Towards an HBV Cure
Towards an HBV Cure
To broaden the therapeutic landscape in chronic hepatitis B management and to ultimately achieve a reliable HBV cure, novel antivirals with original mechanisms of action are needed. Drug development thus focuses on strategies targeting cccDNA either by preventing cccDNA formation, eliminating cccDNA or silencing cccDNA transcription. Control of cccDNA should be achievable by either capsid disassembly, inhibition of rcDNA entry into the nucleus, inhibition of conversion of rcDNA to cccDNA, physical elimination of cccDNA, inhibition of cccDNA transcription (epigenetic control) or inhibition of viral or cellular factors contributing to cccDNA stability/formation. Drugs with such activity could be DAAs targeting the virus or host-targeting agents (HTAs), including inhibitors of key host factors required for the viral replication cycle and immunomodulatory agents (Table 4).
Among the emerging DAAs against HBV currently in the pipeline are novel polymerase inhibitors, capsid inhibitors, rcDNA–cccDNA conversion inhibitors, DNA cleavage enzymes and small interfering RNA (siRNA)-based strategies. While the prospects of novel polymerase inhibitors remain to be determined, capsid inhibitors, inhibitors of cccDNA formation and DNA cleavage enzymes have great potential as novel antiviral strategies that could directly impact cccDNA pools. The recent achievement to produce recombinant HBV polymerase at a large scale may be instrumental to design novel improved polymerase inhibitors. However, these novel inhibitors, whether they inhibit the priming, the RNA-dependent or DNA-dependent DNA synthesis, or the RNAseH activity of the polymerase, will have to show significant advantage over the existing nucleos(t)ide analogues with a high antiviral potency and a high barrier to resistance. The HBV capsid plays a central role in the viral life cycle. It is essential for HBV genome packaging, reverse transcription, intracellular trafficking and maintenance of chronic infection as encapsidated HBV genomes are imported into the nucleus. The first family of nucleocapsid inhibitors, phenylpropenamide derivatives, were shown to interfere with HBV RNA packaging, leading to the formation of empty capsids and reducing the amount of intracellular immature capsids, mature viral particles and intracellular cccDNA pools, without affecting HBcAg levels. Interestingly, a synergistic antiviral activity between phenylpropenamide derivatives and polymerase inhibitors, as well as a lack of cross-resistance, was reported in vitro, highlighting the potential of combining capsid inhibitors with other antivirals for therapy. Heteroaryldihydropyrimidines (HAPs) constitute another family of capsid inhibitors. HAPs were shown to bind to core particles in a specific but reversible manner and to reduce both HBV DNA and HBcAg levels, the latter due to degradation by the proteasome pathway. In addition to inducing capsid disassembly, HAPs have been shown to enhance viral assembly and to favour assembly of aberrant particles, indicating that HAPs interfere with capsid formation/stability in a complex manner. Similar to phenylpropenamide derivatives, HAPs are able to efficiently inhibit viral variants that are resistant to current polymerase inhibitors. Moreover, a short-term study using HBV-infected human liver-chimeric mice demonstrated an HAP-induced reduction of viral load, with virus production increasing again following discontinuation of the drug. Morphothiadine mesilate (GLS4) is the first member of this family of compounds having entered early clinical development; phase I and II clinical trials have been conducted in China. Furthermore, HBV capsid proteins can traffic to the nucleus of infected cells and exert additional biological functions by repressing the transcription of several IFN-stimulated genes or activating the transcription of viral genes from cccDNA; these nuclear functions may represent additional targets for drug development. Interestingly, it was also suggested that HAPs might directly affect cccDNA stability.
