Treatment of chronic hepatitis B virus infection using small molecule

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Treatment of chronic hepatitis B virus infection using small molecule modulators of nucleocapsid assembly: recent advances and perspectives Li Yang, Feifei Liu, Xiankun Tong, Daniel Hoffmann, Jianping Zuo, and mengji lu ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00337 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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ACS Infectious Diseases

Treatment of chronic hepatitis B virus infection using small molecule modulators of nucleocapsid assembly: recent advances and perspectives Li Yang1#, Feifei Liu1,2#, Xiankun Tong1, Daniel Hoffmann3, Jianping Zuo1, Mengji Lu4 1. Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China; 2. University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China 3. Institute of Bioinformatics, University Duisburg Essen, Essen, Universitätsstraße 1, 45117, Essen, Germany 4. Institute of Virology, University Hospital Essen, University Duisburg Essen, Hufelandstrasse 55, 45122 Essen, Germany #

These authors contributed equally to this work.

Corresponding author: Prof. Dr. Mengji Lu Institute of Virology University Hospital Essen Hufelandstrasse 55 45122 Essen, Germany Email:[email protected] Phone: 00492017233530 Fax: 00492017235929

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Based on the recent advance of basic research on molecular biology of hepatitis B virus (HBV) infection, novel antiviral drugs targeting various steps of HBV life cycle are developed in recent years. HBV nucleocapsid assembly is now recognized as a hot target for anti-HBV drug development. Structural and functional analysis of HBV nucleocapsid allowed rational design and improvement of small molecules with the ability to interact with the components of HBV nucleocapsid and modulate the viral nucleocapsid assembly process. Prototypes of small molecule modulators targeting HBV nucleocapsid assembly are being pre-clinically tested or have moved forward in clinical trials, with promising results. This review summarizes the recent advances in the approach to develop antiviral drugs based on the modulation of HBV nucleocapsid assembly. The antiviral mechanisms of small molecule modulators beyond the capsid formation and the potential implications will be discussed. KEYWORDS: Hepatitis B virus, HBV core protein, HBcAg, capsid assembly, capsid assembly modulators (CAMs), antiviral therapy, clinical trials


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Hepatitis B virus (HBV) is a member of the family Hepadnaviridae and causes acute and chronic hepatitis in humans1, 2. Although the general vaccination programs effectively reduced the cases of new HBV infections, there are estimated 240 million of chronically HBV-infected patients worldwide3.Those chronically HBV-infected patients are at high risks to develop into cirrhosis or hepatocellular carcinoma3-5. The treatment with interferon α and/or nucleos(t)ide analogues can effectively suppress HBV replication and successfully slow the progression of liver diseases. However, it is still hampered by various limitations and does not eventually cure the majority of patients1, 6, 7. Therefore, the millions of chronically HBV infected patients need more potent antiviral drugs for cure8. The researches on the molecular virology of HBV and host immune responses to HBV have achieved significant advances and led to identification of suitable viral and host targets for therapies and development of anti-HBV agents9-11. So far, there are several non-nucleoside antiviral agents targeting different steps of HBV life cycle, such as virus entry and nucleocapsid assembly, and the stimulation of host innate and adaptive immune responses10,

12-17.

A type of

potential antiviral drugs called capsid assembly modulators (CAMs) or capsid assembly effectors (CAEs) disrupts or interferes with the process of HBV nucleocapsid assembly12,

16, 18.

These molecules cause inaccurate and premature

assembly of HBV nucleocapsids and prevent HBV pregenomic RNA (pgRNA) encapsidation or misdirect core protein dimers to assemble non-capsid polymers, thereby suppressing HBV replication. This review summarizes the current status of



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CAMs development, with focusing on drug candidates in late preclinical development and clinical testing. HBV life cycle The infectious HBV virion, commonly termed Dane particle19, is a spherical enveloped capsid containing a single copy of the partially double stranded, relaxed circular (rc) DNA genome with a size of about 3200 bp20, 21and the HBV polymerase (HBVPol)

22, 23.

The viral envelope is made up by surface antigens which mediate

specific binding to the host hepatocytes. The HBV life cycle begins with the attachment and entry step into hepatocytes, which occurs through a low-affinity interaction between heparan sulfate proteoglycans (HSPG)24 on hepatocytes and the antigenic loop (AGL) of the HBV envelope proteins25 and then followed by a high-affinity interaction of the PreS1 domain of HBV large surface protein with its liver-specific receptor, sodium-taurocholate cotransporting polypeptide (NTCP) 26-30.

7, 13,

Then, the virion is internalized and de-enveloped to release naked nucleocapsid

into the cytoplasm via not yet fully understood mechanisms31, 32. HBV capsid breaks off in the nuclear pore complex, resulting in a release of rcDNA into the nucleus31, where the 5’flap structure of rcDNA is cleaved by flap-endonuclease 1 (FEN1)33and the gaps on rcDNA are repaired by cellular DNA polymerase κ (POLK) ligase 1/3(LIG 1/3)

35.

34

and DNA

This process leads to the formation of so called covalently

closed circular DNA (cccDNA). HBV cccDNA serves as a template for transcription of the major viral mRNA species36-38.


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A number of RNA transcripts are generated from HBV cccDNA, including 0.7 kb, 2.4/2.1 kb, and 3.5 kb HBV RNAs21. The small 0.7 kb RNA encodes HBV x protein (HBx), which may exert diverse regulatory functions39, 40 and is likely essential for the transcription of the cccDNA by modulating the recruitment of chromatin modifying enzymes onto HBV minichromosome41. The 2.4 /2.1 kb HBV RNAs are transcribed from the open reading frame encoding the hepatitis B surface antigen (HBsAg) and translated to three types of glycoproteins of the envelope of HBV virions: the small surface protein (S-HBsAg), the middle surface protein (M-HBsAg), and the large surface protein (L-HBsAg)

42-45.

The 3.5 kb precore RNA encodes HBV e antigen

(HBeAg), a secretory protein and an important serological marker of HBV infection46. The 3.5 kb pgRNA is an overlength transcript containing a second copy of the direct repeat 1 (DR1), the ε signal and a poly-A tail, serving as a transcript for the translation of the 90 kDa HBVPol and the 21 kDa core protein (HBcAg)44. The pgRNA along with HBVPol is specifically packaged into the nucleocapsid. The encapsidated pgRNA is reverse-transcribed by HBVPol into the minus-strand DNA, and then the plus-strand DNA after removing the pgRNA template by RNase H activity of HBVPol47. The mature nucleocapsids containing rcDNA are then transferred to post-endoplasmic reticulum (ER) or pre-Golgi membranes, enveloped into virions and released from hepatocytes48, or recycled to the nucleus to amplify the cccDNA pool49(Figure 1A). The immature capsids, such as empty capsids50,

51

and

capsids with incomplete HBV RNA/DNA fragments52, could be either released as naked capsids in the form of capsid-antibody complexes (CAC)53 from hepatocytes or



enveloped to form virus-like particles (Figure 1B). Recently, HBV virion production was found to be associated with autophagy54-57. Many steps in HBV life cycle represent targets of direct antiviral drugs including the entry, DNA synthesis and RNAse H activity (Myrcludex B58 and tenofovir59). Of all the steps in the HBV replication cycle, the core protein interaction and nucleocapsid assembly are recognized as potential targets for HBV, and will be discussed in the following sections.

HBV core protein (HBcAg) and nucleocapsid assembly The nucleocapsid assembles from 120 dimers of core protain with T=4 icosahedral symmetry60 (Figure 2A). The crystal structure of the T= 4 capsid has been solved at 3.3A°resolution, revealing a largely helical protein fold that is unusual for icosahedral viruses61. The full-length core protein consists of 183 or 185 amino acid residues depending on the genotype62. The primary sequence contains two distinct domains connected by a hinge region. The N-terminal α helix-rich domain with first 140 amino acid residues is sufficient for the self-assembly of capsids63. The assembly domain has 5 α-helices connected by loops 61. Helices 1, 2 and 5 are part of the chassis of capsid. Helices 3 and 4 form a four helix bundle with corresponding helices from the other monomer. The dimerization of HBcAg is primarily driven by hydrophobic interactions and is aided by flanking salt bridges and hydrogen bonds. The disulfide bridge between cysteine residues 61 in helix 3 of two monomers is not required for


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assembly64; indeed, the C61-C61 disulfide bond may promote the capsid disassembly65. The inter-dimer contact is located near the junction where helix 5 emerges from the four helix bundle and is filled by the helix-turn-extended structure61, 66.

It is a tongue-and-groove contact and is mediated by burial of hydrophobic

surface66. The residues Tyr-132 contributes about 10% of the buried surface66, and the residues Val-124, Arg-127, Ile-129 and Ile-139 are also play important roles61. The burial of hydrophobic surface creates sufficient hydrophobic interactions to form stable capsids. HBcAg dimers are associated by this weak protein-protein interactions, which allows ‘thermodynamic editing” to remove incorrectly and weakly associated dimers67. An engineered mutant within the assembly domain (V124W) increases buried hydrophobic surface and leads to an increased rate of capsid assembly and defects in pgRNA packaging68. The other mutant (Y132A) decreases buried surface and is not able to form capsids61(Figure 2B). This peculiar pocket is recognized as an important drug target for antiviral agents. The C-terminal 150-183/185 amino acids domain (CTD) connects to the assembly domain by a tight proline-rich loop66, 69. The CTD is designated the protamine domain, which is rich in arginine residues and dispensable for the assembly of empty capsids70 (Figure 3). Four clusters of three or four arginine residues (ARD I, II, III, IV) within CTD are required for the packaging of the pgRNA / HBVPol complex and reverse transcription71. The pgRNA encapsidation or/and rcDNA synthesis are significantly reduced, when these arginine clusters are deleted, or partially replaced by alanines, glycines, or lysines. The formation of nucleocapsid also requires CTD



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phosphorylation. Though seven conserved serines and a threonine that are located in the CTD can be phosphorylated, phosphoserine of serines 155, 162, and 170 in subtype ayw (corresponding to 157, 164, and 172 in ayw) are necessary and sufficient for the pgRNA encapsidation and rcDNA synthesis72,

73.

