findings have showed that NTCP inhibitors
may offer a promising novel therapeutic option for HCC.
However, since homo NTCP was cloned 24 years ago, only a few human NTCP inhibitors have been identified [21,22]. To date,
the only recognized NTCP inhibitor is Myrcludex B (MyrB, phase II of clinical trials had been conducted), a myristoylated synthetic
lipopeptide comprising 47 amino acids derived from the pre-S1 domain of the HBV large surface protein. In the past few years, several
FDA-approved drugs (Fig. 1A) were identified as NTCP inhibitors via a combination of computational and in vitro approaches [23],
such as Fluvastatin [24], Macitentan [25], Bosentan [26] and Irbesartan [27]. Nevertheless, most of them have problems with target
selectivity and side effects. For instance, because of inhibition of the uptake transporters NTCP and OATP/Oatp, Fasiglifam may affect
bile acid and bilirubin homeostasis, causing hyperbilirubinemia and cholestatic hepatotoxicity [28]. Besides, another posed challenge is
that the full-size 3D crystal structure of NTCP is still not available, as a result, few NTCP inhibitors relied on reasonable structure-based
de novo drug design are reported. Therefore, the objective of the present study is to develop a novel NTCP inhibitor. Herein, we eventually
discovered a novel NTCP inhibitor B7 that exhibited good anti-proliferative activity and induced apoptosis in HepG2 cells. This work
reveals the potential of B7 as an NTCP inhibitor for future treatment of HCC.

Fig. 1. (A) Several FDA-approved drugs identified as NTCP inhibitors. (B) The workflow of the virtual screening protocol for NTCP inhibitors.
Given the challenges in pharmacologically inhibiting NTCP, we developed a hybrid strategy combining computational and
experimental approaches to screen potential NTCP inhibitors (Fig. 1B), homology modeling (Fig. S1 in Supporting information) was
carried out to obtain the model structure of NTCP for followed screening. Next, the active site of inhibition is predicted and defined as
the space where the helical structures of amino acids 84-87, 157-165 and 267 are embedded in view of the previous functional studies of
NTCP from different species [29]. Then, we virtually docked more than 150,000 compounds from the ZINC library by using LibDock
based on the predicted site pocket of NTCP according to Lipinski’s Rule of Five. Subsequently, the top 200 hits were further screened
using the CDOCKER protocol. Resultant 10 hits with better CDOCKER interaction energies and LibDock scores were selected and
purchased (Fig. S2 in Supporting information) on the basis of the top-ranked NTCP-compound binding models. Among these 10 hits,
AK-968/12572207 showed the best anti-proliferative activity with an IC50 of 22.913 ± 0.3 μmol/L as the leading compound for further
structural modifications.
Next, according to molecular modeling, we analyzed the hydrophobicity of amino acid residues around the AK-968/12572207-
NTCP binding pocket. We synthesized a series of analogs, A1–A24, containing different terminal substituent on the phenyl rings of
benzamides or benzene sulfonamides on both sides (Scheme 1A) in order to fit the hydrophobic cavity at the side chain of Y108 and
obtain suitable substitution vectors for improving activity. It was found that the anti-proliferation ability was improved when 4-position
substituent of the phenyl rings was cyano or trifluoromethyl (IC50 = 20.922 ± 0.8 μmol/L and 14.970 ± 0.5 μmol/L), and both are electron-
withdrawing groups. Intriguingly, while 4-position substituent group was replaced with other electron-withdrawing groups such as
fluorine atom and trifluoromethoxy, the anti-proliferation ability was lower than that of the lead compound, which might suggest that the
anti-proliferation ability was not completely determined by the electrophilicity of substituent groups. Meanwhile, compound A14
exhibited better anti-HBV activities than A6 and was therefore chosen for next optimization. Besides, we furtherly noticed the atmosphere
enriched with a negative charge around N119 (Fig. S3 in Supporting information). Accordingly, we hypothesized that introduction of an
appropriate positive charged group at the side chain of N119 may enhance the binding affinity. Therefore, B1–B8 with different positive
charged groups were synthesized (Scheme 1B). Indeed, we obtained B2 and B7 through introduction of dibutylamine and 1-
bocpiperazine, respectively, which exhibited better anti-proliferation ability with IC50 values of 10.881 ± 0.2 µmol/L and 7.360 ± 0.3
µmol/L, respectively (Table 1). Piperazinyl is a synergistic unit, which often appear in structures of anti-cancer drugs [30]. This may
explain the excellent anti proliferation ability of B7. However, the other compounds of B series did not return increased feedback of the
anti-proliferation ability, though the same positive charged groups were introduced, such as B4 and B5. We speculated that the accuracy
of the homology modeling of NTCP was responsible for the difference. On the other hand, the anti-HBV activities of B series compounds
were stronger than those of A series compounds, which may owe to the unique skeleton structure. Also, B2 and B7 also exhibited
moderate anti-HBV activities, compared with Tenofovir as the positive antiviral control.

