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Virus Research 213 (2016) 205–213

Contents lists available at ScienceDirect

Virus Research

j ourna l h o mepa ge: www.elsev ier .com/ locate /v i rusres

eview

ecent developments in antivirals against hepatitis B virus

a-Juan Wang., Li Yang ∗, Jian-Ping Zuo ∗

hanghai Institute of Materia Medica, Chinese Academy of Sciences, Zuchongzhi Road 555, Shanghai, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 13 November 2015eceived in revised form1 December 2015ccepted 21 December 2015vailable online 28 December 2015

a b s t r a c t

Chronic hepatitis B virus (HBV) infection (CHB) is a major cause of cirrhosis and hepatocellular carcinoma(HCC). Although the availability of HBV vaccines effectively reduces the incidence of HBV infection, thehealthcare burden from CHB remains high. Several antiviral agents, such as (pegylated-) interferon-� andnucleos(t)ide analogs are approved by US FDA for chronic HBV infection management. Entecavir (ETV)and tenofovir disoproxil fumarate (TDF) have been recommended as the first-line anti-HBV drugs for

eywords:epatitis Bntiviral therapy

nhibitory drugsost immune regulators

excellent viral suppression with a low risk of antiviral resistance, but the cost and need for essentiallylife-long treatment are considerable challenges. And none of these current treatments can eradicate theintracellular virus. Given these issues, there is still an unmet medical need for an efficient HBV cure. Wesummarize here the key developments of antivirals against hepatitis B virus, including HBV replicationcycle inhibitors and host immune regulators.

© 2015 Elsevier B.V. All rights reserved.

ontents

1. HBV prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2052. HBV molecular biology and replication cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2053. Current available therapies of hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2064. Emerging antivirals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

4.1. HBV replication cycle inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2064.1.1. HBV entry inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2064.1.2. HBV replication inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2084.1.3. Competent HBV nucleocapsid formation Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2084.1.4. HBV secretion inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

4.2. Host immune regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2095. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

. HBV prevalence

Worldwide there are more than 248 million chronic hepati-is B virus (HBV) carriers (Mortality and Causes of Death, 2015).ccording to the global burden of Disease (GBD) study in 2013, hep-titis B infection led to 300,000 deaths globally (Global Burden of

Since 1981, a safe and effective vaccine against HBV infection,most of which in use are made from recombinant DNA that expressHBsAg only, has been available to induce immunity in unexposedpeople (Trepo et al., 2014). The vaccine led to a significant decreasein the incidence of HBV infection. In Taiwan, the proportion of child

isease Cancer et al., 2013). The clinical outcome of HBV infection istrongly influenced by the age of infection. Over 90% of newbornsrom HBeAg-positive mothers while less than 10% of adults withcute HBV progress to chronic infection (Locarnini et al., 2015).

∗ Corresponding author.E-mail addresses: yangli@simm.ac.cn (L. Yang), jpzuo@simm.ac.cn (J.-P. Zuo).

ttp://dx.doi.org/10.1016/j.virusres.2015.12.014168-1702/© 2015 Elsevier B.V. All rights reserved.

HBsAg carriers decreased from 10% in 1984 (Chen, 2009), to 0·9% in2009 (Ni et al., 2012). In the US, during the period 1999–2006, theprevalence of HBV infection decreased in people aged 6–19 years(from 1.9–0.6%; P < 0.01) and 20–49 years of age (5.9–4.6%; P < 0.01)(Sundaram and Kowdley, 2015; Wasley et al., 2010), and the age-adjusted prevalence of HBsAg (0.27%) in 2013 (Schweitzer et al.,2015) was not statistically different from what they were in 2006

(Wasley et al., 2010).

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. HBV molecular biology and replication cycle

HBV, a member of Hepadnaviridae family, is a DNA virus withn envelope surrounding an icosahedral capsid. The capsid con-ains HBV genome DNA and the viral polymerase (Pol). HBV genomeomprises a relaxed-circular, partially double-stranded 3.2 kb DNArcDNA), which contains four open-reading frames and encodes theurface protein, the core protein/the hepatitis B e antigen (HBeAg),he viral polymerase and the viral X protein (Liang, 2009).

