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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Antiviral Res. 2020 May 5;179:104815. doi: 10.1016/j.antiviral.2020.104815

HBV replication inhibitors

Claire Pierra Rouviere a, Cyril B Dousson a,*, John E Tavis b,*
PMCID: PMC7293572  NIHMSID: NIHMS1590956  PMID: 32380149

Abstract

Chronic Hepatitis B Virus infections afflict >250 million people and kill nearly 1 million annually. Current non-curative therapies are dominated by nucleos(t)ide analogs (NAs) that profoundly but incompletely suppress DNA synthesis by the viral reverse transcriptase. Residual HBV replication during NA therapy contributes to maintenance of the critical nuclear reservoir of the HBV genome, the covalently-closed circular DNA, and to ongoing infection of naive cells. Identification of next-generation NAs with improved efficacy and safety profiles, often through novel prodrug approaches, is the primary thrust of ongoing efforts to improve HBV replication inhibitors. Inhibitors of the HBV ribonuclease H, the other viral enzymatic activity essential for viral genomic replication, are in preclinical development. The complexity of HBV’s reverse transcription pathway offers many other potential targets. HBV’s protein-priming of reverse transcription has been briefly explored as a potential target, as have the host chaperones necessary for function of the HBV reverse transcriptase. Improved inhibitors of HBV reverse transcription would reduce HBV’s replication-dependent persistence mechanisms and are therefore expected to become a backbone of future curative combination anti-HBV therapies.

Keywords: Chronic hepatitis B, Hepatitis B virus, Nucleos(t)ide analogs, Reverse transcriptase inhibitors, Ribonuclease H inhibitors

1. Introduction

1.1. HBV virus and its medical impact.

Hepatitis B Virus (HBV) is an enveloped, partially-double stranded DNA virus that replicates by reverse transcription (Seeger et al., 2013; Summers and Mason, 1982). Approximately 257 million people in the world are chronically infected (Polaris Observatory Collaborators, 2018). HBV has 10 genotypes (A-J) and many sub-genotypes (Kramvis, 2014) that have different geographical distributions and are associated with variable clinical progression (Tian and Jia, 2016). More than 60 million chronically infected people are at risk of major complications from chronic hepatitis type B (CHB), including liver cirrhosis and liver cancer, which cause over 880,000 deaths globally each year (Trepo et al., 2014). Disease burden will continue to rise at least until 2035, especially in developing countries, with more than one million of people newly infected each year (World Health Organization, 2019). Therefore, there is an urgent need of safe, effective, and accessible cures to benefit as many people living with CHB as possible.

1.2. HBV replication.

HBV reverse transcription 130 (Seeger et al., 2013) within hepatocytes begins with binding of the viral polymerase protein (P) to an RNA stem-loop termed ε at the 5’ end of the viral pregenomic RNA (pgRNA, the RNA phase of the viral genome) in a chaperone-dependent reaction. Formation of the P:ε complex triggers encapsidation of the pgRNA and P into viral capsids. Reverse transcription (Fig. 1) within capsids is primed by tyrosine 96 in the terminal protein domain of P using sequences within a bulge in ε as a template. DNA elongation extends for 3–4 nt before the first of three strand transfers shifts the nascent minus-polarity DNA strand to an element called DR1 near the 3’ end of the pgRNA. Minus-polarity DNA is then elongated by the viral reverse transcriptase (RT) activity of P, with the pgRNA being concomitantly degraded by the viral ribonuclease H activity (RNaseH) located in the C-terminal domain of P. DNA elongation extends to the 5’ end of the pgRNA to create full-length minus-polarity DNA with a short terminal duplication containing a second copy of DR1. RNA cleavage terminates 15–18 nt prior to the end of the pgRNA, presumably reflecting the distance between the RT and RNaseH active sites on P. This RNA oligomer is then transferred to a repeat element called DR2 near the 5’ end of the minus-polarity DNA strand based in part on homology between DR1 and DR2. Plus-polarity DNA strand synthesis is then primed by the RNA oligomer, and DNA elongation by the RT extends until it reaches the 5’ end of the minus-polarity DNA strand. The final strand transfer then shifts the 3’ end of the plus-polarity DNA strand to homologous sequences at the 3’ end of the minus-polarity DNA strand. Plus-polarity DNA elongation is then resumed by the RT, but it terminates at random sites approximately 50% around the 3,200 bp genome. This yields the mature, partially double-stranded DNA genome found in virions.

Fig. 1. HBV reverse transcription.

Fig. 1.

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The HBV reverse transcription pathway is shown with the nucleic acids in an extended form for clarity. In capsids, the three strand transfer reactions are promoted by specific conformations of the nucleic acids. Red, RNA; Blue, DNA; Green, protein. R, the long terminal repeat in the pregenomic RNA; r, the shorter terminal repeat in the minus-polarity DNA strand. 1, direct repeat 1; 2, direct repeat 2. Modified from (Tavis et al., 1994).

The newly synthesized viral genomes have two fates. They may be enveloped and secreted as virions, or they may be “recycled” to the nucleus (Seeger et al., 2013). The HBV DNA is repaired within the nucleus by cellular enzymes to an episomal covalently-closed circular DNA (cccDNA) that is the template for viral transcription. Three features of the cccDNA are critical with respect to curative anti-HBV therapies. First, all cccDNA molecules are produced by reverse transcription. Second, cccDNA levels are regulated to maintain them at 5 –12 copies per cell (Ko et al., 2018). Third, cccDNA persists even during highly effective nucleos(s)tide analog (NA) therapy that suppresses viremia below the limit of detection. The durability of the cccDNA appears to have two causes, an apparently long half-life and ongoing replenishment of the cccDNA pool via the recycling pathway (Ko et al., 2018). Replenishment is revealed by sequential accumulation of resistance mutations to NAs in the absence of detectable viremia (Ghany and Liang, 2007; Monto et al., 2010; Zoulim and Locarnini, 2009), and is confirmed by analyses of viral DNA in the liver showing replenishment of the cccDNA via reverse transcription even without detectable viremia (Coffin et al., 2011).