Destruction of these

phosphorylation sites resulted in capsids formation containing a defective short spliced RNA but lacking full-size DNA74. During HBV life cycle, HBV nucleocapsid formation starts when HBcAg dimers bind to the complex of HBV Pol and pgRNA. The capsid assembly process occurs within cytoplasm and is exquisitely timed and regulated, so any disruption could be devastating to virus replication. The CTD, as a nucleic acid chaperone, plays an important role in these processes, and may be a potential target for developing antivirals against HBV.

HBV capsid assembly modulators (CAMs), chemistry, and mode of action Recently, the process of HBV capsid assembly has been recognized as a suitable target for antiviral drugs. Several chemotypes of small molecules were found to be able to disrupt capsid formation and subsequently inhibit HBV pgRNA encapsidation, reverse transcription, and DNA synthesis. The first identified small molecule which misdirect

HBV

capsid

assembly

5,50-bis[8-(phenylamino)-1-naphthalenesulfonate]

was

a

fluorescent

(BisANS)75.

dye

BisANS-bound

HBcAg dimer had an increased propensity for formation of non-capsid polymers. Up to date, these new anti-HBV drug candidates have been termed as CAMs and draw


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attention of basic and pharmaceutic research. At least 10 different chemotypes of CAMs were identified and now undergoing preclinical and clinical testing. The chemical structures of representative CAMs are given in Figure 4. Some chemical compounds disrupt the capsid assembly by specific interaction with the hydrophobic pocket located at HBcAg dimer interface, which induce allosteric conformational changes in core protein subunits76. These compounds are defined as HBV core protein allosteric modulators (CpAMs) and mainly fall in two categories: Class-I CpAMs lead to misassembled non-capsid polymers and Class-II CpAMs induce the formation of morphologically normal capsids that are devoid of HBV genome (Figure 1B). The Class-I CpAMs are heteroaryldihydropyrimidines (HAPs), which have been identified as highly potent assembly effectors, acting by allosterically inducing assembly-active states at stoichiometric levels and stabilizing non-capsid polymers at higher concentrations77,

78.

The representative Class-II

CpAMs are phenylpropenamide (PPA) derivatives and sulfamoyl benzamide (SBA) derivatives. The compounds of PPA family lead to accumulation of genome-free capsids without changing HBV capsid morphology79, 80, which will be discussed later in this review. SBA derivatives as HBV replication inhibitors were discovered from a library consisting of 26,900 small molecules through a high throughput screening. Mechanistic analyses revealed that the compounds significantly reduced the amount of cytoplasmic HBV DNA by inhibiting HBV pgRNA encapsidation but without inhibitory effect on the replication of other members of the hepadnaviral family, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV)81. The two



series of compounds displayed the same mode of action, which reduced encapsidated pgRNA and capsid-associated HBV DNA but did not significantly alter the amounts of total HBV capsids. Moreover, new structural analysis suggests that Class-II CpAMs bind to the same hydrophobic pocket at the inter-dimer interface like HAP (Class-I CpAM) but favor different quasi-equivalent locations from that favored by HAP82-84, so have different effects on HBV biology. This pocket is a promiscuous drug-binding site and a highly attractive target for different CpAMs with a broad range of mechanisms of activity82. Core variants with mutations in this pocket can impact anti-HBV response of CpAMs. Consistent with their differences in binding site interactions, CpAMs from different chemical series could have different resistance profiles in vitro.

Biochemical studies using recombinant core protein

show that CpAMs from HAP family are less active against HBV with Y118F, T109M and T109I changes in assembly domain85. NVR 3-778 belonging to SBA derivatives shows reduced activities against I105T and Y118F, while it remains similarly active against the T109M and T109I85, 86. In addition to PPA derivatives and SBA derivatives, there are other compounds that may also belong to Class-II CpAMs. 371187, a pyridazinone derivative, can inhibit HBV replication by inducing the formation of HBV genome-free capsids with faster electrophoresis mobility. BA-53038B76, a benzamide derivative, can induce morphologically “normal” empty capsid with lower electrophoresis mobility. Although there is not no evidence to support that the two compounds bind to the HAP pocket directly, mutation at V124 can alter the effects of 3711 and BA-53038B on


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capsid assembly, which suggest that they most likely modulate capsid assembly by binding to HAP pocket76, 87. Another HBV CAMs, such as NZ-488 and BCM-59989, do not bind to the peculiar hydrophobic HAP pocket, but can also interfere with the process of HBV nucleocapsid assembly. NZ-4 is identified as a novel HBV CAM targeting the ARD domain of CTD90. NZ-4 is a derivative of bis-heterocycle tandem pairs derived from the natural product leucamide A88 and shows to modulate HBV capsid assembly by decreasing pgRNA encapsidation in dependent on the positively charged amino acid residues of the ARD I90. BCM-599, a 2-Amino-N-(2,6-dichloropyridin-3-yl) acetamide derivative, has high affinity to the groove structure that is surrounded by amino acids 21–35, 101–120 and 136–140 of HBcAg protein, and inhibit HBV production by blocking capsid assembly and increasing the amount of free HBcAg dimers89.

CAMs under preclinical development Currently, a number of CAM candidates were designed, synthetized and subjected to preclinical test76,

91-93,

such as PPAs and pyridazinone derivatives. All of these

compounds have demonstrated strong anti-HBV activity in in vitro studies, which are being developed by different companies and institutes (Table 1). PPA derivatives



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PPA derivatives, including two compounds named AT‑6179 and AT‑13080, have been shown to inhibit HBV pgRNA packaging. Earlier publications described the identification of PPAs as HBV inhibitors and suggested that the propenamide double bond of these molecules has an E configuration (trans). However, single crystal X-ray crystallography studies revealed that the activity of this chemotype of molecules resides in the Z configuration (cis). Further structure–activity relationship studies on the active Z isomer identified compounds with potent antiviral activity against HBV with IC90 value of approximately 0.5μM in vitro94. PPAs, as assembly accelerators, increase the rate of capsid assembly and induce the formation of defective capsids lacking genetic material without changing capsids morphology95. The in vitro studies using the truncated HBcAg with the N-terminal 149 amino acid residues (Cp149) showed that At-130 drives HBV capsid assembly by kinetic trapping of assembly intermediates96, 97. The crystal structure of the HBV capsid in complex with AT-130 revealed that AT-130 binds to the same hydrophobic pocket at the inter-dimer interface like HAPs, but favors a different quasi-equivalent location82,

98.

While

AT-130 bound the HAP hydrophobic pocket, the tertiary and quaternary structural changes were induced. The compensatory tertiary structural changes likely allowed the formation of the observed normal capsids rather than the noncapsid polymers observed with HAPs82, 83. Pyridazinone derivatives The representative pyridazinone derivatives, 371187 and 516799, inhibit HBV replication by inducing the formation of genome-free capsids in cell culture systems.


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The 3711 treatment induced changes of the physical profile of HBV capsids, including electrophoresis mobility and buoyant density profile, but no significant morphological changes. The genome-free capsid had a lower buoyant density and showed slightly slower sedimentation in isopycnic CsCl gradient centrifugation compared with normal HBV nucleocapsid.

The subsequent structure-activity

relationships study and optimization resulted in a lead candidate named 5167 with potent antiviral activity (50% inhibitory concentration (IC50) = 87 nM), low cytotoxicity (CC50 = 90.6 μM), sensitivity of nucleoside analogue-resistant HBV mutants, and synergistic anti-HBV effect with ETV in HepG2.2.15 cells. Though PPAs and pyridazinone derivatives have different chemistries, they most likely fit into the same HAP pocket at the inter-dimer interface near the C-termini of the HBV core protein subunits, with contributions from two neighboring dimers, increasing the buried hydrophobic surface. The pocket is only partially exposed on the capsid interior and not visible from the capsid exterior. A mutant HBcAg Cp V124 was found to strengthen HBV capsid inter-dimer interactions98. At one side, the V124W mutation could fill the HAP pocket and block activity of HAPs, PPAs, and pyridazinone derivatives, suggesting that 3711 and AT-130 affect HBV capsid assembly in a similar fashion by interacting with the same pocket of HBcAg. On the other side, the Cp V124W mutation could mimic the activity of 3711 and AT-130 on HBV capsid assembly87. EP-027367



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EP-027367 is a novel CpAM, which modulates HBV capsid assembly by blocking pgRNA encapsidation. It accelerates the assembly of HBcAg dimers into genome deficient capsids and does not cause HBcAg aggregation. It shows a potent inhibition of HBV replication in HepG2.2.15, HepAD38, and HepDE19 cells with IC50s of 20, 24, and 40 nM, respectively. It is active across HBV genotypes A-H and its antiviral activity is not affected by mutations related to resistance to nucleos(t)ide analogues. Combinations of EP-027367 with nucleos(t)ide analogues display additive or synergistic antiviral activities. Additionally, EP-027367 is able to inhibit the formation of new HBV cccDNA and thereby prevent de novo HBV infection of susceptible cell lines. Given orally at 50, 100 and 200 mg/kg b.i.d. for 28 days, EP-027367 reduced HBV DNA level by 2.2, 2.7, and 3.0 log 10 copies/ml in a human liver chimeric mouse model, respectively100. CAMs under clinical trial More than 10 drug candidates are currently under clinical testing, including BAY41-4109,

GLS4JHS,

RO-7049389,

NVR

3-778,

JNJ6379/

JNJ0440,

AB-423/AB-506, ABi-H0731/ ABi-H2158, NZ-4, RG7907and QL-007 (Table 2). In a phase I trial, the anti-HBV activity, resistance profile, pharmacokinetics (PK), safety, and tolerability of these drug candidates are assessed. Five drug candidates (GLS4JHS, RO-7049389, NVR 3-778, JNJ6379, and Abi-H0731) are found to be well-tolerated in patients, and now are entering phase II clinical trials for evaluation of their anti-HBV activity, as monotherapy or in combination with nucleos(t)ide analogs or peg-IFNα. To compare these drug candidates, the results of phase Ib 28-Day studies in