A complementary approach to the development of capsid inhibitors with direct impact on cccDNA pools is the design of enzymes targeting cccDNA formation or decay. Recently, a small-molecule library screen was conducted to uncover compounds inhibiting cccDNA synthesis. This led to the discovery of disubstituted sulfonamide (DSS) compounds as inhibitors of cccDNA in cell-based assays. DSS did not appear to directly promote the degradation of rcDNA or cccDNA but rather to inhibit de novo cccDNA formation by interfering with rcDNA conversion into cccDNA. Furthermore, DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases, TAL effector nucleases and CRISPR-associated system 9 proteins, specifically targeting the cccDNA are currently being engineered. These enzymes can be delivered as genes within viral vectors to target hepatocytes. Computational modelling studies suggested that several enzymes may have to be administered concomitantly in order to avoid selection of resistant viruses. Of note, enzyme-based strategies are currently also being evaluated in other viral infections, including HIV infection, where this approach is used in order to modify the viral receptors CCR5 and CXCR4 on T cells ex vivo (reviewed in ref.). Further studies are needed to evaluate the potential of these novel antiviral strategies against HBV infection. Noteworthy, cccDNA transcription can be silenced to some extent using small molecules targeting different classes of chromatin-modifying enzymes, similar to the epigenetic silencing of cccDNA by IFN-α, which would lead to functional, although transient, HBV cure.
Silencing HBV gene expression using RNAi constitutes another original antiviral approach against HBV. Polymer formulations enable efficient delivery of siRNA to hepatocytes. ARC-520 is a combination of siRNAs directed against conserved HBV RNA sequences and efficiently knocks down HBV RNA, proteins and DNA levels. ARC-520 is currently being evaluated in a phase II clinical trial (ClinicalTrials.gov identifier NCT02065336). Other siRNAs are also at the preclinical stage. Interestingly, HBV gene silencing could also be combined with IFN induction in the liver. Indeed, 5'-triphosphate (3p) siRNAs directed against HBV can bind and activate cytosolic helicase retinoic acid-inducible protein I to induce expression of type I IFNs. The antiviral activity of these bifunctional, HBV-specific, 3p-siRNAs was more efficient and sustained for a longer time than 3p-RNAs without silencing capacity or siRNAs that targeted identical sequences but did not contain 3p.
In addition to interfering with cccDNA formation and stability, future drugs aiming at curing HBV infection may target other host cell pathways to interfere with the viral replication cycle and/or restore anti-HBV immune responses (reviewed in ref.). The recent clinical development of HTAs for the treatment of chronic hepatitis C highlights the promise of this approach to address unmet needs in the treatment of virus-induced liver disease (reviewed in ref.). In contrast to HCV, few HTAs targeting the HBV replication cycle have been described. These include inhibitors of the recently uncovered HBV receptor NTCP and inhibitors of HBV envelope protein maturation and secretion (reviewed in refs.). Small-molecule compounds binding to NTCP, including cyclosporine A and ezetimibe, have been shown to inhibit HBV/HDV entry in cell culture models, and although licensed for other clinical settings, none of these compounds has so far been tested in vivo against HBV. In contrast, the well-known HBV pre-S1-derived lipopeptide Myrcludex-B that competes with HBV/HDV for binding to NTCP efficiently prevents HBV/HDV entry both in vitro and in human liver-chimeric uPA-SCID mice. Furthermore, this lipopeptide was also able to impair viral dissemination when administered subsequent to viral inoculation in this mouse model. Myrcludex-B is currently being evaluated in a phase II clinical trial in Russia.
The HBV secretory pathway is another potential target for novel antivirals, as inhibiting HBV secretion and budding should decrease the release of progeny subviral particles and virions. This could not only decrease HBV DNA levels but also interfere with HBsAg-mediated immunosuppression, thereby restoring antiviral immunity. Several inhibitors of HBV secretion have been described so far, including iminosugar derivatives of butyldeoxynojirimycin and related glycolipids, α-glucosidase inhibitors, triazol-o-pyrimidine derivatives and a benzimidazole compound. The benzimidazole BM601 has recently been reported to selectively inhibit intracellular re-localisation of the HBV surface protein to the Golgi apparatus. Thereby, it decreases HBsAg and HBV release without affecting HBeAg secretion or induces the release of cellular proteins with an original mechanism of action compared with previously described inhibitors of HBV maturation and secretion. Moreover, amphipathic DNA polymers such as phosphorothioate oligonucleotides have been shown to inhibit HBsAg release, thereby contributing to immunological control of HBV infection. Noteworthy, such compounds exhibit broad antiviral activities and are also evaluated as HIV and HCV fusion inhibitors (reviewed in ref.). As potential disadvantages, HBsAg accumulation could lead to storage diseases and the block of mature virion synthesis could increase cccDNA copy number.