1.3. Challenges to curing HBV.

HBV infections are extremely challenging to cure owing to the virus’ high genetic diversity, persistence of the cccDNA mini-chromosome in human liver cells, integration of HBV DNA into the cellular genome, and HBV’s negative impact on the host immune system. A complete sterilizing cure would require achieving undetectable hepatitis B surface antigen levels (HBsAg) in serum and complete eradication of HBV DNA (including the cccDNA and integrated HBV DNA). This is likely to be unachievable, at least in the foreseeable future, because even natural clearance of an acute infection fails to achieve a sterilizing cure in many patients. Persistence of replication-competent HBV following apparent clearance is revealed by resurgence of HBV viremia in some patients undergoing immunosuppressive therapies (Sagnelli et al., 2014). Instead of a sterilizing cure, the scientific community has accepted a functional cure of hepatitis B virus infections as an achievable step for next generation therapies (Lok et al., 2017). The consensus definition of a functional cure is sustained, undetectable HBsAg and HBV DNA in serum, with or without development of anti-hepatitis B surface antibodies after completion of a finite course of treatment, resolution of residual liver injury, and a decrease in risk of hepatocellular carcinoma (Lok et al., 2017; Yip and Lok, 2019).

1.4. Current therapies for HBV.

Two types of treatment are currently approved for HBV infections. Interferon α, usually administered as pegylated interferon α (IFN-α−2a/b and more recently peg-IFN-α−2a), stimulates innate immune responses against the virus. This treatment has a ≤ 20% functional cure rate (Kim, 2018), but therapy can cause severe side effects that often limit its use (Trepo et al., 2014). HBV genotype affects responses to pegylated INFα, with genotype A patients responding best and D responding the worst (Kramvis, 2014).

The second class of drugs consists of NAs that inhibit the RT activity of the HBV P protein. The NAs are phosphorylated to their triphosphate (TP) forms by cellular kinases. The NA-TPs are substrates for the RT during reverse transcription, where they are chain terminators that block synthesis of both the plus- and minus-polarity HBV DNA strands. Eight NAs have been approved against HBV (Fig. 2) The first was lamivudine (1, 3TC, LMV, Epivir®, Zeffix®, Heptodin® or Hepitec®), approved in the United States in 1998. Subsequently, adefovir dipivoxil (2, bis(POM)PMEA, ADV, Hepsera® or Preveon®; 2002), entecavir (3, BMS-200475–01, ETV, Baraclude®; 2005), telbivudine (4, LdT, TBV, Tyzeka™ or Sebivo®; 2006), tenofovir disoproxil fumarate (5, bis(POC)PMPA fumarate, TDF, Viread®; 2008), and tenofovir alafenamide hemifumarate (6, GS-7340–03; TAF hemifumarate, Vemlidy®; 2016) were approved. Although clevudine (7, L-FMAU, CLV, Levovir® or Revovir®; 2006) was approved in South Korea and the Philippines, its approval was revoked due to drug-related skeletal myopathy resulting from mitochondrial dysfunction (Park et al., 2017). More recently, besifovir dipivoxil maleate (8, ANA-380 / LB80380 maleate, BSV dipivoxyl maleate, Besivo®; 2017) was approved in South Korea.

Fig. 2. Nucleos(t)ide analogs approved for treatment of HBV infections as DNA polymerase inhibitors.

Fig. 2.

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Trade names and developers are indicated for each drug.

NAs are orally available (either by themselves or as prodrugs), have favorable pharmacokinetics with generally no food restrictions, limited drug-drug interaction, and low pill burden (one pill once daily). The NAs can suppress viremia to clinically undetectable levels in up to 76% of HBV e antigen (HBeAg)-positive patients and up to 93% of HBeAg-negative patients after one year of therapy (Trepo et al., 2014), and they can help block mother-to-child transmission (Jia et al., 2020). They normalize serum alanine transferase levels that reflect ongoing liver damage in up to 78% of patients, but death from HBV-induced hepatocellular carcinoma is reduced only 3- to- 4-fold by long-term NA therapy (Liaw, 2013). They have very good safety profiles, and resistance to ETV and tenofovir (TFV) is either manageable or clinically negligible (Trepo et al., 2014). HBV genotype can affect response to NAs, with genotype A and D patients being significantly more likely to achieve a functional cure than genotype B and C patients when treated with TFV analogs (Bayliss et al., 2017). Unfortunately, their functional cure rate is < 10% over many years of follow-up (Kim, 2018), due in part to insufficient efficacy, liver targeting, and/or low barrier to resistance. NA treatment is therefore essentially life-long (Terrault et al., 2016), although drug termination is becoming feasible in some HBeAg-negative patients (Liaw, 2019).

Currently recommended first-line NAs include ETV and the two prodrugs of TFV, TDF and TAF hemifumarate. These drugs achieve excellent virological suppression in a large proportion of patients, with favorable safety and tolerability profiles and low rates of virological breakthroughs owing to their high barrier to resistance (Buti et al., 2016; Chang et al., 2010; Marcellin et al., 2013).

1.5. Need for developing new HBV replication inhibitors.

Despite the existence of NA drugs which can suppress plasma viremia, induce HBeAg seroconversion in a minority of patients, and occasionally cause HBsAg loss or seroconversion , new therapies to further suppress viral replication are still needed and will contribute substantially to future curative therapies. Three observations based on the drawbacks of currently available treatments support this thesis.

