Hepatitis B Virus Capsid Assembly Research Articles

TRPV4 promotes HBV replication and capsid assembly via methylation modification of H3K4 and HBc ubiquitin.

Hepatitis B virus (HBV) infection poses a significant burden on global public health. Unfortunately, current treatments cannot fully alleviate this burden as they have limited effect on the transcriptional activity of the tenacious covalently closed circular DNA (cccDNA) responsible for viral persistence. Consequently, the HBV life cycle should be further investigated to develop new anti-HBV pharmaceutical targets. Our previous study discovered that the host gene TMEM203 hinders HBV replication by participating in calcium ion regulation. The involvement of intracellular calcium in HBV replication has also been confirmed. In this study, we found that transient receptor potential vanilloid 4 (TRPV4) notably enhances HBV reproduction by investigating the effects of several calcium ion-related molecules on HBV replication. The in-depth study showed that TRPV4 promotes hepatitis B core/capsid protein (HBc) protein stability through the ubiquitination pathway and then promotes the nucleocapsid assembly. HBc binds to cccDNA and reduces the nucleosome spacing of the cccDNA-histones complex, which may regulate HBV transcription by altering the nucleosome arrangement of the HBV genome. Moreover, our results showed that TRPV4 promotes cccDNA-dependent transcription by accelerating the methylation modification of H3K4. In conclusion, TRPV4 could interact with HBV core protein and regulate HBV during transcription and replication. These data suggest that TRPV4 exerts multifaceted HBV-related synergistic factors and may serve as a therapeutic target for CHB.

Synthesis and evaluation of highly potent HBV capsid assembly modulators (CAMs)

Chronic hepatitis B virus (HBV) infection remains a major global health burden. It affects more than 290 million individuals worldwide and is responsible for approximately 900,000 deaths annually. Anti-HBV treatment with a nucleoside analog in combination with pegylated interferon are considered first-line therapy for patients with chronic HBV infection and liver inflammation. However, because cure rates are low, most patients will require lifetime treatment. HBV Capsid Assembly Modulators (CAMs) have emerged as a promising new class of compounds as they can affect levels of HBV covalently closed-circular DNA (cccDNA) associated with viral persistence. SAR studies around the core structure of lead HBV CAM GLP-26 (Fig. 1B) was performed and led to the discovery of non-toxic compound 10a displaying sub-nanomolar anti-HBV activity. Advanced toxicity and cellular pharmacology profiles of compounds 10a were also established and the results are discussed herein.

Design, Synthesis, and Biological Evaluation of Novel Thioureidobenzamide (TBA) Derivatives as HBV Capsid Assembly Modulators.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a promising therapeutic approach for the treatment of HBV infection. In this study, we designed and synthesized five series of benzamide derivatives based on a multisite-binding strategy at the tolerant region and diversity modification in the solvent-exposed region. Among them, thioureidobenzamide compound 17i exhibited significantly increased anti-HBV activity in HepAD38 (EC50 = 0.012 μM) and HBV-infected HLCZ01 cells (EC50 = 0.033 μM). Moreover, 17i displayed a better inhibitory effect on the assembly of HBV capsid protein compared with NVR 3-778 and a inhibitory effect similar to the clinical drug GLS4. In addition, 17i showed moderate metabolic stability in human microsomes, had excellent oral bioavailability in Sprague-Dawley (SD) rats, and inhibited HBV replication in the HBV carrier mice model, which could be considered as a promising candidate drug for further development.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Pharmacophore-Based Virtual Screening and Structural Modification of Novel Benzamide Derivatives as HBV Capsid Assembly Modulators.

Hepatitis B virus (HBV) infection is the most common cause of death from liver disease worldwide. The use of capsid assembly modulators is considered a prominent strategy for the development of novel anti-HBV therapies. We performed a pharmacophore-based virtual screening strategy, and a benzamide scaffold hit, WAI-5, was chosen for further structural optimization. A series of novel HBV capsid assembly modulators (CAMs) were found. Compared with the lead hit, the representative compounds 11g and 11n exhibited a 10-fold increase in anti-HBV activity with 50% effective concentration (EC50) values of 1.74 and 1.90 µM, respectively.

