Past, Current, and Future Developments of ... - ACS Publications

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Past, Current, and Future Developments of Therapeutic Agents for Treatment of Chronic Hepatitis B Virus Infection Yameng Pei,†,§ Chunting Wang,†,§ S. Frank Yan,*,‡ and Gang Liu*,† †

School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China Molecular Design and Chemical Biology, Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai 201203, China

‡

ABSTRACT: For decades, treatment of hepatitis B virus (HBV) infection has been relying on interferon (IFN)-based therapies and nucleoside/nucleotide analogues (NAs) that selectively target the viral polymerase reverse transcriptase (RT) domain and thereby disrupt HBV viral DNA synthesis. We have summarized here the key steps in the HBV viral life cycle, which could potentially be targeted by novel anti-HBV therapeutics. A wide range of next-generation direct antiviral agents (DAAs) with distinct mechanisms of actions are discussed, including entry inhibitors, transcription inhibitors, nucleoside/ nucleotide analogues, inhibitors of viral ribonuclease H (RNase H), modulators of viral capsid assembly, inhibitors of HBV surface antigen (HBsAg) secretion, RNA interference (RNAi) gene silencers, antisense oligonucleotides (ASOs), and natural products. Compounds that exert their antiviral activities mainly through host factors and immunomodulation, such as host targeting agents (HTAs), programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors, and Toll-like receptor (TLR) agonists, are also discussed. In this Perspective, we hope to provide an overview, albeit by no means being comprehensive, for the recent development of novel therapeutic agents for the treatment of chronic HBV infection, which not only are able to sustainably suppress viral DNA but also aim to achieve functional cure warranted by HBsAg loss and ultimately lead to virus eradication and cure of hepatitis B.

1. EPIDEMIOLOGY OF HEPATITIS B VIRUS INFECTION Hepatitis B virus (HBV) infection remains a major health concern, with an estimated 350 million chronically infected patients worldwide, and it is particularly prevalent in Southeast Asia and sub-Sahara Africa.1 Chronic infection develops in approximately 90% of patients who contract the virus during infancy, while it develops in 30−50% of children infected before the age of six years old.2 For healthy adults, however, less than 5% of those infected will develop the chronic disease. Furthermore, of the chronically infected adults, about 20−30% develop cirrhosis and/or liver cancer.3 Although HBV vaccines induce over 95% protective responses in humans nowadays, chronic HBV infection remains a major cause of cirrhosis, which ultimately leads to hepatocellular carcinoma (HCC). It is currently estimated that HBV infection and the associated liver diseases result in close to one million deaths every year worldwide.1−3 HBV is known to primarily replicate within hepatocytes, while a steady low rate of virus replication was also found in the lymphatic system using the woodchuck hepatitis virus (WHV) animal model.4 Hepatitis B virus does not cause any direct damage to the host liver cells; however, viral infection does induce a host immune response, which not only helps the host eliminate the virus but more importantly is responsible for the unwanted, detrimental inflammatory damages inflicted on the liver.5 Long-term and repeated liver damages due to inflammation caused by chronic HBV infection can result in fibrosis, cirrhosis, and the eventual HCC.1−3 © 2017 American Chemical Society

2. HEPATITIS B VIRUS GENOME AND LIFE CYCLE 2.1. Hepatitis B Virus Genome Structure. HBV is a member of the hepadnaviridae family of viruses, having a 3.2-kb circular, partially double-stranded DNA genome containing four partially overlapping open reading frames (ORFs) that code for a total of seven viral proteins.6 The precore gene codes for the nonstructural, secretory HBV e antigen (HBeAg), while the core gene on the same open reading frame codes for the HBV core antigen (HBcAg), which is the primary protein component of the viral nucleocapsid. The polymerase gene codes for the viral polymerase that includes the reverse transcriptase (RT) domain, the ribonuclease H (RNase H) domain, and the terminal protein (TP) domain. The HBV surface antigen (HBsAg) gene codes for three HBV surface proteins, namely the large (L), middle (M), and small (S) surface proteins, which are part of the virus lipid envelope. Those surface proteins are synthesized from different promoters and are implicated in viral attachment, maturation, secretion, and immunogenicity.6 The X gene codes for the small regulatory HBV X protein (HBx), which is known to be a multifaceted protein involved in however yet unknown functions related to virus life cycle, virus−host interactions, and most importantly contributing to the etiology of HCC.7−9 Received: September 28, 2016 Published: April 6, 2017 6461

