Recent Advances in Hepatitis B Treatment

pharmaceuticals

Review Recent Advances in Hepatitis B Treatment

Georgia-Myrto Prifti 1, Dimitrios Moianos 1 , Eroﬁli Giannakopoulou 1, Vasiliki Pardali 1, John E. Tavis 2 and Grigoris Zoidis 1,*

1 Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; [email protected] (G.-M.P.); [email protected] (D.M.); [email protected] (E.G.); [email protected] (V.P.) 2 Molecular Microbiology and Immunology, Saint Louis University, Saint Louis, MO 63104, USA; [email protected] * Correspondence: [email protected]; Tel.: +30-210-7274809

Abstract: Hepatitis B virus infection affects over 250 million chronic carriers, causing more than 800,000 deaths annually, although a safe and effective vaccine is available. Currently used antiviral agents, pegylated interferon and nucleos(t)ide analogues, have major drawbacks and fail to completely eradicate the virus from infected cells. Thus, achieving a “functional cure” of the infection remains a real challenge. Recent ﬁndings concerning the viral replication cycle have led to development of novel therapeutic approaches including viral entry inhibitors, epigenetic control of cccDNA, immune modulators, RNA interference techniques, ribonuclease H inhibitors, and capsid assembly modulators. Promising preclinical results have been obtained, and the leading molecules under development have entered clinical evaluation. This review summarizes the key steps of the HBV life cycle, examines the currently approved anti-HBV drugs, and analyzes novel HBV treatment regimens.

Citation: Prifti, G.-M.; Moianos, D.; Keywords: hepatitis B; hepatitis B virus; HBV; antiviral agents; HBV inhibitors; cccDNA; HBV life Giannakopoulou, E.; Pardali, V.; Tavis, cycle; HBV treatment; nucleoside analogues; antiviral therapy J.E.; Zoidis, G. Recent Advances in Hepatitis B Treatment. Pharmaceuticals 2021, 14, 417. https://doi.org/10.3390/ph14050417 1. Introduction Hepatitis B is a liver disease caused by the Hepatitis B Virus (HBV). HBV belongs to Academic Editor: Juan Carlos Saiz the Hepadnaviridae family and is classiﬁed into ten genotypes (A to J) [1]. It is transmitted by exposure to infectious blood or other body ﬂuids (e.g., semen, vaginal secretions—sexual Received: 30 March 2021 intercourse) as well as perinatally from infected mothers to infants [2]. The acute phase Accepted: 23 April 2021 Published: 1 May 2021 of the infection can be either symptomatic or asymptomatic. Acute infections can either spontaneously resolve or proceed to chronic infections. Chronic HBV infection is among

Publisher’s Note: MDPI stays neutral the leading causes of hepatic cirrhosis and is the single largest cause of hepatocellular with regard to jurisdictional claims in carcinoma (HCC). According to the World Health Organization (WHO), over 250 million published maps and institutional affil- people are chronically infected, and HBV caused 887,000 deaths in 2015 [3]. The highest iations. epidemic prevalence is present in SE Asian, African, and Western Pacific countries [4]. The hepatitis B surface antigen (HBsAg), originally known as “Australia antigen” (AusAg), was firstly identified in the serum of indigenous Australians by Baruch Samuel Blumberg in 1965 [5]. This antigen was later related with viral hepatitis [6]. The goal of the current therapeutic development is a “functional cure” defined as Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. sustained undetectable levels of HBsAg and HBV DNA in serum, with or without sero- This article is an open access article conversion to hepatitis B surface antibodies (anti-HBs) after the end of the treatment [7]. distributed under the terms and This reduction has been associated with an improved clinical condition and significantly conditions of the Creative Commons decreased the chance of infection rebound. Other important HBV biomarkers include Attribution (CC BY) license (https:// serum HBV DNA, hepatitis B core antigen (HBcAg), and its antibody anti-HBc, hepati- creativecommons.org/licenses/by/ tis B e antigen (HBeAg), and anti-HBe antibody [8–10]. HBeAg is a secreted variant of 4.0/). HBcAg, and viral infections are classified either as HbeAg-positive or HbeAg-negative,

Pharmaceuticals 2021, 14, 417. https://doi.org/10.3390/ph14050417 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 27