HBV virion enters the hepatocytes by binding with the sodium-aurocholate cotransporting polypeptide (NTCP) receptor (step A)nd is uncoated (step B). The naked nucleocapsid is transportedo the nucleus, and the HBV genome is integrated into the hostenome (step C), or converted into a covalently closed circular DNAcccDNA), which serves as the transcriptional template (step D).fter HBV RNAs transcription (step E) and HBV proteins synthe-is (step F), the nucleocapsid is formed in the cytosol, and duringhis process a pregenomic RNA (pgRNA) and viral polymerase (Pol)re incorporated into the assembling core (step G). Once pgRNAs encapsidated, reverse transcription begins and (−) DNA strands synthesized from the pgRNA template by reverse transcriptasectivity of Pol (step H), followed by pgRNA degradation by RNase Hctivity of Pol and synthesis of the (+) DNA strand by the DNA poly-erase activity of Pol (step I). Mature nucleocapsid is then either

irected to the secretory pathway for envelopment with L, M, and surface proteins (step J), or recycled into the nucleus to amplifyhe cccDNA pool (step K). The envelope protein can be secreted asmall, non-infectious subviral particles, and the precore protein isecreted as HBeAg (step L) (Fig. 1).

The complex steps of HBV replication cycle, including hepato-yte entry, replication, nucleocapsid formation, and release, are allotential targets for antivirals. And given the importance of host

mmune regulation in HBV cure, agents targeting innate or adaptivemmunity are currently undergoing development for viral controlBaumert et al., 2015).

. Current available therapies of hepatitis B

Several antiviral agents are approved by the United States Foodnd drug administration (FDA) for the management of chronic HBVnfection (CHB): interferon-� (IFN-�) and (pegylated-) IFN-�, andve nucleos(t) ide analogues (NAs), including lamivudine (LMV),defovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), andenofovir disoproxil fumarate (TDF) (Fig. 2) (Trepo et al., 2014).Pegylated-) IFN-� has a weaker antiviral activity than NAs, buts associated with a higher rate of HBeAg and HBsAg loss. The NAsan efficiently inhibit HBV replication by targeting the viral reverseranscriptase. Although the resistance problem to the NAs has beenolved by TDF and to a lesser extent by ETV, all the NAs have noirect effect on viral transcription, translation or cccDNA, whicheans that the NAs are highly effective at suppressing viral repli-

ation, but rarely lead to the loss of HBsAg and the eradicationf the intracellular virus (Boettler et al., 2014; Gish et al., 2015).t was reported that CHB patients who have achieved a serologicesolution of infection (loss of HBsAg, undetectable serum HBVNA, appearance of anti-HBs) can experience reactivation of theirisease as a consequence of immunosuppression or the use of anti-

nflammatory medications (Perrillo et al., 2015; Reddy et al., 2015;eetharam et al., 2014). Therefore, the development of new classesf HBV inhibitors and therapeutic strategies, including combina-ion therapies, will be needed to ultimately cure the majority of

HB patients (Zeisel et al., 2015). In this review, we attempt toummarize the current advances in the field of antivirals againstBV and their targets, including HBV replication cycle inhibitorsnd host immune regulators (Table 1).

ch 213 (2016) 205–213

4. Emerging antivirals

4.1. HBV replication cycle inhibitors

4.1.1. HBV entry inhibitorsMaintenance of chronic HBV infection is thought to depend on

a dynamic turnover of infected hepatocytes that are cleared by theimmune system and cells that become newly infected. Previousstudies suggested that hepatocyte turnover is much faster in HBV-infected liver than in healthy hepatocytes. HBV entry inhibitorsmay contribute to the eventual clearance of the virus with pro-longed therapy by reducing newly infected hepatocyte (Volz et al.,2013).