DOI: 10.1021/acs.jmedchem.6b01442 J. Med. Chem. 2017, 60, 6461−6479

Journal of Medicinal Chemistry

Perspective

Figure 1. Hepatitis B virus life cycle. The infectious HBV Dane particles attach to the cell surface HSPGs and bind to the putative HBV receptor, NTCP. Following viral particle endocytosis, the nucleocapsid is released into the cytoplasm from the endosome. The HBV partially double-stranded genomic rcDNA is then transported into the nucleus and is repaired to form cccDNA. Four distinct viral transcripts including pgRNA and preS1, preS2, and X RNAs are generated from cccDNA, which are responsible to produce all seven viral proteins. Viral polymerase and pgRNA are encapsulated through self-assembly of HBcAg to form the nascent nucleocapsid, within which the second-generation viral DNA is synthesized by reverse transcription catalyzed by viral polymerase using pgRNA as the template. The mature, rcDNA-containing nascent HBV nucleocapsid is either able to re-enter the nucleus for recycling or ready for secretion. After possible post-translational modifications within the endoplasmic reticulum and/or Golgi apparatus, the enveloped, mature viral particles are secreted out of the infected hepatocyte. A great number of noninfectious SVPs, which completely lack a functional nucleocapsid but display large number of different HBsAg proteins, are also secreted out of the hepatocyte. Every step of the HBV life cycle could potentially be a therapeutic target for treatment of HBV infection. cccDNA, covalently closed circular DNA; HBcAg, hepatitis B virus core antigen; HBeAg, hepatitis B virus e antigen; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HSPG, heparan sulfate proteoglycan; NTCP, sodium taurocholate cotransporting polypeptide; pgRNA, pregenomic RNA; rcDNA, relaxed circular DNA; SVP, subviral particle.

viral transcription and viral protein production.11 Specifically, cccDNA is known to generate four distinct viral transcripts, including the pregenomic RNA (pgRNA) and three subgenomic RNAs (preS1, preS2, and X RNAs), which are responsible to produce all seven viral proteins. Namely, the core protein (HBcAg) and viral polymerase are translated from pgRNA, while the secretory HBeAg is produced, subject to posttranslational proteolysis, from the precore RNA that is only slightly longer upstream to the pgRNA. The regulatory HBx protein is produced from the X RNA, and the three HBsAg surface envelope proteins are translated from their respective subgenomic RNAs (L protein from preS1 RNA and M and S proteins from preS2 RNA). It is noted, however, that both host factors, such as CCAAT/enhancer-binding protein (C/EBP) and hepatocyte nuclear factors (HNFs), and viral proteins including HBcAg and HBx are involved in the HBV transcriptional regulation.12 Followed by viral transcription and translation initiated through HBV cccDNA, the pgRNA and viral polymerase are encapsulated through self-assembly of the core proteins in the cytoplasm, forming the nascent viral nucleocapsid. Within the nucleocapsid, the second-generation viral DNA is synthesized

2.2. Hepatitis B Virus Life Cycle. Figure 1 depicts the HBV life cycle, from viral particle entry into the hepatocyte to the secretion of newly produced virus out of the infected liver cell. Of course, every step of the HBV life cycle could potentially be a target for disruption and thereby as a therapeutic target subject to pharmaceutical modulation as treatment of HBV infection. As shown in Figure 1, first, the infectious hepatitis B virus particles, referred as the Dane particles, bind to the hepatocyte through a reversible, nonspecific attachment to the cell surface heparan sulfate proteoglycans (HSPGs), together with the specific recognition of the preS1 domain of the viral surface protein HBsAg by the putative HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP).10 The viral particle then internalizes into the endosome through endocytosis, and the nucleocapsid is subsequently released into the cytoplasm. The HBV genomic material inside the nucleocapsid, the relaxed circular DNA (rcDNA), is then transported into the nucleus. Taking advantage of the host DNA repair machinery, the partially double-stranded viral rcDNA is repaired to form covalently closed circular DNA (cccDNA). Although cccDNA exists only in very small amounts within the infected hepatocyte, it is believed to be stable and serve as the master template for all 6462



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Table 1. Selected Therapeutics in Clinical Development for Treatment of Chronic Hepatitis B Virus Infection name

a

mechanism of action

status

myrcludex-B

entry inhibitor

phase II

tenofovir alafenamide fumarate (TAF, GS-7340) CMX-157 morphothiadin (GLS4) NVR 3-778

viral polymerase RT inhibitor, NA viral polymerase RT inhibitor, NA nucleocapsid inhibitor nucleocapsid inhibitor

approved phase II phase I phase I/II

REP 2139 vesatolimod (GS-9620)

nucleic acid polymer TLR7 agonist

phase II phase II

ARC-520 ARC-521 ALN-HBV IONIS-HBVRx (GSK3228836) IONIS-HBVLRx (GSK3389404)

RNAi DPC RNAi DPC RNAi GalNAc antisense oligonucleotide antisense oligonucleotide

terminated terminated phase I/II phase I phase I

clinical trial registry no.a NCT02888106 NCT02637999 NCT02957994 NCT02710604 ChiCTR-IIR-16008284 NCT02112799 NCT02401737 NCT02565719 NCT01590641 NCT01590654 NCT02738008 NCT02797522 NCT02826018 NCT02981602 NCT02647281

NCT number is a United States National Clinical Trial registry number; ChiCTR is a Chinese Clinical Trial Register identification number.

