NTCP/SLC10A1
B
Elucidation of Molecular Mechanism of Hepatitis B Virus Infection
and Drug Discovery Targeting Bile Acid Transporter, NTCP/SLC10A1
27
Fukano, Kento
2
...................................................................................................... 4
............................................................................................................. 6
............................................................................................................ 12
B HBV
................................. 13
............................................................................................. 13
HBV ..... 14
NTCP
................................................................................... 16
NTCP ........... 19
.................................................................................................... 22
NTCP HBV
......................................................................... 24
............................................................................................. 24
RaPID HBV
..................................................................... 25
HBV ............. 29
HBV ....................... 31
................................................................. 33
HBV ......... 36
3
.................................................................................................... 38
HBV .................................................. 40
............................................................................................. 40
Troglitazone HBV
.............................................................................................. 41
Troglitazone PPARγ HBV
.......................................................................................... 45
Troglitazone HBV .................................... 47
.................................................................................................... 51
Troglitazone HBV
.................................................................................................. 53
............................................................................................. 53
NTCP NTCP
............................................................................ 54
HBV NTCP ................. 58
.................................................................................................... 61
............................................................................................................ 62
........................................................................................................ 64
..................................................................................................... 73
............................................................................................................ 77
4
aa : amino acids
cccDNA : covalently closed circular deoxyribonucleic acid
CsA : cyclosporin A
DAPI : 4’,6-diamidino-2-phenylindole
DMSO : dimethyl sulfoxide
FBS : fetal bovine serum
FDA : food and drug administration
GEq : genome equivalent
HA : hemagglutinin
HBc : hepatitis B virus core protein
HBs : hepatitis B virus surface protein
HBV : hepatitis B virus
HBx : hepatitis B virus x protein
HCV : hepatitis C virus
HDV : hepatitis D virus
His : histidine
IFN : interferon
LHBs : large hepatitis B virus surface protein
MHBs : middle hepatitis B virus surface protein
MTT : methylthiazole tetrazolium
NMDG : N-methyl-D-glucamin
NTCP : sodium taurocholate cotransporting polypeptide
PBS : phosphate buffered saline
5
PEG : polyethylene glycol
pgRNA : pregenomic RNA
PLA : proximity ligation assay
PPAR : peroxisome proliferator-activated receptor
PPREs : PPAR response elements
PVDF : polyvinylidene difluoride
RaPID : random peptide integrated discovery
RT-PCR : reverse transcription-polymerase chain reaction
RXR : retinoid X receptor
SHBs : small hepatitis B virus surface protein
SLC : solute carrier
TAMRA : 6-carboxytetramethylrhodamine
TBS : tris-buffered saline
TCA : taurocholic acid
6
7
B HBV 2 6 100
1,2)HBV
interferon-α IFNα IFNα
IFN lamivudine adefovir entecavir tenofovir
3,4)IFN
HBV HBV
HBV
HBV
HBV
Fig. 15,6)
HBV
HBV
large HBV surface antigen LHBs sodium
