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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