First, current NAs are not effective enough to suppress genomic recycling, allowing intracellular replenishment of the cccDNA even in the absence of detectable serum viremia (Ghany and Liang, 2007; Monto et al., 2010; Zoulim and Locarnini, 2009). Consequently, the NAs have little to no effect on the cccDNA reservoir in the nucleus of infected hepatocytes. The immune modulation induced by IFN-α containing therapies can suppress cccDNA levels, but the reduction is small in most cases (Takkenberg et al., 2011). The failure of current therapies to abrogate cccDNA maintenance is a major contributor to viral persistence during therapy (Revill et al., 2016; Wong et al., 2013). This indicates that suppressing reverse transcription enough to block recycling would result in a decline in cccDNA levels due to death of infected cells and intracellular decay of the cccDNA.

Second, the NAs work by essentially the same mechanism, and the first generation molecules are chemically similar to one another, indicating that potential pharmacological limitations to their uptake, phosphorylation to their TP forms, or heterogeneity among hepatocytes in their ability to utilize these drugs may limit their effectiveness. Additionally, their selectivity for the HBV RT can be limited to some degree by the similarity of the RT to cellular DNA polymerases.

Finally, the early generations of NAs, such as LAM and ADV, have low barriers to resistance (2017), and some NA-resistance mutations can potentiate resistance to other NAs [pre-existing LMV mutations sharply reduce the barrier to resistance to ETV; (Terrault et al., 2016)].

These limitations open the door to development of novel replication inhibitors, both new HBV P inhibitors and their prodrugs, and drugs against other as-of-yet unexploited targets essential for viral replication. This review provides an overview of efforts to develop novel replication inhibitors, particularly against the two viral enzymatic activities needed for viral reverse transcription, the RT and RNaseH, because they are the most advanced. We provide an update of the status, advantages, and limitations of inhibitors targeting these enzymes. We also take the opportunity to clarify and homogenize the commonly used NA abbreviations.

2. Nucleos(t)ide analog reverse transcriptase inhibitors

2.1. Nucleos(t)ide analogs as key tools against HBV.

Oral NA therapy is the most commonly used anti-HBV treatment worldwide. The NAs must be phosphorylated in cells into their pharmacologically active TPs or equivalent metabolites (Kim et al., 2010; Michailidis et al., 2012). The NAs profoundly inhibit HBV DNA synthesis and frequently reduce serum HBV DNA to levels below the limit of quantification. NA treatment for more than 5 years can also reduce the amount of intrahepatic cccDNA, the key molecule in HBV persistence. This was demonstrated by a study of patients who had been enrolled in international NA clinical trials (ETV vs. LMV; TBV vs. LMV, and CLV vs. ADV) who had liver biopsies at baseline, one year after treatment, and again after continuous treatment for 72 to 145 months. At the third biopsy, the median levels of cccDNA declined by 2.9 log from baseline, with 49% patients having cccDNA levels below the detection limit (Lai et al., 2017).

2.2. Nucleos(t)ide analog associated resistance profiles.

Differences exist among the NAs with respect to potency, safety, and resistance profiles. The most important difference is the propensity for drug resistance mutations to emerge following long-term therapy (Table 1) (2017; Manzoor et al., 2015; Zoulim, 2004). The most common resistance mutations affect the YMDD motif in the RT active site on the P protein. Mutation of methionine at position 204 of the RT domain to either valine (M204V, YVDD mutation) or isoleucine (M204I, YIDD mutation) can reduce susceptibility to monotherapy with NAs having low barriers to resistance. ETV, TDF and TAF hemifumarate, which have medium to high barriers to resistance (Chang et al., 2010; Liu et al., 2017), are thus the recommended first-line monotherapies, while LMV, ADV, TBV, and CLV, which have low barriers to resistance evolution, are not recommended (EASL, 2017; Lok et al., 2016; Terrault et al., 2016). Although resistance to ETV is not common in treatment-naive patients, it occurs in half of the patients who have used LMV. Thus, ETV is not recommended for patients pretreated with LMV, TBV or CLV. No mutations have yet been reported to confer TFV resistance in vivo (EASL, 2017). Finally, combination of TFV with ETV has been evaluated in several clinical studies and appears to be a safe option as a rescue therapy in patients with multidrug resistance (Park et al., 2016; Zoulim et al., 2016).

Table 1.

Resistance profiles, advantages and limitations of currently approved NAs for HBV treatment.