HBV entry is essential for the initiation, spread, and maintenanceof viral infection. The process involves the interaction between viralenvelope proteins and cellular receptors. The establishment of Hep-aRG cell line (Gripon et al., 2002) and systems based on primaryhuman hepatocytes and primary Tupaia belangeri hepatocyteshave facilitated investigations of the cellular and viral determi-nants involved in HBV entry (Wang and Chen, 2014). It has beenwell documented that the N-terminus (amino acids 1–47) of HBVpreS1 domain of the large viral envelope protein (L protein) is anessential element for viral entry (Schieck et al., 2013). The evidencesuggests that Myrcludex-B, a synthetic myristoylated lipopeptideof this region, sequence-specifically and acylation-dependently tar-gets hepatocytes and efficiently blocks de novo HBV infection bothin vitro (Barrera et al., 2005; Glebe et al., 2005; Schulze et al., 2010)and in vivo (Petersen et al., 2008). Moreover, because upon viralentry, HBV genome is released into the cell nucleus, then the rcDNAis converted into the cccDNA, Myrcludex-B can efficiently hinderthe amplification of the cccDNA pool in initially infected hepato-cytes. Therefore, Myrcludex-B both sheds light on the molecularmechanism of HBV entry into hepatocytes and provides a basis forthe development of potent hepadnaviral entry inhibitors as a noveltherapeutic concept for the treatment of hepatitis B (Volz et al.,2013). Currently Myrcludex-B is being evaluated in phase II clinicaltrials (Haefeli et al., 2012).

Heparan sulfate proteoglycan (HSPG) is believed to be involvedin the initial binding process of HBV entry (Leistner et al., 2008;Schulze et al., 2007), but a more specific and high-affinity cel-lular receptor on hepatocytes should be involved in HBV stricttissue specificity. Recently, through a combined approach ofusing biochemical and proteomic analyses with primary cul-tures of treeshrew hepatocytes, sodium/bile acid cotransporter(SLC10A1, also known as human sodium taurocholate cotrans-porting polypeptide or NTCP) was discovered to be involved inHBV (mainly the pre-S1 domain) and host interaction (Ezzikouriet al., 2014; Ni et al., 2014; Yan et al., 2015a). The identification ofNTCP as a functional receptor for HBV may lead to novel inhibitorsagainst the viral infection in the future (Yan et al., 2015a; Yuenand Lai, 2015). Chemicals that inhibit hNTCP metabolic functionshave been tested for their efficiency in blocking HBV infection.For example, cyclosporine A (CsA) and its analogs can block HBVentry by cyclophilin-independent interference with the bindingbetween NTCP and large envelope protein in vitro (Nkongolo et al.,2014; Watashi et al., 2014), and cyclosporine B shows the highestpotency for inhibition of HBV infection among cyclosporine analogs(Iwamoto et al., 2014). Moreover, Irbesartan (Ko et al., 2015; Wanget al., 2015a), ezetimibe (Konig et al., 2014; Lucifora et al., 2013),and ritonavir, three FDA approved therapeutics with inhibitorypotential on the metabolic function of hNTCP, can prevent infec-tion at an early stage of the viral replication cycle (Blanchet et al.,

2014). Another new inhibitor of HBV entry is a flavonoid presentin green tea extract, epigallocatechin-3-gallate (EGCG), which canblock the endocytosis/fusion step via clathrin-dependent endocy-

Y.-J. Wang. et al. / Virus Research 213 (2016) 205–213 207

Fig. 1. Overview of the key steps of the hepatitis B virus replication cycle and potential drug targets. The numbered steps are discussed in the text.

US FD

ttsVr

Fig. 2. Nucleos(t) ide drugs approved by the

osis of NTCP (Huang et al., 2014). Entry inhibitors targeting NTCP is

hought to be clinically achievable without significant effect on bilealt transport by NTCP (Liang et al., 2015) (Slijepcevic et al., 2015;az et al., 2015). Moreover, entry inhibitors targeting NTCP, a keyeceptor for both HBV and its satellite hepatitis D virus (HDV), can

A for the treatment of chronic HBV infection.

be used for the clinical evaluation in patients who are co-infected

with HBV and HDV(Blanchet et al., 2014; Yan and Li, 2015; Yan et al.,2015a), for which there is currently no specific antiviral exceptIFN-�.