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HBV-infected patients are summarized in Table 3. There is only limited information available; thus, the summary in the present review is largely based on presentations on scientific meetings. BAY41-4109 The HAP chemotype represents a well-studied series of CpAMs. An early example in this chemotype is the compound BAY41-4109101. In in vitro studies, BAY41-4109 inhibites HBV DNA replication in HepG2.2.15 cells with an IC50 of 0.05 μM, and reduces the amount of HBV core protein101. In in vivo studies, oral administration of BAY 41-4109 b.i.d. demonstrates a statistically significant reduction of viral DNA at 15 and 30 mpk in HBV transgenic mice102 and showes approximately 1 log drop in viral load at 25 mpk in Alb-uPA/SCID mice transplanted with human hepatocytes and infected with HBV103. However, BAY41-4109 shows hepatotoxicity at high doses in rats104. BAY41-4109 currently is undergoing a phase I study by AiCuris in Germany, but the clinical result is unclear105. GLS4JHS GLS4JHS,

(ethyl

4-[2-bromo-4-fluorophenyl]-6-[morpholinomethyl]-2-[2-thiazolyl]-1,4-dihydro-pyrim idine-5-carboxylate), is a HAP inhibitor and now being tested in clinical phase II. Its molecular structure and mode of anti-HBV action are similar to Bay41-4109. The IC50 value for GLS4JHS is 0.06 μM in HepAD38 cells. Because GLS4JHS can induce the CYP3A4 enzyme, administrating GLS4JHS alone cannot achieve prospective blood



concentrations of antiviral effect. Its anti-HBV effect is limited by the short half-life. Ritonavir is a protease inhibitor that can inhibit hepatic enzymes to improve the exposure of GLS4JHS. Combination with Ritonavir, the dosage and frequency of GLS4JHS are reduced, and the anti-HBV effect is improved. In a phase I trial, the anti-HBV activity, resistance profile, pharmacokinetics (PK), safety, and tolerability of combination of GLS4JHS and Ritonavir are assessed. Twenty four chronic HBV infected (CHB) patients are randomized to receive a 28-day course. The results showed that the mean decline from baseline in HBV DNA is -1.42 log10 IU/mL and in HBsAg is -0.06 log10 IU/mL, and in HBeAg is -0.25 log10 IU/mL at combination of GLS4JHS 120 mg, q.d. and Ritonavir 100 mg, q.d. group. However, the majority of patients experience viral rebound after termination of the treatment. The PK profile is supportive of once-daily dosing with mean peak plasma concentrations at 2.5 hours post dose and means terminal half-life of 64.3 hours after last dosage in 120 mg group. A steady state is achieved following 6 days of daily dosing. The accumulation rate is similar among the different groups (1.64–1.98), indicating a low accumulation of the drug.

GLS4JHS was tolerated in all dose groups. The combination of GLS4JHS and

Ritonavir is well tolerated and produced a rapid and substantial decrease in HBV-DNA levels in patients chronically infected with HBV. On the basis of these results, GLS4JHS 120 mg is recommended for further evaluation in phase II trials106. RO-7049389 RO-7049389 is a new generation of HAP analogues and now being investigated in phase II clinical trials. Compared with the earlier generation (4-methyl HAP


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analogues)107, RO-7049389 does not exhibit an inhibitory activity against five major CYP enzymes 3A4, 2D6, 2C9, 2C19, and 1A2. In vitro, the anti-HBV activity and cytotoxicity of RO-7049389 were evaluated in stably transfected cell lines and transient transfection system, respectively. RO-7049389 decreased HBV DNA levels by 50% (IC50) at 6±1nM in HepG2.2.15 cells. RO-7049389 also exhibited antiviral activity against the HBV genotypes A, B, C, and D as well as nucleos(t)ide analog-resistant HBV mutants in transfected Huh7 cells or primary human hepatocytes. In the AAV-HBV/mouse model, RO-7049389 suppressed serum HBV DNA after 8-week treatment with 20 mg/kg dosing per day, as well as HBsAg and HBeAg level108. Notably, all of the virologic markers remained at low levels during a 3-week follow-up period after treatment. The safety, tolerability, pharmacokinetics (PK), and anti-HBV activity of RO7049389 had been investigated in phase I study. In a phase Ia trial, RO7049389 was rapidly absorbed and eliminated from plasma. It was well tolerated in healthy adult volunteers at single ascending doses from 150 to 1000 mg and at multiple ascending doses from 1000 to 2000 mg during a blinded safety evaluation. In a phase Ib trial, six untreated CHB patients completed 28 days dosing period of RO7049389 at a dose of 200 mg b.i.d. Robust and continued HBV DNA declines from pre-dosing levels were observed, with median (maximal) decline being 2.7 (3.4) log10 IU/ml and below the limits of detection in three patients. No on-treatment virologic rebound was observed108.



NVR 3-778 NVR 3-778 belongs to SBA CpAMs and is now in clinical phase 2 trials in combination with other antiviral agents and interferon. In the HBV-infected humanized uPA/SCID mouse model, NVR 3-778 was given alone (402 mg/kg, b.i.d) or in combination with pegylated interferon alpha (peg-IFNα) or entecavir for 6 weeks. The results showed that the combination of NVR3–778 and peg-IFNα inhibited HBV DNA and RNA to a greater extent than each compound alone, while combining NVR 3-778 with ETV did not improve HBV suppression109, 110. In a phase Ia trial, NVR 3-778 showed a good safety profile in healthy adult volunteers111. Different doses of NVR 3–778 were well tolerated in a phase Ib clinical trial enrolling 36 HBeAg positive CHB patients. Significant HBV-DNA decline was observed only with the high dose at 1200 mg (600 mg, b.i.d.)112. Currently, NVR 3–778 in combination with peg-IFN, as well as with nucleoside analogs, is being explored. In a 28-day interim analysis, NVR 3–778 (400 and 600 mg, b.i.d.) plus peg-IFNα was associated with reduction of both HBV DNA (1.97 log10 IU/mL) and HBeAg but not HBsAg reduction, which is likely due to the short treatment duration113. JNJ6379/JNJ0440 JNJ632114 and JNJ6379 (JNJ56136379) are SBA derivatives. Administration of JNJ632 in HBV genotype D infected chimeric mice resulted in a 2.77 log reduction of the HBV DNA viral load during this 28 day study, while no reduction of HBeAg and HBsAg levels was observed115. Recently, the phase I trial of JNJ6379 has been


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completed. JNJ6379 was well-tolerated in healthy volunteers at single oral doses of up to 600 mg and at multiple doses of 150 mg for two days followed by 100 mg once a day for 10 days. JNJ6379 administered orally for 28 days at different doses (25 mg, 75 mg or 150 mg, q.d.) demonstrated potent anti-HBV activity by reducing HBV DNA and RNA levels in adult, treatment-naïve CHB patients, however, without relevant changes in HBsAg levels. All three dose regimens were safe and well-tolerated and displayed dose-proportional pharmacokinetics. A phase IIa study in treatment-naïve and virologically-suppressed HBeAg positive and negative CHB patients has been initiated to evaluate JNJ6379 alone or in combination with nucleos(t)ide analogs (NCT03361956)115-117 . AB-423 AB-423118 is a HBV CpAM of the SBA in phase I clinical trials. AB-423 inhibits HBV pgRNA encapsidation and does not cause aggregation of HBcAg. Molecular docking study predicts that AB-423 likely binds to HBcAg dimer-dimer interface that is also the binding site of HAPs and SBA. In a de novo HBV infection model of C3AhNTCP cells, AB-423 is able to prevent the conversion of encapsidated rcDNA to cccDNA presumably by interfering with the process of capsid assembly. In cell culture models, AB-423 has shown a potent inhibition of HBV replication (IC50/IC90 = 0.08-0.27 μM/0.33-1.32 μM) with no significant cytotoxicity (CC50 >10 μM), and inhibits HBV genotypes A through D and nucleos/tide-resistant variants.

The

combination studies with AB-423 and anti-HBV agents such as nucleos(t)ide analogs, RNAi agents, or IFN α result in additive to synergistic antiviral activity. In vivo, a



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7-day b.i.d. administration of AB-423 in a hydrodynamic injection mouse model of HBV resulted in a dose-dependent reduction in serum HBV DNA levels. Combinations with entacavir or RNAi agent ARB-1467 resulted in a trend towards greater antiviral activity than either agent alone118,

119.

The phase Ia clinical results

support that AB-423 is safe and well-tolerated at a single dose of 200 mg per day and displays dose-proportional pharmacokinetics. The safety, tolerability, and PK of AB-423 at higher single doses and multiple doses are now being tested. Further evaluation for antiviral activity in CHB patients will be performed in phase Ib clinical trials. Abi-H0731/ ABi-H2158 Abi-H0731 is a new chemotype of oral therapeutics for the treatment of HBV infection and shows antiviral activity against all major HBV genotypes as well as nucleos(t)ide analog-resistant HBV variants.

Its phase I trial has been completed

successfully. The Phase Ia study of Abi-H0731 showed favorable results on the safety, tolerability, and pharmacokinetics in healthy volunteers120. The phase Ib trial study indicated that ABI-H0731 is able to reduce HBV DNA with once daily dosing for 28 days, is generally safe and well tolerated, and exhibits increasing plasma exposures with increasing dose in both HBeAg positive and negative patients. At 100 mg per day, the lowest dose tested, HBV mean declines of 1.3 and 2.2 log10 IU/mL were observed in six HBeAg positive patients and four negative patients, respectively. Declines up to approximately 4 logs in two HBeAg negative patients were observed following administration of 400 mg per day. HBV RNA reductions were generally


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proportional to reductions of plasma HBV DNA. No serious adverse events (AEs) and no dose limiting laboratory toxicities were observed121. In 2018, the U.S. Food and Drug Administration (FDA) has granted Fast Track designation to ABi-H0731 for the treatment of patients with chronic Hepatitis B infection. ABI-H0731 is being evaluated in two global phase IIa proofs of concept studies to provide the opportunity for preliminary evaluation of combination therapy of ABI-H0731 with currently approved antiviral treatment for CHB. Base on ABi-H0731, the management of Assembly Biosciences Company develops the second generation candidate, ABi-H2158. Phase Ia clinical trial initiation in healthy volunteers is performing now. NZ-4 NZ-4 is a 2,2-bis-heterocycle tandem derivative and modulates HBV capsid assembly by decreasing enapsidated pgRNA88. NZ-4 inhibited wild-type HBV replication in an in vitro cell setting, maintained activity against nucleotide analogue-resistant mutants, and demonstrated suppression of DHBV and WHV replication in vivo. By contrast, HAPs, PPAs, and pyridazinone derivatives did not inhibit DHBV replication both in cultured cells and DHBV-infected ducks. Mechanistically, NZ-4 allows a new insight on developing HBV nucleocapsid assembly effector. NZ-4 was shown to induce a portion of defect HBV capsids, which was loss of viral genomic material and exerted faster electrophoretic mobility behavior on agarose gel. Based on negative stain transmission electron microscopy (EM), the morphology of drug-induced capsids was



subjected. Almost all capsids (ca 90-95%) displayed the “empty” appearance of capsids with normal morphology. Interestingly, the action of NZ-4 on capsid formation was dependent on the presence of the arginine cluster amino acid residues 150-152 (ARD I) of CTD, which indicated that this domain was probably a promiscuous site for different assembly effectors. This property of NZ-4 is unique compared with other assembly effectors described above. Currently, NZ-4 is being evaluated in phase I clinical trials in China.