In order to circumvent the systemic side effects of IFN-α that limit its clinical use, efforts are ongoing to uncover other means of inducing intrahepatic antiviral immune responses in the infected host. Most recently, an original mechanism to activate antiviral immune responses has been described using antibodies directed against the lymphotoxin-β receptor (LTβR). Similarly to members of the IFN or TNF family of cytokines, antibodies activating LTβR reduced HBV DNA, HBsAg and cccDNA levels in relevant cell culture models in the absence of detectable hepatotoxicity. Antibody-mediated activation of LTβR appeared to have a dual mechanism of action, targeting both HBV replication and cccDNA stability via induction of deamination and apurinic/apyrimidinic site formation in cccDNA and upregulation of the expression of nuclear APOBEC3 deaminases. This approach may however imply a significant problem as the increased mutation rate may support generation of resistant variants as it was shown for HIV. The potential synergistic effect of combinations of LTβR agonists and current polymerase inhibitors remains to be assessed.
Other immunomodulatory compounds exhibiting activity against HBV in clinical development include TLR7 agonists (ClinicalTrials.gov identifier NCT02166047), thymosin α1 (ClinicalTrials.gov identifier NCT00291616) and nitazoxanide, which induce the production of IFN and/or activation of B and T cells. Furthermore, recombinant IL-7 and IFN-λ have also been considered to enhance immune functions against HBV (ClinicalTrials.gov identifier NCT01027065 and NCT01204762). Interestingly, the immunomodulatory properties of some of these compounds have also been suggested to be of interest in treating other infectious diseases, including chronic HCV infection. However, it remains to be demonstrated whether they can clear viral infection. Finally, therapeutic vaccines designed to trigger both humoral and cellular immune responses against HBV are also currently being evaluated in clinical trials (ClinicalTrials.gov identifier NCT01943799, NCT01023230 and NCT00536627) and may be potentiated with the use of NUC and/or TLR-9 agonists that induce the formation of intrahepatic myeloic cell aggregates involved in T cell expansion and support HBV clearance by favouring local cytotoxic T lymphocyte expansion, at least in mouse models of HBV infection.
Drug Discovery: Towards an HBV Cure
To broaden the therapeutic landscape in chronic hepatitis B management and to ultimately achieve a reliable HBV cure, novel antivirals with original mechanisms of action are needed. Drug development thus focuses on strategies targeting cccDNA either by preventing cccDNA formation, eliminating cccDNA or silencing cccDNA transcription. Control of cccDNA should be achievable by either capsid disassembly, inhibition of rcDNA entry into the nucleus, inhibition of conversion of rcDNA to cccDNA, physical elimination of cccDNA, inhibition of cccDNA transcription (epigenetic control) or inhibition of viral or cellular factors contributing to cccDNA stability/formation. Drugs with such activity could be DAAs targeting the virus or host-targeting agents (HTAs), including inhibitors of key host factors required for the viral replication cycle and immunomodulatory agents (Table 4).