208 Y.-J. Wang. et al. / Virus Research 213 (2016) 205–213

Table 1Anti-HBV agents development.

Targets Compounds Development status

HBV replication cycle inhibitors Entry Myrcludex-B (Barrera et al., 2005; Glebe et al.,2005; Haefeli et al., 2012; Petersen et al., 2008;Schulze et al., 2010; Volz et al., 2013)Cyclosporine A (Nkongolo et al., 2014),Ezetimibe (Konig et al., 2014)EGCG (Huang et al., 2014)

Phase IIFDA approved but not tested for HBVPreclinical

Replication Nucleotide analoguesa

Disubstituted sulfonamide compounds (Caiet al., 2012)DNA cleavage enzymes (Bloom et al., 2013;Chen et al., 2014)RNA interference (Lanford et al., 2013a,b;Schluep et al., 2013; Yuen et al., 2014)

FDA approvedPreclinicalPreclinicalARC-520 in phase II

Nucleocapsid formation BisANS (Zlotnick et al., 2002)HAPs (Bourne et al., 2008; Deres et al., 2003;Stray et al., 2005; Stray and Zlotnick, 2006)PPA (Delaney et al., 2002; Feld et al., 2007)Pyridazinone derivatives (Wang et al., 2015b)Isothiafludine (Yang et al., 2014)

PreclinicalBAY41-4109 in phase I (Liu et al., 2015)GLS4 in phase II (Wang et al., 2012; Wuet al., 2013; Zhou et al., 2013a; Zhouet al., 2013b)Preclinical and early clinical phasePreclinicalNZ-4 in phase I

Secretion Iminosugar derivatives ofButyldeoxynojirimycin and related glycolipids(Zeisel et al., 2015)�-glucosidase inhibitors (Zeisel et al., 2015)Amphipathic DNA polymers (APs)Benzimidazole derivative (Xu et al., 2014)

PreclinicalPreclinicalREP 9 AC in phase II (Mahtab et al.,2011)Preclinical

Host immune regulators Innate and adaptive immune responses IFN-� and pegylated IFN-�a

LT�R (Lucifora et al., 2014)Toll-like receptor 7 agonist (Fosdick et al.,2014; Gane et al., 2015; Lanford et al., 2013;Lawitz et al., 2014; Lopatin et al., 2013; Menneet al., 2015)Thymosin �1 (Goldstein and Goldstein, 2009)PD1 blockade (Bengsch et al., 2014; Liu et al.,2014)Therapeutic vaccines (Fontaine et al., 2015;Godon et al., 2014; Michel et al., 2011; Yoon

l., 201

FDA approvedPreclinicalPhase IIPhase IVPhase I/II for HCCGS-4774 in phase II; DV-601 in phase I;DNA vaccine pCMVS2.S in phase I/II

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a Drugs approved by FDA. FDA, US Food and Drug Administration; HCC, hepatoce

.1.2. HBV replication inhibitorsFollowing infection into target cells, HBV virion undergoes

ncoating by the lipid bilayer and dissolution of the nucleocap-id. HBV nucleocapsid then shuttles from cytoplasm to nucleus andelease rcDNA (relaxed circular DNA) into the cell nucleus (Li et al.,010). Inside the nucleus, rcDNA is converted into covalently closedircular DNA (cccDNA). HBV cccDNA acts as the transcription tem-late for pregenome RNA (pgRNA) and viral mRNAs. pgRNA acts as

template to synthesize viral genome DNA by the function of theiral polymerase. Newly assembled capsid can either be envelopednd secreted or reimported into the nucleus to form additionalccDNA (Seeger and Mason, 2000; Seeger and Mason, 2015). SoccDNA is thought to be responsible for the persistent infection ofepatocytes.

As described above, 5 orally administered NAs (Fig. 2), whichnhibit HBV replication by targeting HBV polymerase, have beenpproved by FDA for the treatment of chronic hepatitis B infection.ssentially, due to high efficacy and high barrier to drug resistance,TV and TDF have been recommended as the first-line anti-HBVrugs (Cholongitas et al., 2015; Papatheodoridis et al., 2012).