taurocholate cotransporting polypeptide / solute carrier family 10 member 1
NTCP/SLC10A1
HBV
DNA relaxed circular DNA rcDNA DNA
DNA covalently closed circular DNA cccDNA
8
cccDNA RNA
3.5 kb RNA pregenomic RNA pgRNA
pgRNA HBc
pgRNA pgRNA
pgRNA
DNA RNaseH
pgRNA DNA
rcDNA cccDNA
HBV
HBV 2012 Yan
HBV HBV
NTCP/SLC10A1
7)NTCP SLC10
1 2
8)NTCP
349 56 kDa 7 9
NTCP HBV
preS1 HBV
9
NTCP NTCP HBV
NTCP HBV LHBs
preS1 preS1 2 48
myrcludex-B FDA
cyclosporin A irbesartan ezetimibe ritonavir
propranolol NTCP HBV
9-17)HBV
HBV NTCP
NTCP
NTCP
18)
19)myrcludex-B
20)
NTCP
HBV NTCP
HBV
in vitro
NTCP HBV
HBV
10
HBV NTCP
HBV
HBV
11
Figure 1. HBV
HBV NTCP
cccDNA cccDNA 4
RNA RNA
Core Pol 3.5 kb RNA pgRNA
pgRNA cDNA pgRNA
DNA DNA
cccDNA
rcDNA
Core
Pol
SHBs
MHBs
LHBs
attachment
entry
nuclear import
cccDNAformation
transcription
0.7 kb
2.1 kb
2.4 kb
3.5 kbpgRNA translation
reverse transcription
DNA synthesis
release
trafficking
HBx
capsid formation
cytoplasm
nucleus
NTCP(SLC10A1)
translation
12
13
B HBV
HBV NTCP/SLC10A1
LHBs preS1
7)NTCP HBV
NTCP HBV
9-17)HBV
NTCP
NTCP
18)NTCP
21)
19)NTCP
NTCP
HBV
HBV
14
HBV
HBV HBV
A CsA Scynexis
HBV HBV
HepG2-hNTCP-C4 8 µM HBV 16
13 HBs ELISA HBc
HBV22)
MTT HBV HBV
HBV preS1
2-48 preS1 peptide
11 CsA SCY806 SCY446 SCY450
SCY995 4 HBs HBc
Fig. 2A-C
HBV HBe
CsA 4 HBV 4
CsA HBV IC50 0.5 2.0
µM Fig. 2D
15
Figure 2. CsA HBV
A-C HepG2-hNTCP-C4 DMSO 0.16% preS1 peptide
100 nM CsA 8 µM HBV 16
HBV 12
HBs A HBc C B
Ca HBc
HBc Ca Lumina Vision image analysis software
Cb D CsA SCY806 SCY446 SCY450 SCY995
HBV 0.25
0.5 1 2 4 8 µM A-C HBV
HBe HBV IC50 Mean ± SD
*P < 0.05 **P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
A
0
2
4
6
8
HBs
C
020406080
100120
HBc
co
ntr
ol
DM
SO
pre
S1
pe
pti
de
Cs
A
SC
Y8
06
SC
Y5
82
SC
Y6
51
SC
Y6
60
SC
Y4
46
SC
Y1
98
SC
Y4
50
SC
Y4
53
SC
Y9
95
SC
Y5
06
SC
Y6
40
B cell viability
OD
45
0 (
fold
)
co
ntr
ol
DM
SO
pre
S1
pe
pti
de
Cs
A
SC
Y8
06
SC
Y5
82
SC
Y6
51
SC
Y6
60
SC
Y4
46
SC
Y1
98
SC
Y4
50
SC
Y4
53
SC
Y9
95
SC
Y5
06
SC
Y6
40
HB
s (
IU/m
l)
co
ntr
ol
DM
SO
pre
S1
pe
pti
de
Cs
A
SC
Y8
06
SC
Y5
82
SC
Y6
51
SC
Y6
60
SC
Y4
46
SC
Y1
98
SC
Y4
50
SC
Y4
53
SC
Y9
95
SC
Y5
06
SC
Y6
40
HB
c a
rea
(% o
f c
on
tro
l)
**
***
****
****
**** ** ** ** **preS1 peptide CsAcontrol
SCY446 SCY450SCY806
SCY995
red
blue
HBc
nucleus
(a) (b)
D
0
10
20
30
0 2 4 6 8
IC50: 1.27 ± 0.43 µM
concentration (µM)
HB
e
S/C
O
1.05 ± 0.33 µM
0
5
10
15
20
0 2 4 6 8
0.54 ± 0.13 µM
0
5
10
15
20
0 2 4 6 8
0.91 ± 0.19 µM
0
5
10
15
20
0 2 4 6 8
1.97 ± 0.04 µM
0
5
10
15
20
0 2 4 6 8
concentration (µM) concentration (µM) concentration (µM) concentration (µM)
CsA SCY806 SCY446 SCY450 SCY995
NN
O
OHN
NO
HN
HN O
N
O
O
N
O
O
N
N
O
OHN
OH
O
NN
O
OHN
NO
HN
HN O
N
O
O
N
O
O
N
N
O
OHN
OH
OH
O O
NN
O
OHN
NO
HN
HN O
N
O
O
N
O
O
N
N
O
OHN
OH
O
R
O
NN
O
OHN
NO
HN
HN O
N
O
O
N
O
O
N
N
O
OHN
OH
OH
O
O
O
NN
O
OHN
NO
HN
HN O
N
O
O
N
O
O
N
N
O
OHN
OH
O
R
S
NMe2
O
16
NTCP
CsA HBV HBV
CsA preS1 NTCP
HBV CsA HBV
preS1 PreS1 peptide
TAMRA preS1 40 µM
HepG2-hNTCP-C4 preS1
HBV SCY806 SCY446
SCY450 SCY995 preS1
Fig. 3A HBV SCY582
CsA preS1
HBV
CsA myrcludex-B ezetimibe irbesartan ritonavir vanitaracin
A NTCP HBV
HBV NTCP
CsA
NTCP
CsA
CsA CsA NTCP
Na+
Na+
6.25 12.5 25 µM [3H]-taurocholic acid TCA 0.1 µM
HepG2-hNTCP-C4 [3H]
17
NTCP
CsA Na+
TCA
Fig. 3B CsA HBV SCY446
CsA TCA
SCY995 TCA
HBV
18
Figure 3. CsA HBV NTCP
Aa preS1 Ab c HBV
preS1 HepG2-hNTCP-C4 preS1
40 nM 40 µM Ab
preS1
preS1 Ab Dynamic Cell Count KEYENCE
Ac B Na+
6.25 12.5 25 µM [3H]-taurocholic acid TCA 0.1 µM
HepG2-hNTCP-C4 5 [3H]
Mean ± SD *P < 0.05 **P < 0.01
preS1-cell binding
red
blue
preS1 probe
nucleus
control preS1 peptide CsA
SCY806 SCY446 SCY450
SCY995 SCY582
A
0
25
50
75
100
125
co
ntr
ol
pre
S1
pe
pti
de
Cs
A
SC
Y8
06
SC
Y4
46
SC
Y4
50
SC
Y9
95
SC
Y5
82
preS1-cell binding
flu
ore
scen
ce a
rea
(% o
f c
on
tro
l)
**** **
**** **
B
00.20.40.60.8
11.21.4
NTCP transporter activity
TC
A u
pta
ke
(fo
ld)
CsA SCY446 SCY995
Na+: +– + + + + + + + + +c
on
tro
l
*
** * ** * *
LHBs
NTCP
preS1-probe
peptides
NTCP
preS1 preS2 S
389aa
LHBs
108 164
myr
2 48
myr
TAMRA
2
preS1-probe
(a) (b)
(c)
19
NTCP
NTCP CsA SCY446 SCY995
NTCP
NTCP
NTCP
NTCP
[3H]-TCA
Fig.
