Current treatments of chronic hepatitis B are limited to IFN therapy and nucleos(t)ide analogue reverse transcriptase inhibitors, all of which are used for prolonged periods but cure the infection in only a small minority of cases and display no benefit when used in combination (16
). The underlying cause of HBV chronicity is the intracellular persistence of a multicopy episomal form of the virus genome, cccDNA, which is inherently stable in the nuclei of infected hepatocytes and is only indirectly affected by current therapies (25
). In order to clear the infection, a durable, curative antiviral therapy that directly reduces the level of HBV cccDNA without killing infected hepatocytes is needed.
It is generally acknowledged that cccDNA formation and persistence involve many viral and cellular factors, and there are likely to be many molecular opportunities for intervention, ranging from biosynthesis to maintenance of cccDNA. The putative approaches to cccDNA inhibition include, but are not limited to, the following. The first is inhibition of cccDNA establishment. Such an inhibitor would work by abrogating any of the several steps that lead from mature cytoplasmic rcDNA to the presence of cccDNA in the nucleus (12
). The second is inhibition of cccDNA maintenance factors. Although it is still unclear whether a cccDNA-specific maintenance factor(s) exists, a small molecule may specifically recognize and alter the stability of the cccDNA minichromosome. The third is chemical alteration of cccDNA. Compared to the host chromatin, cccDNA should exhibit increased sensitivity to DNA damage due to the very dense coding of the HBV genome (30
). The fourth is epigenetic silencing of cccDNA transcriptional activity (32
). The fifth is induction of the intracellular innate defense. It has been demonstrated that clearance of cccDNA during acute infection can occur through a noncytolytic mechanism that is largely independent of adaptive immune function (9
), indicating that cccDNA can be reduced by innate cellular defenses that may be activated by a biologically active stimulator or a small-molecule compound.
In this study, by employing a cell-based screening strategy that monitors the HBV cccDNA level through proportional expression of HBeAg, we discovered two structurally related sulfonamide compounds that significantly reduced the levels of HBV cccDNA in cell cultures. Further mechanistic studies revealed that the DSS compounds directly block the conversion of HBV rcDNA into cccDNA, rather than inhibiting the production of rcDNA as existing HBV drugs do. Therefore, we have provided the first proof-of-concept evidence that it is feasible to develop small-molecule inhibitors that prevent the formation of cccDNA from the rcDNA precursor, an essential but unexploited step in the HBV replication cycle.
The exact target(s) of DSS compounds is still unclear, considering that there are many molecular details yet to be elucidated in the understanding of cccDNA formation and metabolism. cccDNA is formed from both the incoming viral genome during initiation of infection and newly synthesized mature viral DNA, through conversion of the viral rcDNA genome, most possibly by employing the host DNA repair machinery in the nucleus (1
). In one attempt to identify the potential intermediate(s) bridging rcDNA-to-cccDNA conversion, a linear woodchuck hepatitis virus genome that contains a terminal duplication of the cohesive region (between DR1 and DR2) from rcDNA by displacement synthesis through the cohesive overlap was proposed to be a cccDNA precursor by serving as a substrate for DNA repair through legitimate recombination. The authors found that some integrated viral DNA appeared to have this linear DNA as a precursor; however, the extrachromosomal form of such hypothetic DNA species has not been directly detected and thus remains mysterious (44
). Our recent studies focusing on a DP-rcDNA molecule which loses the covalently bonded viral polymerase revealed that DP-rcDNA is a bona fide precursor of cccDNA formation, albeit at low efficiency (11
). In addition, a model of the molecular pathway of cccDNA formation has been proposed in which the completion of viral plus-strand DNA inside the nucleocapsid triggers the removal of viral polymerase from minus-strand DNA. The deproteinization reaction is tightly associated with a nucleocapsid structural shift, resulting in exposure of the NLS at the C terminus of capsid protein, which in turn initiates the karyopherin-mediated nuclear import of capsid/DP-rcDNA complexes. Finally, DP-rcDNA is released from the capsid in the nucleus and is converted into cccDNA presumably through DNA repair (11
). Interestingly, DSS compounds reduce DP-rcDNA, as well as cccDNA, without directly affecting viral DNA replication, thus providing another line of evidence that DP-rcDNA is a precursor of cccDNA formation. Nevertheless, it is still possible that DSS compounds may inhibit the production of another undiscovered cccDNA precursor(s) or DNA repair mechanism(s) involved in cccDNA formation.