Compound Resistance mutations Advantages Limitations
Lamivudine
(LAM)		 M204V
M204I
L180M + M204V
L180M + M204V/I ± I169T ± V173L ± M250V
L180M + M204V/I ± T184G ± S202I/G		 Oral.
Safe with negligible side effects.
Effective and safe in pregnancy.
Least expensive.		 Long-term treatment is necessary.
Low barrier to resistance.
Adefovir dipivoxil (ADV) A181T/V
N236T		 Oral.
Low resistance.
Effective in LMV-resistant CHB patients.
Long-term ADV use promotes viral suppression, regression of fibrosis, and reversal of cirrhosis.		 Long-term treatment is necessary.
Poorly absorbed.
Associated with a high level of nephrotoxicity.
Less potent than other NAs.
Entecavir
(ETV)		 L180M + M204V/I ± I169T ± V173L ± M250V
L180M + M204V/I ± T184G ± S202I/G		 Oral.
Potent viral suppression.
Undetectable HBV DNA levels upon long-term treatment.
Safe with negligible side effects.
Medium barrier to resistance.
3 Mutations must be present for ETV resistance.
Switch to ETV recommanded in case of TDF or TAF resistance.
Recommanded in combination with TDF or TAF as a rescue therapy.
Preferred treatment option with TDF or TAF in patients with decompensated disease.
Can be used in children.		 Long-term treatment is necessary.
ETV monotherapy can cause HIV resistance mutations Cross-resistance to LMV resistants.
Resistance to ETV is 57% at 6 years in LMV-refractory patients.
Telbivudine
(TBV)		 M204I
L180M + M204V
L180M + M204V/I ± I169T ± V173L ± M250V
L180M + M204V/I ± T184G ± S202I/G		 Oral.
Viral suppression superior to LMV and ADV.
Renoprotective effects: prevents ADV-induced nephrotoxicity and improves kidney function in liver transplant patients.
Effective and safe in pregnancy.		 Long-term treatment is necessary.
Low barrier to resistance.
Tenofovir disoproxyl
fumarate
(TDF)		 No clinical evidence for TDF resistance Oral.
More potent than LMV and ADV.
Undetectable HBV DNA upon long-term treatment.
Safe with negligible side effects.
High barrier to resistance.
Switch to TDF recommanded in case of LMV, TBV, ETV, or ADV resistance.
Recommended in combination with ETV as a rescue therapy.
Preferred treatment option with ETV in patients with decompensated disease.
TDF is recommended in pregnant women with CHB and advanced fibrosis or cirrhosis.
Can be used in children.		 Long-term treatment is necessary.
TDF monotherapy can cause HIV resistance mutations. Associated with bone and kidney toxicity.
Tenofovir alafenamide
hemifumarate
(TAF)		 No clinical evidence for TAF resistance Oral.
High barrier to resistance.
Undetectable HBV DNA levels upon long-term treatment.
Switch to TAF recommanded in case of LMV, TBV, ETV, or ADV resistance.
Recommanded in combination with ETV as a rescue therapy.
Better kidney and bone safety than TDF.
Can be used in children.		 TAF monotherapy can cause HIV resistance mutations.
Clevudine
(CLV)		 M204I
L80I		 Oral.
Long intracellular half-life of CLV-TP.
Can suppress HBV replication for an extended periods after withdrawal.		 Low barrier resistance.
Resistance in 2–14% of LMV naive patients, and 40% of patients previously treated with LMV.
No longer developed for treatment of CHB due to skeletal myopathy and mitochondrial dysfunction.
Besifovir dipivoxyl
maleate
(BSV)		 No identified resistance after 2 years treatment Reduced bone and renal toxicity at 48 weeks us. TDF.
Potent viral suppressing effect in patients with wild type and LMV- resistance strains.		 Carnitine depletion.
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2.3. Nucleos(t)ide prodrug approaches.

Recent efforts have focused on improving tissue targeting, tolerability and safety of NA drugs through development of prodrugs that could be selectively delivered or activated in the liver. Prodrugs of TFV and adefovir (PMEA) are in the most advanced stages of development.

TAF hemifumarate (6), an improved prodrug of TFV (5) (Fig. 2), is a second-generation prodrug of TFV designed to optimize antiviral potency and improve safety. Metabolism studies showed that TAF is efficiently taken up into hepatocytes, in part via uptake transporters such as OATP1B1 and OATP1B2, but also by high passive permeability. It is rapidly hydrolyzed within hepatocytes by carboxylesterase 1 (CES1) to yield TFV, which is subsequently phosphorylated to its metabolites (TFV-MP and TFV-DP) (Murakami et al., 2015). Pharmacokinetic studies in dogs demonstrated that TAF has a hepatic extraction ratio of 65% (Babusis et al., 2013). It is noteworthy that a non-negligible fraction of TAF is also delivered non-selectively to peripheral blood monocellular cells (PBMCs), as TAF is also used to treat HIV infections in T lymphocytes (Antela et al., 2016). Unlike TFV, TAF does not induce renal transporter (e.g., organic anion transporters, OAT1 and OAT3)-dependent cytotoxicity, which partially explains its improved safety profile (Bam et al., 2014). In a phase Ib 28 day safety study, 25 mg daily administration of TAF had similar efficacy as 300 mg TDF but had improved kidney and bone safety due to reduced systematic circulation of TFV and increased exposure of active forms of TFV in liver (Agarwal et al., 2015). TAF (25 mg) was therefore selected for further development in phase III trials in CHB patients (Buti et al., 2016; Chan et al., 2016), and subsequently approved for treatment of CHB (GS-7340–03, TAF hemifumarate salt, Vemlidy®) (Byrne et al., 2018). However, according to recent data from Scherer de Fraga et al, (de Fraga et al., 2020), the greater safety profile of TAF towards TDF may have been overestimated, as also mentioned in the Hill et al. meta-analysis which compared both drugs in HIV and HBV therapy (Hill et al., 2018).

TFV exalidex (9, CMX-157, TXL™, Fig. 3) is a lipid conjugated liver-targeted prodrug of TFV designed to use lipid uptake pathways to increase tissue penetration and reduce circulating TFV (Painter et al., 2007). Preclinical characterization revealed that this hexadecyloxypropyl ester prodrug of TFV is about 100-fold more potent than TFV in vitro. In addition, TXL™ is efficiently extracted by the liver in rats and is rapidly converted to the active forms of TFV in primary human hepatocytes. Phase I studies have shown excellent tolerance and good pharmacokinetics. A phase II trial has been completed in which HBV-infected subjects were administered doses up to 100 mg of the prodrug for 28 days (Chatsiricharoenkul et al., 2017). Thus, TXL™ has achieved clinical proof-of-concept (POC) for antiviral activity, and displayed excellent safety, tolerability, and pharmacokinetic profiles. Its formulation has been optimized to further enhance drug delivery in order to move towards a phase III trial.