3Aa CsA SCY446 0 5 10 µM TCA
5 10 20 40 80 160 240 µM CsA SCY446
TCA
Fig. 4Ab c CsA SCY446 NTCP
NTCP
NTCP HBV preS1
CsA SCY446
SCY995 6.25 12.5 25 µM preS1
40 80 160 320 nM preS1 CsA SCY446
SCY995 preS1
Fig. 4B CsA SCY446 SCY995
preS1 NTCP
HBV
CsA SCY446 NTCP
20
SCY995
21
Figure 4. CsA NTCP
Aa
Ab c CsA SCY446 0 5 10 µM
TCA 5 10 20 40 80 160 240 µM
NTCP
B preS1 preS1
preS1
Mean ± SD
B
CsA SCY446
C
preS1-TAMRA
SCY995
excess amount of fluorescent-labeled preS1red: preS1 probeblue: nucleus
HepG2HepG2-hNTCP-C4
control
0
4000
8000
12000
16000
0 50 100 150 200 250
SCY446
0
2000
4000
6000
8000
10000
12000
0 50 100 150 200 250
CsA
[3H
]-T
CA
up
tak
e
non-competitive
control competitive
maximum-effect
TCA concentration TCA concentration
< mode of inhibition >
A
B
(a) (b) (c)
22
B IFN
IFN
HBV HBV
HBV HBV
HBV
HBV CsA
CsA HBV
4 HBV
NTCP NTCP HBV
myrcludex-B CsA irbesartan ezetimibe ritonavir vanitaracin A
9-17,23,24)HBV
NTCP
4 CsA NTCP HBV
SCY995 NTCP
SCY995
HBV
HBV HBV
23
NTCP
24
NTCP HBV
NTCP
HBV
in vitro RaPID Random Peptide
Integrated Discovery25)
NTCP
NTCP HBV
25
RaPID HBV
RaPID
NTCP 10
10
HBV
100 µM 2 HBV 16
HBV 12
HBs HBV
HBV
CsA 10
Fig. 5C HBs
Fig. 5B
1st HBV D2
D4 PX(I/L)H(I/L)F P
L1 L4 D1 3 (T/L)W(T/I)W
W 2
NTCP
P 88 W 179 NTCP
12
12 HBV
Fig. 5A 10 µM
26
HBV 12 10
Fig. 6B HBV
7 Fig. 6A
WD1 WL2 WL4 3 HBV
27
Figure 5. HBV 1st
A HBV
HepG2-hNTCP-C4 2
HBV 16 HBV
12 HBs B C
B C CsA
100 µM A HBV Mean
± SD *P < 0.05 **P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
00.20.40.60.8
11.21.4
B
C
co
ntr
ol
Cs
A L1
L2
D1
D2
D3
D4
L5
L6
L3
L4
cell viability
OD
45
0 (
fold
)H
Bs
(fo
ld)
HBs
*
** ** ** ****
** ** ** ** **
co
ntr
ol
Cs
A L1
L2
D1
D2
D3
D4
L5
L6
L3
L4
A
HBV
peptide
2 h 16 h 12 days
peptide HBV infection + peptide
wash detection
28
Figure 6. HBV 2nd
A B A HepG2-hNTCP-C4 B
CsA 10 µM Fig. 5A
HBV HBe A
B Mean ± SD *P < 0.05 **P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
0
0.2
0.4
0.6
0.8
1
1.2
PL
1
A
Bcell viability
co
ntr
ol
Cs
A
WL
1
WL
2
D1
D2
D4
L1
L4
WD
1
WD
2
WD
3
WD
4
WL
5
WL
6
WL
3
WL
4
OD
45
0 (
fold
)
**
*
HBe
***
****
*
** ****
** **
first hit peptides second hit peptides
* **
HB
e (
fold
)
PD
1
PL
1
co
ntr
ol
Cs
A
WL
1
WL
2
D1
D2
D4
L1
L4
WD
1
WD
2
WD
3
WD
4
WL
5
WL
6
WL
3
WL
4
first hit peptides second hit peptides
PD
1
29
HBV
3 WD1 WL2 WL4 HBV
HepG2-hNTCP-C4 HBV
HBs HBc
CsA HBs HBc HBV
entecavir Fig. 7A B
Fig. 5A HBV HBV
3 WD1 WL2
WL4 Fig. 7C HBs HBc
Fig. 7A B HBV
D1 D2
FBS
HBV CsA
WD1 WL2 WL4 HBs Fig.
7D IC50 3.17 ± 0.30 0.85 ± 0.01 2.54 ± 0.11 0.66 ± 0.03
CsA
5 HBV
30
Figure 7. 3 HBV
A-C HepG2-hNTCP-C4 preS1 peptide 100 nM CsA
30 µM HBV 16
HBV 12 HBs
A HBc B C B
HBc D
HepG2-hNTCP-C4 0.3 1 3 10 µM A-C
HBV HBs Mean ± SD
N.S.; not significant *P < 0.05 **P < 0.01
HB
s (
fold
)
co
ntr
ol
Cs
A
WL
2
WD
1
WL
4
en
tec
av
ir
**
N.S.