To ultimately achieve a reliable HBV cure, novel antiviralsargeting cccDNA are developing, including preventing cccDNA for-

ation, eliminating cccDNA or silencing cccDNA transcription. Two

tructurally related disubstituted sulfonamide (DSS) compounds,ermed CCC-0975 and CCC-0346 (Fig. 3), was reported to reduce theevels of HBV cccDNA and the putative precursor of cccDNA, depro-einized relaxed circular DNA (DP-rcDNA). DSS compounds did not

5)

carcinoma; IFN, interferon; LT�R, lymphotoxin-� receptor.

promote the intracellular decay of HBV DP-rcDNA and cccDNA,suggesting that the compounds interfere primarily with rcDNAconversion into cccDNA (Cai et al., 2012). DNA cleavage enzymes,which can cleave sequence-specific DNA targets and specificallytarget the conserved regions of the viral genomic DNA, are currentlybeing engineered (Bloom et al., 2013; Chen et al., 2014). Strategybased on RNA interference to reduce viral RNAs, proteins, and viralDNA is also discussed (Klein et al., 2003; Lanford et al, 2013a,b;McCaffrey et al., 2003; Morrissey et al., 2005; Schluep et al., 2013;Yuen et al., 2014).

4.1.3. Competent HBV nucleocapsid formation InhibitorsIt is now well known that HBV capsid plays a vital role in

HBV replication cycle. HBV capsids mostly compose of 120 coreprotein (Cp) homo-dimers arranged with T = 4 icosahedral sym-metry (Zlotnick et al., 1996). HBV Cp encompasses 183 or 185amino acid depending on the genotype and is comprised of an N-terminal assembly domain (amino acids 1–149) and a C-terminal150-183/185 amino acids domain (CTD). Cp not only forms the shellof capsid, but also modulates almost every step of the viral lifecycle,including genome packaging, reverse transcription, intracellulartrafficking, and maintenance of a stable infection. The capsid

assembly process is exquisitely timed and regulated, which is char-acterized by slow nucleation rate and weak pairwise dimer–dimerassociation energy, any disruption could be devastating to virusreplication (Tan et al., 2015; Zlotnick et al., 2015). It has been shown

Y.-J. Wang. et al. / Virus Research 213 (2016) 205–213 209

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hat a variety of small molecules can target Cp and have pleiotropichenotypic effects in the HBV replication cycle.

The first identified small molecule which misdirect HBV capsidssembly was a fluorescent dye, 5,50-bis[8-(phenylamino)-1-aphthalenesulfonate] (Bis-ANS) (Fig. 4). BisANS-bound Cp dimerad an increased propensity for formation of non-capsid polymersZlotnick et al., 2002).

Heteroaryldihydropyrimidines (HAPs) (Fig. 4) have been iden-ified as highly potent HBV inhibitors acting as assembly effectors.APs were shown to inhibit HBV replication both in vitro and in vivo

Bourne et al., 2008; Stray and Zlotnick, 2006). HAPs act by alloster-cally inducing assembly-active states at stoichiometric levels andtabilizing non-capsid polymers at higher concentrations (Bournet al., 2008; Stray et al., 2005). Crystal structures of HBV capsido-crystallized with HAP1 suggested that HAPs-binding site was aydrophobic pocket at the dimer–dimer interface (Bourne et al.,006; Katen et al., 2013; Tan et al., 2013). HAPs were also reportedo directly interfere with cccDNA transcription and stability (Bellonit al., 2014). BAY41-4109 currently is undergoing a phase I studyy AiCuris in Germany, but the clinical result is unclear (Liu et al.,015). Morphothiadine mesilate (GLS4) is currently undergoinglinical trials in China (Wang et al., 2012, 2013; Zhou et al., 2013a,b).