1 (myrcludex-B), a myristoylated peptide consisting of 47 N-terminal amino acids of the preS1 domain of the HBV L protein, could inhibit HBV infection with a 50% inhibitory concentration (IC50) of 8 nM in an HBV-susceptible HepaRG cell line.17 Susceptibility of both HepaRG cells and primary human hepatocytes (PHHs) to HBV was reduced when these cells were preincubated with 1 peptide even for a short period of time, suggesting that 1 might play a role in preventing HBV from infecting human hepatocytes by blocking HBV entry.17 In addition, using immunodeficient urokinase-type plasminogen activator (uPA) chimeric mouse models transplanted with either primary human hepatocytes or Tupaia belangeri (northern treeshrew) hepatocytes, 1 was shown to be able to block both HBV and woolly monkey hepatitis B virus (WMHBV) infection.18 It is noteworthy that 1 was now shown to be an inhibitor of NTCP.19 Phase I clinical evaluation of 1 demonstrated an acceptable tolerability and safety profile in healthy volunteers, and a phase IIa clinical trial was recently completed in chronically infected HBV patients with promising results.20 Most recently, 1 is being further investigated in phase IIb clinical trials (NCT02888106 and NCT02637999; NCT, National Clinical Trial). An additional number of NTCP inhibitors has been reported, such as 2 (cyclosporin A, Figure 2) and its derivatives,21,22 progesterone, propranolol, bosentan, oxysterols,23 3 (irbesartan, Figure 2),15,24,25 ezetimibe,15,26 and ritonavir.27 For example, 2, which primarily targets cellular peptidyl prolyl cis/transisomerase (PPIase) and cyclophilins28 and inhibits membrane transportation,29 was also identified to be a novel NTCP inhibitor with an IC50 value of 50 μM in HepG2.2.15 cells.94 It was also shown that 44 had no effects on the secretion of HBeAg, α-1-acid glycoprotein, and α-1-antitrypsin but did impact the levels of secreted viral and subviral particles, suggesting possible specific inhibition of HBsAg secretion.94 Extensive SAR studies have led to a more potent HBsAg secretion inhibitor, 45 (PBHBV-2-15, Figure 9), with a substantially reduced EC50 of 1.4 μM and a minimum selectivity index of 36.95 3.8. Host Targeting Agents. Therapeutic agents that directly act on the HBV viral proteins have been at the center of the discovery effort for novel treatments of HBV. On the other hand, an increasing volume of evidence has indicated that the close interaction between HBV and the host is an indispensable component of the viral life cycle,12 together with the realization of the crucial role that the human immune system plays in the pathogenesis and disease prognosis of HBV infection.96−98 Besides direct antiviral agents (DAAs), an alternative approach to treat HBV infection will be the application of host targeting agents (HTAs), which target the host proteins that may directly interact with the viral factors and/or are involved in the host immune response. For example, it was reported that heat shock protein 90 (Hsp90) facilitated HBV capsid formation and stabilization, and inhibition and/or down-regulation of Hsp90 decreased HBV production in HepG2.2.15 cells.99 In this case, targeting Hsp90 could be explored as an alternative, new approach to treat HBV infection. 6468



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Figure 10. Distinct host targeting agents, cccDNA inhibitors, and nucleic acid polymer for treatment of HBV infection. 46, TLR7 agonist; 47, cIAP antagonists; 48, Hsc70 inhibitor; 49, Hsp90 inhibitor; 50 and 51, putative cccDNA inhibitor; 53, NAP.

trials,115,116 and it has been in phase II clinical investigations for the treatment of chronic HBV infection (NCT01590641 and NCT01590654). Although no TLR7 agonists have been approved for HBV treatment, this mechanism of action represents an alternative, new approach by inducing broad, protective immunity against viral infection. Development of such compounds is currently of great interest to the pharmaceutical industry.107,108 When treated with polyinosinic:polycytidylic acid [poly(I:C)], an TLR3 agonist,117 both parenchymal and nonparenchymal liver cells produced IFN-β and inhibited the replication of HBV.118 In addition, viral load reduction was observed in an HBV hydrodynamic mouse model when poly(I:C) was administered by intraperitoneal (IP) injection,118 suggesting potential application of TLR3 agonists as an anti-HBV treatment. 3.8.2. Antagonizing Cellular Inhibitor of Apoptosis Proteins (cIAPs). Recent studies have shown that cIAP antagonists, such as 47 (birinapant, Figure 10) and other second mitochondria-derived activator of caspases (SMAC) mimetics, are able to reduce the serum levels of HBV DNA and HBsAg and promote elimination of hepatocytes containing HBcAg in an HBV hydrodynamic mouse model.119−121 It is believed that those SMAC mimetics induce cell death in a TNF-mediated fashion, which does not intrinsically discriminate between infected or normal liver cells. This may raise significant safety concerns for this new class of compounds in treating chronic HBV patients, and careful clinical investigations