In phase Ia clinical trials, the toleration

and pharmacokinetics of NZ-4 were under investigation, however, the results of the initial phase Ib testing are not available yet90. Perspective In the past few years, the development of HBV CAMs became a hot topic in basic and pharmaceutic research. The new data emerging in this research field are encouraging and promising. A number of chemotypes of chemical compounds were discovered to effectively interfere with HBV capsid assembly and now already under clinical testing. The results of clinical trials published so far demonstrated that these compounds are well-tolerated and are able to effectively decrease HBV loads in patients in short term experiments. Yet, none of these compounds is tested in phase III clinical trials and proven to be useful in long term treatment of chronically HBV infected patients. As entecavir and tenofovir are extremely successful in the clinical use and give excellent results regarding the antiviral potency and drug resistance profile, new drugs based on HBV CAMs need to pass strict evaluations to become accepted. One critical issue is whether HBV CAMs may have the ability to target HBV cccDNA formation that is


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not sufficiently blocked by nucleotide analogues. As HBV CAMs induce polymerization of HBcAg and thereby limit the availability of HBcAg in infected cells, they may also interfere with some functions of HBcAg in HBV life cycle, for example, binding to HBV cccDNA and regulation of cccDNA stability and viral transcription63, 122. It would also be highly interesting to investigate the fate of HBV pgRNAs if HBcAg is not available in a sufficient amount. Would HBV pgRNA then be exposed to the host surveillance of pathogen associated molecular patterns123? This could be an important mechanism to enhance host immune control. Combinations of drug candidates based on HBV CAMs with IFN and other drugs showed that there may be a great potential for synergistic antiviral effect. Besides, other therapeutic approaches like therapeutic vaccines may need to be combined with antiviral treatment. HBV CAMs represent new candidates in addition to already existing nucleotide analogues. Acknowledgement This study is supported by a grant of DFG TRR60 to M.L. and National Science Foundation of Shanghai (17ZR1436400).



References 1. Trepo, C.; Chan, H. L.; Lok, A.(2014) Hepatitis B virus infection. Lancet., 384 (9959), 2053-63. DOI: 10.1016/S0140-6736(14)60220-8. 2. Tang, L. S. Y.; Covert, E.; Wilson, E.; Kottilil, S.(2018) Chronic Hepatitis B Infection: A Review. JAMA., 319 (17), 1802-1813. DOI: 10.1001/jama.2018.3795. 3. Schweitzer, A.; Horn, J.; Mikolajczyk, R. T.; Krause, G.; Ott, J. J.(2015) Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. DOI: 10.1016/S0140-6736(15)61412-X. 4. Fitzmaurice, C.; Dicker, D.; Pain, A.; Hamavid, H.; Moradi-Lakeh, M.; MacIntyre, M. F.; Allen, C.; Hansen, G.; Woodbrook, R.; Wolfe, C.; Hamadeh, R. R.; Moore, A.; Werdecker, A.; Gessner, B. D.; Te Ao, B.; McMahon, B.; Karimkhani, C.; Yu, C.; Cooke, G. S.; Schwebel, D. C.; Carpenter, D. O.; Pereira, D. M.; Nash, D.; Kazi, D. S.; De Leo, D.; Plass, D.; Ukwaja, K. N.; Thurston, G. D.; Yun Jin, K.; Simard, E. P.; Mills, E.; Park, E. K.; Catala-Lopez, F.; deVeber, G.; Gotay, C.; Khan, G.; Hosgood, H. D., 3rd; Santos, I. S.; Leasher, J. L.; Singh, J.; Leigh, J.; Jonas, J.; Sanabria, J.; Beardsley, J.; Jacobsen, K. H.; Takahashi, K.; Franklin, R. C.; Ronfani, L.; Montico, M.; Naldi, L.; Tonelli, M.; Geleijnse, J.; Petzold, M.; Shrime, M. G.; Younis, M.; Yonemoto, N.; Breitborde, N.; Yip, P.; Pourmalek, F.; Lotufo, P. A.; Esteghamati, A.; Hankey, G. J.; Ali, R.; Lunevicius, R.; Malekzadeh, R.; Dellavalle, R.; Weintraub, R.; Lucas, R.; Hay, R.; Rojas-Rueda, D.; Westerman, R.; Sepanlou, S. G.; Nolte, S.; Patten, S.; Weichenthal, S.; Abera, S. F.; Fereshtehnejad, S. M.; Shiue, I.; Driscoll, T.; Vasankari, T.; Alsharif, U.; Rahimi-Movaghar, V.; Vlassov, V. V.; Marcenes, W. S.; Mekonnen, W.; Melaku, Y. A.; Yano, Y.; Artaman, A.; Campos, I.; MacLachlan, J.; Mueller, U.; Kim, D.; Trillini, M.; Eshrati, B.; Williams, H. C.; Shibuya, K.; Dandona, R.; Murthy, K.; Cowie, B.; Amare, A. T.; Antonio, C. A.; Castaneda-Orjuela, C.; van Gool, C. H.; Violante, F.; Oh, I. H.; Deribe, K.; Soreide, K.; Knibbs, L.; Kereselidze, M.; Green, M.; Cardenas, R.; Roy, N.; Tillman, T.; Li, Y.; Krueger, H.; Monasta, L.; Dey, S.; Sheikhbahaei, S.; Hafezi-Nejad, N.; Kumar, G. A.; Sreeramareddy, C. T.; Dandona, L.; Wang, H.; Vollset, S. E.; Mokdad, A.; Salomon, J. A.; Lozano, R.; Vos, T.; Forouzanfar, M.; Lopez, A.; Murray, C.; Naghavi, M.(2015) The Global Burden of Cancer 2013. JAMA Oncol., 1 (4), 505-27. DOI: 10.1001/jamaoncol.2015.0735. 5. Chan, S. L.; Wong, V. W.; Qin, S.; Chan, H. L.(2016) Infection and Cancer: The Case of Hepatitis B. J Clin Oncol., 34 (1), 83-90. DOI: 10.1200/JCO.2015.61.5724. 6. Boettler, T.; Moradpour, D.; Thimme, R.; Zoulim, F.(2014) Bridging basic science and clinical research: the EASL Monothematic Conference on Translational Research in Viral Hepatitis. J Hepatol., 61 (3), 696-705. DOI: 10.1016/j.jhep.2014.05.016. 7. Liang, T. J.; Block, T. M.; McMahon, B. J.; Ghany, M. G.; Urban, S.; Guo, J. T.; Locarnini, S.; Zoulim, F.; Chang, K. M.; Lok, A. S. (2015) Present and future therapies of hepatitis B: From discovery to cure. Hepatology., 62 (6), 1893-908. DOI: 10.1002/hep.28025. 8. Zeisel, M. B.; Lucifora, J.; Mason, W. S.; Sureau, C.; Beck, J.; Levrero, M.; Kann, M.; Knolle, P. A.; Benkirane, M.; Durantel, D.; Michel, M. L.; Autran, B.; Cosset, F. L.; Strick-Marchand, H.; Trepo, C.; Kao, J. H.; Carrat, F.; Lacombe, K.; Schinazi, R. F.; Barre-Sinoussi, F.; Delfraissy, J. F.; Zoulim, F.(2015) Towards an HBV cure: state-of-the-art and unresolved questions--report of the ANRS workshop on HBV cure. Gut., 64 (8), 1314-26. DOI: 10.1136/gutjnl-2014-308943.


