See Article, pages 522–534
Chronic HBV infection is a major cause of advanced liver disease and cancer world-wide, posing a global health challenge. While approved therapies efficiently control HBV viremia, viral elimination is rare. The HBV life cycle displays several unique features that enable the virus to persist in hepatocytes. By replicating its genome exclusively in the neo-formed nucleocapsids, HBV evades recognition by cellular antiviral immune responses. Moreover, HBV also establishes a stable episome in the nucleus of infected cells. This episome, called cccDNA (covalently closed circular DNA) has also been termed the “minichromosome”. Except for the lack of a replication origin, the HBV cccDNA molecule is indeed organized into a chromatin-like structure that displays a typical beads-on-a-string arrangement on electron microscopy.1 The HBV cccDNA serves as a template for transcription of all HBV RNAs by the cellular RNA polymerase II. Modulation of its transcriptional activity results in the recruitment of histones, histone-modifying enzymes and several cellular transcription factors.2, 3, 4, 5, 6 Even though long debated, the role of the viral protein HBx in the control of HBV transcription became clearer in the last decade (for review see7,8). Collectively, several different independent studies have shown that HBx is required for the initiation and maintenance of cccDNA-driven transcription of HBV RNAs.6,9, 10, 11 HBx most likely exerts this key role through different and complementary mechanisms: HBx promotes the degradation of the structural maintenance of chromosomes complex Smc5/6, which was recently identified as a host restriction factor that suppresses RNA transcription from cccDNA.12, 13, 14 It has also been shown that HBx is directly recruited to cccDNA. Absence of HBx results in decreased acetylation of cccDNA-bound histones, impaired recruitment of the transcriptional coactivator p300, and to recruitment of histone deacetylases3 and heterochromatin protein factor 1 (HP1).6 Furthermore, numerous studies (although often performed in less physiological overexpression systems) have reported an effect of HBx on the perturbation of host cellular pathways such as apoptosis, calcium signaling, and the cell cycle, which are associated with liver diseases and hepatocarcinogenesis (for review see8,15). Since HBx plays a central role in HBV infection and is most likely relevant for virus-induced liver disease, it is an interesting target for new therapies to treat chronic HBV infection. This concept is in line with other approaches aiming for “functional cure”, which is characterised by sustained loss of HBsAg (with or without anti-HBs seroconversion) and is associated with improved clinical outcomes.16 In order to identify compounds targeting HBx, Cheng et al.17 screened a library of small molecules for their ability to inhibit HBx expression. Using this approach, they identified dicoumarol, which dose-dependently reduces HBx protein expression without affecting the levels of HBx mRNAs. Among several properties, dicoumarol is a competitive inhibitor of NAD(P)H:quinone oxidoreductase (NQO1).18 Loss- and gain-of -function assays showed that NQO1 binds to HBx and promotes its stability by preventing its degradation by the proteasome in a ubiquitin-independent manner. Using state-of-the-art cell-based HBV infection models, Cheng et al.17 showed that both NQO1 knockdown as well as treatment with dicoumarol decrease the levels of all HBV parameters except for cccDNA. The antiviral effect of dicoumarol was confirmed in vivo in 2 different mouse models. From a mechanistic point of view, Cheng et al.17 showed that overexpression of NQO1, and the subsequent increase in the levels of HBx and its association with cccDNA, leads to increased HBV RNA transcription from cccDNA. Reciprocally, inhibition of NQO1 (by genetic or chemical approaches) decreases the association of HBx with cccDNA, increases repressive marks on cccDNA-associated histones and decreases the levels of HBV RNAs both in vitro and in vivo. Finally, Cheng et al. observed a partial rescue of the levels Smc5/Smc6 proteins in cells transfected with a vector expressing HBx and treated with dicoumarol. Why this was not observed in HBV-infected cells remains to be determined. Of note, the higher expression of NQO1 in HBV permissive cancer cell lines, such as HepG2, compared to normal hepatocytes19 may explain some of the discrepancies observed regarding the role of HBx and the intensity of HBV transcription/replication in different cell types.
According to Cheng et al.,17 the antiviral effect of dicoumarol seemed to be mainly due to its activity against NQO1, since genetic perturbation of NQO1 levels leads to drug-equivalent phenotypes against HBV. NQO1 is a protein with multiple protectives roles including some that extend beyond its catalytic function as a quinone reductase. Indeed, besides its antioxidant functions, NQO1 was shown to bind to selective proteins such as p53 and prevent their degradation by the proteasome (for review see20
). The data from Cheng et al.17 suggest that HBx benefits from this NQO1 “gatekeeper” role. Unfortunately, the detailed mechanisms explaining NQO1 selectivity regarding the protection of given proteins have not yet been elucidated. Collectively, increasing HBx degradation by decreasing NQO1 activity is an interesting option to block HBV transcription and thereby the production of HBV proteins, as well as to block HBx-induced host dysregulation that can lead to carcinogenesis.8 However, it should be considered that reduced NQO1 activity has been associated with a higher risk of cancer development.21 NQO1 activity was also shown to be increased in some forms of cancer, including liver cancer,19
and blockage of NQO1 by dicoumarol inhibits cell growth and seeding efficiency in soft agar of pancreatic cancer cells.22
In this regard, efforts have been made to identify other molecules that selectively inhibit NQO1 (for review see23 ), however the clinical translation of this approach for cancer prevention or treatment remains to be determined.
Beside its activity against NQO1, dicoumarol has several other pleiotropic effects. Originally dicoumarol was uncovered as a natural anticoagulant (based on its chemical similarity to vitamin K) and was in clinical use for decades as an oral drug to prevent thrombogenesis.18 Interestingly, dicoumarol also has been described to exert antimicrobial and antiviral activities.18
However, safety issues such as hemorrhage, anemia, neurotoxicity or periodontal toxicity18 have resulted in replacement of dicoumarol with next generation vitamin K antagonists. Even though Cheng et al.17 estimated that the human equivalent dose of dicoumarol required for anti-HBV properties may be in a range for therapeutic use as an anticoagulant, it remains to be determined if the drug would fulfil modern safety requirements for long-term treatment. Moreover, since dicoumarol targets the HBx protein and not the cccDNA itself, withdrawal of the treatment would likely lead to rebound of viral transcription and replication.
In summary, Cheng et al.17 provided new insights into the regulation of HBx by cellular factors as well as the proof of concept that targeting HBx by chemical compounds is a valuable antiviral strategy. While the clinical application of dicoumarol may be challenging in this setting, because of its safety profile, the identification of new molecules specifically targeting the interaction between HBx and NQO1, without fully inhibiting the physiological activities of NQO1, may provide a new approach to enlarge our armamentarium against HBV.作者: StephenW 时间: 2021-2-16 18:19