3.8.1. Toll-like Receptor (TLR) Agonists. It was shown that the pattern recognition receptors (PRRs), such as TLRs, on the liver nonparenchymal cells, including Kupffer cells, stellate cells, and sinusoidal endothelial cells, could recognize HBV and initiate the host innate immune responses, resulting in cytokine production and subsequent suppression of viral infection.96,98,100−102 Indeed, several TLR agonists have been shown to suppress HBV replication both in vitro103−106 and in vivo.96,97 Particularly, a number of TLR7 agonists that induce type I IFNs by activating pDCs have been evaluated for their antiviral activities in both preclinical and clinical settings.107,108 As shown in Figure 10, 46 (vesatolimod, GS-9620) is a partially selective TLR7 agonist.109−111 In an HEK293 cellbased assay, the EC50 values of 46 for TLR7 and TLR8 were 0.29 and 9.0 μM, respectively.111 This level of selectivity between TLR7 and TLR8 activation corroborated with the difference between IFN-α induction (TLR7-driven) and tumor necrosis factor (TNF)-α induction (TLR8-driven) observed from a human peripheral blood mononuclear cell (hPBMC)based assay.112 Menne et al. also reported that 46 reduced serum viral DNA, cccDNA, and RNA in woodchucks chronically infected with WHV.113 It was suggested that 46 might not induce a systemic IFN-α production due to its highly localized activation of TLR7, which may instead lead to activation of pDCs in gut-associated lymphoid tissues and/or the liver.114 46 has been reported to demonstrate solid PK and safety profiles as well as efficacy signals in phase I clinical 6469



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Interestingly, blockage of PD-1/PD-L1 checkpoint resulted in restoration of exhausted CD8+ T cell function in chronic lymphocytic choriomeningitis virus (LCMV) infection,133 and this was also observed in the case of HIV infection,134−136 suggesting a possible application of PD-1/PD-L1 inhibition in treating viral infection. It becomes increasingly clear that chronic HBV infection is also at least in part an immune disorder; for example, T cells, especially CD8+ T cells, are the key contributor in control of HBV replication.137,138 On the other hand, chronic HBV infection often results in CD8+ T cell exhaustion and is associated with upregulation of PD-1.133,139 It is noteworthy that HBV NA treatment was shown to reduce PD-1 expression, presumably enhancing immunity against HBV.140,141 Possibly, a combination of NAs, PD-1/PD-L1 blockers, and/or other immunomodulatory agents could be a useful strategy in treating HBV infection. Indeed, in woodchucks that have been chronically infected with WHV, one such combination of a PD-L1 blocker with a therapeutic vaccine was shown to enhance virus-specific immunity.142 It should be noted, however, that safety evaluation needs to be carefully carried out for immunotherapies such as PD-1/PD-L1 inhibitors for chronic diseases such as HBV infection. 3.9. Targeting cccDNA. HBV cccDNA is believed to be highly persistent within the infected hepatocyte, and, albeit in a minute amount, is the master template for all viral transcription and protein synthesis. Because of its central function in HBV life cycle, exceptionally long half-life, and sheer low copy number, cccDNA becomes the principal culprit of chronic HBV infection and the dominant factor that chronic infection is extremely difficult to clear.11 Clearance of cccDNA would represent eradication of the virus and ultimate cure of hepatitis B. However, the underlying foundational biology of cccDNA dynamics is not clear, and it has been immensely challenging to discover therapeutic agents that can eliminate and/or silence cccDNA. Using secreted HBeAg as a surrogate biomarker for cccDNA, Cai et al. screened a small compound library by employing an HepDE19 cell-based assay.143 Two disubstituted sulfonamide (DSS) compounds (50 and 51 in Figure 10) were identified to inhibit cccDNA production at low μM range.143 In addition, those DSS compounds were shown to inhibit HBV cccDNA without affecting intracellular HBV rcDNA or viral polymerase activity; furthermore, 50 was also able to reduce DHBV cccDNA biosynthesis in primary duck hepatocytes (PDHs).143 The mechanism of action of the DSS compounds is however yet to be elucidated. In addition, one ought to keep in mind that HBeAg is, for lack of a better choice, a surrogate biomarker of cccDNA, given the challenges to accurately measure the cellular HBV cccDNA. 3.10. RNA Interference and Antisense Therapies. 3.10.1. RNA Interference Therapy and Delivery. RNA interference is an evolutionarily conserved mechanism to suppress specific genes through neutralization of targeted mRNA molecules, which has immense potential as a therapeutics for targeted inhibition of disease relevance with high specificity and potency at the mRNA level.144 However, significant challenges related to for example cell and tissue delivery and toxicity have prevented RNAi therapeutics from becoming an otherwise much broader clinical application.145,146 Recent technological advancement in delivery and chemical conjugation of large molecules, such as lipid-based nanoparticles, N-acetylgalactosamine (GalNAc) conjugates, and dynamic polyconjugates