Identification of novel tetrahydroquinoxaline derived phenyl ureas as modulators of the hepatitis B virus nucleocapsid assembly

A key step of hepatitis B virus (HBV) replication is the selective packaging of pregenomic RNA (pgRNA) by core protein (Cp) dimers, forming a nucleocapsid where the reverse transcriptional viral DNA replication takes place. One approach in the development of new anti-HBV drugs is to disrupt the assembly of HBV nucleocapsids by misdirecting Cp dimers to assemble morphologically normal capsids devoid of pgRNA. In this study, we built upon our previous discovery of benzamide-derived HBV capsid assembly modulators by exploring fused bicyclic scaffolds with an exocyclic amide that is β, γ to the fused ring, and identified 1,2,3,4-tetrahydroquinoxaline derived phenyl ureas as a novel scaffold. Structure-activity relationship studies showed that a favorable hydrophobic substitution can be tolerated at the 2-position of the 1,2,3,4-tetrahydroquinoxaline core, and the resulting compound 88 demonstrated comparable or improved antiviral potencies in mouse and human hepatocyte-derived HBV-replicating cell lines compared to our previously reported benzamide compound, 38017 (8). In addition, a novel bis-urea series based on 1,2,3,4-tetrahydroquinoxaline was also found to inhibit HBV DNA replication with sub-micromolar EC50 values. The mode of action of these compounds is consistent with specific inhibition of pgRNA encapsidation into nucleocapsids in hepatocytes.

Energetics and Kinetic Assembly Pathways of Hepatitis B Virus Capsids in the Presence of Antivirals.

Capsid assembly modulators (CAMs) are antiviral molecules that disturb the formation of icosahedral viral capsids, in particular, those of the Hepatitis B virus (HBV). We report an integrated, physics-driven study elucidating quantitatively the effects of two classes of CAMs on the HBV capsid assembly. Time-resolved small-angle X-ray scattering measurements revealed accelerated self-assembly processes that implied the increase of subunit binding energy from 9- up to 18-fold the thermal energy due to CAMs. Cryotransmission electron microscopy images showed that both classes induce various changes in capsid morphology: from a slight elongation, unrecognized in previous work, to a strong deformation with a capsid size more than twice as large. The observed capsid morphologies were closely reproduced in coarse-grained simulations by varying the Föppl-von-Kármán number, thus pointing out the role of CAMs in altering the capsid elastic energy. Our results illuminate the mechanisms of action of CAMs on HBV capsid assembly at high spatiotemporal resolution and may bring perspectives on virus-derived nanocapsules with tunable morphologies.

Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division.