Scheme 1. General procedures for the synthesis of compounds A1-A24 (A) and B1-B8 (B). Reagents and conditions: (a) Corresponding substituted
benzenesulfonyl chloride or benzoyl chloride, CH Cl , -5 ℃, 1 h; (b) Corresponding substituted benzenesulfonyl chloride, CH Cl , -5 ℃, 1 h; (c) Chloroacetic
CN, 80 ℃, reflux, 6 h.
2
2
2
2
2 2 2 3
chloride, CH Cl , -5 ℃, 1 h; (d) NHR , CH
Table 1
a
Anti-proliferative activities and anti-HBV activities of the compounds.
Anti-proliferative
activity (IC50, μmol/L)
4.237 ± 0.5
-
22.913 ± 0.3
>50
>50
>50
>50
>50
20.922 ± 0.8
>50
29.250 ± 1.6
>50
39.930 ± 0.7
24.522 ± 0.9
>50
25.183 ± 0.7
14.970 ± 0.5
>50
19.540 ± 0.9
>50
26.913 ± 0.3
24.044 ± 0.1
46.025 ± 0.6
>50
>50
41.382 ± 0.2
>50
HBsAg
(ng/mL)
-
HBeAg
(ng/mL)
-
Compd.
Cisplatin
Tenofovir
AK-968/12572207
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
B1
B2
B3
B4
B5
B6
B7
B8
1.92 ± 0.7
4.47 ± 0.5
9.46 ± 0.1
8.97 ± 0.3
7.13 ± 0.3
9.84 ± 0.8
7.88 ± 0.7
5.57 ± 0.2
7.67 ± 0.3
6.82 ± 0.4
9.04 ± 0.2
6.24 ± 0.3
5.29 ± 0.2
7.45 ± 0.4
6.26 ± 0.2
5.26 ± 0.3
8.62 ± 0.2
6.07 ± 0.5
8.80 ± 0.6
7.26 ± 0.7
7.09 ± 0.8
7.74 ± 0.7
9.46 ± 0.6
8.97 ± 0.8
7.48 ± 0.6
9.33 ± 0. 5
5.09 ± 0.2
4.07 ± 0.4
7.06 ± 0.2
7.73 ± 0.5
5.24 ± 0.2
7.58 ± 0.1
3.24 ± 0.2
7.06 ± 0.5
4.90 ± 0.2
17.88 ± 0.4
33.02 ± 0.7
29.33 ± 2.0
32.26 ± 0.9
27.68 ± 1.2
26.62 ± 0.5
23.02 ± 1.0
25.26 ± 0.8
17.38 ± 0.2
30.26 ± 0.5
24.46 ± 0.7
18.86 ± 0.5
25.59 ± 1.2
17.03 ± 1.3
14.59 ± 0.4
20.08 ± 1.0
15.69 ± 1.5
29.72 ± 0.8
21.02 ± 0.5
20.94 ± 1.8
22.25 ± 1.5
25.34 ± 2.1
27.23 ± 0.6
19.05 ± 0.4
28.89 ± 0.7
13.07 ± 0.6
6.46 ± 0.6
14.52 ± 1.7
15.34 ± 2.1
9.64 ± 1.0
15.30 ± 0.5
7.64 ± 0.5
13.09 ± 1.0
13.233 ± 1.3
10.881 ± 0.2
>50
>50
14.592 ± 1.5
41.317 ± 1.7
7.360 ± 0.3
>50
a
Each compound was tested in triplicate with Cisplatin as a positive control for anti-proliferative activity and Tenofovir as a positive control for anti-HBV activity.
Cellular Thermal Shift Assay (CETSA) has been developed as a popular method to monitor and quantify the extent to which a drug
candidate reaches and directly binds to a protein target of interest within a cell since several years ago [31]. And as a result, plenty of
researches have been utilizing CETSA for target verification [32]. And CETSA assessment by western blotting has revealed an increase
in the melting temperature of NTCP (Figs. 2B and C), which confirmed the interaction of B7 (Fig. 2A) and NTCP. In addition, to
demonstrate the stability of B7/NTCP complex, we also performed a 100 ns MD simulation on the B7/NTCP complex. The low root-
mean-square deviation (RMSD) fluctuations and the convergence of the energies, temperatures, and pressures of the system indicated