will be required to understand the benefit and risk for application of SMAC mimetics in a chronic disease such as hepatitis B. 3.8.3. Lymphotoxin (LT) β Receptor (LTβR) Agonists. TNF-α was known to control HBV replication in a noncytopathic fashion.122 Interestingly, activation of another signaling pathway within the TNF superfamily, the LTβ pathway, using either a superagonistic tetravalent bispecific LTβR antibody (BS1) or a bivalent agonistic anti-LTβR monoclonal antibody (CBE11), resulted in decreases in nearly all HBV viral markers, particularly cccDNA and HBeAg, with an EC50 of 0.01 μg/mL in HBV infected differentiated HepaRG cells.123 It was proposed that the resultant upregulation of nuclear APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3) deaminases (A3Bs) from LTβR activation might be responsible for the antiviral activities.123 Specifically, A3Bs were suggested to recognize and bind to HBV cccDNA possibly mediated by HBV core protein and subsequently to form apurinic/ apyrimidinic (AP) sites within the cccDNA.123 These AP sites are then recognized by cellular AP endonucleases, eventually leading to cccDNA degradation and clearance of HBV. Because removal and/or silence of cccDNA is a necessary step for the ultimate eradication of HBV, LTβR agonists thereby represent an attractive approach for a potential cure of the disease; nonetheless, it is still in early discovery stage and significant efforts will be required for identifying a viable clinical candidate. 3.8.4. Inhibitors of Heat Stress Cognate 70 (Hsc70) and Heat Shock Protein 90. Hsc70 has been shown to be a necessary host factor for the viral reverse transcription process in experiments using duck hepatitis B virus (DHBV) DNA polymerase.124,125 In addition, RNA interference (RNAi)-based knockdown of Hsc70 could block HBV replication without obvious cytotoxicity,126 suggesting that Hsc70 could be a potential target for treating HBV infection. A selective Hsc70 inhibitor, 48 (oxymatrine) shown in Figure 10, was found to suppress de novo synthesis of both the wild-type HBV strain and strains resistant to 3TC, ADV, and ETV, presumably through destabilization of Hsc70 mRNA.127 In HepG2.2.15 cells, 48 exhibited an EC50 of 0.031 mg/mL and a CC50 of 3.12 mg/mL, with an SI of over 100.126 Hsp90 can interact with the viral reverse transcriptase to facilitate ribonucleoprotein (RNP) complex formation between the polymerase and an RNA ligand. Hu and Seeger showed that an Hsp90 monoclonal antibody could block RNP formation and small molecule antibiotics 49 (geldanamycin, Figure 10) could also selectively inhibit Hsp90 function through directly blocking the interactions between Hsp90 and p23, resulting in both decreased RNP formation in vitro and RNA packaging in vivo.128 It is noteworthy that p23 is a ubiquitously expressed phosphoprotein required for Hsp90 function.129 In all, Hsc70 and Hsp90 inhibition might provide an alternative approach to overcome the drug resistance that is associated with the current NA treatment. 3.8.5. Programmed Cell Death Protein 1 (PD-1)/ Programmed Death Ligand 1 (PD-L1) Inhibitors. Immune checkpoint inhibitors, best exemplified by three recently approved PD-1/PD-L1 blocking drugs (nivolumab, pembrolizumab, and atezolizumab),130 as well as many similar compounds that are currently in clinical development, represent a novel therapeutic approach to treat diseases by actively modulating the immune system.131,132 Although we have witnessed the tremendous success of those PD-1/PD-L1 inhibitors in treating many different cancers, this type of immune checkpoint inhibitors has not been widely applied in other diseases. 6470



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analogue, REP 2139, albeit without a disclosed structure, was claimed to be in a phase II clinical trial (NCT02565719) for the treatment of HBV and HBV/HDV coinfection both as monotherapy and combination therapy with IFNs and has demonstrated reduction in both HBsAg and HDV RNA in Caucasian patients.165−168 If confirmed in clinical evaluations, NAP-based therapy could be another alternative, novel treatment option for HBV and HBV/HDV coinfected patients. 3.12.2. Natural Products. Natural product is a great source for drug discovery.169 Helioxanthin analogues as shown in Figure 3, presumed HBV viral transcription inhibitors, were discovered from a natural source. Many natural products have been reported to show inhibitory activities against HBV with however often weak ones.170−175 As shown in Figure 11,