Hepatitis B virus (HBV) capsid assembly is traditionally thought to occur predominantly in the cytoplasm, where the virus gains access to the virion egress pathway. To better define sites of HBV capsid assembly, we carried out single cell imaging of HBV Core protein (Cp) subcellular trafficking over time under conditions supporting genome packaging and reverse transcription in Huh7 hepatocellular carcinoma cells. Time-course analyses including live cell imaging of fluorescently tagged Cp derivatives showed Cp to accumulate in the nucleus at early time points (~24 h), followed by a marked re-distribution to the cytoplasm at 48 to 72 h. Nucleus-associated Cp was confirmed to be capsid and/or high-order assemblages using a novel dual label immunofluorescence strategy. Nuclear-to-cytoplasmic re-localization of Cp occurred predominantly during nuclear envelope breakdown in conjunction with cell division, followed by strong cytoplasmic retention of Cp. Blocking cell division resulted in strong nuclear entrapment of high-order assemblages. A Cp mutant, Cp-V124W, predicted to exhibit enhanced assembly kinetics, also first trafficked to the nucleus to accumulate at nucleoli, consistent with the hypothesis that Cp's transit to the nucleus is a strong and constitutive process. Taken together, these results provide support for the nucleus as an early-stage site of HBV capsid assembly, and provide the first dynamic evidence of cytoplasmic retention after cell division as a mechanism underpinning capsid nucleus-to-cytoplasm relocalization. IMPORTANCE Hepatitis B virus (HBV) is an enveloped, reverse-transcribing DNA virus that is a major cause of liver disease and hepatocellular carcinoma. Subcellular trafficking events underpinning HBV capsid assembly and virion egress remain poorly characterized. Here, we developed a combination of fixed and long-term (>24 h) live cell imaging technologies to study the single cell trafficking dynamics of the HBV Core Protein (Cp). We demonstrate that Cp first accumulates in the nucleus, and forms high-order structures consistent with capsids, with the predominant route of nuclear egress being relocalization to the cytoplasm during cell division in conjunction with nuclear membrane breakdown. Single cell video microscopy demonstrated unequivocally that Cp's localization to the nucleus is constitutive. This study represents a pioneering application of live cell imaging to study HBV subcellular transport, and demonstrates links between HBV Cp and the cell cycle.

Development of novel HBV capsid assembly modifiers through molecular dynamics, docking, and experiments.

Hepatitis B Virus (HBV) is the leading cause of liver complications, including cirrhosis, hepatocellular carcinoma, and liver failure. Interfering with the self-assembly process of virus nucleocapsid is a promising approach for the development of the novel antiviral agents. Starting from microsecond-scale molecular dynamic simulations of a tetramer of the HBV capsid protein Cp149, we identified three novel conformations for docking based on their pocket volume and principal component analysis of inter-dimer motions. Compounds were selected from the DivV set of NCI and ZINC0.9 databases for experimental testing according to their docking scores for the selected pocket. Combining molecular dynamics simulations and in vitro testing, we discovered several novel compounds with moderate activity. Additionally, transmission electron microscopy and capsid assembly experiments suggest that our compounds possess a novel mechanism for assembly modulation, potentially opening new avenues for HBV inhibition.

Randomised phase 2 study (JADE) of the HBV capsid assembly modulator JNJ-56136379 with or without a nucleos(t)ide analogue in patients with chronic hepatitis B infection

ObjectiveWe present the final analysis results of the phase 2 JADE study (ClinicalTrials.gov Identifier: NCT03361956).Design232 patients with chronic hepatitis B (CHB) not currently treated at study start (NCT) at study...

Synthesis and evaluation of N-sulfonylpiperidine-3-carboxamide derivatives as capsid assembly modulators inhibiting HBV in vitro and in HBV-transgenic mice

The hepatitis B virus (HBV) capsid assembly modulators (CAMs) have been developed as effective anti-HBV agents in the treatment of chronic HBV infection by targeting the HBV core protein and inducing the formation of aberrant or morphologically normal capsid. However, some CAMs have been observed adverse events such as ALT flares and rash. Therefore, finding new CAMs is of great importance. In this report, we synthesized N-sulfonylpiperidine-3-carboxamides (SPCs) derivatives and evaluated their anti-HBV activities. Among the SPC derivatives, compound C-49 notably suppressed HBV replication in HepAD38, HepG2-HBV1.3 and HepG2-NTCP cells. Moreover, treatment with C-49 for 12 days exhibited potent anti-HBV activity (100 mg/kg; 2.42 log reduction of serum HBV DNA) in HBV-transgenic mice without apparent hepatotoxicity. Our findings provided a new SPC derivative as HBV capsid assembly modulator for developing safe and efficient anti-HBV therapy.

Discovery of carboxyl-containing heteroaryldihydropyrimidine derivatives as novel HBV capsid assembly modulators with significantly improved metabolic stability.

