Revill PA, Chisari FV, Block JM, et al. (2019) A global scientific strategy to cure
hepatitis B. Lancet Gastroenterol Hepatol 4: 545-58.
Stanaway JD, Flaxman AD, Naghavi M, et al. (2016) The global burden of viral
hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study
2013. Lancet 388: 1081-88.
Yuen MF, Chen DS, Dusheiko GM, et al. (2018) Hepatitis B virus infection. Nat
Rev Dis Primers 4: 18035.
Song A, Lin X, Chen X. (2021) Functional cure for chronic hepatitis B:
accessibility, durability, and prognosis. Virol J 18: 114.
Yan H, Zhong G, Xu G, et al. (2012) Sodium taurocholate cotransporting
polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1:
e00049.
Le Seyec J, Chouteau P, Cannie I, Guguen-Guillouzo C, Gripon P. (1999)
Infection process of the hepatitis B virus depends on the presence of a defined
sequence in the pre-S1 domain. J Virol 73: 2052-7.
Iwamoto M, Saso W, Sugiyama R, et al. (2019) Epidermal growth factor receptor
is a host-entry cofactor triggering hepatitis B virus internalization. Proc Natl Acad
Sci U S A 116: 8487-92.
Karayiannis P. (2017) Hepatitis B virus: virology, molecular biology, life cycle
and intrahepatic spread. Hepatol Int 11: 500-08.
Tsukuda S, Watashi K. (2020) Hepatitis B virus biology and life cycle. Antiviral
Res 182: 104925.
10 Konig A, Doring B, Mohr C, et al. (2014) Kinetics of the bile acid transporter and
hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in
hepatocytes. J Hepatol 61: 867-75.
21
517
11
518
519
520
521
12
522
523
13
524
525
14
526
527
528
15
529
530
531
16
532
533
534
17
535
536
537
18
538
539
19
540
541
542
20
543
544
21
545
546
22
547
548
549
550
551
23
Watashi K, Sluder A, Daito T, et al. (2014) Cyclosporin A and its analogs inhibit
hepatitis B virus entry into cultured hepatocytes through targeting a membrane
transporter, sodium taurocholate cotransporting polypeptide (NTCP). Hepatology
59: 1726-37.
Lucifora J, Esser K, Protzer U. (2013) Ezetimibe blocks hepatitis B virus infection
after virus uptake into hepatocytes. Antiviral Res 97: 195-7.
Huang HC, Tao MH, Hung TM, et al. (2014) (-)-Epigallocatechin-3-gallate
inhibits entry of hepatitis B virus into hepatocytes. Antiviral Res 111: 100-11.
Ko C, Park WJ, Park S, et al. (2015) The FDA-approved drug irbesartan inhibits
HBV-infection in HepG2 cells stably expressing sodium taurocholate cotransporting polypeptide. Antivir Ther 20: 835-42.
Tsukuda S, Watashi K, Hojima T, et al. (2017) A new class of hepatitis B and D
virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the
viral large surface proteins. Hepatology 65: 1104-16.
Fukano K, Tsukuda S, Oshima M, et al. (2018) Troglitazone Impedes the
Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of
Hepatitis B Virus Into Hepatocytes. Front Microbiol 9: 3257.
Kirstgen M, Lowjaga K, Muller SF, et al. (2020) Selective hepatitis B and D virus
entry inhibitors from the group of pentacyclic lupane-type betulin-derived
triterpenoids. Sci Rep 10: 21772.
Mak LY, Seto WK, Yuen MF. (2021) Novel Antivirals in Clinical Development
for Chronic Hepatitis B Infection. Viruses 13.
European Association for the Study of the Liver. Electronic Address EEE,
European Association for the Study of The L. (2018) EASL Recommendations on
Treatment of Hepatitis C 2018. J Hepatol 69: 461-511.
Seca AML, Pinto D. (2019) Biological Potential and Medical Use of Secondary
Metabolites. Medicines (Basel) 6.