Fig. 3. New nucleos(t)ide analog prodrugs recently published or in clinical development for treating HBV infections.

Fig. 3.

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AGX-1009 (10, Fig. 3) is a lipid-based TFV prodrug similar to TFV exalidex (Zhuorong et al., 2012). Preliminary pharmacodynamics, pharmacokinetics and toxicity studies showed that AGX-1009 strongly inhibits HIV-1 and HBV with a low toxicity profiles and high oral bio-availability (Asia Pacific Biotech News, 2010). AGX-1009 related assets were sold to Cinkate Pharmaceuticals in 2014, and phase I study was initiated in China. No recent reports of development have been released and it appears that development of AGX-1009 against HBV has been halted.

ADV (2, Fig. 2), a diester prodrug of the active phosphonate PMEA, is effective in LMV-resistant CHB patients (Marcellin et al., 2008; Murakami et al., 2014). Long-term ADV treatment helps achieve and maintain viral suppression, and it promotes regression of fibrosis and reversal of cirrhosis in most patients (Wang et al., 2014). However, PMEA is actively transported into the renal proximal tubules and causes nephrotoxicity at ADV doses of 30 – 120 mg/day (Lee et al., 2010). Hence, ADV is used at suboptimal dose of 10 mg to limit renal toxicity.

Pradefovir (PDV) mesylate (11, MB-06886 mesylate, Remofovir mesylate®, Hepavir B®, Fig. 3), is a cyclic 1-aryl-1,3-propanyl type prodrug of PMEA with high plasma stability that was designed to specifically target the liver and reduce risks to secondary tissues (especially the kidneys). This novel prodrug, originally developed by Metabasis Therapeutics, uses a cytochrome oxidation-based technology (HepDirect™) to provide higher liver delivery with reduced systemic distribution. Unlike ADV, PDV conversion to PMEA is catalyzed by cytochrome P450 isozyme 3A4, which is predominantly expressed in the liver, reducing nonhepatic activation of the prodrug (Lin et al., 2006). Consequently, PDV has a 12-fold improvement in the liver/kidney ratio over ADV in rats (Reddy et al., 2008). This superiority was confirmed by a 48-week phase II trial analysis demonstrating that PDV mesylate was well tolerated, and significantly more active than ADV (proportions of patients with HBV DNA < 400 c/mL were 45%, 63%, 56% and 71% for the PDV 5 mg, 10 mg, 20 mg and 30 mg groups, respectively, and 36% for the ADV 10 mg group) (Lee et al., 2006). Although a phase II trial of PDV mesylate was discontinued in USA and Europe (Slusarczyk et al., 2018; Tillmann, 2007), development is still progressing in China, where a recent study is evaluating the tolerability, pharmacokinetics, and antiviral activity of PDV mesylate in CHB patients (Zhang et al., 2020). Oral single doses were well tolerated at 10 – 120 mg/day without causing significant kidney impairment in healthy subjects, and the maximum concentration (Cmax) and area under the curve from time 0 to 48 h (AUC0–48) of serum PMEA ranges from 18 to 312 ng/mL and 72 to 1095 ng*h/mL, respectively (Ding et al., 2017). PDV mesylate was safe and well tolerated by CHB patients after 28 days of therapy. Its antiviral activity was similar to TDF as exemplified by the change in HBV DNA in the 60–120 mg PDV group, compared to the 300 mg TDF group. Moreover, PDV mesylate was associated with 4- to 4.5-fold reduced circulating serum PMEA levels compared to ADV (Zhang et al., 2020). Based on these findings, a dose close to 45 mg was selected for a phase II clinical trial. Later 30, 45 and 60 mg QD pradefovir were recommended to be appropriate dosages in phase II clinical trial (Zhang et al., 2020).

Similar liver-targeting prodrug approaches have also been used on some molecules in late stage clinical development. For example, BSV dipivoxyl maleate (8, Fig. 2) is a dipivoxyl prodrug of a TFV analog (2-aminopurine base acyclic phosphonate, LB80331), which is activated in both intestine and liver. This prodrug is converted to its parent drug LB80331 via hydrolysis and is then further oxidized at the 6-position of the purine base to form its active metabolite (LB80317), an acyclic nucleotide analog of guanosine monophosphate. A 48-week phase IIb trial showed that BSV dipivoxyl maleate at 90 and 150 mg daily doses was as effective as ETV at a 0.5 mg daily dose and did not elicit renal toxicity (Lai et al., 2014). A rollover study extending to 96 weeks confirmed the equivalent efficacy of BSV dipivoxyl maleate with ETV with no identifiable HBV drug resistance (Yuen et al., 2015). Clinical trials showed effective antiviral activity against both wild type and LMV-resistant strains (L180M/V and M204I/V) in HBeAg positive chronic HBV patients (Clinical Trials.gov, 2013; Yuen et al., 2015; Yuen et al., 2010). Phase III studies in South Korea have shown that BSV dipivoxyl maleate (150 mg) has antiviral efficacy comparable to that of TDF (300 mg) after 48 weeks of treatment, with durable effects for 96 weeks, and significantly reduced bone and renal toxicities (Ahn et al., 2019). The only limitation of BSV was depletion of L-carnitine in 87% of patients receiving 90 mg and in all patients receiving 150 mg, requiring carnitine supplementation (Yuen et al., 2015). Based on these data, BSV dipivoxyl maleate was approved in 2017 in South Korea (LB80380 maleate, Besivo®).