D1
D2
C
B
D
HBs
0
0.2
0.4
0.6
0.8
1
1.2
****
**
**
ce
llu
lar
DN
A (
fold
)
co
ntr
ol
Cs
A
WL
2
WD
1
WL
4
en
tec
av
ir
D1
D2
cell viability
00.20.40.60.8
11.21.4
HBc
control entecavir CsA
D1 D2
WL2 WL4WD1
A
0
0.2
0.4
0.6
0.8
1
1.2
co
ntr
ol
HB
s (
fold
)
WL2WD1 WL4CsA
**
**
****
**
*
**
**
**
****
**
*
**
****
HBs
Red: HBc, Blue: nucleus
100 µm
31
HBV
WD1 WL2 WL4 HBV
HBV
cccDNA RNA
Fig. 1 WD1 WL2
WL4 Hep38.7-Tet
Hep38.7-Tet tetracycline HBV
NTCP HBV26,27)
Hep38.7-Tet 30 µM tetracycline 6
HBV DNA real-time PCR
entecavir
HBV DNA Fig. 8A 3 WD1
WL2 WL4 HBV DNA
HBV preS1
CsA 3
WD1 WL2 WL4 preS1
Fig. 8B C WD1
WL2 WL4 NTCP preS1 HBV
32
Figure 8. HBV
A HBV entecavir 100 nM
30 µM tetracycline 6 Hep38.7-Tet
HBV DNA real-time PCR B
C HBV preS1
HepG2-hNTCP-C4 preS1 40 nM CsA
1 3 10 30 µM B
preS1
preS1 B Dynamic Cell Count KEYENCE
C Mean ± SD **P < 0.01
WL
4
B
0
0.2
0.4
0.6
0.8
1
1.2
**
HB
V D
NA
(fo
ld)
co
ntr
ol
WL
2
en
tec
av
ir
A HBV replication assay
(Hep38.7-Tet cells)
WD
1
flu
ore
sc
en
ce
are
a (
fold
)
co
ntr
ol
WL2WD1 WL4CsA
C
0
0.2
0.4
0.6
0.8
1
1.2
CsA
WL2
WL4
WD1
control
preS1-cell binding
Red: preS1 probe, Blue: nucleus
100 µm
preS1-cell binding
33
HBV
D HDV C HCV
HDV HBV
HBV NTCP28)
HCV
HBV HDV NTCP
29)HDV HepG2-hNTCP-C4 30 µM
16 6 HDV RNA
real-time RT-PCR WD1 WL2 WL4
HDV RNA Fig. 9A HCV Huh-7.5.1
30 µM HCV 4
72
HCV HDV 3
HCV Fig. 9B
WD1 WL2 WL4 NTCP
WD1 WL2 WL4 NTCP NTCP
[3H]-TCA
3 10 30 µM 0.1 µM [3H]-TCA HepG2-hNTCP-C4
[3H] 3
CsA HBV CsA
30 µM NTCP
34
Fig. 9C WD1 WL2 WL4
HBV
NTCP
35
Figure 9. NTCP
A HepG2-hNTCP-C4 preS1 peptide 100 nM
30 µM HDV 16 HDV
6 HDV RNA real-time
RT-PCR B Huh-7.5.1 bafilomycin A1
20 nM 30 µM HCV 4
72
HCV C CsA 3
10 30 µM [3H]-TCA 0.1 µM Na
+
HepG2-hNTCP-C4 15 [3H]
Mean ± SD N.S.; not significant **P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
00.20.40.60.8
11.21.4
A
** ** ** **
HCV(NTCP-independent infection)
luc
ife
ras
e a
cti
vit
y (
fold
)
**
N.S.
N.S.N.S.
co
ntr
ol
WL
2
WD
1
WL
4
bafi
lom
ycin
A1
HD
V R
NA
(fo
ld)
HDV(NTCP-dependent infection)
pre
S1
pe
pti
de
co
ntr
ol
WL
2
WD
1
WL
4
B
[3H
]-T
CA
up
tak
e (
fold
)
00.20.40.60.8
11.21.4
N.S.
CsA WL2WD1 WL4
co
ntr
ol
N.S.N.S.
**** **
CNTCP transporter activity
36
HBV
HBV HBV
WD1 WL4 30 µM
A B C HBV entecavir HBV C
L180M/S202G/M204V30)
HBV C G145R
31)HBV HBs
HBV D
HBV Fig. 7 WD1 WL4 A B C
HBV Fig. 10 entecavir
HBV HBV Fig. 10
HBV
37
Figure 10. HBV
30 µM Fig. 5A
A B C HBV entecavir HBV C
L180M/S202G/M204V HBV C G145R
HBV HBs
Mean ± SD **P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
0
0.2
0.4
0.6
0.8
1
1.2
HB
s (
fold
)
genotype A
co
ntr
ol
WD
1
WL
4
** **0
0.2
0.4
0.6
0.8
1
1.2
genotype B
co
ntr
ol
WD
1
WL
4
**
**
genotype C
0
0.2
0.4
0.6
0.8
1
1.2
co
ntr
ol
WD
1
WL
4
** **
genotype C
(L180M/S202G/M204V)
co
ntr
ol
WD
1
WL
4
**
**
genotype C
(G145R)
0
0.2
0.4
0.6
0.8
1
1.2
co
ntr
ol
WD
1
WL
4
****
38
HBV NTCP HBV
HBV NTCP
NTCP R252H
21)NTCP
18)HBV
myrcludex-B
100
20)NTCP
NTCP
HBV
SCY995
SCY995 CsA
NTCP
NTCP
32)NTCP
HBV NTCP
NTCP
HBV33)
NTCP
3 WD1 WL2
39
WL4 NTCP HBV
NTCP
HBV
34)
HBV myrcludex-B B
D35)
Myrcludex-B
NTCP
47
myrcludex-B
HBV
HBV HBV
HBV
40
HBV
NTCP NTCP
HBV
HBV
HBV NTCP HBV
HBV
41
Troglitazone HBV
HBV HepaRG
HBV troglitazone Fig. 11A
Troglitazone HBV HepG2-hNTCP-C4
HepG2-hNTCP-C4 2
HBV 16 Fig. 11B HBV
12 HBs
HBe HBV DNA cccDNA HBc
preS1 peptide
Fig. 11C-G Troglitazone
Fig. 11H HBs Fig. 11C HBe Fig. 11D HBV
DNA Fig. 11E cccDNA Fig. 11F HBc Fig. 11G
troglitazone HBV
Troglitazone HBV
Troglitazone
Fig. 12A Fig. 12A 2
7
1 Fig. 12A Fig. 11B
HBV pioglitazone
HBV Fig. 12B
trolox HBV
HBV ciglitazone troglitazone
42
Fig. 12B HBV
Troglitazone ciglitazone HBV
troglitazone
ciglitazone 3 10 30 µM Fig. 11B HBV
Troglitazone ciglitazone Fig.