that it was a stable system (Fig. S4 in Supporting information). Furtherly, to explore the binding mode, the ligand structure with the most
favorable binding free energies and reasonable orientations was selected as the optimal docked conformation, the result showed that the
binding stability was mainly determined by hydrogen bonds (Fig. 2D). Thus, these results indicated that B7 was an NTCP inhibitor.
Fig. 2. B7 binds to NTCP. (A) Chemical structure of B7. (B) CETSA assessment by western blotting. (C) Statistics of NTCP/β-actin. (D) Cartoon representation
of NTCP model with docked B7.
Compound B7 exhibited a favorable anti-proliferation effect on HepG2 cells and no significant toxicity in normal liver cells (Fig.
S5A in Supporting information). Furtherly, to detect the effect of B7 on HCC cells, we firstly determined its effect on cell proliferation.
B7 showed a robust dose-dependent anti-proliferation effect on HepG2 cells after 24 h (Fig. 3A). Next, we found a significant increase
of apoptosis after B7 treatment in HepG2 cells. Condensed chromatin with bright fluorescence was observed in HepG2 cells after B7
treatment with Hoechst 33258 staining (Fig. 3B). Additionally, Annexin-V/PI double staining also confirmed that B7 induced dose-
dependent apoptosis (Figs. 3C and E). Next, we evaluated the JNK regulated classic mitochondrial apoptosis pathway and found B7
induced obviously up-regulation of JNK, p-JNK, caspase-3, Bax (Fig. 3F), and down-regulation of Bcl-2. Moreover, B7 could decrease
the expression of NTCP which contributes to the progression of HCC (Fig. 3D). These results demonstrated that B7 exhibited an anti-
proliferation effect on HepG2 cells by inducing apoptosis.

Fig. 3. B7 induced apoptosis in HepG2 cells. (A) The cell inhibition rates were measured after being treated with different concentrations of B7 for 24 h. (B)
Morphologic changes were observed under fluorescence microscopy with Hoechst 33258 staining after 24 h exposure to different concentrations of B7. Scale bar
=
50 μm. (C) The apoptosis ratios were determined by flow cytometry analysis with annexin-V/PI double staining after 24 h exposure to different concentrations
** ***
of B7. ns: not significance; P < 0.01, P < 0.001 vs. control. (D) The expressions of NTCP, JNK, p-JNK, caspase-3, Bax, and Bcl-2 were detected by western
blot analysis after different concentrations of B7 treatment for 24 h. β-actin was used as the loading control. P<0.01, P<0.001 vs. control. (E) The cell death
rate was measured after treated with different concentrations of B7. ns: not significance; P < 0.01, P < 0.001 vs. control. (F) Ratio of Bax/β-actin after cells
were treated with different concentrations of B7. ns: not significance; P < 0.01, P < 0.001 vs. control.
**
***
**
***
**
***
HCC induced by HBV infection remains one of the most prevalent yet undertreated public health problems. Furthermore, given the
unmet therapeutic drug diversity and the emergence of drug-resistant of present agents, developing new small-molecules with novel
targets represents an opportunity in HCC therapies, and thus, NTCP inhibitors may offer an alternative option for HCC treatment after
taking the relationship between NTCP and HCC into consideration.
In summary, combining in silico high-throughput screening, chemical synthesis and anti-proliferative activity screening, we
discovered a novel NTCP inhibitor B7 with favorable anti-proliferative activity against HepG2 cells. Moreover, we demonstrated that
B7 could bind to NTCP and trigger apoptosis. These results could provide recommendable guidelines for the future development of
NTCP-targeting small molecules and the treatment of HCC.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 81922064, 81874290, 81673290, 81803347, and
81903502) and the Natural Science Foundation of Guangdong Province (No. 2018A030313707). Post-Doctor Research Project, West
China Hospital, Sichuan University (No. 2019HXBH034).

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