Xiong X, Tang N, Lai X, et al. (2021) Insights Into Amentoflavone: A Natural
Multifunctional Biflavonoid. Front Pharmacol 12: 768708.
Samec D, Karalija E, Dahija S, Hassan STS. (2022) Biflavonoids: Important
Contributions to the Health Benefits of Ginkgo (Ginkgo biloba L.). Plants (Basel)
11.
Ito T, Yokota R, Watarai T, et al. (2013) Isolation of six isoprenylated
biflavonoids from the leaves of Garcinia subelliptica. Chem Pharm Bull (Tokyo)
61: 551-8.
22
552
24
553
554
555
25
556
557
558
26
559
560
27
561
562
28
563
564
29
565
566
30
567
568
569
31
570
571
572
32
573
574
575
33
576
577
578
34
579
580
581
35
582
583
36
584
585
586
587
37
Yu S, Yan H, Zhang L, et al. (2017) A Review on the Phytochemistry,
Pharmacology, and Pharmacokinetics of Amentoflavone, a Naturally-Occurring
Biflavonoid. Molecules 22.
Lin YM, Anderson H, Flavin MT, et al. (1997) In vitro anti-HIV activity of
biflavonoids isolated from Rhus succedanea and Garcinia multiflora. J Nat Prod
60: 884-8.
Lin YM, Flavin MT, Schure R, et al. (1999) Antiviral activities of biflavonoids.
Planta Med 65: 120-5.
Li F, Song X, Su G, et al. (2019) Amentoflavone Inhibits HSV-1 and ACVResistant Strain Infection by Suppressing Viral Early Infection. Viruses 11.
Indrasetiawan P, Aoki-Utsubo C, Hanafi M, et al. (2019) Antiviral Activity of
Cananga odorata Against Hepatitis B Virus. Kobe J Med Sci 65: E71-E79.
Cheng Z, Sun G, Guo W, et al. (2015) Inhibition of hepatitis B virus replication
by quercetin in human hepatoma cell lines. Virol Sin 30: 261-8.
Parvez MK, Ahmed S, Al-Dosari MS, et al. (2021) Novel Anti-Hepatitis B Virus
Activity of Euphorbia schimperi and Its Quercetin and Kaempferol Derivatives.
ACS Omega 6: 29100-10.
Srividhya M, Hridya H, Shanthi V, Ramanathan K. (2017) Bioactive Amento
flavone isolated from Cassia fistula L. leaves exhibits therapeutic efficacy. 3
Biotech 7.
Iwamoto M, Watashi K, Tsukuda S, et al. (2014) Evaluation and identification of
hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane
transporter NTCP. Biochem Biophys Res Commun 443: 808-13.
Ogura N, Watashi K, Noguchi T, Wakita T. (2014) Formation of covalently
closed circular DNA in Hep38.7-Tet cells, a tetracycline inducible hepatitis B
virus expression cell line. Biochem Biophys Res Commun 452: 315-21.
Shimura S, Watashi K, Fukano K, et al. (2017) Cyclosporin derivatives inhibit
hepatitis B virus entry without interfering with NTCP transporter activity. J
Hepatol 66: 685-92.
Asami J, Kimura KT, Fujita-Fujiharu Y, et al. (2022) Structure of the bile acid
transporter and HBV receptor NTCP. Nature 606: 1021-26.
Nkongolo S, Ni Y, Lempp FA, et al. (2014) Cyclosporin A inhibits hepatitis B
and hepatitis D virus entry by cyclophilin-independent interference with the
NTCP receptor. J Hepatol 60: 723-31.
Wilsky S, Sobotta K, Wiesener N, et al. (2012) Inhibition of fatty acid synthase by
amentoflavone reduces coxsackievirus B3 replication. Arch Virol 157: 259-69.
23
588
38
589
590
39
591
592
593
40
594
595
41
596
597
598
42
599
600
601
602
43
44
603
604
605
45
606
607
608
46
609
610
611
612
47
613
614
48
615
616
617
49
618
619
620
621
622
50
Ryu YB, Jeong HJ, Kim JH, et al. (2010) Biflavonoids from Torreya nucifera
displaying SARS-CoV 3CL(pro) inhibition. Bioorg Med Chem 18: 7940-7.