12D HBs Fig. 12C
IC50 9.7 ± 1.9 4.7 ± 2.6 µM
ciglitazone troglitazone HBV
43
Figure 11. Troglitazone HBV
A Troglitazone B HBV
HepG2-hNTCP-C4 2
HBV 16 HBV
12 HBV C-G C-H
HepG2-hNTCP-C4 preS1 peptide 100 nM troglitazone 25 µM
B HBV HBs
C HBe D HBV DNA E cccDNA F HBc
G H G
HBc Mean ± SD *P < 0.05 **P < 0.01
S NH
O
O
OO
CH3
CH3
HO
H3C
CH3
0
1
2
3
0
10
20
30
40
0
2
4
6
8
10
HB
s (
ng
/mL
)
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
**
HBs
**
troglitazonepreS1 peptidecontrol
red
blue
HBc
nucleus
HBc
HBV
compound
2 h 16 h 12 days
compound HBV infection + compound
wash detection
B
C
0
0.2
0.4
0.6
0.8
1
1.2
OD
45
0 (
fold
)
cell viability
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
HG
A
troglitazone
HB
V D
NA
(× 1
07 c
op
ies/w
ell)
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
**
HBV DNA
**
E
HB
e (
ng
/mL
)
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
**
HBe
**
D
0
0.5
1
1.5
2
2.5
cc
cD
NA
(× 1
05 c
op
ies/w
ell)
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
**
cccDNA
**
F
44
Figure 12. Troglitazone
A Troglitazone B HepG2-hNTCP-C4
preS1 peptide 100 nM troglitazone 25 µM Fig. 11B
HBV HBs
C D preS1 peptide 100 nM troglitazone
ciglitazone 3 10 30 µM Fig. 11B HBV
HBs C D
Mean ± SD *P < 0.05 **P < 0.01
troglitazone analogs
0
0.2
0.4
0.6
0.8
1
1.2
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
cig
lita
zo
ne
ros
iglita
zo
ne
pio
glita
zo
ne
ba
lag
lita
zo
ne
da
rglita
zo
ne
ne
tog
lita
zo
ne
mit
og
lita
zo
ne
tro
lox
HB
s (
fold
)
**
**
**
** **
HBV infection
(HepG2-hNTCP-C4 cells)
B
****
**
0
0.2
0.4
0.6
0.8
1
1.2
troglitazone
pre
S1
pe
pti
de
co
ntr
ol
HB
s (
fold
)
****
**
**
**
HBV infection
(primary human hepatocytes)
IC50: 9.7 ± 1.9 µM
ciglitazone
4.7 ± 2.6 µM
C
**
0
0.2
0.4
0.6
0.8
1
1.2
OD
45
0 (
fold
)
cell viability
(primary human hepatocytes)
D
troglitazone
pre
S1
pe
pti
de
co
ntr
ol
ciglitazone
A
S NH
O
O
O
CH3
ciglitazone
S NH
O
O
O
N
N
O
CH3
balaglitazone
S NH
O
O
O
N
O
H3C
mitoglitazone
S NH
O
O
OO
CH3
CH3
HO
H3C
CH3
troglitazone
S NH
O
O
ON
H3C
pioglitazone
S NH
O
O
O
F
netoglitazone
S NH
O
O
ONN
CH3
rosiglitazone
S NH
O
O
O
N
O
CH3
darglitazone
OHO
H3C
CH3
HO
H3C
CH3 O
trolox
45
Troglitazone PPARγ HBV
Troglitazone pioglitazone
PPARγ peroxisome
proliferator-activated receptor γ 36)
Fig. 11 12 troglitazone HBV PPARγ
PPARγ RXRα retinoid X receptor α
PPAR PPAR response elements: PPREs
PPREs
PPARγ RXRα
Fig. 13A HepG2-hNTCP-C4
24 PPARγ
Troglitazone pioglitazone
PPARγ Fig. 13B HBV troglitazone
Fig. 13D troglitazone PPARγ
PPARγ SR-202 Fig.
13C troglitazone HBV Fig. 13E
PPARγ troglitazone HBV
46
Figure 13. PPARγ HBV
A-C PPAR PPRE
PPARγ RXRα
A HepG2-hNTCP-C4 troglitazone 25 µM
pioglitazone 25 µM B troglitazone 25 µM troglitazone 25 µM
SR-202 1mM C 24
D E HepG2-hNTCP-C4 troglitazone 25 µM
pioglitazone 25 µM D troglitazone 25 µM troglitazone 25 µM
SR-202 1mM E Fig. 11B HBV
HBs Mean ± SD N.S.; not
significant **P < 0.01
0
0.2
0.4
0.6
0.8
1
1.2
0
2
4
6
0
2
4
6
8
anti-HBV activity
PPRE luc
RXRαPPARγ
luc
ife
ras
e a
cti
vit
y (
fold
)
PPARγ activity
tro
glita
zo
ne
co
ntr
ol
tro
glita
zo
ne
+ S
R-2
02
HB
s (
fold
)
tro
glita
zo
ne
co
ntr
ol
tro
glita
zo
ne
+ S
R-2
02
anti-HBV activity
tro
glita
zo
ne
co
ntr
ol
luc
ife
ras
e a
cti
vit
y (
fold
)
pio
glita
zo
ne
PPARγ activity
A
B C
D E
N.S.
0
0.4
0.8
1.2
1.6
HB
s (
fold
)
tro
glita
zo
ne
co
ntr
ol
pio
glita
zo
ne
N.S.