Interfering with the assembly of hepatitis B virus (HBV) capsid is a promising approach for treating chronic hepatitis B (CHB). In order to enhance the metabolic stability and reduce the strong hERG inhibitory effect of HBV capsid assembly modulator (CAM) GLS4, we rationally designed a series of carboxyl-containing heteroaryldihydropyrimidine (HAP) derivatives based on structural biology information combined with medicinal chemistry strategies. The results from biological evaluation demonstrated that compound 6a-25 (EC50 = 0.020 μM) exhibited greater potency than the positive drug lamivudine (EC50 = 0.09 μM), and was comparable to the lead compound GLS4 (EC50 = 0.007 μM). Furthermore, it was observed that 6a-25 reduced levels of core protein (Cp) and capsid in cells. Preliminary assessment of drug-likeness revealed that 6a-25 exhibited superior water solubility (pH 2.0: 374.81 μg mL-1; pH 7.0: 6.85 μg mL-1; pH 7.4: 25.48 μg mL-1), liver microsomal metabolic stability (t1/2 = 108.2 min), and lower hERG toxicity (10 μM inhibition rate was 72.66%) compared to the lead compound GLS4. Overall, compound 6a-25 holds promise for further investigation.

Design, synthesis, and biological evaluation of novel sulfamoylbenzamide derivatives as HBV capsid assembly modulators

Capsid assembly modulators (CAMs) represent a novel class of antiviral agents targeting hepatitis B virus (HBV) capsid to disrupt the assembly process. NVR 3-778 is the first CAM to demonstrate antiviral activity in patients infected with HBV. However, the relatively low aqueous solubility and moderate activity in the human body halted further development of NVR 3-778. To improve the anti-HBV activity and the drug-like properties of NVR 3-778, we designed and synthesized a series of NVR 3-778 derivatives. Notably, phenylboronic acid-bearing compound 7b (EC50 = 0.83 ± 0.33 µM, CC50 = 19.4 ± 5.0 µM) displayed comparable anti-HBV activity to NVR 3-778 (EC50 = 0.73 ± 0.20 µM, CC50 = 23.4 ± 7.0 µM). Besides, 7b showed improved water solubility (328.8 µg/mL, pH 7) compared to NVR 3-778 (35.8 µg/mL, pH 7). Size exclusion chromatography (SEC) and quantification of encapsidated viral RNA were used to demonstrate that 7b behaves as a class II CAM similar to NVR 3-778. Moreover, molecular dynamics (MD) simulations were conducted to rationalize the structure–activity relationships (SARs) of these novel derivatives and to understand their key interactions with the binding pocket, which provide useful indications for guiding the further rational design of more effective anti-HBV drugs.

Multiscale Modeling of Hepatitis B Virus Capsid Assembly and Its Dimorphism.

Hepatitis B virus (HBV) is an endemic, chronic virus that leads to 800000 deaths per year. Central to the HBV lifecycle, the viral core has a protein capsid assembled from many copies of a single protein. The capsid protein adopts different (quasi-equivalent) conformations to form icosahedral capsids containing 180 or 240 proteins: T = 3 or T = 4, respectively, in Caspar-Klug nomenclature. HBV capsid assembly has become an important target for recently developed antivirals; nonetheless, the assembly pathways and mechanisms that control HBV dimorphism remain unclear. We describe computer simulations of the HBV assembly, using a coarse-grained model that has parameters learned from all-atom molecular dynamics simulations of a complete HBV capsid and yet is computationally tractable. Dynamical simulations with the resulting model reproduce experimental observations of HBV assembly pathways and products. By constructing Markov state models and employing transition path theory, we identify pathways leading to T = 3, T = 4, and other experimentally observed capsid morphologies. The analysis shows that capsid polymorphism is promoted by the low HBV capsid bending modulus, where the key factors controlling polymorphism are the conformational energy landscape and protein-protein binding affinities.

Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model.