Page 24 of 46

Page 25 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9. Zhang, E.; Kosinska, A.; Lu, M.; Yan, H.; Roggendorf, M.(2015) Current status of immunomodulatory therapy in chronic hepatitis B, fifty years after discovery of the virus: Search for the "magic bullet" to kill cccDNA. Antiviral Res., 123, 193-203. DOI: 10.1016/j.antiviral.2015.10.009. 10. Durantel, D.; Zoulim, F.(2016) New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus. J Hepatol., 64 (1 Suppl), S117-31. DOI: 10.1016/j.jhep.2016.02.016. 11. Mitra, B.; Thapa, R. J.; Guo, H.; Block, T. M.( 2018) Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B. Antiviral Res., 158, 185-198. DOI: 10.1016/j.antiviral.2018.08.014. 12. Cole, A. G.(2016) Modulators of HBV capsid assembly as an approach to treating hepatitis B virus infection. Curr Opin Pharmacol., 30, 131-137. DOI: 10.1016/j.coph.2016.08.004. 13. Li, W.; Urban, S. (2016) Entry of hepatitis B and hepatitis D virus into hepatocytes: Basic insights and clinical implications. J Hepatol., 64 (1 Suppl), S32-S40. DOI: 10.1016/j.jhep.2016.02.011. 14. Bengsch, B.; Thimme, R. (2018) For whom the interferons toll - TLR7 mediated boosting of innate and adaptive immunity against chronic HBV infection. J Hepatol., 68 (5), 883-886. DOI: 10.1016/j.jhep.2018.01.025. 15. Du, K.; Liu, J.; Broering, R.; Zhang, X.; Yang, D.; Dittmer, U.; Lu, M. (2018) Recent advances in the discovery and development of TLR ligands as novel therapeutics for chronic HBV and HIV infections. Expert Opin Drug Discov., 13 (7), 661-670. DOI: 10.1080/17460441.2018.1473372. 16. Yang, L.; Lu, M. (2018) Small Molecule Inhibitors of Hepatitis B Virus Nucleocapsid Assembly: A New Approach to Treat Chronic HBV Infection. Curr Med Chem., 25 (7), 802-813. DOI: 10.2174/0929867324666170704121800. 17. Liu, H.; Hou, J.; Zhang, X. (2018) Targeting cIAPs, a New Option for Functional Cure of Chronic Hepatitis B Infection? Virol Sin., 33 (5), 459-461. DOI: 10.1007/s12250-018-0062-x. 18. Pei, Y.; Wang, C.; Yan, S. F.; Liu, G. (2017) Past, Current, and Future Developments of Therapeutic Agents for Treatment of Chronic Hepatitis B Virus Infection. J Med Chem., 60 (15), 6461-6479. DOI: 10.1021/acs.jmedchem.6b01442. 19. Dane, D.; Cameron, C. H.; Briggs, M. (1970) Virus-Like Particles in Serum of Patients with Australia-Antigen-Associated Hepatiti. . The Lancet ., 295 (7649), 695-698. 20. Summers, J.; Mason, W. S. (1982) Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell ., 29 (2), 403-15. DOI: 0092-8674(82)90157-X. 21. Seeger, C.; Mason, W. S. (2015) Molecular biology of hepatitis B virus infection. Virology., 479-480, 672-86. DOI: 10.1016/j.virol.2015.02.031. 22. Hu, J.; Seeger, C. (2015) Hepadnavirus Genome Replication and Persistence. Cold Spring Harb Perspect Med., 5 (7), a021386. DOI: 10.1101/cshperspect.a021386. 23. Revill, P.; Locarnini, S.(2016) Antiviral strategies to eliminate hepatitis B virus covalently closed circular DNA (cccDNA). Curr Opin Pharmacol., 30, 144-150. DOI: 10.1016/j.coph.2016.08.015.



24. Leistner, C. M.; Gruen-Bernhard, S.; Glebe, D. (2018) Role of glycosaminoglycans for binding and infection of hepatitis B virus. Cell Microbiol., 10 (1), 122-33. DOI: 10.1111/j.1462-5822.2007.01023.x. 25. Salisse, J.; Sureau, C. (2009) A Function Essential to Viral Entry Underlies the Hepatitis B Virus "a" Determinant. J Virol., 83 (18), 9321-9328. 26. Glebe, D.; Urban, S.; Knoop, E. V.; Cag, N.; Krass, P.; Grun, S.; Bulavaite, A.; Sasnauskas, K.; Gerlich, W. H. (2005) Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes. Gastroenterology., 129 (1), 234-45. DOI: S0016508505008954. 27. Yan, H.; Zhong, G.; Xu, G.; He, W.; Jing, Z.; Gao, Z.; Huang, Y.; Qi, Y.; Peng, B.; Wang, H.; Fu, L.; Song, M.; Chen, P.; Gao, W.; Ren, B.; Sun, Y.; Cai, T.; Feng, X.; Sui, J.; Li, W. (2012) Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife., 1, e00049. DOI: 10.7554/eLife.00049. 28. Yan, H.; Liu, Y.; Sui, J.; Li, W. (2015) NTCP opens the door for hepatitis B virus infection. Antiviral Res., 121, 24-30. DOI: 10.1016/j.antiviral.2015.06.002. 29. Donkers, J. M.; Zehnder, B.; van Westen, G. J. P.; Kwakkenbos, M. J.; AP, I. J.; Oude Elferink, R. P. J.; Beuers, U.; Urban, S.; van de Graaf, S. F. J. (2017) Reduced hepatitis B and D viral entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP. Sci Rep ., 7 (1), 15307. DOI: 10.1038/s41598-017-15338-0. 30. Liu, C.; Xu, G.; Gao, Z.; Zhou, Z.; Guo, G.; Li, D.; Jing, Z.; Sui, J.; Li, W. (2018) The p.Ser267Phe variant of sodium taurocholate cotransporting polypeptide (NTCP) supports HBV infection with a low efficiency. Virology., 522, 168-176. DOI: 10.1016/j.virol.2018.07.006. 31. Blondot, M. L.; Bruss, V.; Kann, M. (2016) Intracellular transport and egress of hepatitis B virus. J Hepatol., 64 (1 Suppl), S49-59. DOI: 10.1016/j.jhep.2016.02.008. 32. Osseman, Q.; Gallucci, L.; Au, S.; Cazenave, C.; Berdance, E.; Blondot, M. L.; Cassany, A.; Begu, D.; Ragues, J.; Aknin, C.; Sominskaya, I.; Dishlers, A.; Rabe, B.; Anderson, F.; Pante, N.; Kann, M. (2018) The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids. J Hepatol., 68 (3), 441-448. DOI: 10.1016/j.jhep.2017.10.032. 33. Kitamura, K.; Que, L.; Shimadu, M.; Koura, M.; Ishihara, Y.; Wakae, K.; Nakamura, T.; Watashi, K.; Wakita, T.; Muramatsu, M. (2018) Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus. PLoS Pathog., 14 (6), e1007124. DOI: 10.1371/journal.ppat.1007124. 34. Qi, Y.; Gao, Z.; Xu, G.; Peng, B.; Liu, C.; Yan, H.; Yao, Q.; Sun, G.; Liu, Y.; Tang, D.; Song, Z.; He, W.; Sun, Y.; Guo, J. T.; Li, W. (2016) DNA Polymerase kappa Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus. PLoS Pathog., 12 (10), e1005893. DOI: 10.1371/journal.ppat.1005893. 35. Long, Q.; Yan, R.; Hu, J.; Cai, D.; Mitra, B.; Kim, E. S.; Marchetti, A.; Zhang, H.; Wang, S.; Liu, Y.; Huang, A.; Guo, H. (2017) The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. PLoS Pathog., 13 (12), e1006784. DOI: 10.1371/journal.ppat.1006784. 36. Liang, T. J. (2009) Hepatitis B: the virus and disease. Hepatology., 49 (5 Suppl), S13-21. DOI: 10.1002/hep.22881.


Page 26 of 46

Page 27 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37. Quasdorff, M.; Protzer, U. (2010) Control of hepatitis B virus at the level of transcription. J Viral Hepat., 17 (8), 527-36. DOI: 10.1111/j.1365-2893.2010.01315.x. 38. Nassal, M. (2015) HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut., 64 (12), 1972-84. DOI: 10.1136/gutjnl-2015-309809. 39. Andrisani, O. M. (2013) Deregulation of epigenetic mechanisms by the hepatitis B virus X protein in hepatocarcinogenesis. Viruses., 5 (3), 858-72. DOI: 10.3390/v5030858. 40. Slagle, B. L.; Bouchard, M. J. (2016) Hepatitis B Virus X and Regulation of Viral Gene Expression. Cold Spring Harb Perspect Med., 6 (3), a021402. DOI: 10.1101/cshperspect.a021402. 41. Belloni, L.; Pollicino, T.; De Nicola, F.; Guerrieri, F.; Raffa, G.; Fanciulli, M.; Raimondo, G.; Levrero, M. (2009) Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A., 106 (47), 19975-9. DOI: 10.1073/pnas.0908365106. 42. Stibbe, W.; Gerlich, W. H. (1983) Structural relationships between minor and major proteins of hepatitis B surface antigen. J Virol., 46 (2), 626-8. 43. Heermann, K. H.; Goldmann, U.; Schwartz, W.; Seyffarth, T.; Baumgarten, H.; Gerlich, W. H. (1984) Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol., 52 (2), 396-402. 44. Schädler, S.; Hildt, E. (2009) HBV Life Cycle: Entry and Morphogenesis. Viruses., 1, 185-209. DOI: 10.3390/v1020185. 45. Zhao, J. H.; Zhang, Y. L.; Zhang, T. Y.; Yuan, L. Z.; Cheng, T.; Chen, P. J.; Yuan, Q.; Xia, N. S. (2017) A novel toolbox for the in vitro assay of hepatitis D virus infection. Sci Rep., 7, 40199. DOI: 10.1038/srep40199. 46. Lamontagne, R. J.; Bagga, S.; Bouchard, M. J. (2016) Hepatitis B virus molecular biology and pathogenesis. Hepatoma Res., 2, 163-186. DOI: 10.20517/2394-5079.2016.05. 47. Beck, J.; Nassal, M. (2007) Hepatitis B virus replication. World J Gastroenterol., 13 (1), 48-64. DOI: 10.3748/wjg.v13.i1.48 48. Huovila, A. P.; Eder, A. M.; Fuller, S. D. (1992) Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol., 118 (6), 1305-20. 49. Zhang, Y. Y.; Zhang, B. H.; Theele, D.; Litwin, S.; Toll, E.; Summers, J. (2003) Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. Proc Natl Acad Sci U S A., 100 (21), 12372-7. DOI: 10.1073/pnas.2033898100. 50. Hu, J.; Liu, K. (2017) Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses., 9 (3). DOI: 10.3390/v9030056. 51. Ning, X.; Luckenbaugh, L.; Liu, K.; Bruss, V.; Sureau, C.; Hu, J. (2018) Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions. J Virol., 92 (14). DOI: 10.1128/JVI.00272-18. 52. Bai, L.; Zhang, X.; Li, W.; Wu, M.; Liu, J.; Kozlowski, M.; Chen, L.; Zhang, J.; Huang, Y.; Yuan, Z. (2018) Extracellular HBV RNAs are heterogeneous in length and circulate as capsid-antibody-complexes in addition to virions in chronic hepatitis B patients. J Virol. DOI: 10.1128/JVI.00798-18. 53. Bardens, A.; Doring, T.; Stieler, J.; Prange, R. (2011) Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner. Cell Microbiol., 13 (4), 602-19. DOI: 10.1111/j.1462-5822.2010.01557.x.