(DPCs), has led an resurgence of RNAi-based therapies in clinical trials.147,148 Multiple attempts have been made for using RNAi to treat HBV infection; although no drugs have been approved to date, some of the candidates are in advanced clinical evaluations.148,149 For example, 52 (ARC-520, structure not disclosed), which is based on a proprietary DPC delivery technology, was in phase II clinical trial for chronic HBV infection.150−152 In preclinical animal studies, 52 significantly decreased HBV DNA, HBsAg, and HBeAg in chimpanzees, hydrodynamic injection HBV mice, and HBV transgenic mice.152,153 However, 52 was very recently discontinued (NCT02738008), together with ARC-521 (structure not disclosed) (NCT02797522) and ARC-ATT (structure not disclosed), all of which used the same DPC-based technology, citing potential toxicity liabilities associated with the delivery vehicle. ALN-HBV (structure not disclosed) is another investigational RNAi drug that is based on the GalNAc conjugate delivery approach for chronic HBV infection. In preclinical studies, it was shown to significantly reduce the HBsAg level in rodent HBV models and be well tolerated in GLP (good laboratory practice) toxicology studies with rats and nonhuman primates.154 ALN-HBV is now in phase I/II clinical investigations with both healthy volunteers and HBV patients (NCT02826018). 3.10.2. Antisense Oligonucleotide (ASO) Therapy. Antisense oligonucleotide is either a RNA- or DNA-based short synthetic fragment which is able to bind to targeted RNA sequences with high specificity to generate RNA:RNA duplexes and DNA:RNA hybrids, respectively.155 Multiple preclinical studies have reported that ASOs were able to reduce HBV viral load and HBsAg and HBeAg levels; however, challenges such as stability and intracellular delivery remain to be major hurdles for developing broadly applied ASO-based HBV treatment.156−162 One advancement over the traditional ASO therapy is the antisense locked nucleic acid (LNA) technology, which could greatly improve nucleic acid in vitro and in vivo stabilities; for example, Deng et al. reported that in an HBV transgenic mouse model by using antisense LNA serum HBV DNA and HBsAg levels were reduced by 53% and 73%, respectively.163 To further improve cellular delivery, lipid-based delivery systems were developed for antisense LNA against HBV and it has demonstrated superior activities than RNAi in blocking HBV replication and HBV protein production in HepG2.2.15 cells.163 A number of ASOs for treatment of chronic HBV infection are in early clinical development including IONIS-HBVRx (GSK3228836) (NCT02981602) and IONIS-HBVLRx (GSK3389404) (NCT02647281), whose structures are not disclosed. 3.12. Other Approaches for Treating Chronic Hepatitis B Virus Infection. 3.12.1. Nucleic Acid Polymers (NAPs). Taking advantage of the sequence-independent amphipathic properties of the phosphorothioate oligonucleotides (Figure 10), NAPs are suggested to interfere with the amphipathic interactions often involved in viral entry and thereby potentially be a novel class of broad-spectrum antivirals.164 In the case of HBV infection, a number of NAPs have shown entry and post entry antiviral activities both in vitro and in vivo in PDHs and DHBV infected ducks, respectively.164 For example, when used prophylactically, 53 (REP 2055, Figure 10) was shown activities against DHBV infection at a low dose of 1 mg/kg with good tolerability.164 Furthermore, there were also reports claiming that NAP analogues were able to suppress serum HBsAg in ̈ Bangladeshi HBV patients.165 A leading NAP treatment-naive

Figure 11. Natural products for treatment of HBV with respective antiviral activities.

a number of examples including 54 (robustaflavone)171 and 55 (caudatin)175 demonstrated in vitro activities against HBV DNA replication in HepG2.2.15 cells with IC50 values from sub-μM to double-digit μM range. Moreover, 56 (alisol A)172 and 57 (vanitaracin A)170 were shown to inhibit HBsAg with IC50 values of 39 and 10.5 μM in HepG2.2.15 and HepG2-hNTCP-C4 cells, respectively (Figure 11). It is noted, however, that the mechanisms of actions of most anti-HBV natural products are not clearly elucidated. Furthermore, it is worthwhile to point out that although natural products often present interesting starting points for medicinal chemistry, it is also critically important to have a clear, practical optimization strategy early in the program to address key challenges specific to natural products such as chemical synthesis in order to be able to achieve viable clinical candidates in a relatively timely fashion. 3.12.3. Drug Repositioning. Developing new indications, sometimes seemingly distant ones, for existing drugs is probably the most efficient approach to bring a new therapy to the market; indeed, it has achieved a number of successful cases such as sildenafil for erectile dysfunction and bevacizumab for age-related macular degeneration.176 It has been found that HBV infection induces cellular DNA damage responses, during which process the signaling pathways involving ataxia telangiectasia and Rad3-related protein (ATR), ataxia 6471



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lifetime treatment only for virus DNA suppression. Since the approval of the first NA drug, lamivudine, also known as 3TC, over 20 years ago, incremental changes have been made to this class of drugs, the latest approved one being tenofovir alafenamide fumarate late last year. Step changes are absolutely and urgently needed for HBV treatment options. Currently, major efforts are being made in developing therapies that can achieve functional cure of the disease, i.e., finite treatment to accomplish HBsAg loss with or without seroconversion. None of the current drugs are able to accomplish predictable HBsAg loss in any given HBV patient, the best of which is IFN-based therapies that, despite significant side effects, have a functional cure rate of as high as 12%. The ultimate goal to cure hepatitis B is of course eradication of HBV, which, because of the nature of the Hepadnaviridae family of viruses, entails clearance and/or silence of cccDNA, a mounting task given relatively poor understanding of the cccDNA foundational biology. Hepatitis B virus life cycle is reasonably well understood, and a large number of direct antiviral agents with distinct mechanisms of actions targeting specific steps of the HBV life cycle are being actively discovered and some of which are already being tested in HBV patients. This extensive list of different DAAs covers nearly the entire HBV life cycle, including entry inhibitors, viral transcription inhibitors, viral polymerase inhibitors (both RT and RNase H domains), nucleocapsid assembly modulators, and HBsAg secretion inhibitors. The most advanced DAAs except the existing NA class of drugs are probably the compounds that interfere with HBV core protein functions for which the basic biology of nucleocapsid assembly from core protein dimers is quite well elucidated on a molecular level. In addition, the function of the elusive HBx protein is yet to be clarified, which probably involves multiple interactions with an array of host proteins. Furthermore, given the increasing realization of the indispensable roles that host factors play in the pathogenesis of HBV infection, the effort to discover host targeting agents for treating hepatitis B has been substantially increased, particularly for those involving the immune system. HBV HTAs display a wide range of mechanisms, reflecting the high complexity involved in the host−virus interactions, which includes TLR agonists, cIAP antagonists, LTβR agonists, Hsc70 inhibitors, as well as immune checkpoint inhibitors such as PD-1/PD-L1 blockers. An increasing number of HTAs with novel mechanisms of actions is constantly being identified on a regular basis; however, it remains to be seen in clinical trials whether any of the HTAs, especially as monotherapy, will be a viable treatment option for hepatitis B. Gene therapies including RNAi and ASO approaches are now in clinical evaluations for efficacy and safety against HBV, while breakthrough in delivery technologies is likely to be a key determinant in this case. Combination therapy is a rather common approach for infectious diseases such as in the cases of HIV and HCV treatments. Multiple attempts over the years to combine existing NAs and IFNs have not yielded any meaningful benefits; that being said, the current choices for combinations are very much limited with merely two classes of drugs. It is foreseeable that given advancement in clinical development of drug candidates with different, new mechanisms of actions, the number of options for various combination strategies will be significantly increased. Soon we will be able to experiment with combination regimens involving DAAs, HTAs, and even gene therapies for