Capsid assembly modulators (CAMs) have emerged as a promising class of antiviral agents. We studied the effects of twenty-one newly designed and synthesized CAMs including heteroaryldihydropyrimidine compounds (HAPs), their analogs and standard compounds on hepatitis B virus (HBV) capsid assembly. Cytoplasmic expression of the HBV core (HBc) gene driven by the exogenously delivered recombinant alphavirus RNA replicon was used for high level production of the full-length HBc protein in mammalian cells. HBV capsid assembly was assessed by native agarose gel immunoblot analysis, electron microscopy and inhibition of virion secretion in HepG2.2.15 HBV producing cell line. Induced fit docking simulation was applied for modelling the structural relationships of the synthesized compounds and HBc. The most efficient were the HAP class compounds—dihydropyrimidine 5-carboxylic acid n-alkoxyalkyl esters, which induced the formation of incorrectly assembled capsid products and their accumulation within the cells. HBc product accumulation in the cells was not detected with the reference HAP compound Bay 41-4109, suggesting different modes of action. A significant antiviral effect and substantially reduced toxicity were revealed for two of the synthesized compounds. Two new HAP compounds revealed a significant antiviral effect and a favorable toxicity profile that allows these compounds to be considered promising leads and drug candidates for the treatment of HBV infection. The established alphavirus based HBc expression approach allows for the specific selection of capsid assembly modulators directly in the natural cell environment.

JNJ-73763989 pharmacokinetics and safety: Liver-targeted siRNAs against hepatitis B virus, in Japanese and non-Japanese healthy adults, and combined with JNJ-56136379 and a nucleos(t)ide analogue in patients with chronic hepatitis B.

JNJ-73763989 comprises two hepatitis B virus (HBV)-specific, liver-targeted N-galactosamine-conjugated short interfering RNA triggers, JNJ-73763976 and JNJ-73763924. JNJ-73763989 pharmacokinetics, safety and tolerability were assessed in two phase 1 studies: Japanese (NCT04002752), and non-Japanese healthy participants and chronic hepatitis B (CHB) patients also receiving the HBV capsid assembly modulator JNJ-56136379 and a nucleos(t)ide analogue (NA) (NCT03365947). Healthy participant cohorts were double-blind and randomized to receive a single subcutaneous JNJ-73763989 dose (non-Japanese participants, 35, 100, 200, 300 or 400mg; Japanese participants, 25, 100 or 200mg) or placebo. JNJ-73763976 and JNJ-73763924 plasma concentrations were assessed over 48h. CHB patients received JNJ-73763989 200mg every 4weeks plus daily oral JNJ-56136379 250mg and NA in an open-label fashion. Safety and tolerability were assessed through Day 28 (healthy participants) or Day 112 (patients). Thirty non-Japanese (n = 4/dose; placebo, n = 10) and 24 Japanese healthy participants (n = 6/dose; placebo, n = 6) were randomized. JNJ-73763976 and JNJ-73763924 exposure generally increased in a dose-proportional manner. Mean plasma half-life was 4-9h. No differences between pharmacokinetic parameters were apparent between non-Japanese and Japanese healthy participants. In the 12 CHB patients, mean JNJ-73763976, JNJ-73763924 and JNJ-56136379 plasma concentrations 2h post-dose on Day 29 were 663, 269 and 14,718ng/mL, respectively. In both studies, all adverse events were mild/moderate. JNJ-73763976 and JNJ-73763924 had short plasma half-lives and exposure generally increased in a dose-proportional manner; there were no pharmacokinetic differences between Japanese and non-Japanese healthy adults. JNJ-73763989 with or without JNJ-56136379 and NA was generally safe and well tolerated.

Discovery of SHR5133, a Highly Potent and Novel HBV Capsid Assembly Modulator.

Capsid assembly modulators (CpAMs) represent a new class of antivirals targeting hepatitis B virus (HBV) core protein to disrupt the assembly process. In this work, a novel chemotype featuring a fused heterocycle amide was discovered through pharmacophore exploration. Lead optimization resulted in compound 8 with an EC50 value of 511 nM, and then methyl substitution on the piperazine was found to improve the in vitro potency remarkably. Further SAR studies established the key compound SHR5133, which showed high in vitro antiviral potency, favorable pharmacokinetic profiles across species, and robust in vivo efficacy.

