N-Hydroxypiridinedione: A Privileged Heterocycle for Targeting the HBV RNase H.

Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of N-hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg2+ ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC50 values in the low µM range (1.1-7.7 µM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure-activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents.

Exploration of 1-(indolin-1-yl)-2-(thiazol-2-yl)ethan-1-one derivatives as novel anti-HBV agent with potential TLR7-agonistic effect.

Hepatitis B virus (HBV) infection, as a serious global public health issue, is closely related to the immune dysfunction. Herein, thirty-seven 1-(indolin-1-yl)-2-(thiazol-4-yl)ethan-1-one derivatives were prepared as potential immunomodulatory anti-HBV agents. Anti-HBV activity evaluation confirmed compound 11a could significantly suppress the HBV DNA replication in both wild and resistant HBV stains, with IC50 values of 0.13 µM and 0.36 µM, respectively. Preliminary action mechanism studies showed that 11a had an inhibitory effect on cellular HBsAg secretion and could effectively activate TLR7, thereby inducing the secretion of TLR7-regulated cytokines IL-12, TNF-α and IFN-α in human PBMC cells. SPR analysis confirmed that 11a could bind to TLR7 protein with an affinity of 7.06 µM. MD simulation predicted that 11a could form tight interactions with residues in the binding pocket of TLR7. Physicochemical parameters perdition and pharmacokinetic analysis indicated that 11a displayed relatively favorable drug-like properties. Considering all the results, compound 11a might be a promising lead for developing novel immunomodulatory anti-HBV agents.

Current Status and Challenges in Anti-Hepatitis B Virus Agents Based on Inactivation/Inhibition or Elimination of Hepatitis B Virus Covalently Closed Circular DNA.

There has been over half a century since the discovery of hepatitis B virus (HBV) to now, but approximately 300 million patients with chronic hepatitis B (CHB) still live in the world, resulting in about one million deaths every year. Although currently approved antivirals (e.g., nucleoside analogues) are effective at reducing HBV replication, they have almost no impact on the existing HBV covalently closed circular DNA (cccDNA) reservoir. HBV cccDNA is a critical obstacle to the complete elimination of the virus via antiviral therapy. The true cure of HBV infection requires the eradication of viral cccDNA from HBV-infected cells; thus, the development of new agents directly or indirectly targeting HBV cccDNA is urgently needed due to the limitations of current available drugs against HBV infection. In this regard, it is the major focus of current anti-HBV research worldwide via different mechanisms to either inactivate/inhibit (functional cure) or eliminate (complete cure) HBV cccDNA. Therefore, this review discussed and summarized recent advances and challenges in efforts to inactivate/silence or eliminate viral cccDNA using anti-HBV agents from different sources, such as small molecules (including epigenetic drugs) and polypeptides/proteins, and siRNA or gene-editing approaches targeting/attenuating HBV cccDNA via different mechanisms, as well as future directions that may be considered in efforts to truly cure chronic HBV infection. In conclusion, no breakthrough has been made yet in attenuating HBV cccDNA, although a number of candidates have advanced into the phase of clinical trials. Furthermore, the overwhelming majority of the candidates function to indirectly target HBV cccDNA. No outstanding candidate directly targets HBV cccDNA. Relatively speaking, CCC_R08 and nitazoxanide may be some of the most promising agents to clear HBV infection in small molecule compounds. Additionally, CRISPR-Cas9 systems can directly target HBV cccDNA for decay and demonstrate significant anti-HBV activity. Consequently, gene-editing approaches targeting HBV cccDNA may be one of the most promising means to achieve the core goal of anti-HBV therapeutic strategies. In short, more basic studies on HBV infection need to be carried out to overcome these challenges.

Pimobendan Inhibits HBV Transcription and Replication by Suppressing HBV Promoters Activity.

Current anti-HBV therapeutic strategy relies on interferon and nucleos(t)ide-type drugs with the limitation of functional cure, inducing hepatitis B surface antigen (HBsAg) loss in very few patients. Notably, the level of HBsAg has been established as an accurate indicator to evaluate the drug efficacy and predict the disease prognosis, thus exploring a novel drug targeting HBsAg will be of great significance. Herein, by screening 978 compounds from an FDA-approved drug library and determining the inhibitory function of each drug on HBsAg level in HepG2.2.15 cells supernatant, we identified that pimobendan (Pim) has a powerful antiviral activity with relatively low cytotoxicity. The inhibitory effect of Pim on HBsAg as well as other HBV markers was validated in HBV-infected cell models and HBV-transgenic mice. Mechanistically, real-time PCR and dual-luciferase reporter assay were applied to identify the partial correlation of transcription factor CAAT enhancer-binding protein α (C/EBPα) with the cccDNA transcription regulated by Pim. This indicates Pim is an inhibitor of HBV transcription through suppressing HBV promoters to reduce HBV RNAs levels and HBsAg production. In conclusion, Pim was identified to be a transcription inhibitor of cccDNA, thereby inhibiting HBsAg and other HBV replicative intermediates both in vitro and in vivo. This report may provide a promising lead for the development of new anti-HBV agent.