telangiectasia mutate (ATM), and checkpoint kinase 1 (Chk1, ATR substrate) are activated.177−179 Interestingly, known ATR/ATM inhibitors 58 (caffeine) and 59 (theophylline), as well as Chk1 inhibitor 60 (UCN-01), were demonstrated to suppress HBV DNA in HBV-infected HL7702 cells (Figure 12).178 In particular, 59, a drug for treating respiratory

Figure 12. Repositioned drugs as therapeutics for HBV infection.

diseases, was further shown to significantly reduce the levels of HBV DNA, HBsAg, and HBeAg at 2.5 mM in HepG2.2.15 cells as well as achieving decreased levels of serum HBV DNA and HBsAg in HBV transgenic mice.180 Of course, significant amount of translational research is still required before theophylline could be considered for clinical investigation in HBV patients. The antiviral activity of 61 (nitazoxanide, Figure 12), a broad-spectrum antiparasitic, was discovered serendipitously when treating AIDS patients for cryptosporidium diarrhea; incidentally, those patients were also coinfected with HBV and/ or hepatitis C virus (HCV).181−184 It was later confirmed that 61 and its active metabolite 62 (tizoxanide, Figure 12) demonstrated extracellular HBV DNA inhibition in HepG2.2.15 cells with IC50 values of 0.12 and 0.15 μM, respectively.185 Furthermore, they were also able to reduce the levels of extracellular HBsAg and HBeAg and intracellular HBcAg in HepG2.2.15 cells with however no effects on HBV transcription, and in an Huh7-based transient transfection assay, they further exhibited activities against several 3TC-resistant HBV strains and one ADV-resistant strain.185 Mechanistic studies elucidated that 61 induces protein kinase RNA-activated (PKR) activation, which in turn causes an elevated level of intracellular phosphorylated eukaryotic initiation factor 2α (eIF2α), a known critical cellular factor against viral infection.183,185−189 Interestingly, early clinical investigations showed that 61 was effective in chronic HBV patients, demonstrating potential to promote HBsAg and HBeAg seroconversion.183,190

4. CONCLUSION AND PERSPECTIVES Chronic hepatitis B virus infection remains to be a great healthcare burden worldwide, particularly in China, Southeast Asia, and sub-Sahara Africa. Together with associated liver diseases such as cirrhosis and hepatocellular carcinoma, HBV infection can result in nearly one million deaths worldwide on an annual basis. Treatment for chronic hepatitis B has evolved rather slowly, which still largely relies on nucleoside/nucleotide analogue drugs with a single mechanism of action, needing 6472



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functional cure of hepatitis B. Furthermore, clearance and/or silence of cccDNA is the ultimate goal for eradication of HBV and cure of the disease, while a significant volume of basic research is still required not only for better understanding of the foundational biology of cccDNA but also for development of practical, sensitive detection method. In all, deeper understanding of the biological underpinnings of hepatitis B virus life cycle, cccDNA dynamics, and host−virus interactions will lay the foundation for discovering and developing novel therapeutic agents and innovative treatment strategies that can not only accomplish functional cure of the disease but also achieve eventual eradication of the virus, cure of hepatitis B, and decreased rate of associated liver diseases including cirrhosis and hepatocellular carcinoma.

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ACKNOWLEDGMENTS

G.L. gratefully acknowledges the funding support of grant from the National 863 Program of China (no. 2012AA020303).