Lee WP, Lan KL, Liao SX, et al. (2018) Inhibitory Effects of Amentoflavone and
Orobol on Daclatasvir-Induced Resistance-Associated Variants of Hepatitis C
Virus. Am J Chin Med 46: 835-52.
Zembower DE, Lin YM, Flavin MT, Chen FC, Korba BE. (1998) Robustaflavone,
a potential non-nucleoside anti-hepatitis B agent. Antiviral Res 39: 81-8.
Glebe D, Urban S, Knoop EV, et al. (2005) Mapping of the hepatitis B virus
attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia
hepatocytes. Gastroenterology 129: 234-45.
Gripon P, Cannie I, Urban S. (2005) Efficient inhibition of hepatitis B virus
infection by acylated peptides derived from the large viral surface protein. J Virol
79: 1613-22.
Kang C, Syed YY. (2020) Bulevirtide: First Approval. Drugs 80: 1601-05.
Donkers JM, Zehnder B, Van Westen GJP, et al. (2017) Reduced hepatitis B and
D viral entry using clinically applied drugs as novel inhibitors of the bile acid
transporter NTCP. Sci Rep 7: 15307.
Yu Y, Li S, Liang W. (2018) Bona fide receptor for hepatitis B and D viral
infections: Mechanism, research models and molecular drug targets. Emerg
Microbes Infect 7: 134.
Tsukuda S, Watashi K, Iwamoto M, et al. (2015) Dysregulation of retinoic acid
receptor diminishes hepatocyte permissiveness to hepatitis B virus infection
through modulation of sodium taurocholate cotransporting polypeptide (NTCP)
expression. J Biol Chem 290: 5673-84.
Thongsri P, Pewkliang Y, Borwornpinyo S, et al. (2021) Curcumin inhibited
hepatitis B viral entry through NTCP binding. Sci Rep 11: 19125.
Chen B, Wang X, Zhang Y, et al. (2020) Improved solubility, dissolution rate, and
oral bioavailability of main biflavonoids from Selaginella doederleinii extract by
amorphous solid dispersion. Drug Deliv 27: 309-22.
Bhujbal SV, Mitra B, Jain U, et al. (2021) Pharmaceutical amorphous solid
dispersion: A review of manufacturing strategies. Acta Pharm Sin B 11: 2505-36.
Pandi P, Bulusu R, Kommineni N, Khan W, Singh M. (2020) Amorphous solid
dispersions: An update for preparation, characterization, mechanism on
bioavailability, stability, regulatory considerations and marketed products. Int J
Pharm 586: 119560.
623
24
624
Fig. 1. Amentoflavone inhibits HBV infection. Anti-HBV activity and cytotoxicity of
25
625
amentoflavone on HepG2-hNTCP-C4. (A) Levels of HBsAg production in culture
626
supernatants of HepG2-hNTCP-C4 cells were determined by ELISA. (B) To monitor
627
cytotoxic levels of amentoflavone, viability assessment (left) and cell count (right) were
628
performed. Cell viability was determined by XTT assay. HepG2-hNTCP-C4 cells in a 24-
629
well plate were treated with different concentrations of amentoflavones at 37°C for 24 h.
630
After the removal of amentoflavone, cells were incubated in medium without
631
amentoflavone for 7 days. Living cells were manually counted. Data are expressed as
632
mean ± SD from triplicate wells. (C to I) HBV was inoculated onto HepG2-hNTCP-C4
633
cells in the presence of amentoflavone or 200 nM preS1 peptide at 37°C for 18 h.