Lagociclovir valactate (12, MIV-210, Fig. 3) is a prodrug of the nucleoside analog 3’-fluoro-2’,3’-dideoxyguanosine (FLG) with high oral bioavailability in humans and potent activity against HBV. Preclinical in vitro and in vivo data revealed that MIV-210 was a good candidate for further testing as an agent against HBV. A study to determine the dose-related antiviral efficacy and safety of MIV-210 in chronically infected woodchucks showed that oral administration of MIV-210 at 20 or 60 mg/kg/day induced a rapid virological response, reducing serum woodchuck hepatitis virus (WHV) DNA levels by 4.75 log10 and 5.72 log10, respectively after 2 weeks (Michalak et al., 2009). Further, a 10 mg/kg/day dose decreased the WHV load by 400-fold after 4 weeks of treatment, and a dose of 5 mg/kg/day was sufficient to maintain this antiviral effect during the following 6-week period. MIV-210 at 20 or 60 mg/kg/day for a 10-week period reduced the liver WHV DNA load 200- to 2,500-fold from pretreatment levels and, importantly, led to a 2.0 log10 drop in the hepatic content of WHV cccDNA (Michalak et al., 2009). Following favorable plasma levels of MIV-210 and good oral bioavailability in phase I studies in healthy volunteers, a phase IIa clinical trial was initiated in South Korea (Wang and Chen, 2014). Although MIV-210 had competitive antiviral activity relative to existing drugs, it did not show superiority and its development was halted.

Although CLV (7, Fig. 2) is no longer being developed for the treatment of CHB because of drug-related skeletal myopathy observed during a phase III clinical trial (Park et al., 2017), its TP is of interest due to its action as a competitive non-substrate inhibitor of the HBV RT and its good intracellular half-life (T1/2 = 11 h) (Jones et al., 2013; Niu et al., 2008). CLV-TP is able to suppress HBV replication for several months after treatment (Anderson, 2009; Cheng et al., 2012). It has also been found to inhibit HBV infection at multiple steps of its replication cycle, and cause significant reduction of cccDNA in animal models (Anderson, 2009; Cheng et al., 2012). Based on this data, prodrugs of CLV analogs were recently re-investigated by Emory University (De La Rosa et al., 2017) then by Antios, to reduce systemic exposure to CLV and skeletal myopathy. The lead candidate, ATI-2173 (5’-phosphoramidate prodrug of CLV, licensed to Antios Therapeutics, 13, Fig. 3) has shown in vitro efficacy against HBV, with significant reduction of cccDNA in the woodchuck HBV model and inhibition of all stages of DNA synthesis by the active TP (unpublished). A first-in-human phase I clinical trial for ATI-2173 was recently initiated in 35 healthy subjects as a randomized, double-blind, placebo-controlled single-ascending dose study in healthy volunteers to evaluate the compound’s safety, tolerability and pharmacokinetic profile (Clinical Trials.gov, 2020).

More recently, liver-specific delivery prodrug approaches (similar to PDV) have been applied by Zhejiang Palo Alto Pharmaceuticals Inc. on ETV (14, Fig. 3) (Zhijian et al., 2019a) and on TFV/PMEA acyclic phosphonate analogs (Zhijian et al., 2019b) (15, Fig. 3). Shenzhen TargetRx Inc. also patented novel prodrugs of acyclic nucleotide phosphonate analogs (16, 17, Fig. 3) for treatment of HBV (Wang and Zhao, 2019a; Wang and Zhao, 2019b). In parallel, new phosphoramidate prodrugs of TFV were claimed to have equivalent anti-HBV activity as TAF with anti-liver fibrosis effects (18, Fig. 3) (Xinglu et al., 2019), and other new prodrugs were reported to have higher lipid solubility, higher bioavailability, higher activity and lower toxicity than TDF and TAF (19, Fig. 3) (Honghai, 2019). A series of bis(L-amino acid) ester prodrugs of TFV were recently designed and synthesized by Wang et al. as new anti-HBV agents. The most active compound, a bis(L-valine) ester prodrug of TFV had in vivo efficacy which was not inferior to TDF in the Duck Hepatitis B Virus model (20, Fig. 3) (Wang et al., 2019). Prodrugs of BSV have been recently patented by Sichuan Haisco Pharmaceutical Co. They concern either lipid-based prodrugs of BSV (21, Fig. 3) (Yi et al., 2017a) or phosphoramidate type BSV prodrugs (22, Fig. 3) (Yi et al., 2017b). Chu et al. discovered 2’-fluoro-6’-methylene carbocyclic adenosine (FMCA) and more recently, its phosphoramidate prodrug (23, FMCAP, Fig. 3), which may be suitable for use in combination therapies for drug-resistant CHB, and more particularly LMV/ETV triple mutants (L180M + S202G + M204V) (Singh et al., 2018, 2019). Finally, AIB-001, a new liver targeted orally-available inhibitor of the HBV polymerase, has been recently reported by Ai-biopharma (unpublished; structure undisclosed).

Although non-curative in the vast majority of patients, extended treatment durations with currently available NAs can reduce levels of the intra-hepatic cccDNA, the viral reservoir responsible of viral relapse after discontinuation of treatment. This observation confirms the potential of NAs to contribute substantially to curative HBV therapies. It strengthens the rationale for discovery of new and improved NAs, and development of novel prodrugs with better design, improved liver-targeting and barrier to resistance profiles. Thus, improved NAs are likely to become the backbone of future curative combination therapies due to their advanced state of development, excellent safety profiles, and high antiviral efficacy.