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ABBREVIATIONS USED ADV, adefovir dipivoxil; AIDS, acquired immune deficiency syndrome; AP, apurinic/apyrimidinic; APOBEC3, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3; A3B, apolipoprotein B mRNA editing enzyme catalytic polypeptidelike 3 deaminase; ASO, antisense oligonucleotide; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; BDCA-2, blood dendritic cell antigen 2; CAdA, 4′-C-cyano-2-amino-2′-deoxyadenosine; CC50, 50% cytotoxic concentration; cccDNA, covalently closed circular DNA; CdG, 4′-C-cyano-2′-deoxyguanosine; C/EBP, CCAAT/ enhancer-binding protein; Chk1, checkpoint kinase 1; ChiCTR, Chinese Clinical Trial Register; cIAP, cellular inhibitor of apoptosis protein; DAA, direct antiviral agent; DHBV, duck hepatitis B virus; DPC, dynamic polyconjugate; DSS, disubstituted sulfonamide; EC50, 50% effective concentration; EGCG, epigallocatechin-3-gallate; eIF2α, eukaryotic initiation factor 2α; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility-shift assay; ER, endoplasmic reticulum; ETV, entecavir; FTF, fetoprotein transcription factor; GalNAc, N-acetylgalactosamine; GLP, good laboratory practice; HAP, heteroaryldihydropyrimidine; HBcAg, hepatitis B virus core antigen; HBeAg, hepatitis B virus e antigen; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; HIV, human immunodeficiency virus; HNF, hepatocyte nuclear factor; HSPG, heparan sulfate proteoglycan; Hsc70, heat stress cognate 70; Hsp90, heat shock protein 90; HTA, host targeting agent; IC50, 50% inhibitory concentration; IFN, interferon; kb, kilobase; LCMV, lymphocytic choriomeningitis virus; LNA, locked nucleic acid; LTβR, lymphotoxin β receptor; NAP, nucleic acid polymer; NA, nucleoside/nucleotide analog; NCT, national clinical trial; NTCP, sodium taurocholate cotransporting polypeptide; ORF, open reading frame; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; PD-1, programmed cell death protein 1; PDH, primary duck hepatocyte; PD-L1, programmed death ligand 1; pgRNA, pregenomic RNA; PHH, primary human hepatocyte; PK, pharmacokinetic; PKR, protein kinase RNA-activated; PPAR, peroxisome proliferator-activated receptor; PPIase, peptidyl prolyl cis/trans-isomerase; PRR, pattern recognition receptor; rcDNA, relaxed circular DNA; RNase H, ribonuclease H; RNAi, RNA interference; RNP, ribonucleoprotein; RT, reverse transcriptase; SAR, structure−activity relationship; SI, selectivity index; SMAC, second mitochondria-derived activator of caspases; SOCS-1, suppressor of cytokine signaling-1; SVP, subviral particle; TAF, tenofovir alafenamide fumarate; TDF, tenofovir disoproxil fumarate; TLR, Toll-like receptor; TNF, tumor necrosis factor; TP, terminal protein; uPA, urokinasetype plasminogen activator; WHV, woodchuck hepatitis virus; WMHBV, woolly monkey hepatitis B virus

AUTHOR INFORMATION

Corresponding Authors

*For G.L.: phone, +86 10 62797740; E-mail, gangliu27@ tsinghua.edu.cn. *For S.F.Y.: phone, +86 21 29846888; E-mail, [email protected]. ORCID

Gang Liu: 0000-0001-5549-5686 Author Contributions §

Y.P. and C.W. contributed equally to this work.

Notes

The authors declare no competing financial interest. Biographies Yameng Pei is a Ph.D. candidate in Chemical Biology at School of Pharmaceutical Sciences, Tsinghua University, China, under the supervision of Prof. Gang Liu. Her scientific interests include developing innovative screen assays for identifying novel anti-HBV compounds as well as the molecular mechanisms of pattern recognition receptors relevant to hepatocellular carcinoma. Chunting Wang is a Ph.D. candidate at School of Pharmaceutical Sciences, Tsinghua University, China, under the supervision of Prof. Gang Liu. He is interested in the design and synthesis of chemical compounds against hepatitis B virus and tumor metastasis. S. Frank Yan received his Ph.D. in Structural Chemistry from New York University, United States, under the supervisions of Prof. Suse Broyde, Prof. Robert Shapiro, and Prof. Nicholas Geacintov in the field of chemical carcinogenesis. He then started his industry career in Novartis and later joined Roche R&D China as the Head of Molecular Design and Biostructure in 2008. He has extensive experience in various disease areas including infectious diseases, oncology, metabolic diseases, and neuroscience. His scientific contributions include over 40 journal publications and patents. Gang Liu received his Ph.D. in Chemistry from Beijing Medical University, China, and obtained his postdoctoral training in Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, China, and Scripps Research Institute, United States. He is currently Chang Jiang Scholar, Peking Union Scholar, and 100 Top Talent Program Professor at School of Pharmaceutical Sciences, Tsinghua University, China. His research interests include developing synthetic methodologies of small molecule heterocyclic compound libraries, natural products, and peptide/glycopeptide mimics and analytic assay methodologies for high throughput screening. His lab is focused on discovering, optimizing, and developing compounds for cancer and infectious diseases.

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