**
**
** **
47
Troglitazone HBV
Troglitazone HBV HBV
Fig. 14A HBV
troglitazone
HepG2.2.15.7 HepG2.2.15.7 HBV
DNA
HBV NTCP
HBV26,27)
troglitazone
25 µM 6 HepG2.2.15.7 HBV DNA
real-time PCR
entecavir HBV DNA troglitaazone
Fig. 14B
HDV HCV troglitazone
HDV HepG2-hNTCP-C4 troglitazone 25 µM HDV
16 6 HDV RNA real-time
RT-PCR Troglitazone HDV RNA
Fig. 14C Huh-7.5.1 troglitazone 25 µM
HCV 4 72
HCV HDV
HCV Fig. 14D
troglitazone NTCP
48
troglitazone HBV preS1
preS1 peptide
preS1 Fig. 14E
troglitazone preS1
HBV HepG2-hNTCP-C4 HBV
4ºC HBV troglitazone 25 µM
16 Fig. 14Fa
PBS HBV DNA HBV
11)Troglitazone HBV DNA
Fig. 14Fb preS1
HBV preS1 troglitazone
17)HepG2-hNTCP-C4 troglitazone pioglitazone 25 µM
preS1 40 nM 37ºC 8 Fig. 14Fc
HBV pioglitazone
preS1
Fig. 14Fd-2 8 troglitazone preS1
Fig. 14Fd-5 HBV
Fig. 14Fb
8 preS1
NTCP
Fig. 14Fd-1 3 Troglitazone preS1 NTCP
Fig. 14Fd-4 6 troglitazone HBV
49
Figure 14. Troglitazone
A B-F HBV B
HBV entecavir 100 nM troglitazone
25 µM 6 HepG2.2.15.7 HBV DNA
real-time PCR C HepG2-hNTCP-C4
preS1 peptide 100 nM troglitazone 25 µM HDV
16 HDV 6
HDV RNA real-time RT-PCR D
Huh-7.5.1 bafilomycin A1 10 nM troglitazone 25 µM
HCV 4 72
0
0.2
0.4
0.6
0.8
1
1.2
HD
V R
NA
(fo
ld)
tro
glita
zo
ne
pre
S1
pe
pti
de
co
ntr
ol
**
HDV
**
0
0.2
0.4
0.6
0.8
1
1.2
HB
V D
NA
(fo
ld)
tro
glita
zo
ne
en
tec
av
ir
co
ntr
ol
**
HBV replication
(HepG2.2.15.7 cells)
B
C
red
blue
preS1 probe
nucleus
control troglitazonepreS1 peptide
preS1-cell binding
E
F
HBV
compound
3 h 16 h
HBV treatment wash detection
4°C! 37°C
HBV internalization
BC
E
F
entecavir
preS1 peptide
troglitazone
D
0
0.2
0.4
0.6
0.8
1
1.2
luc
ife
ras
e a
cti
vit
y (
fold
)
tro
glita
zo
ne
bafi
lom
ycin
A1
co
ntr
ol
HCV
**
HB
V D
NA
(fo
ld)
tro
glita
zo
ne
co
ntr
ol
**
0
0.2
0.4
0.6
0.8
1
1.2
A
green
red
blue
NTCP
preS1 probe
nucleus
control
NT
CP
pre
S1
pro
be
me
rge
troglitazone pioglitazone
preS1 internalization
preS1 probe
compound
8 h
treatment detection
37°C!
(a)
(b)
(c)
(d)
1)
2)
3)
4)
5)
6)
7)
8)
9)
50
HCV E
HBV preS1
HepG2-hNTCP-C4 preS1 40 nM preS1 peptide
100 nM troglitazone 25 µM
preS1 F a HBV
HepG2-hNTCP-C4 HBV 4ºC 3
HBV troglitazone 25 µM
16 PBS
HBV DNA HBV b
c preS1 HepG2-hNTCP-C4
troglitazone 25 µM pioglitazone 25 µM preS1 40 nM
37ºC 8 NTCP d
d NTCP preS1
Mean ± SD **P < 0.01
51
HBV
troglitazone
HBV
34)AIDS C
HBV
HBV
HBV
NTCP troglitazone
HBV
HBV
(–)-epigallocatechin-3-gallate
37)
chloroquine
38,39)
arbidol HCV
40)Silybum marianum
silibinin HCV
52
41-44)C
45-47)
HBV B
53
Troglitazone HBV
HBV
HBV troglitazone
HBV
SLC EAAT2 excitatory amino acid
transporter; SLC1A2 NBCe1-A Na+-HCO3
– co-transporter; SLC4A4 GAT1
GABA transporter; SLC6A1 GlyT2 glycine transporter; SLC6A5 ASBT
apical sodium dependent bile acid transporter; SLC10A2 hNKCC2
Na+-K
+-2Cl
– co-transporter; SLC12A1 hNKCC1 SLC12A2
48-55)GAT1 GlyT2
54,55)
DAT dopamine transporter; SLC6A3
56)
NTCP57)
NTCP
HBV
54
NTCP NTCP
PLA NTCP
Myc NTCP HA NTCP
HepG2 Myc
HA PLA
NTCP PLA Fig. 15Aa-c
NTCP HBV
NTCP
preS1 peptide NTCP PLA
Fig. 15Ad-f PLA
preS1 preS1
NTCP
NTCP
349 NTCP 20
34
His NTCP His-NTCP
HCV E1 aa 111-140 preS1 aa 2-48 34 NTCP
4ºC 4
NTCP His-NTCP preS1
peptide HCV E1 peptide
Fig. 15Ba
55
preS1 peptide
Fig. 15Bb NTCP aa 221-240 aa