54. Sir, D.; Tian, Y.; Chen, W. L.; Ann, D. K.; Yen, T. S.; Ou, J. H. (2010) The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication. Proc Natl Acad Sci U S A., 107 (9), 4383-8. DOI: 10.1073/pnas.0911373107. 55. Lin, Y.; Deng, W.; Pang, J.; Kemper, T.; Hu, J.; Yin, J.; Zhang, J.; Lu, M. (2017) The microRNA-99 family modulates hepatitis B virus replication by promoting IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy. Cell Microbiol., 19 (5). DOI: 10.1111/cmi.12709. 56. Doring, T.; Zeyen, L.; Bartusch, C.; Prange, R. (2018) Hepatitis B Virus Subverts the Autophagy Elongation Complex Atg5-12/16L1 and Does Not Require Atg8/LC3 Lipidation for Viral Maturation. J Virol., 92 (7). DOI: 10.1128/JVI.01513-17. 57. Lin, Y.; Wu, C.; Wang, X.; Kemper, T.; Squire, A.; Gunzer, M.; Zhang, J.; Chen, X.; Lu, M. (2018) Hepatitis B virus is degraded by autophagosome-lysosome fusion mediated by Rab7 and related components. Protein Cell. DOI: 10.1007/s13238-018-0555-2. 58. Uhl, P.; Helm, F.; Hofhaus, G.; Brings, S.; Kaufman, C.; Leotta, K.; Urban, S.; Haberkorn, U.; Mier, W.; Fricker, G. (2016) A liposomal formulation for the oral application of the investigational hepatitis B drug Myrcludex B. Eur J Pharm Biopharm., 103, 159-166. DOI: 10.1016/j.ejpb.2016.03.031. 59. Murakami, E.; Wang, T.; Park, Y.; Hao, J.; Lepist, E. I.; Babusis, D.; Ray, A. S. (2015) Implications of efficient hepatic delivery by tenofovir alafenamide (GS-7340) for hepatitis B virus therapy. Antimicrob Agents Chemother., 59 (6), 3563-9. DOI: 10.1128/AAC.00128-15. 60. Dryden, K. A.; Wieland, S. F.; Whitten-Bauer, C.; Gerin, J. L.; Chisari, F. V.; Yeager, M. (2006) Native hepatitis B virions and capsids visualized by electron cryomicroscopy. Mol Cell., 22 (6), 843-50. DOI: 10.1016/j.molcel.2006.04.025. 61. Wynne, S. A.; Crowther, R. A.; Leslie, A. G. (1993) The crystal structure of the human hepatitis B virus capsid. Mol Cell., 3 (6), 771-80. DOI: S1097-2765(01)80009-5. 62. Chain, B. M.; Myers, R., Variability and conservation in hepatitis B virus core protein. BMC Microbiol 2005, 5, 33. DOI: 10.1186/1471-2180-5-33. 63. Zlotnick, A.; Venkatakrishnan, B.; Tan, Z.; Lewellyn, E.; Turner, W.; Francis, S. (2015) Core protein: A pleiotropic keystone in the HBV lifecycle. Antiviral Res., 121, 82-93. DOI: 10.1016/j.antiviral.2015.06.020. 64. Nassal, M.; Rieger, A.; Steinau, O. (1992) Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking. J Mol Biol., 225 (4), 1013-25. DOI: 0022-2836(92)90101-O. 65. Selzer, L.; Katen, S. P.; Zlotnick, A. (2014) The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability. Biochemistry., 53 (34), 5496-504. DOI: 10.1021/bi500732b. 66. Venkatakrishnan, B.; Zlotnick, A. (2016) The Structural Biology of Hepatitis B Virus: Form and Function. Annu Rev Virol., 3 (1), 429-451. DOI: 10.1146/annurev-virology-110615-042238. 67. Ceres, P.; Zlotnick, A. (2002) Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids. Biochemistry., 41 (39), 11525-11531. 68. Packianathan, C.; Katen, S. P.; Dann, C. E., 3rd; Zlotnick, A. (2010) Conformational changes in the hepatitis B virus core protein are consistent with a role for allostery in virus assembly. J Virol., 84 (3), 1607-15. DOI: 10.1128/JVI.02033-09.


Page 28 of 46

Page 29 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


69. Zlotnick, A.; Cheng, N.; Stahl, S. J.; Conway, J. F.; Steven, A. C.; Wingfield, P. T. (1997) Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA. Proc Natl Acad Sci U S A., 94 (18), 9556-61. 70. Nassal, M. (1992) The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol., 66 (7), 4107-16. 71. Chu, T. H.; Liou, A. T.; Su, P. Y.; Wu, H. N.; Shih, C. (2014) Nucleic acid chaperone activity associated with the arginine-rich domain of human hepatitis B virus core protein. J Virol., 88 (5), 2530-43. DOI: 10.1128/JVI.03235-13. 72. Gazina, E. V.; Fielding, J. E.; Lin, B.; Anderson, D. A. (2000) Core protein phosphorylation modulates pregenomic RNA encapsidation to different extents in human and duck hepatitis B viruses. J Virol., 74 (10), 4721-8. 73. Melegari, M.; Wolf, S. K.; Schneider, R. J. (2005) Hepatitis B virus DNA replication is coordinated by core protein serine phosphorylation and HBx expression. J Virol., 79 (15), 9810-20. DOI: 10.1128/JVI.79.15.9810-9820.2005. 74. Kock, J.; Nassal, M.; Deres, K.; Blum, H. E.; von Weizsacker, F. (2004) Hepatitis B virus nucleocapsids formed by carboxy-terminally mutated core proteins contain spliced viral genomes but lack full-size DNA. J Virol., 78 (24), 13812-8. DOI: 10.1128/JVI.78.24.13812-13818.2004. 75. Zlotnick, A.; Ceres, P.; Singh, S.; Johnson, J. M. (2002) A small molecule inhibits and misdirects assembly of hepatitis B virus capsids. J Virol., 76 (10), 4848-54. DOI: 10.1128/JVI.76.10.4848-4854.2002 76. Zhang, X.; Cheng, J.; Ma, J.; Hu, Z.; Wu, S.; Hwang, N.; Kulp, J.; Du, Y.; Guo, J. T.; Chang, J. (2018) Discovery of novel hepatitis B virus nucleocapsid assembly inhibitors. ACS Infect Dis. DOI: 10.1021/acsinfecdis.8b00269. 77. Stray, S. J.; Bourne, C. R.; Punna, S.; Lewis, W. G.; Finn, M. G.; Zlotnick, A. (2005) A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly. Proc Natl Acad Sci U S A., 102 (23), 8138-43. DOI: 10.1073/pnas.0409732102. 78. Bourne, C.; Lee, S.; Venkataiah, B.; Lee, A.; Korba, B.; Finn, M. G.; Zlotnick, A. (2008) Small-molecule effectors of hepatitis B virus capsid assembly give insight into virus life cycle. J Virol., 82 (20), 10262-70. DOI: 10.1128/JVI.01360-08. 79. King, R. W.; Ladner, S. K.; Miller, T. J.; Zaifert, K.; Perni, R. B.; Conway, S. C.; Otto, M. J. (1998) Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-L-2',3'-dideoxy-3'-thiacytidine. Antimicrob Agents Chemother., 42 (12), 3179-86. 80. Delaney, W. E. t.; Edwards, R.; Colledge, D.; Shaw, T.; Furman, P.; Painter, G.; Locarnini, S. (2002) Phenylpropenamide derivatives AT-61 and AT-130 inhibit replication of wild-type and lamivudine-resistant strains of hepatitis B virus in vitro. Antimicrob Agents Chemother., 46 (9), 3057-60. 81. Campagna, M. R.; Liu, F.; Mao, R.; Mills, C.; Cai, D.; Guo, F.; Zhao, X.; Ye, H.; Cuconati, A.; Guo, H.; Chang, J.; Xu, X.; Block, T. M.; Guo, J. T. (2013) Sulfamoylbenzamide derivatives inhibit the assembly of hepatitis B virus nucleocapsids. J Virol., 87 (12), 6931-42. DOI: 10.1128/JVI.00582-13.



82. Katen, S. P.; Tan, Z.; Chirapu, S. R.; Finn, M. G.; Zlotnick, A. (2013) Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure. Structure., 21 (8), 1406-16. DOI: 10.1016/j.str.2013.06.013. 83. Tan, Z.; Maguire, M. L.; Loeb, D. D.; Zlotnick, A. (2013) Genetically altering the thermodynamics and kinetics of hepatitis B virus capsid assembly has profound effects on virus replication in cell culture. J Virol., 87 (6), 3208-16. DOI: 10.1128/JVI.03014-12. 84. Zhou, Z.; Hu, T.; Zhou, X.; Wildum, S.; Garcia-Alcalde, F.; Xu, Z.; Wu, D.; Mao, Y.; Tian, X.; Zhou, Y.; Shen, F.; Zhang, Z.; Tang, G.; Najera, I.; Yang, G.; Shen, H. C.; Young, J. A.; Qin, N. (2017) Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms. Sci Rep., 7, 42374. DOI: 10.1038/srep42374. 85. Klumpp, K.; Lam, A. M.; Lukacs, C.; Vogel, R.; Ren, S.; Espiritu, C.; Baydo, R.; Atkins, K.; Abendroth, J.; Liao, G.; Efimov, A.; Hartman, G.; Flores, O. A. (2015) High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein. Proc Natl Acad Sci U S A., 112 (49), 15196-201. DOI: 10.1073/pnas.1513803112. 86. Lam, A. M.; Espiritu, C.; Vogel, R.; Ren, S.; Lau, V.; Kelly, M.; Kuduk, S. D.; Hartman, G. D.; Flores, O. A.; Klumpp, K. (2018) Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator Against the Hepatitis B Virus. Antimicrob Agents Chemother. DOI: 10.1128/AAC.01734-18. 87. Wang, Y. J.; Lu, D.; Xu, Y. B.; Xing, W. Q.; Tong, X. K.; Wang, G. F.; Feng, C. L.; He, P. L.; Yang, L.; Tang, W.; Hu, Y. H.; Zuo, J. P. (2015) A novel pyridazinone derivative inhibits hepatitis B virus replication by inducing genome-free capsid formation. Antimicrob Agents Chemother., 59 (11), 7061-72. DOI: 10.1128/AAC.01558-15. 88. Yang, L.; Shi, L. P.; Chen, H. J.; Tong, X. K.; Wang, G. F.; Zhang, Y. M.; Wang, W. L.; Feng, C. L.; He, P. L.; Zhu, F. H.; Hao, Y. H.; Wang, B. J.; Yang, D. L.; Tang, W.; Nan, F. J.; Zuo, J. P. (2014) Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation. Acta Pharmacol Sin., 35 (3), 410-8. DOI: 10.1038/aps.2013.175. 89. Cho, M. H.; Jeong, H.; Kim, Y. S.; Kim, J. W.; Jung, G. (2013) 2-amino-N-(2,6-dichloropyridin-3-yl)acetamide derivatives as a novel class of HBV capsid assembly inhibitor. J Viral Hepat., 21 (12), 843-52. DOI: 10.1111/jvh.12214. 90. Yang, L.; Wang, Y. J.; Chen, H. J.; Shi, L. P.; Tong, X. K.; Zhang, Y. M.; Wang, G. F.; Wang, W. L.; Feng, C. L.; He, P. L.; Xu, Y. B.; Lu, M. J.; Tang, W.; Nan, F. J.; Zuo, J. P. (2016) Effect of a hepatitis B virus inhibitor, NZ-4, on capsid formation. Antiviral Res., 125, 25-33. DOI: 10.1016/j.antiviral.2015.11.004. 91. Tang, J.; Huber, A. D.; Pineda, D. L.; Boschert, K. N.; Wolf, J. J.; Kankanala, J.; Xie, J.; Sarafianos, S. G.; Wang, Z. (2019) 5-Aminothiophene-2,4-dicarboxamide analogues as hepatitis B virus capsid assembly effectors. Eur J Med Chem., 164, 179-192. DOI: 10.1016/j.ejmech.2018.12.047. 92. Li, X.; Zhou, K.; He, H.; Zhou, Q.; Sun, Y.; Hou, L.; Shen, L.; Wang, X.; Zhou, Y.; Gong, Z.; He, S.; Jin, H.; Gu, Z.; Zhao, S.; Zhang, L.; Sun, C.; Zheng, S.; Cheng, Z.; Zhu, Y.; Zhang, M.; Li, J.; Chen, S. (2017) Design, Synthesis, and Evaluation of Tetrahydropyrrolo[1,2-c]pyrimidines as