634
Secretion of HBeAg (C) in culture supernatants and HBV RNA (D) and cccDNA (E)
635
levels in infected cells were determined by ELISA and qPCR, respectively. (F) HepG2-
636
hNTCP-C4 cells were infected with HBV in the presence of amentoflavone (80 µg/ml),
637
preS1 peptide (200 nM), or 0.2% DMSO as the untreated control. Infected cells were
638
stained with anti-HBs (green) and anti-HBc (red) antibodies. Nuclear was stained with
639
Hoechst 33342 (blue). Scale bar, 100 µM. (G) HBV-infected HepG2-hNTCP-C4 cells
640
were stained with anti-HBs antibody, as shown in (F). The number of HBsAg-positive
641
cells from seven randomly selected areas was counted. The percentages of HBsAg-
642
positive cells by the compounds compared to the untreated control are shown. Data show
643
mean ± SD from two independent experiments. (H, I) Anti-HBV activity and cytotoxicity
644
of amentoflavone on PXB-cells. The HBsAg levels in culture supernatants of PXB-cells
645
were determined by ELISA (H) and cell viability was determined by the XTT assay (I).
646
(J) HepG2-hNTCP-C4 cells were infected with HBV genotype C in the presence of
647
amentoflavone (80 µg/ml), preS1 peptide (200 nM), or 0.2% DMSO. After the removal
648
of amentoflavone, cells were incubated in medium without amentoflavone for 10 days.
26
649
HBV RNA levels in the cells were quantified by qPCR. Data show mean ± SD from two
650
independent experiments. All data except (C) and (F) are expressed as mean ± SEM from
651
two or three independent experiments. *P < 0.05, **P < 0.01. AM: amentoflavone.
652
653
654
655
656
Fig. 2. Amentoflavone inhibits HBV entry step. (A) A schematic of time-of-addition
657
experiments. Pre-treatment: cells were pretreated with compounds for 2 h before HBV
658
infection. After removing compounds, pretreated cells were challenged with HBV
659
infection for 18 h in the absence of compounds. Co-addition: compounds were added to
660
cell cultures for 18 h during virus inoculation. Post-infection: cells were infected with
661
HBV for 18 h in the absence of compounds. After washing out unbound virus, the
27
662
infected cells were treated with compounds for 24 h. HBsAg levels in culture
663
supernatants at day 8 p.i. were determined by ELISA. The gray and white square show
664
the periods of treatment and nontreatment of compounds. (B) HepG2-hNTCP-C4 cells
665
were pretreated with amentoflavone (80 µg/ml), preS1 peptide (200 nM), or 0.2%
666
DMSO as negative control for 2 h at 37°C. After washing out compounds, pretreated
667
cells were inoculated with HBV without compounds (pretreatment). HBV was
668
inoculated onto HepG2-hNTCP-C4 cells in the presence of amentoflavone (80 µg/ml),
669
preS1 peptide (200 nM), or 0.2% DMSO for 18 h at 37°C (co-addition). Cells were
670
inoculated with HBV without compounds for 18 h at 37°C. After washing out free
671
virus, cells were treated with amentoflavone (80 µg/ml), preS1 peptide (200 nM), or
672
0.2% DMSO for 24 h at 37°C (post-infection). Levels of HBsAg in culture supernatants
673
were determined by ELISA. AM: amentoflavone. Values show mean ± SD from two
674
independent experiments.
675
676
677
678
679
680
28
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
Fig. 3. Amentoflavone blocks HBV adsorption via preS1 binding. (A) Virucidal
700
activity of amentoflavone. A mixture of HBV and amentoflavone was preincubated at
701
37°C for 2 h. After removal of free amentoflavone with centrifugal filter device, residual
702
virus infectivity was titrated and expressed as a percentage relative to the untreated
703
control. (B) Effect of amentoflavone on HBV adsorption. HBV- amentoflavone mixture
704
was preincubated for 2 h at 37°C, then inoculated onto pre-chilled HepG2-hNTCP-C4
29
705
cells for 90 min at 4°C to allow HBV adsorption. After extensive washing with cold PBS,
706
cells were incubated without compounds for 10 days at 37°C. HBV RNA was extracted
707
and quantified by qPCR analysis. (C) Effect of amentoflavone on virus internalization.