271-290 2 preS1 peptide
His-NTCP Fig. 15B
NTCP 2 aa 221-240 aa 271-290 NTCP
2
NTCP Fig. 15Ca
NTCP aa 221-240 NTCP aa 271-290
preS1 peptide NTCP PLA
Fig. 15Cb-4 vs. 8 10 15Cc NTCP
aa 221-240 NTCP aa 271-290 NTCP
56
Figure 15. NTCP
A Myc NTCP HA NTCP
HepG2 preS1 40 nM 4ºC 30
Myc HA Duolink PLA
AmergePLA signals
pre
S1
pro
be
(−)
pre
S1
pro
be
(+)
NTCP oligomerization
B
pre
S1
pe
pti
de
1-2
0
11
-30
21
-40
41
-60
51
-70
61
-80
no
-pe
pti
de
HC
V E
1 p
ep
tid
e
NTCP peptide (aa)
pre
S1
pe
pti
de
31
-50
71
-90
81
-10
0
91
-11
0
10
1-1
20
111
-13
0
no
-pe
pti
de
HC
V E
1 p
ep
tid
e
12
1-1
40
13
1-1
50
14
1-1
60
15
1-1
70
16
1-1
80
17
1-1
90
18
1-2
00
19
1-2
10
NTCP peptide (aa)
pre
S1
pe
pti
de
20
1-2
20
211
-23
0
22
1-2
40
23
1-2
50
24
1-2
60
25
1-2
70
no
-pe
pti
de
HC
V E
1 p
ep
tid
e
26
1-2
80
27
1-2
90
28
1-3
00
29
1-3
10
30
1-3
20
311
-33
0
32
1-3
40
33
0-3
49
NTCP peptide (aa)
pull down
(NTCP peptide - recombinant NTCP)(a)
0
0.5
1
1.5
2
pre
S1
pe
pti
de
22
1-2
40
27
1-2
90
(b)
NTCP peptide (aa)
de
ns
ity
(fo
ld)
1-2
011
-30
21
-40
41
-60
51
-70
61
-80
31
-50
71
-90
81
-10
09
1-1
10
10
1-1
20
111
-13
01
21
-14
01
31
-15
01
41
-16
01
51
-17
01
61
-18
01
71
-19
01
81
-20
01
91
-21
02
01
-22
02
11
-23
0
23
1-2
50
24
1-2
60
25
1-2
70
26
1-2
80
28
1-3
00
29
1-3
10
30
1-3
20
311
-33
03
21
-34
03
30
-34
9
0.8
0
60
120
180
PL
A s
ign
als
/ce
ll
preS1 probe: – + + +
1-2
0
co
ntr
ol
22
1-2
40
(c) PLA signals
+
27
1-2
90
NTCP peptide (aa)
* N.S.*
*
preS1 probe
a)
d)
b)
e)
c)
f)
C
me
rge
PL
A s
ign
als
control 1-20 221-240 271-290
NTCP peptide (aa)
green
red
blue
PLA signals
preS1 probe
nucleus
(a)
(b)
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
preS1 probe: +– + + +
green
red
blue
PLA signals
preS1 probe
nucleus
+ NTCP peptide
HBV
NTCP (oligomerization) NTCP peptide
57
NTCP PLA
preS1 B His
NTCP HCV E1 aa
111-140 preS1 aa 2-48 34 NTCP 4ºC 4
NTCP
a preS1 peptide
b C
A NTCP aa 1-20 aa 221-240 aa271-290
NTCP (a) Myc NTCP HA
NTCP HepG2
NTCP aa 1-20 aa 221-240 aa271-290 preS1
40 nM b PLA
preS1 PLA
Dynamic Cell Count KEYENCE c Mean
± SD N.S.; not significant *P < 0.05
58
HBV NTCP
HBV NTCP NTCP aa
221-240 NTCP aa 271-290 NTCP
preS1 HBV Fig. 16Aa
HepG2-hNTCP-C4 NTCP aa 1-20 aa 221-240 aa271-290
preS1 40 nM 37ºC 8
NTCP aa 1-20 preS1
Fig. 16Ab-1 2 NTCP aa
221-240 NTCP aa 271-290
preS1 NTCP Fig. 16Ab-3 4
2 NTCP HBV Fig. 16Ac
NTCP HBV
troglitazone NTCP
PLA Fig. 16B HBV
preS1 pioglitazone PLA
NTCP HBV
59
Figure 16. NTCP HBV
A Fig. 14Fc d NTCP aa 1-20 aa 221-240
aa271-290 preS1 (a b) HepG2-hNTCP-C4
NTCP aa 1-20 aa 221-240 aa271-290 preS1
40 nM b b NTCP
preS1 c
HepG2-hNTCP-C4 NTCP aa 1-20 aa 221-240 aa271-290
Fig. 11B HBV HBs
B Fig. 15A NTCP
Myc NTCP HA NTCP
HepG2 troglitazone
pioglitazone preS1 40 nM a a
PLA preS1
B
me
rge
green
red
blue
PLA signals
preS1 probe
nucleus
PL
A s
ign
als
control troglitazone pioglitazone
preS1 probe:
control aa 1-20
aa 221-240 aa 271-290
green
red
blue
NTCP
preS1 probe
nucleus
preS1 internalization
0
0.5
1
1.5
HB
s (
fold
)
HBV infection
pre
S1
pe
pti
de
22
1-2
40
27
1-2
90
co
ntr
ol
1-2
0
**
*
**
NTCP peptide (aa)
A
(b) (c)
(a)
(a)
0
60
120
180
*
PL
A s
ign
als
/ce
ll
preS1 probe: – + + +
tro
glita
zo
ne
co
ntr
ol
pio
glita
zo
ne
(b)PLA signals
N.S.