An in silico study to unveil potential drugs and vaccine chimera for HBV capsid assembly protein: combined molecular docking and dynamics simulation approach.

Humans are a major reservoir of the hepatitis B virus (HBV), therefore promising treatment and control vaccination strategies are needed to eradicate the virus. Though promising drugs and vaccines are available against HBV, still efforts are required to enrich the therapy options. Herein, the HBV assembly protein was explored to identify novel targets for future use against HBV. Computer-aided drug designing and immune-informatics techniques were employed for the identification of putative inhibitors and vaccine ensemble against HBV using capsid assembly protein. The identified drug molecule binds with high affinity to the active pocket of the protein, and several epitopes are scanned in the protein sequence. The drug molecule, besides being a good putative inhibitor, has acceptable drug-like properties. A multi-epitope vaccine is also constructed to overcome the limitations of weakly immunogenic epitopes. In contrast to the MHC II level, the set of predicted epitopes has been recognized to interact with significant numbers of HLA alleles of MHC I. Selected epitopes are extremely virulent, antigenic, nontoxic, nonallergic, have suitable affinity to bind with the prevailing DRB*0101 allele, and also spectacle 86% mediocre population coverage. A multi-epitope peptide-based vaccine chimera having 73 amino acids was designed. It emerged as substantially immunogenic, thermally stable, robust in producing cellular as well as humoral immune responses, and had competent physicochemical properties to analyze in vitro and in vivo studies. The capsid assembly protein is a in more stable nature in the presence of the drug molecule compared to the TLR3 receptor in the vaccine presence. These particulars were confirmed by exposing the docked molecules to absolute and relative binding free energy approaches of MMGBSA/PBSA. The purpose to investigate the interactions between the vaccine and a representative TLR3 immune receptor can reveal the intermolecular affinity and possible presentation mechanism of the vaccine by TLR3 to the host immune system. It was revealed that the vaccine is showing a very good affinity of binding for the TLR3 and forming a network of hydrophobic and hydrophilic interactions. Overall, the findings of this study are promising and might be useful for further experimental validations.

Influence of assembly modulators on the assembly kinetics and structure of hepatitis b virus capsid

Hepatitis B virus (HBV) is an enveloped virus with an icosahedral nucleocapsid. The constituent of the capsid is the Core protein, whose 149 N-terminal residues (Cp149) can self-assemble in vivo but also in vitro into capsids containing mainly 120 dimers. CpAMs (for Capsid Assembly Modulators), which are new antiviral molecules currently in clinical trials, can disrupt HBV capsid assembly. They can either lead to the formation of seemingly normal icosahedral capsids (class I CpAMs, e.g., JNJ-632), or to the formation of aberrant (larger and malformed) assemblies (class II CpAMs, e.g., BAY 41-4109).

4-Oxooctahydroquinoline-1(2H)-carboxamides as hepatitis B virus (HBV) capsid core protein assembly modulators

Hepatitis B virus (HBV) core protein, the building block of the HBV capsid, plays multiple roles in viral replication, and is an attractive target for development of antiviral agents with a new mechanism of action. In addition to the heteroaryldihydropyrimidines (HAPs), sulfamoylbenzamides (SBAs), dibenzothiazepine derivatives (DBTs), and sulfamoylpyrrolamides (SPAs) that inhibit HBV replication by modulation of viral capsid assembly and are currently under clinical trials for the treatment of chronic hepatitis B (CHB), other chemical structures with activity to modulate HBV capsid assembly have also been explored. Here we describe our continued optimization of a benzamide originating from our high throughput screening. A new bicyclic carboxamide lead featuring an electron deficient non-planar core structure was discovered. Evaluations of its ADMET (absorption, distribution, metabolism, excretion and toxicity) and pharmacokinetic (PK) profiles demonstrate improved metabolic stability and good bioavailability.