Compounds of phenylpropenamide (PPA) family, AT-61 and AT-30 (Fig. 4), were also found to inhibit HBV replication in vitroDelaney et al., 2002; King et al., 1998). It was reported thatPAs lead to accumulation of genome-free capsids, but withoutnfluence on capsid morphology (Feld et al., 2007). By studies onn vitro Cp149 capsid assembly, AT-130 was found to accelerateapsid assembly, stabilize capsids, and cause tertiary and qua-ernary structural changes of capsid but does not disrupt capsidtructure (Katen et al., 2010; Li et al., 2013). Moreover, it waseported that AT-130 binds the same hydrophobic pocket at thenter-dimer interface as HAP, but favors a different quasiequiva-ent location from the one favored by HAP1 (Katen et al., 2013; Tant al., 2015). Clinical trials of PPAs are still required.

Recently, new classes of HBV inhibitors of competent nucle-capsid assembly are emerging. One compound of pyridazinoneerivatives, 3711 (Fig. 4), was discovered to inhibit HBV replicationy inducing genome-free capsid formation in cell culture system.711 treatment induced capsid physical profile changes, includinglectrophoresis mobility and buoyant density profile (Wang et al.,015b). The effect of 3711 on capsid formation can be mimickedy an HBV Cp mutant, which is insensitive to HAPs by increas-

ng the buried hydrophobic surface at the dimer–dimer interface.tudies on HAPs, PPAs and 3711 provide evidence that the inter-imer interface plays a critical role in regulating capsid assemblynd replication.

Another reported HBV inhibitor acting by decreasing enapsi-ated pgRNA is a derivative of bis-heterocycle tandem pairs derived

rom the natural product leucamide A, isothiafludine (NZ-4) (Fig. 4)Yang et al., 2014). NZ-4 induces a portion of incompetent HBVapsids formation, which exerts faster electrophoretic mobilityehavior on agarose gel. Interestingly, compared with assembly

uted sulfonamide (DSS) compounds.

effectors described above, the effect of NZ-4 on capsid formationis dependent on the presence of the arginine cluster amino acidresidues 150-152 (ARD I) of CTD (Yang et al., 2015). It is well stud-ied that the arginines in ARD I is essential for the packaging ofpgRNA/HBV (Hatton et al., 1992), and the study of NZ-4 indicatesthat this domain is probably a promiscuous site for assembly effec-tors development. Currently, NZ-4 is being evaluated in phase Iclinical trials in China.

A series of sulfamoylbenzamide (SBA) derivatives represent anovel chemical entity which can significantly reduce the amount ofcytoplasmic HBV DNA (Cho et al., 2013). Structures of representa-tive compounds, DVR-01 and DVR-23, are presented in Fig. 4. SBAspotently inhibit the formation of pgRNA-containing nucleocapsidsof HBV but not WHV and DHBV. The differential sensitivity of HBVand WHV to the SBAs provides a unique opportunity to identifythe viral target of the compounds. Through a heterologous geneticcomplementation study of the Cp, DNA polymerase, and pgRNAbetween HBV and WHV, Campagna et al. suggested that HBV Cpis the molecular target of the SBAs. Due to its favorable pharma-cological properties, SBAs is warranted for further development asnovel antiviral therapeutics for the treatment of chronic hepatitisB (Campagna et al., 2013).

4.1.4. HBV secretion inhibitorsHBV secretion and budding process is another potential target

for antivirals (Wang and Chen, 2014). To date, multiple compoundshave been described (Zeisel et al., 2015). An iminosugar, N-nonyl-deoxy-galactonojirimycin (N-nonyl-DGJ) (Fig. 5) exert an antiviralaction at a point before viral envelopment and perhaps preventthe proper encapsidation of the HBV pgRNA (Mehta et al., 2002).�-glucosidase inhibitor (N-butyl-deoxy-nojirimycin, N-butyl-DNJ)(Fig. 5) treatment results in production of HBV virions with alteredmolecular composition and infectivity (Lazar et al., 2007). REP 9AC, a 40-nucleotide polycytidine (alternating adenosine) amphi-pathic DNA polymer (AP), is currently in a phase I/II clinical trial(Mahtab et al., 2011). A benzimidazole derivative, BM601 (Fig. 5),has recently been reported to selectively inhibit the secretion ofHBV virions and HBsAg. It acts by interfering surface protein aggre-gation in trans-Golgi apparatus, without triggering cellular stressresponse or affecting HBeAg or host protein secretion (Xu et al.,2014). Inhibiting HBV virion and HBsAg secretion could not onlydecrease HBV DNA levels, but also interfere with HBsAg-mediatedimmunosuppression. These compounds could be used as modeldrugs to investigate the secretion pathway of HBV (Liu et al., 2015).However, the induced intracellular HBsAg accumulation could leadto storage diseases, and blockage of virion synthesis could increaseintracellular recirculation of rcDNA, leading to increased cccDNAcopy numbers (Gish et al., 2015; Zeisel et al., 2015).