3. Non-nucleos(t)ide analog reverse transcriptase inhibitors

Non-nucleos(t)ide analog inhibitors of the HIV RT (NNRTIs) typically bind to allosteric pockets distinct from the enzyme active site. Little work has been done to identify HBV NNRTIs, primarily due to difficulties producing recombinant RT with substantial DNA elongation activity. This limitation has recently been partially overcome and an assay to detect inhibitors of RT activity suitable for mid-throughput screening has been developed (Nakajima et al., 2019). This assay was used to identify a stilbene derivative, piceatannol, as a specific inhibitor of HBV. Derivative analysis identified the compound PDM2 that inhibited HBV replication with a 50% effective concentration (EC50) value of 14.4 ± 7.7 μM. PDM2 was shown to bind directly to the RT domain of P, and it was effective against LMV/ETV-resistant RTs (Nakajima et al., 2019). Further development of this screening assay may open the door to identification of compounds that inhibit the RT by as-of-yet unexploited mechanisms.

4. Ribonuclease H inhibitors

4.1. The HBV RNaseH as a drug target.

The HBV RNaseH degrades the pgRNA after the viral RT has copied it into minus-polarity DNA. RNaseHs usually have two Mg++ ions in the active site that promote RNA cleavage by activating a water molecule that then cleaves the RNA backbone through a nucleophilic attack (Klumpp et al., 2003), and all evidence to date implies that the HBV enzyme also employs this two ion cleavage mechanism. Blocking RNaseH function causes minus-polarity DNA elongation to stall prematurely, causes accumulation of extensive RNA:DNA heteroduplexes within viral capsids, and prevents synthesis of the viral plus-polarity DNA strand (Hu et al., 2013; Tavis et al., 2013). This prevents formation of mature HBV genomes.

The RNaseH has not been targeted for drug discovery until recently for two reasons. First, the RNaseH is very difficult to express as a functional recombinant enzyme, preventing high throughput screening and complicating mechanistic studies of RNaseH inhibitors. Second, the immediate effect of inhibiting the RNaseH is to block production of the viral plus-polarity DNA strand, but significant amounts of truncated minus-polarity DNA are made which cause a high background in screening assays that measure HBV DNA production as their readout. Both of these problems have been partially resolved recently by the recent production of active recombinant HBV RNaseH (Tavis et al., 2013; Villa et al., 2016), and development of a strand-preferential quantitative PCR assay that selectively detects the viral plus-polarity DNA strand (Cai et al., 2014). Both assays have limitations that prevent their use in high-throughput screening, but they are suitable for hypothesis driven low- and mid-throughput screening.

4.2. Identification of HBV RNaseH inhibitors.

Compound screening against the HBV RNaseH has focused on chemotypes that inhibit the HIV RNaseH (Tavis et al., 2013). Over 150 HBV replication inhibitors that work by blocking the viral RNaseH have been identified by screening over 3,000 compounds (Edwards et al., 2017; Edwards et al., 2019; Huber et al., 2017; Lomonosova et al., 2017; Lu et al., 2015; Tavis et al., 2013). Mechanism of action against the HBV RNaseH has been confirmed in biochemical assays and by detecting RNA:DNA heteroduplex accumulation in capsids in the presence of the inhibitors (Edwards et al., 2017; Hu et al., 2013; Tavis et al., 2013). These inhibitors are primarily found in four chemotypes, the α-Hydroxytropolones, N-Hydroxyisoquinolinediones, N-Hydroxypyridinediones, and NHydroxynapthyridinones. Fifty-one compounds have sub-micromolar EC50 values, with the best compounds having EC50s of approximately 100 nM, 50% cytotoxic concentrations (CC50) in hepatoma cells in the 25–100 μM range, and therapeutic indexes (CC50/EC50) of > 300. Example HBV RNaseH inhibitors from each of the four chemotypes are in Fig. 4.

Fig. 4. Example HBV ribonuclease H inhibitors.

Fig. 4.

Open in a new tab

The HID, HPD, HNO, and αHT chemotypes are indicated.

4.3. Mechanism of inhibition.

HIV RNaseH active site inhibitors (Cao et al., 2014; Ilina et al., 2012) work by coordinating the catalytic Mg++ ions in the RNaseH active site with a trident of adjacent oxygen atoms or electron donors (Cao et al., 2014; Davies et al., 1991). All known HBV RNaseH inhibitors have Mg++-coordinating tridents. Disrupting the trident ablates inhibition [(Cai et al., 2014; Lu et al., 2015) and unpublished] and inhibition efficacy is dependent on Mg++ concentration (unpublished), so the HBV RNaseH inhibitors also appear to act by coordinating the catalytic divalent cations. Ongoing biochemical analyses imply that the RNaseH inhibitors are mixed-mode inhibitors whose coordination of the active-site Mg++ ions both interferes with substrate binding and suppresses catalysis (unpublished).

This metal-coordinating mechanism is very similar to that used by the HIV integrase inhibitors Bictegravir, Dolutegravir, Elvitegravir, and Raltegravir. In addition, the Influenza Virus PA cap-snatching endonuclease inhibitor Baloxavir marboxil was just approved by the FDA. Baloxavir inhibits the viral PA endonuclease using the same mechanism used by the HIV and HBV RNaseH active-site inhibitors (Omoto et al., 2018). Therefore, metal chelation is a well-established drug mechanism, and the challenge for HBV RNaseH drug development is one of achieving adequate selectivity for HBV. This challenge is significant given the number of metalloenzymes in a cell, but is similar to challenges faced by drugs that bind to enzymes whose active sites are well conserved among large enzyme families, such as the nucleic acid polymerases or protein kinases that have been successfully targeted.