Design and synthesis of novel quinazolinone derivatives as anti-HBV agents with TLR8 agonist effect.

Hepatitis B virus (HBV) infection is a worldwide threat to public health. In this work, a series of novel quinazolinone derivatives (5a-q) were synthesized and evaluated as novel anti-HBV agents. Among them, compound 5l exhibited potent inhibitory effect on HBV DNA replication in both wild type and drug resistant (lamivudine and entecavir) HBV strains with IC50 values of 0.15 and 0.10 µM, respectively. Notably, the selective index value of 5l was high above 66.67, indicating the favorable safety profile. Molecular docking study indicated that compound 5l well fitted into the binding pocket of TLR8 protein-protein interface. Dual-luciferase reporter gene assay further confirmed that compound 5l could dose-dependently activate TLR8, thus effectively inducing the activity of TLR8-dependent NF-κB. Collectively, compound 5l displayed potent anti-HBV activities and TLR8 agonist effect in vitro, and might be a potential immunomodulatory anti-HBV agent to warrant further investigation.

Synthesis and evaluation of new phenyl acrylamide derivatives as potent non-nucleoside anti-HBV agents.

As a continuation of our previous work, a series of new phenyl acrylamide derivatives (4Aa-g, 4Ba-t, 5 and 6a-c) were designed and synthesized as non-nucleoside anti-HBV agents. Among them, compound 4Bs could potently inhibit HBV DNA replication in wild-type and lamivudine (3TC)/entecavir resistant HBV mutant strains with IC50 values of 0.19 and 0.18 µM, respectively. Notably, the selective index value of 4Bs was above 526, indicating the favorable safety profile. Interestingly, unlike nucleoside analogue 3TC, 4Bs could significantly inhibit 3.5 kb pgRNA expression. Molecular docking study revealed that 4Bs could fit well into the dimer-dimer interface of HBV core protein by hydrophobic, π-π and H-bond interactions. Considering the potent anti-HBV activity, low toxicity and diverse anti-HBV mechanism from that of nucleoside anti-HBV agent 3TC, compound 4Bs might be a promising lead to develop novel non-nucleoside anti-HBV therapeutic agents, and warranted further investigation.

Discovery of novel quinazolinone derivatives as potential anti-HBV and anti-HCC agents.

As a continuation of earlier works, a series of novel quinazolinone derivatives (5a-s) were synthesized and evaluated for their in vitro anti-HBV and anti-hepatocellular carcinoma cell (HCC) activities. Among them, compounds 5j and 5k exhibited most potent inhibitory effect on HBV DNA replication in both drug sensitive and resistant (lamivudine and entecavir) HBV strains. Interestingly, besides the anti-HBV effect, compound 5k could significantly inhibit the proliferation of HepG2, HUH7 and SK- cells, with IC50 values of 5.44, 6.42 and 6.75 µM, respectively, indicating its potential anti-HCC activity. Notably, the in vitro anti-HCC activity of 5k were more potent than that of positive control 5-fluorouracil and sorafenib. Further studies revealed that compound 5k could induce HepG2 cells apoptosis by dose-dependently upregulating Bad and Bax expression and decreasing Bcl-2 and Bcl-xl protein level. Considering the potent anti-HBV and anti-HCC effect, compound 5k might be a promising lead to develop novel therapeutic agents towards HBV infection and HBV-induced HCC.

Nobiletin, a novel inhibitor, inhibits HBsAg production and hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a serious problem due to its extensive worldwide distribution and poor prognosis including cirrhosis and/or hepatocellular carcinoma. The hepatitis B surface antigen(HBsAg) is a vital serum marker in HBV infection and a major obstacle for effective and subsequently virus clearance. However, Current anti-HBV drugs, such as nucleos(t)ide analogs (NA) and PegIFN, do not meet ideal result of sustained HBsAg loss (defined as functional cure). Therefore, there is an urgent need to identify a new compound targeting HBsAg. In this study, nobiletin was screened out from 1500 compounds due to its low cytotoxicity and high antiviral activity. The effect of nobiletin on HBV was determined in HepG2.2.15 and HepG2-NTCP cells. Furthermore, the antiviral capability of nobiletin was also verified in vivo. Unlike entecavir (ETV) therapy, which reduced HBV DNA but do not lead to an effective reduction in HBsAg, nobiletin significantly reduced the level of HBsAg as well as lowered HBV DNA in vivo and in vitro. Meanwhile, combination of nobiletin and ETV led to broad reductions of both HBV DNA and HBsAg level. This study may shed light on the development of a novel class of anti-HBV agents.

Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B virus inhibitors.