4.2. Host immune regulators

After 36 years of experience, IFN-� has long been used forCHB treatment (Scaglione and Lok, 2012). It has both antiviral (Pei

210 Y.-J. Wang. et al. / Virus Research 213 (2016) 205–213

Fig. 4. The chemical structures of some HBV nucleocapsid formation Inhibitors.

Y.-J. Wang. et al. / Virus Research 213 (2016) 205–213 211

s of so

eit(i

HgRbttrLriTembcY

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HredHrtui

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Fig. 5. The chemical structure

t al., 2014; Yan et al., 2015b) and immunomodulatory activity,ts main disadvantages are the need for parenteral administra-ion and the frequent adverse effects. Compared to standard IFN-�,pegylated-) IFN-� has the advantage of more convenient admin-stration (Locarnini et al., 2015).

Due to the importance of host immune regulation inBV cure, efforts are ongoing to uncover different strate-ies on inducing host intracellular antiviral immune responses.ecently, anti-LT�R (lymphotoxin-� receptor) antibodies haveeen described for HBV cure. Antibodies activating LT�R promotehe apurinic/apyrimidinic site formation in cccDNA and upregulatehe expression of nuclear APOBEC3 deaminases, which lead to theeduction of HBV DNA, HBsAg and cccDNA levels. The potential ofT�R agonists in combination with current polymerase inhibitorsemains to be evaluated (Lucifora et al., 2014). Other agents exhibit-ng immunomodulatory activity were also being studied, includingoll-like receptor 7 agonist(GS-9620)(Gane et al., 2015; Lanfordt al., 2013a,b; Lopatin et al., 2013; Menne et al., 2015), thy-osin �1(Goldstein and Goldstein, 2009; Korba et al., 2008), PD-1

lockade(Bengsch et al., 2014; Liu et al., 2014), and therapeutic vac-ines (Fontaine et al., 2015; Godon et al., 2014; Michel et al., 2011;oon et al., 2015)

. Conclusions and perspectives

Current HBV therapies have offered clear benefits to chronicBV patients. Effective antiviral treatment not only inhibits HBV

eplication, reduces serum viral load and accelerates hepatitis B antigen (HBeAg) serum conversion, but also reduces the inci-ence of liver fibrosis, cirrhosis, hepatic decompensation and HCC.owever, current treatments for HBV infection can only suppress

eplication but cannot eradicate the virus. And attempts to improvehe response by administering the current regimens have beennsuccessful. So new antiviral drugs targeting a number of steps

n the HBV replication cycle are now being developed.The ultimate goal of future therapies for HBV infection involves

he complete elimination of all viral DNA from the body, includinghe removal of all cccDNA and integrated virus. This goal may beifficult or impossible to prove and may turn out to be unachievableGish et al., 2015). A more realistic goal is to achieve “functionalure”, in which the treatment either eliminate the virus from theiver or durable immune control of hepatitis B virus (HBV) infectionn the absence of chronic therapy (Fletcher and Delaney, 2013).

This review summarized recent achievement of antiviralsgainst HBV targeting different steps in the HBV replication cycle,ncluding entry, replication, capsid formation, secretion processes.hese novel compounds currently being evaluated in preclinical or

me HBV secretion inhibitors.

clinical evaluation should offer additional and improved choices forfinite duration treatment in the near future.

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