4.4. Selectivity.

Selectivity of RNaseH inhibitors for HBV over other common microbial pathogens has been measured by counter-screening against growth of the fungus Cryptococcus neoformans (Donlin et al., 2017) and bacteria including Escherichia coli and Staphylococcus spp. (Cao et al., 2018). The HBV RNaseH compounds are overall poorly active against these microbes. For example, A24 (Fig. 4, EC50 = 0.29 μM vs. HBV) is 41-fold less active against C. neoformans than HBV and is inactive against the bacteria. Biochemical selectivity of the inhibitors for the HBV RNaseH has been measured compared to human RNaseH 1 (huRH1), the human enzyme most similar to the HBV RNaseH, and compared to the E. coli RNaseH. Less than 10% of the > 600 compounds screened from the library from which the HBV RNaseH inhibitors were found inhibited the E. coli enzyme at ≤ 100 μM, with only two having IC50s < 10 μM. Similarly, the 540 compounds tested to date have an average 50% inhibitory concentrations (IC50) against huRH1 of 345 μM (range = 4.0 to > 500 μM) (unpublished). Unfortunately, limitations to the recombinant HBV RNaseH prevent determining quantitative IC50 values for evaluation during drug discovery, preventing calculation of a rigorous selectively index. Together, these experiments indicate that RNaseH inhibitors can be selective for HBV at both the microbial and enzymatic levels. Off-target effects of RNaseH inhibitors on human enzymes and on the microbiota must be closely monitored, but selectivity barriers are unlikely to be insurmountable.

4.5. Validation of the HBV RNaseH as a drug target.

HBV RNaseH active site inhibitors are equally effective against recombinant HBV RNaseHs from clinical isolates of 3 genotypes (Lu et al., 2016), indicating that HBV’s high genetic diversity is unlikely to excessively complicate RNaseH drug development. RNaseH inhibitors are synergistic with the nucleoside analog drug LMV and also with RNaseH inhibitors from other chemotypes in HBV replication inhibition experiments with dose-reduction indexes up to 9.5, and they are additive with the capsid assembly modifier Hap12 (Edwards et al., 2019; Lomonosova et al., 2017),.

Two HBV RNaseH inhibitors, the HPD 208 and the αHT 110 (Fig. 4), were tested against HBV replication in chimeric mice (Allweiss and Dandri, 2016) carrying humanized livers. Mice were infected with HBV, treated intraperitoneally daily for 2 weeks, and HBV plasma titers were determined. 208 suppressed HBV replication by 1.3 log10 (p = 0.00003), the αHT 110 reduced HBV titers by 0.4 log10 (p = 0.001), and viral titers rebounded following drug withdrawal as expected for replication antagonists (Long et al., 2018). Although toxicity was observed, it was in part due to formulation as vehicle-treated animals lost 12% of their body weight while on therapy compared to 15% for 208-treated mice. This POC experiment with unoptimized primary screening hits demonstrates that RNaseH inhibitors can work in vivo.

5. Other targets for directly inhibiting HBV replication

HBV’s complex reverse transcription pathway (Fig. 1) presents many unexploited targets for directly inhibiting HBV replication. These include blocking the chaperone-mediated binding of HBV P to ε on the pgRNA, inhibiting protein-priming of reverse transcription, and suppressing any of the 3 strand transfer reactions. Hemin and related porphyrin compounds have been reported to block protein priming (Lin and Hu, 2008), and some efforts have gone into identifying chaperone inhibitors with sufficient efficacy and selectivity for HBV therapy (Wang et al., 2010; Zhou et al., 2012). However, these targets remain poorly explored, in part due to difficulty in designing specific screening assays.

6. Prospects for HBV replication inhibitors to improve patient care

The goal of current HBV drug discovery is to achieve a functional cure (Lok et al., 2017) which allows treatment to be stopped with no risk of virological relapse and no risk of liver disease progression. The role of direct inhibitors of HBV replication in curative therapies will be to eliminate the low-level residual viremia present during current NA therapy to block infection of new cells and to abrogate the recycling pathway to prevent replenishment of the cccDNA in infected cells. It is likely that improved NAs with higher efficacy, high resistance barrier, and outstanding pharmacological and toxicology profiles will be a major part of this effort. However, it is unlikely that targeting just the RT active site will be able to achieve the profound suppression needed to terminate viral replication for the months to years that curative therapies are envisioned to require, or that there will be a “one size fits all” curative therapy in the foreseeable future. This is because HBV patients are very heterogeneous with respect to HBV genotype and strains, disease stage and presentation, co-infection with HIV and Hepatitis C Virus (HCV), geographic location, and economic status.

Curing HBV will almost certainly need combinations of powerful, safe and pan-genotypic antiviral agents with multiple modes of action (Alter et al., 2017; Revill et al., 2019), similar to the multiple cocktails used to treat HIV and HCV. The drug combinations are likely to consist of viral-targeting agents that profoundly suppress viral load and production of viral proteins, coupled with agents that suppress HBsAg levels and help restore the host immune system’s efficacy against HBV. Clinical trials of the new generations of anti-HBV drugs are beginning, and as expected, there have been many failures already, but these trials have provided POC that combining powerful HBV replication inhibitors with host immune modulators can have profound effects on controlling HBV replication (Peters and Locarnini, 2017).

Highlights.

  • NA inhibitors currently dominate HBV therapy

  • Improvement of NAs focuses on efficacy, safety, resistance and prodrug delivery

  • HBV ribonuclease inhibitors are in preclinical development

  • Other approaches for directly inhibiting HBV reverse transcription are possible

Acknowledgements

We thank Arbutus Biopharma for synthesizing the RNaseH inhibitors A23 and A24.

JET is an inventor on patents covering use of RNaseH inhibitors as anti-HBV drugs and is a shareholder and scientific advisor to Casterbridge Pharmaceuticals, Inc. CBD and CPR are shareholders of Ai-biopharma and inventors of HBV inhibitors. JET’s effort in writing this review was supported by grants from the National Institutes of Health [R01 AI122669 and R21 AI124672] and a grant from the US Department of Defense [W81XWH-18-1-0307]. CBD and CPR have nothing to disclose.

Footnotes

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