Among the emerging DAAs against HBV currently in the pipeline are novel polymerase inhibitors, capsid inhibitors, rcDNA–cccDNA conversion inhibitors, DNA cleavage enzymes and small interfering RNA (siRNA)-based strategies. While the prospects of novel polymerase inhibitors remain to be determined, capsid inhibitors, inhibitors of cccDNA formation and DNA cleavage enzymes have great potential as novel antiviral strategies that could directly impact cccDNA pools. The recent achievement to produce recombinant HBV polymerase at a large scale may be instrumental to design novel improved polymerase inhibitors. However, these novel inhibitors, whether they inhibit the priming, the RNA-dependent or DNA-dependent DNA synthesis, or the RNAseH activity of the polymerase, will have to show significant advantage over the existing nucleos(t)ide analogues with a high antiviral potency and a high barrier to resistance. The HBV capsid plays a central role in the viral life cycle. It is essential for HBV genome packaging, reverse transcription, intracellular trafficking and maintenance of chronic infection as encapsidated HBV genomes are imported into the nucleus. The first family of nucleocapsid inhibitors, phenylpropenamide derivatives, were shown to interfere with HBV RNA packaging, leading to the formation of empty capsids and reducing the amount of intracellular immature capsids, mature viral particles and intracellular cccDNA pools, without affecting HBcAg levels. Interestingly, a synergistic antiviral activity between phenylpropenamide derivatives and polymerase inhibitors, as well as a lack of cross-resistance, was reported in vitro, highlighting the potential of combining capsid inhibitors with other antivirals for therapy. Heteroaryldihydropyrimidines (HAPs) constitute another family of capsid inhibitors. HAPs were shown to bind to core particles in a specific but reversible manner and to reduce both HBV DNA and HBcAg levels, the latter due to degradation by the proteasome pathway. In addition to inducing capsid disassembly, HAPs have been shown to enhance viral assembly and to favour assembly of aberrant particles, indicating that HAPs interfere with capsid formation/stability in a complex manner. Similar to phenylpropenamide derivatives, HAPs are able to efficiently inhibit viral variants that are resistant to current polymerase inhibitors. Moreover, a short-term study using HBV-infected human liver-chimeric mice demonstrated an HAP-induced reduction of viral load, with virus production increasing again following discontinuation of the drug. Morphothiadine mesilate (GLS4) is the first member of this family of compounds having entered early clinical development; phase I and II clinical trials have been conducted in China. Furthermore, HBV capsid proteins can traffic to the nucleus of infected cells and exert additional biological functions by repressing the transcription of several IFN-stimulated genes or activating the transcription of viral genes from cccDNA; these nuclear functions may represent additional targets for drug development. Interestingly, it was also suggested that HAPs might directly affect cccDNA stability.
A complementary approach to the development of capsid inhibitors with direct impact on cccDNA pools is the design of enzymes targeting cccDNA formation or decay. Recently, a small-molecule library screen was conducted to uncover compounds inhibiting cccDNA synthesis. This led to the discovery of disubstituted sulfonamide (DSS) compounds as inhibitors of cccDNA in cell-based assays. DSS did not appear to directly promote the degradation of rcDNA or cccDNA but rather to inhibit de novo cccDNA formation by interfering with rcDNA conversion into cccDNA. Furthermore, DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases, TAL effector nucleases and CRISPR-associated system 9 proteins, specifically targeting the cccDNA are currently being engineered. These enzymes can be delivered as genes within viral vectors to target hepatocytes. Computational modelling studies suggested that several enzymes may have to be administered concomitantly in order to avoid selection of resistant viruses. Of note, enzyme-based strategies are currently also being evaluated in other viral infections, including HIV infection, where this approach is used in order to modify the viral receptors CCR5 and CXCR4 on T cells ex vivo (reviewed in ref.). Further studies are needed to evaluate the potential of these novel antiviral strategies against HBV infection. Noteworthy, cccDNA transcription can be silenced to some extent using small molecules targeting different classes of chromatin-modifying enzymes, similar to the epigenetic silencing of cccDNA by IFN-α, which would lead to functional, although transient, HBV cure.
Silencing HBV gene expression using RNAi constitutes another original antiviral approach against HBV. Polymer formulations enable efficient delivery of siRNA to hepatocytes. ARC-520 is a combination of siRNAs directed against conserved HBV RNA sequences and efficiently knocks down HBV RNA, proteins and DNA levels. ARC-520 is currently being evaluated in a phase II clinical trial (ClinicalTrials.gov identifier NCT02065336). Other siRNAs are also at the preclinical stage. Interestingly, HBV gene silencing could also be combined with IFN induction in the liver. Indeed, 5'-triphosphate (3p) siRNAs directed against HBV can bind and activate cytosolic helicase retinoic acid-inducible protein I to induce expression of type I IFNs. The antiviral activity of these bifunctional, HBV-specific, 3p-siRNAs was more efficient and sustained for a longer time than 3p-RNAs without silencing capacity or siRNAs that targeted identical sequences but did not contain 3p.