*
1)
3)
2)
4)
+ ++–
NTCP(oligomer)
internalize
HBV
internalize
NTCP peptide
+ NTCP peptide
60
PLA Dynamic Cell Count
KEYENCE b Mean ± SD N.S.; not significant
*P < 0.05 **P < 0.01
61
NTCP SLC
48-55,57)
GAT1 GlyT2
54,55)NTCP
NTCP
NTCP troglitazone NTCP
NTCP–HBV preS1 HBV
NTCP HBV
Troglitazone NTCP
HBV
HBV
62
63
NTCP
HBV CsA SCY995
WD1 WL2 WL4
HBV myrcludex-B
HBV troglitazone
NTCP HBV
troglitazone HBV
HBV
HBV
B
HBV
64
65
DMEM/F-12, GlutaMAX Opti-MEM, Reduced Serum Medium TaqMan Gene
Expression Master Mix Thermo Fisher Scientific penicillin G
potassium streptomycin sulfate Meiji Seika Pharma G418
Disulfate Aqueous Solution nacalai tesque CsA abcam
PreS1 peptide preS1 Scrum
[3H]-TCA American Radiolabeled Chemicals Entecavir
Santa Cruz Biotechnology Bafilomycin A1 Wako
Troglitazone AdooQ BioScience Ciglitazone
mitoglitazone trolox Cayman Chemical Rosiglitazone
pioglitazone darglitazone netoglitazone SR-202 tetracycline HEPES
PEG8000 Cell Proliferation Kit II (XTT) Proximity Ligation Assay NTCP
Sigma-Aldrich
Luciferase Assay System Promega Balaglitazone
MedChemExpress
HepG2 HepG2-hNTCP-C4 Hep38.7-Tet HepG2.2.15.7 10 mM
HEPES [pH7.4] 100 units/mL penicillin 100 µg/mL streptomycin 10% FBS
5 µg/mL insulin DMEM/F-12 + GlutaMax 5% CO2 37ºC
22,27)HepG2-hNTCP-C4 HepG2.2.15.7 Hep38.7-Tet
500 µg/mL G418 HepG2-hNTCP-C4 400 µg/mL G418
HepG2.2.15.7 Hep38.7-Tet Hep38.7-Tet
66
HBV 400 ng/mL tetracycline
Huh-7 Huh-7.5.1 10 mM HEPES [pH7.4] 100 units/mL penicillin
100 µg/mL streptomycin 10% FBS 0.1 mM non-essential amino acids 1 mM
sodium pyruvate DMEM 5% CO2 37ºC
58)
dHCG 5% CO2 37ºC59)
HBV
HBV Hep38.7-Tet D
HBV
9-30 3 A B
C HBV HBV HBV HepG2
3-9
3 2.3% NaCl 10%
PEG8000 PEG HBV DMEM/F-12
+ GlutaMax 200
HBV HBV 500 12,000 genome equivalent GEq
/cell 4% PEG8000 16
HBV PEG8000 12
HBs HBe HBc HBV DNA cccDNA
HBV
HBs HBe
67
HBs HBe ELISA ELISA PBS
3,846 HBs 8,000 HBe
4ºC 0.2% BSA 0.02% NaN3 1 x PBS
0.05%
Tween20-TBS 2 0.05%
Tween20-TBS HBs
HBs 2 0.05% Tween20-TBS
15 450 nm
HBc
HBc HBV 13 4%
paraformaldehyde 30
1 x PBS 0.3% Triton X-100 1
100 HBc
4ºC Alexa Fluor 594 donkey
anti-rabbit IgG H+L DAPI 1 BZ-X
KEYENCE
Real-time PCR
HBV DNA cccDNA real-time PCR
HBV DNA 5’-AAGGTAGGAGCTGGAGCATTCG-3’
5’-AGGCGGATTTGCTGGCAAAG-3’
5’-AGCCCTCAGGCTCAGGGCATAC-3’ cccDNA
68
5’-CGTCTGTGCCTTCTCATCTGC-3’ 5’-GCACAGCTTGGAGGCTTGAA-3’
5’-CTGTAGGCATAAATTGGT MGB -3’
Real-time PCR 1 x TaqMan Real-Time PCR Master Mix
200 nM forward primer 200 nM reverse primer 100 nM probe
DNA PCR PCR 50ºC 2 94ºC 10
94ºC 15 60ºC 1 50
DNA QIAamp DNA Mini Kit
QIAGEN
Cell Proliferation Kit II XTT
preS1
HepG2-hNTCP-C4 TAMRA preS1 peptide preS1
40 nM 37ºC 30 PBS preS1
4% paraformaldehyde
BZ-X KEYENCE preS1
HBV
Hep38.7-Tet Hep2.2.15.7 3
Hep38.7-Tet tetracycline 6
HBV DNA real-time PCR
69
HBV
HepG2-hNTCP-C4 HBV 12,000 genome equivalent GEq /cell
4% PEG8000 4ºC 3
HBV PEG8000 16
PBS HBV DNA real-time
PCR HBV
preS1
HepG2-hNTCP-C4 preS1 40 nM 37ºC 8
PBS preS1 4%
paraformaldehyde 1 x PBS
0.3% Triton X-100 1
20 NTCP 4ºC
Alexa Fluor 488 donkey anti-mouse IgG H+L
DAPI 1 TCS SP8
Leica preS1
D HDV
HDV Huh-7 HDV pSVLD3 HBV
pT7HB2.7
7-12 260,61)
HDV HDV 5 GEq/cell 5% PEG8000 16
70
HDV PEG8000
6 HDV RNA real-time RT-PCR
HDV
HDV RNA real-time RT-PCR 5’-GGACCCCTTCAGCGAACA-3’
5’-CCTAGCATCTCCTCCTATCGCTAT-3’
5’-AGGCGCTTCGAGCGGTAGGAGTAAGA-3’62)
RNA RNeasy Mini Kit QIAGEN
HCV
Huh-7.5.1 HCV 4
72 PBS Luciferase Assay
Substrate58)
NTCP
HepG2-hNTCP-C4 [3H]-taurocholic acid TCA
136 mM NaCl NMDG 5 mM KCl 1 mM MgCl2 1.1 mM KH2PO4
1.8 mM CaCl2 10 mM D-glucose 10 mM HEPES [pH7.4] 10 µM TCA 0.1 µM
[3H]-TCA 37ºC 5 15
[3H]-TCA 0.05% SDS
PPAR PPRE
71
PPARγ RXRα HepG2-hNTCP-C4
TransIT-LT1 Takara Bio Inc. 48
24 PBS Luciferase
Assay Substrate
PPARγ
His NTCP
4ºC 4
NTCP
2 x sample buffer 100 mM Tris-HCl [pH 6.8] 4% SDS 20%
glycerol 10% 2-mercaptoethanol SDS-PAGE
PVDF 5%
0.05% Tween20-TBS
0.05% Tween20-TBS Canget Signal Immunoreaction Enhancer
Solution 1 NTCP actin His
4ºC 0.05% Tween20-TBS Canget Signal
Immunoreaction Enhancer Solution 2 HRP
HRP 1
EzWestLumi plus
72
Proximity Ligation Assay: PLA
HepG2 Myc NTCP HA NTCP
48 preS1 40 nM
4ºC 30 4% paraformaldehyde 30
Myc HA Duolink PLA
TCS SP8 Leica NTCP
73
74
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