The mechanisms underlying the deproteinization of hepadnavirus rcDNA remain elusive. The viral polymerase is covalently linked to the 5′ phosphate group of the rcDNA minus strand through a tyrosine residue (Y63 for HBV, Y96 for DHBV) in the terminal protein (TP) domain, as a consequence of TP-mediated protein priming during the initial reverse transcription of viral pgRNA (29
). It is conceivable that the removal of polymerase from rcDNA ought to be an essential step in cccDNA formation. Our previous work ruled out the possibility that the viral polymerase is removed by endonuclease cleavage of the sequences proximal to the linkage between the rcDNA terminus and polymerase, based upon the fact that the 5′ end of the DP-rcDNA minus strand is the same as the end of rcDNA (11
). Thus, the anticipated mechanisms of rcDNA deproteinization are narrowed down to the following. (i) The phosphodiester bond is hydrolyzed by a phosphodiesterase, one candidate being a recently discovered human 5′-tyrosyl DNA phosphodiesterase (TDP2) that removes topoisomerase covalently conjugated at the 5′ end of a chromosomal DNA break (3
). (ii) Polymerase is removed by proteolytic digestion, which results in a small peptide, or at least the tyrosine residue, being left on the DP-rcDNA. Therefore, it will be important to evaluate the potential phosphodiesterase or protease inhibitor activity of DSS compounds in the future.
The pool size of cccDNA ranges from 1 to 50 copies per cell, and once established, nascent cccDNA converted from progeny rcDNA, coupled with its stability within the infected hepatocyte, helps to maintain viral persistence. The basis of cccDNA longevity is poorly understood. The precise half-life of HBV cccDNA has been reported to range from days to months in different animal and tissue culture models (47
), and our results herein showed that the half-life of cccDNA was around 9 days in confluent HepDES19 cells under 3TC treatment (). Because cccDNA plays a central role in HBV persistence, elimination of cccDNA is the ultimate goal of antiviral therapy. Unfortunately, current antiviral treatment with nucleos(t)ide analogues fails to eliminate the preexisting cccDNA pool and/or prevent cccDNA formation from trace level wild-type or drug-resistant virus (48
). Although DSS compounds are unable to promote the degradation of the preexisting cccDNA in cell cultures, their mechanism of action is certainly distinct from that of the nucleos(t)ide analogues, and thus, they will likely retain activity in preventing cccDNA formation even when the virus has become resistant to polymerase inhibitors. In addition, DSS compounds may also exhibit synergistic effects in combination therapy with replication inhibitors. It is envisioned that the combination of inhibition of viral replication by nucleos(t)ide analogue with its amelioration in liver function (25
) and the inhibition of de novo
cccDNA formation by a DSS compound may enhance the eventual clearance of cccDNA. The antiviral effect of DSS compounds in combination with a nucleos(t)ide analogue and the potential inhibitory effect of DSS compounds against drug-resistant virus cccDNA formation will be tested in future studies.
We have confirmed that the DSS compounds, especially CCC-0975, also inhibited DHBV cccDNA formation in virus-infected PDHs. This observation indicates a wide spectrum for these compounds in hepadnavirus cccDNA intervention and also provides an opportunity to evaluate their antiviral activity (alone or in combination with nucleos[t]ide analogues) in the duck model. Other hepadnavirus animal models, including humanized uPA/SCID mice and woodchucks (4
), are also available for in vivo
testing of DSS compounds. First, however, the potency of the hit compounds needs to be improved. This work is under way through a SAR study. The structural features of these DSS compounds offer multiple modification opportunities for lead optimization. For example, with the common key sulfonamide moiety being maintained, both linkages and substituents will be structurally explored for SAR (e.g., electronic effect, steric effect, etc.) investigation and to improve antiviral profiles.
In summary, we have, for the first time, discovered two structurally related novel inhibitors of HBV cccDNA biosynthesis through an innovative screening approach. Distinct from those of current antiviral agents, the unique antiviral mechanism of DSS compounds is inhibition of the formation of cccDNA from its rcDNA precursor and may involve the inhibition of rcDNA deproteinization, a possible intermediate step during cccDNA formation. Thus, these two inhibitors also provide a promising research tool to identify a viral/host protein(s) involved in cccDNA formation. Clearance of cccDNA is the ultimate goal for the cure of hepatitis B, and it cannot be reliably accomplished by currently available therapeutics alone; the further development of DSS compounds may ultimately lead to drugs that change the landscape of hepatitis B management.