In addition to interfering with cccDNA formation and stability, future drugs aiming at curing HBV infection may target other host cell pathways to interfere with the viral replication cycle and/or restore anti-HBV immune responses (reviewed in ref.). The recent clinical development of HTAs for the treatment of chronic hepatitis C highlights the promise of this approach to address unmet needs in the treatment of virus-induced liver disease (reviewed in ref.). In contrast to HCV, few HTAs targeting the HBV replication cycle have been described. These include inhibitors of the recently uncovered HBV receptor NTCP and inhibitors of HBV envelope protein maturation and secretion (reviewed in refs.). Small-molecule compounds binding to NTCP, including cyclosporine A and ezetimibe, have been shown to inhibit HBV/HDV entry in cell culture models, and although licensed for other clinical settings, none of these compounds has so far been tested in vivo against HBV. In contrast, the well-known HBV pre-S1-derived lipopeptide Myrcludex-B that competes with HBV/HDV for binding to NTCP efficiently prevents HBV/HDV entry both in vitro and in human liver-chimeric uPA-SCID mice. Furthermore, this lipopeptide was also able to impair viral dissemination when administered subsequent to viral inoculation in this mouse model. Myrcludex-B is currently being evaluated in a phase II clinical trial in Russia.
The HBV secretory pathway is another potential target for novel antivirals, as inhibiting HBV secretion and budding should decrease the release of progeny subviral particles and virions. This could not only decrease HBV DNA levels but also interfere with HBsAg-mediated immunosuppression, thereby restoring antiviral immunity. Several inhibitors of HBV secretion have been described so far, including iminosugar derivatives of butyldeoxynojirimycin and related glycolipids, α-glucosidase inhibitors, triazol-o-pyrimidine derivatives and a benzimidazole compound. The benzimidazole BM601 has recently been reported to selectively inhibit intracellular re-localisation of the HBV surface protein to the Golgi apparatus. Thereby, it decreases HBsAg and HBV release without affecting HBeAg secretion or induces the release of cellular proteins with an original mechanism of action compared with previously described inhibitors of HBV maturation and secretion. Moreover, amphipathic DNA polymers such as phosphorothioate oligonucleotides have been shown to inhibit HBsAg release, thereby contributing to immunological control of HBV infection. Noteworthy, such compounds exhibit broad antiviral activities and are also evaluated as HIV and HCV fusion inhibitors (reviewed in ref.). As potential disadvantages, HBsAg accumulation could lead to storage diseases and the block of mature virion synthesis could increase cccDNA copy number.
In order to circumvent the systemic side effects of IFN-α that limit its clinical use, efforts are ongoing to uncover other means of inducing intrahepatic antiviral immune responses in the infected host. Most recently, an original mechanism to activate antiviral immune responses has been described using antibodies directed against the lymphotoxin-β receptor (LTβR). Similarly to members of the IFN or TNF family of cytokines, antibodies activating LTβR reduced HBV DNA, HBsAg and cccDNA levels in relevant cell culture models in the absence of detectable hepatotoxicity. Antibody-mediated activation of LTβR appeared to have a dual mechanism of action, targeting both HBV replication and cccDNA stability via induction of deamination and apurinic/apyrimidinic site formation in cccDNA and upregulation of the expression of nuclear APOBEC3 deaminases. This approach may however imply a significant problem as the increased mutation rate may support generation of resistant variants as it was shown for HIV. The potential synergistic effect of combinations of LTβR agonists and current polymerase inhibitors remains to be assessed.
Other immunomodulatory compounds exhibiting activity against HBV in clinical development include TLR7 agonists (ClinicalTrials.gov identifier NCT02166047), thymosin α1 (ClinicalTrials.gov identifier NCT00291616) and nitazoxanide, which induce the production of IFN and/or activation of B and T cells. Furthermore, recombinant IL-7 and IFN-λ have also been considered to enhance immune functions against HBV (ClinicalTrials.gov identifier NCT01027065 and NCT01204762). Interestingly, the immunomodulatory properties of some of these compounds have also been suggested to be of interest in treating other infectious diseases, including chronic HCV infection. However, it remains to be demonstrated whether they can clear viral infection. Finally, therapeutic vaccines designed to trigger both humoral and cellular immune responses against HBV are also currently being evaluated in clinical trials (ClinicalTrials.gov identifier NCT01943799, NCT01023230 and NCT00536627) and may be potentiated with the use of NUC and/or TLR-9 agonists that induce the formation of intrahepatic myeloic cell aggregates involved in T cell expansion and support HBV clearance by favouring local cytotoxic T lymphocyte expansion, at least in mouse models of HBV infection.