Page 30 of 46

Page 31 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Capsid Assembly Inhibitors for HBV Treatment. ACS Med Chem., 8 (9), 969-974. DOI: 10.1021/acsmedchemlett.7b00288. 93. Boucle, S.; Lu, X.; Bassit, L.; Ozturk, T.; Russell, O. O.; Amblard, F.; Coats, S. J.; Schinazi, R. F. (2017) Synthesis and antiviral evaluation of novel heteroarylpyrimidines analogs as HBV capsid effectors. Bioorg Med Chem Lett., 27 (4), 904-910. DOI: 10.1016/j.bmcl.2017.01.010. 94. Wang, P.; Naduthambi, D.; Mosley, R. T.; Niu, C.; Furman, P. A.; Otto, M. J.; Sofia, M. J. (2011) Phenylpropenamide derivatives: anti-hepatitis B virus activity of the Z isomer, SAR and the search for novel analogs. Bioorg Med Chem Lett., 21 (15), 4642-7. DOI: 10.1016/j.bmcl.2011.05.077. 95. Feld, J. J.; Colledge, D.; Sozzi, V.; Edwards, R.; Littlejohn, M.; Locarnini, S. A. (2007) The phenylpropenamide derivative AT-130 blocks HBV replication at the level of viral RNA packaging. Antiviral Research., 76 168-177. DOI: 10.1016/j.antiviral.2007.06.014 96. Katen, S. P.; Chirapu, S. R.; Finn, M. G.; Zlotnick, A. (2010) Trapping of hepatitis B virus capsid assembly intermediates by phenylpropenamide assembly accelerators. ACS Chem Biol., 5 (12), 1125-36. DOI: 10.1021/cb100275b. 97. Li, L.; Chirapu, S. R.; Finn, M. G.; Zlotnick, A. (2013) Phase diagrams map the properties of antiviral agents directed against hepatitis B virus core assembly. Antimicrob Agents Chemother., 57 (3), 1505-8. DOI:10.1128/AAC.01766-12. 98. Tan, Z.; Pionek, K.; Unchwaniwala, N.; Maguire, M. L.; Loeb, D. D.; Zlotnick, A. (2015) The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription. J Virol., 89 (6), 3275-84. DOI: 10.1128/JVI.03545-14. 99. Lu, D.; Liu, F.; Xing, W.; Tong, X.; Wang, L.; Wang, Y.; Zeng, L.; Feng, C.; Yang, L.; Zuo, J.; Hu, Y. (2016) Optimization and Synthesis of Pyridazinone Derivatives as Novel Inhibitors of Hepatitis B Virus by Inducing Genome-free Capsid Formation. ACS Infect Dis. DOI: 10.1021/acsinfecdis.6b00159. 100. Croasdell, G. (2018) European Association for the Study of the Liver-53rd International Liver Congress. Drugs of the futrue., 43 (7), 537-543. 101. Deres, K.; Schroder, C. H.; Paessens, A.; Goldmann, S.; Hacker, H. J.; Weber, O.; Kramer, T.; Niewohner, U.; Pleiss, U.; Stoltefuss, J.; Graef, E.; Koletzki, D.; Masantschek, R. N.; Reimann, A.; Jaeger, R.; Gross, R.; Beckermann, B.; Schlemmer, K. H.; Haebich, D.; Rubsamen-Waigmann, H. (2003) Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids. Science., 299 (5608), 893-6. DOI: 10.1126/science.1077215. 102. Weber, O.; Schlemmer, K. H.; Hartmann, E.; Hagelschuer, I.; Paessens, A.; Graef, E.; Deres, K.; Goldmann, S.; Niewoehner, U.; Stoltefuss, J.; Haebich, D.; Ruebsamen-Waigmann, H.; Wohlfeil, S. (2002) Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antiviral Res., 54 (2), 69-78. DOI: S0166354201002169. 103. Brezillon, N.; Brunelle, M. N.; Massinet, H.; Giang, E.; Lamant, C.; DaSilva, L.; Berissi, S.; Belghiti, J.; Hannoun, L.; Puerstinger, G.; Wimmer, E.; Neyts, J.; Hantz, O.; Soussan, P.; Morosan, S.; Kremsdorf, D. (2011) Antiviral activity of Bay 41-4109 on hepatitis B virus in humanized Alb-uPA/SCID mice. PLoS One., 6 (12), e25096. DOI: 10.1371/journal.pone.0025096.



104. Shi, C.; Wu, C. Q.; Cao, A. M.; Sheng, H. Z.; Yan, X. Z.; Liao, M. Y. (2007) NMR-spectroscopy-based metabonomic approach to the analysis of Bay41-4109, a novel anti-HBV compound, induced hepatotoxicity in rats. Toxicol Lett., 173 (3), 161-7. DOI: 10.1016/j.toxlet.2007.07.010. 105. Liu, N., Zhao, F.B., Jia, H.Y., Rai, D., Zhan, P., Jiang, X.M., Liu, X.Y. (2015) Non-nucleoside anti-HBV agents: advances in structural optimization and mechanism of action investigations. Med. Chem. Commun., 6 (4), 521-535. DOI: 10.1039/C4MD00521J. 106. Ding, Y.; Zhang, H.; Niu, J.; Chen, H.; Liu, C.; Li, X.; Wang, F. (2017) Multiple dose study of GLS4JHS, interfering with the assembly of hepatitis B virus core particles, in patients infected with hepatitis B virus. Journal of Hepatology., 66, S27. 107. Qiu, Z.; Lin, X.; Zhou, M.; Liu, Y.; Zhu, W.; Chen, W.; Zhang, W.; Guo, L.; Liu, H.; Wu, G.; Huang, M.; Jiang, M.; Xu, Z.; Zhou, Z.; Qin, N.; Ren, S.; Qiu, H.; Zhong, S.; Zhang, Y.; Wu, X.; Shi, L.; Shen, F.; Mao, Y.; Zhou, X.; Yang, W.; Wu, J. Z.; Yang, G.; Mayweg, A. V.; Shen, H. C.; Tang, G. (2016) Design and Synthesis of Orally Bioavailable 4-Methyl Heteroaryldihydropyrimidine Based Hepatitis B Virus (HBV) Capsid Inhibitors. J Med Chem., 59 (16), 7651-66. DOI: 10.1021/acs.jmedchem.6b00879. 108. Gane, E.; Liu, A.; Yuen, M. F.; Schwabe, C.; Bo, Q.; Das, S. K.; Gao, L.; Zhou, X.; Wang, Y.; Coakley, E.; Jin, Y. (2018) In RO7049389, a core protein allosteric modulator, demonstrates robust anti-HBV activity in chronic hepatitis B patients and is safe and well tolerated, European Association for the Study of the Liver(EASL)-The International Liver Congress, Paris, France, Paris, France. 109. Klumpp, K.; Shimada, T.; Allweiss, L.; Volz, T.; Lutgehetmann, M.; Hartman, G.; Flores, O. A.; Lam, A. M.; Dandri, M. (2018) Efficacy of NVR 3-778, Alone and In Combination With Pegylated Interferon, vs Entecavir In uPA/SCID Mice With Humanized Livers and HBV Infection. Gastroenterology., 154 (3), 652-662 e8. DOI: 10.1053/j.gastro.2017.10.017. 110. Tavis, J. E.; Lomonosova, E. (2018) NVR 3-778 Plus Pegylated Interferon-alpha Treatment for Chronic Hepatitis B Viral Infections: Could 1 + 1 = 3? Gastroenterology., 154 (3), 481-482. DOI: 10.1053/j.gastro.2018.01.011. 111. Gane, E. J., Schwabe, C., Walker K, Flores L, Hartman GD, Klumpp K,; Liaw S, B. N. (2014) Phase 1a safety and pharmacokinetics of NVR 3-778, a potential first-in-class HBV core inhibitor. Hepatology., 60, 1279. 112. Yuen, M. F.; Dong, J. K.; Weilert, F.; Chan, L. Y.; Lalezari, J. P.; Hwang, S. G.; Nguyen, T. T.; Liaw, S.; Brown, N.; Klumpp, K.; Hartman, G. D.; Gane, E. J. (2015) Phase 1b Efficacy and Safety of NVR 3-778, a First-In-Class HBV Core Inhibitor, in HBeAg-Positive Patients with Chronic HBV Infection. Hepatology., 62 (6), 1385A-1386A. 113. Yuen, M. F.; Dong, J. K.; Weilert, F.; Chan, L. Y.; Lalezari, J. P.; Hwang, S. G.; Nguyen, T. T.; Liaw, S.; Brown, N.; Klumpp, K.; Flores, O. A.; Hartman, G. D.; Gane, E. J. (2016) NVR 3-778, a first-inclass HBV core inhibitor, alone and in combination with peg-interferon (PEGIFN), in treatment naive HBeAgpositive patients: early reductions in HBV DNA and HBeAg. J Hepatol., 64 (2), S210-S211. 114. Berke, J. M.; Dehertogh, P.; Vergauwen, K.; Van Damme, E.; Mostmans, W.; Vandyck, K.; Pauwels, F. (2017) Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus. Antimicrob Agents Chemother., 61 (8). DOI: 10.1128/AAC.00560-17.