708
HepG2-hNTCP-C4 cells were exposed with HBV on ice for 3 h in the absence of
709
compounds, and cultures were then transferred to 37°C in the presence of amentoflavone
710
for 20 h to allow viral internalization. After trypsinization and extensive washing of the
711
cells, intracellular HBV DNA were quantified by qPCR. (A-C) Data are expressed as
712
mean ± SEM from two independent experiments. (D) HepG2-hNTCP-C4 cells were
713
incubated with 40 nM TAMRA-labeled preS1 peptide in the presence of 80 µg/ml
714
amentoflavone, 200 nM non-label preS1 peptide, or 0.2% DMSO as a control at 37°C for
715
45 min. The binding of TAMRA-labeled preS1 to the cell surface was observed by
716
fluorescence microscopy. Red and blue signals indicate preS1 probe and the nucleus,
717
respectively. Scale bar, 100 µm. (E) HepG2-hNTCP-C4 cells were incubated with
718
different concentrations of TAMRA-labeled preS1 peptide (TAMRA-preS1) at 37°C for
719
45 min. In a competitive binding experiment, cells were exposed with 40 nM TAMRA-
720
preS1 in the presence or absence of compounds (0.2% DMSO, 200 nM non-label preS1
721
peptide, or 80 µg/ml amentoflavone). The red fluorescence intensity was measured using
722
multimode microplate reader. Data are expressed as mean ± SD from triplicate wells. **P
723
< 0.01, ns: not significant, AM: amentoflavone, a.u.: arbitrary unit
724
30
725
726
727
728
729
730
731
732
733
734
735
Fig. 4. Amentoflavone inhibits NTCP transporter activity. Cells were treated with
736
compounds (amentoflavone (AM), 200 nM preS1 peptide, 10 µM cyclosporin A (CyA)
737
and, 0.2% DMSO) for 37°C for 30 min followed by the addition of TCA for 5 min. Fold
738
reduction of cellular TCA uptake compared to the 0.2% DMSO (Na+) control was
739
calculated. Data are expressed as mean ± SD from triplicate wells. **P < 0.01.
740
31
741
742
Fig. 5. Anti-HBV and cytotoxic activities of amentoflavone derivatives. (A) Chemical
743
structure of amentoflavone and amentoflavone derivatives. Sciadopitysin, cupressflavone,
744
amentoflavone, and robustaflavone consists of two apigenin moieties (rings A-C) linked
32
745
through carbon-carbon. Amentoflavone and sciadopitysin have the same core structure of
746
C3-8 linkage. Robustaflavone has a structure with C3-6 linkage. Cupressflavone consists
747
of two apigenin units linking at each A ring. (B) HBV was inoculated to HepG2-hNTCP-
748
C4 cells in the presence of various concentrations of compounds (sciadopitysin,
749
cupressflavone, amentoflavone, robustaflavone: 40, 60, and 80 µg/ml, apigenin: 20 and
750
40 µg/ml, 200 nM preS1 peptide, 0.2% DMSO) for 18 h. Levels of HBsAg (B) and
751
HBeAg (C) secreted in the culture supernatants on day 7 p.i. were determined by ELISA.
752
Relative values as compared to the 0.2% DMSO control were calculated. (D) HepG2-
753
hNTCP-C4 cells were treated with amentoflavone derivatives (sciadopitysin,
754
cupressflavone, amentoflavone, robustaflavone: 40, 60, and 80 µg/ml, apigenin: 20, 40
755
and 80 µg/ml), 200 nM preS1 peptide or 0.2% DMSO for 18 h. Cell viability relative to
756
the 0.2% DMSO control was shown. Data are expressed as mean ± SEM from two
757
independent experiments. Sci: sciadopitysin, Cup: cupressflavone, AM: amentoflavone,
758
Rob: robustaflavone, Api: apigenin, *P < 0.05, **P < 0.01.
759
760
761
762
763
764
33
...
論文の公開元へ