Hepatitis B virus (HBV) infection is a worldwide public health issue. Search for novel non-nucleoside anti-HBV agents is of great importance. In the present study, a series of quinazolinones derivatives (4a-t and 5a-f) were synthesized and evaluated as novel anti-HBV agents. Among them, compounds 5e and 5f could significantly inhibit HBV DNA replication with IC50 values of 1.54 µM and 0.71 µM, respectively. Interestingly, the selective index values of 5f was higher than that of lead compound K284-1405, suggesting 5f possessed relatively safety profile than K284-1405. Notably, 5e and 5f exhibited remarkably anti-HBV activities against lamivudine and entecavir resistant HBV strain with IC50 values of 1.90 and 0.84 µM, confirming their effectiveness against resistant HBV strain. In addition, molecular docking studies indicated that compounds 5e and 5f could well fit into the dimer-dimer interface of HBV core protein dominated by hydrophobic interactions. Notably, their binding modes were different from the lead compound K284-1405, which may be attributed to the additional substituent groups in the quinazolinone scaffold. Taken together, 5e and 5f possessed novel chemical structure and potent anti-HBV activity against both drug sensitive and resistant HBV strains, thus warranting further research as potential non-nucleoside anti-HBV candidates.

2-Arylthio-5-iodo pyrimidine derivatives as non-nucleoside HBV polymerase inhibitors.

In this study, a series of 2-arylthio-5-iodo pyrimidine derivatives, as non-nucleoside hepatitis B virus inhibitors, were evaluated and firstly reported as potential anti-HBV agents. To probe the mechanism of active agents, DHBV polymerase was isolated and a non-radioisotopic assay was established for measuring HBV polymerase. The biological results demonstrated that 2-arylthio-5-iodo pyrimidine derivatives targeted HBV polymerase. In addition, pharmacophore models were constructed for future optimization of lead compounds. Further study will be performed for the development of non-nucleoside anti-HBV agents.

2'-Fluoro-6'-methylene carbocyclic adenosine and its phosphoramidate prodrug: A novel anti-HBV agent, active against drug-resistant HBV mutants.

Chronic hepatitis B (CHB) is one of the major causes of morbidity and mortality worldwide. Currently, clinically approved nucleos(t)ide analogs (NAs) are very efficient in reducing the load of hepatitis B virus (HBV) with minimum side effects. However, the long-term administration of antiviral drugs promotes HBV for potential drug resistance. To overcome this problem, combination therapies are administered, but HBV progressively altered mutations remain a threat. Therefore, optimally designed NAs are urgently needed to treat drug-resistant HBV. Herein, 2'-fluoro-6'-methylene carbocyclic adenosine (FMCA) and its phosphoramidate (FMCAP) have been discovered, which may be utilized in combination therapies for curing drug-resistant chronic hepatitis B. In preclinical studies, these carbocyclic NAs demonstrated potential anti-HBV activity against adefovir, as well as lamivudine (LMV/LAM) drug-resistant mutants. In vitro, these molecules have demonstrated significant activity against LMV/entecavir (ETV) triple mutants (L180M + S202G + M204V). Also, preliminary studies of FMCA/FMCAP in chimeric mice and female Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse models having the LMV/ETV triple mutant have shown a high rate of reduction of HBV DNA levels compared to ETV. In this review, we have summarized preclinical studies of FMCA and its phosphoramidate prodrug (FMCAP).

Design, synthesis and evaluation of novel phenyl propionamide derivatives as non-nucleoside hepatitis B virus inhibitors.

As an ongoing search for potent non-nucleoside anti-HBV agents with novel structures, we described a series of phenyl propionamide derivatives (3a-b, 4a-e, 7a-g, 8a-h and 9a-b) by pharmacophore fusion strategy in the present work. All the compounds exhibited an anti-HBV activity to some extent. Among them, compounds 8d and 9b displayed most potent anti-HBV activity with IC50 values on HBV DNA replication of 0.46 and 0.14 µM, respectively. And the selective index values of 8d and 9b were more than 217.39 and 153.14, suggesting that 8d and 9b exhibited favorable safety profiles. Interestingly, 8d and 9b possessed significantly antiviral activities against lamivudine and entecavir resistant HBV mutants with IC50 values of 0.77 and 0.32 µM. Notably, preliminary anti-HBV action mechanism studies showed that 8d could inhibit intracellular HBV pgRNA and RT activity of the HBV polymerase. Molecular docking studies suggested that compound 8d could fit into the dimer-dimer interface of HBV core protein by hydrophobic interaction. In addition, in silico prediction of physicochemical properties showed that 8d conformed well to the Lipinski's rule of five, suggesting its potential for use as a drug like molecule. Taken together, 8d possessed significantly anti-HBV activity, low toxicity, diverse anti-HBV mechanism and favorable physicochemical properties, and warranted further investigation as a promising non-nucleoside anti-HBV candidate.

Toward Elimination of Hepatitis B Virus Using Novel Drugs, Approaches, and Combined Modalities.

Hepatitis B virus (HBV) causes significant morbidity and mortality worldwide. The majority of chronically infected individuals do not achieve a functional and complete cure. Treated persons who achieve a long-term sustained virologic response (undetectable HBV DNA), are still at high risk of developing morbidity and mortality from liver complications. This review focuses on novel, mechanistically diverse anti-HBV therapeutic strategies currently in development or in clinical evaluation, and highlights new combination strategies that may contribute to full elimination of HBV DNA and covalently closed circular DNA from the infected liver, leading to a complete cure of chronic hepatitis B.