Page 32 of 46

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115. Vandyck, K.; Rombouts, G.; Stoops, B.; Tahri, A.; Vos, A.; Verschueren, W.; Wu, Y.; Yang, J.; Hou, F.; Huang, B.; Vergauwen, K.; Dehertogh, P.; Berke, J. M.; Raboisson, P. (2018) Synthesis and Evaluation of N-Phenyl-3-sulfamoyl-benzamide Derivatives as Capsid Assembly Modulators Inhibiting Hepatitis B Virus (HBV). J Med Chem., 61 (14), 6247-6260. DOI: 10.1021/acs.jmedchem.8b00654. 116. Yogaratnam, J. Z.; Vandenbossche, J. J.; Jessner, W.; Van den Boer, M.; Biewenga, J.; Rasschaert, F.; Vistuer, C.; Janssen, K.; Smyej, I.; Verstraete, D.; Berke, J.M.; Vandyck, K.; Chanda, S.;Fry, J. (2016) Safety, tolerability and pharmacokinetics of JNJ-56136379, a novel HBV capsid assembly modulator, in healthy subjects. AASLD The Liver Meeting, Boston MA. 117. Zoulim, F.; Yogaratnam, J. Z.; Vandenbossche, J. J.; Lenz, O.; Talloen, W.; Vistuer, C.; Moscalu, I.; Streinu-Cercel, A.; Bourgeois, S.; Buti, M.; Crespo, J.; Pascasio10, J. M.; Sarrazin, C.; Vanwolleghem, T.; Blatt, L.; Fry, J. (2018) In Safety and antiviral activity of novel HBV capsid assembly modulator JNJ-56136379 in non-cirrhotic, treatment-naïve patients with chronic hepatitis B. European Association for the Study of the Liver(EASL)-The International Liver Congress, Paris, France. 118. Mani, N.; Cole, A. G.; Ardzinsk, A.; Cai, D.; Cuconati, A.; Dorsey, B. D.; Guo, F.; Guo, H.; Guo, J. T.; Kultgen, S.; Lee, A. C.; Rijnbrand, R.; Snead, N. M.; Steuer, H.; Wang, X.; Zhao, Q.; Sofia, M. J. (2016) In The HBV capsid inhibitor AB-423 exhibits a dual mode of action and displays additive/synergistic effects in in vitro combination studies. AASLD The Liver Meeting, Boston, MA. 119. Mani, N.; Cole, A. G.; Phelps, J. R.; Ardzinski, A.; Cobarrubias, K. D.; Cuconati, A.; Dorsey, B. D.; Evangelista, E.; Fan, K.; Guo, F.; Guo, H.; Guo, J. T.; Harasym, T. O.; Kadhim, S.; Kultgen, S. G.; Lee, A. C. H.; Li, A. H. L.; Long, Q.; Majeski, S. A.; Mao, R.; McClintock, K. D.; Reid, S. P.; Rijnbrand, R.; Snead, N. M.; Micolochick Steuer, H. M.; Stever, K.; Tang, S.; Wang, X.; Zhao, Q.; Sofia, M. J. (2018) Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation. Antimicrob Agents Chemother., 62 (6). DOI: 10.1128/AAC.00082-18. 120. In Assembly Biosciences Announces Successful Completion of ABI-H0731 Phase 1a Trial and Upcoming Conference Presentations.26th Conference of the Asian Pacific Association for the Study of the Liver (APASL), Shanghai, China, Shanghai, China, 2017. 121. Yuen, M. F.; Agarwal, K.; Gane, E. J.; Schwabe, C.; Ahn, S. H.; Kim, D. J.; Lim, Y. S.; Cheng, W.; Sievert, W.; Visvanathan, K.; Ruby, E.; Liaw, S.; Colonno, R.; Lopatin, U. (2018) In Interim Safety, Tolerability, Pharmacokinetics, and Antiviral Activity of ABI-H0731, a Novel Core Protein Allosteric Modifier (CpAM), in Healthy Volunteers and Non-Cirrhotic Viremic Subjects with Chronic Hepatitis B, European Association for the Study of the Liver(EASL)-The International Liver Congress, Paris, France. 122. Lucifora, J.; Xia, Y.; Reisinger, F.; Zhang, K.; Stadler, D.; Cheng, X.; Sprinzl, M. F.; Koppensteiner, H.; Makowska, Z.; Volz, T.; Remouchamps, C.; Chou, W. M.; Thasler, W. E.; Huser, N.; Durantel, D.; Liang, T. J.; Munk, C.; Heim, M. H.; Browning, J. L.; Dejardin, E.; Dandri, M.; Schindler, M.; Heikenwalder, M.; Protzer, U. (2014) Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science., 343 (6176), 1221-8. DOI: 10.1126/science.1243462.



123. Cui, X.; Clark, D. N.; Liu, K.; Xu, X. D.; Guo, J. T.; Hu, J. (2015) Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes. J Virol., 90 (1), 486-96. DOI: 10.1128/JVI.01263-15. 124. Bassit, L. e. a. (2018) European Association for the Study of the Liver-53rd International Liver Congress Abst PS-026.


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Figures. Figure 1. An illustration of the life cycle of HBV and capsid assembly. A. HBV life cycle consists of receptor binding and entry, transport of nucleocapsids to nuclear pores and release of HBV genomes into the nuclei, cccDNA formation, transcription, pgRNA encapsidation, reverse transcription, DNA synthesis, envelopment, and release of virions and subviral particles. B. The pathway for HBV capsid assembly. Early studies mainly focused on the formation of HBV nucleocapsids with the steps pgRNA encapsidation, reverse transcription, and HBV DNA synthesis. Recently, the process of HBV capsid assembly was found to produce a variety of products including empty capsids47,

48

and capsids with incomplete HBV

RNA/DNA fragments 49. These products could be either released as naked capsids50 from hepatocytes or enveloped to form virion-like particles. HBV CpAMs could induce the formation of irregular and instable capsid structures or empty capsids with the normal morphology.



Figure 1.


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Figure 2. The top view of an HBV Cp149 dimer structure and HBV capsid structure. A. The structure of a T = 4 wild type capsid has the four helix bundle forming the spikes on the surface. The HBcAg dimers form a trimer structure (as shown in the center) as the nucleus for capsid assembly. B. The two halves of the dimer are in blue and gray. The residues Val-124 and Tyr-132 with particular relevance for HBV capsid assembly are colored green.

Figure 2


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Figure 3. The primary structure of HBcAg and corresponding functions. The primary structure of HBcAg contains two functional domains for capsid assembly (NTD) and RNA/DNA binding (CTD). The CTD consists of arginine-rich domains and interacts with HBV pgRNA.

Figure 3



Figure 4. Structure of representative capsid assembly modulators (CAMs).

Figure 4


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Tables.

Table1 HBV CpAMs in preclinical trials Compound codes

Chemotypes

Company/Institute

Reference

EP-027367

NA

Enanta Pharmaceuticals, USA

100

AT-61/AT-130

Phenylpropenamides (PPAs)

NA

79, 80

3711/5167

Pyridazinone derivatives

Shanghai Institute of Materia Medica

87, 99

BCM-599/ BCM-606 2-Amino-N-(2,6-dichloropyridin-3-yl)acetamide derivatives NA

89

BisANS

5,5’-bis[8-(phenylamino)-1-naphthalenesulfonate](BisANS) NA

75

BA-5308B

Benzamide derivative

NA

76

GLP-26

NA

Emory University

124


ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

QL-0A6a

NA

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QILU Pharmaceutical , PR China

NA: information not available


qilu-pharma.com

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Table2 CpAMs in clinical trials Compound codes

Chemotypes

BAY41-4109

Clinical trial

Company

Reference

heteroaryldihydropyrimidine (HAP) Phase I

AiCuris, Germany

101-105

GLS4JHS

heteroaryldihydropyrimidine (HAP) Phase II

HEC Pharm, PR China

106

RO-7049389

heteroaryldihydropyrimidine (HAP) Phase II

Roche

107, 108

NVR 3-778

sulfamoyl benzamide (SBA)

Phase II

Janssen, USA

86, 109-113

JNJ6379/ JNJ0440


Phase II/ Phase I Janssen, USA

114-117

AB-423/AB-506


Phase I/ Phase I

118, 119

Arbutus Biopharma, Canada

Abi-H0731/ Abi-H2185 NA

Phase II/ Phase I Assembly biosciences, USA

121

NZ-4

Phase I

88, 90

Isothiafludine

HaiHe Pharmaceutical, China


ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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RG7907

NA

Phase I

Roche, Switzerland

QL-007

NA

Phase I

QILU Pharmaceutical , PR China qilu-pharma.com

NA: information not available.


roche.com

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Table 3 Phase Ib 28-Day Studies in HBV-infected patients Compound codes

Dosage

HBV DNA Reduction (log10 IU/mL)

Estimated*

GLS4JHS

120mg+RTV 100mg/ q.d.

1.42

NA

RO-7049389

200mg/

b.i.d.

2.7

n=3

NVR 3-778

600mg/

b.i.d

1.97

NA

JNJ6379

75mg/ q.d.

2.0

n=1

Abi-H0731

100mg/ q.d.

1.3-2.2

NA

Estimated*: number of treated patients with HBV DNA level below the limit of detection. NA: information not available.



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