e antigen (HBeAg), and anti-HBe antibody [8–10]. HBeAg is a secreted variant of HBcAg, and viral infections are classified either as HbeAg-positive or HbeAg-negative, with HBeAg-positive patients having higher viral titers and a more frequent and rapid disease Pharmaceuticals 2021, 14, 417 progression [11]. These biomarkers are used to guide treatment decisions following2 of 27 guidelines established by the major hepatology medical societies [12–14]. Despite the existence of a safe and effective vaccine, no therapeutic regimen that rou- tinelywith HBeAg-positiveinduces a “functional patients cure” having for higher chronic viral HBV titers has and been a more identified frequent yet. and rapidThis review summarizesdisease progression the HBV [11 replication]. These biomarkers cycle, the are existing used to guide treatment treatment options decisions and followingtheir significant disadvantages,guidelines established and novel by the therapeutic major hepatology approaches medical that societies are currently [12–14]. the subject of extensive scientificDespite theresearch, existence with of athe safe ultimate and effective goal of vaccine, achieving no therapeutica “functional regimen cure” thatof the dis- routinely induces a “functional cure” for chronic HBV has been identified yet. This review ease. summarizes the HBV replication cycle, the existing treatment options and their significant disadvantages, and novel therapeutic approaches that are currently the subject of extensive 2.scientific HBV Replication research, with Cycle the ultimate goal of achieving a “functional cure” of the disease. 2.1. Virion Structure and Genome 2. HBV Replication Cycle 2.1.HBV Virion particles, Structure and also Genome known as Dane particles (Figure 1A), were firstly identified by Dane and colleagues in 1970 [15]. Their shape is spherical, with a diameter of ∼42 nm. HBV particles, also known as Dane particles (Figure1A), were firstly identified by TheyDane consist and colleagues of an outer in 1970 envelope, [15]. Their which shape is a is host spherical,-derived with lipid a diameter bilayer ofcontaining∼42 nm. three differentThey consist-sized of HBV an outer surface envelope, antigens which (HBsAg is a host-derived or HBs)— lipidlarge bilayer (L-HBs), containing middle three (M-HBs) anddifferent-sized small (S-HBs) HBV— surfacesurrounding antigens the (HBsAg viral ornucleocaps HBs)—largeid. (L-HBs), The nucleocapsid middle (M-HBs) (∼27 and nm diameter)small is (S-HBs)—surroundingicosahedral and comprises the viral the nucleocapsid. HBV core protein The nucleocapsid (HBcAg), (as∼ 27well nm as diameter) the viral DNA genomeis icosahedral and the and viral comprises DNA polymerase the HBV core (P) protein [16,17]. (HBcAg), The virus as also well secretes as the viral a wide DNA range of defectivegenome andparticles the viral (Figure DNA 1B) polymerase, including (P) e [16nveloped,17]. The nucleocapsids virus also secretes that a are wide empty range or con- tainof defective defective particles immature (Figure genomes1B), including and subviral enveloped lipid nucleocapsids particles containing that are emptythe viral or surface antigens.contain defective The subviral immature particles genomes are secreted and subviral along lipidwith particlesthe infectious containing virions the at viral levels that surface antigens. The subviral particles are secreted along with the infectious virions at are thousands of times higher, and they play an important role in suppressing antibody levels that are thousands of times higher, and they play an important role in suppressing responsesantibody responsesto the virus to the [18]. virus [18].

FigureFigure 1. 1. HepatitisHepatitis B B Virus Virus particles. (AA)) InfectiousInfectious HBV HBV virion virion (Dane (Dane particle). particle). The The lipid lipid enve- envelope, bearinglope, bearing three threetypes types of surface of surface proteins proteins—small—small (S- (S-HBs),HBs), mid middledle (M (M-HBs)-HBs) and and large (L-HBs)—(L-HBs)—sur- roundssurrounds the nucleocapsid, the nucleocapsid, consisting consisting of ofHBV HBV relaxed relaxed circular circular DNA DNA (rcDNA), (rcDNA), the the viral viral DNA DNA poly- polymerase (P), and the core protein (HBcAg). (B) Non-infectious HBV particles; enveloped nucleocapsids containing immature or defective DNA/RNA, subviral particles, and naked nucleocapsids.

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merase (P), and the core protein (HBcAg). (B) Non-infectious HBV particles; enveloped nucleocapsids containing immature or defective DNA/RNA, subviral particles, and naked nucleocapsids.

The HBV genome is a 3.2 kb circular, partially double-stranded DNA (relaxed circular DNA; rcDNA). The negative-sense, non-coding (−) DNA strand is complete and complementary to the mRNA transcripts, whereas the positive (+) DNA strand is incomplete

Pharmaceuticals 2021, 14, 417 and has a fixed 5′-end and a variable-size 3′-end [19–321]. of 27 The former contains four overlapping open reading frames (ORFs)—C, P, S, and X (Figure 2). These are transcribed into five RNA transcripts of varying lengths and are subsequently translated into seven func- The HBV genome is a 3.2 kb circular, partially double-stranded DNA (relaxed circular tionalDNA; rcDNA). proteins. The negative-sense, HBcAg is non-coding produced (−) DNA from strand ORF is complete-C, HBeAg and comple- is produced from ORF preC + C, mentary to the mRNA transcripts, whereas the positive (+) DNA strand is incomplete and DNAhas a ﬁxed polymerase 50-end and a variable-size from ORF 30-end [P19,– and21]. The HBV former X contains protein four overlapping (HBx) from ORF X. The ORF S, because ofopen its readingmultiple frames in (ORFs)—C,-frame P, start S, and codons, X (Figure2). encodes These are transcribed the L-HBs, into ﬁve M-HBs, and S-HBs envelope pro- RNA transcripts of varying lengths and are subsequently translated into seven functional teinsproteins. (pre HBcAg-S1 is + produced pre-S2 from + ORF-C,S, pre HBeAg-S2 + is S, produced or S, from respectively) ORF preC + C, DNA [17,19]. This compact nature of the polymerase from ORF P, and HBV X protein (HBx) from ORF X. The ORF S, because of its HBVmultiple genome in-frame start results codons, encodesin approximately the L-HBs, M-HBs, andtwo S-HBs thirds envelope of proteins nucleotides encoding more than one functional(pre-S1 + pre-S2 element + S, pre-S2 +[22,23]. S, or S, respectively) The overlap [17,19]. Thisof more compact than nature of1000 the nucleotides between the P and HBV genome results in approximately two thirds of nucleotides encoding more than one Sfunctional genes elementis the [ 22largest,23]. The overlapgene of overlap more than 1000 of nucleotidesany known between animal the P and S virus [24]. genes is the largest gene overlap of any known animal virus [24].

Figure 2. Hepatitis B Virus genome. Partially double-stranded, relaxed circular DNA (rcDNA) with Figurefour overlapping 2. Hepatitis open reading B framesVirus (ORFs). genome. Partially double-stranded, relaxed circular DNA (rcDNA) with2.2. Viral four Entry overlapping open reading frames (ORFs). The HBV virion binds to the heparan sulfate proteoglycans (HSPGs) cell-surface receptors, via low-affinity and non-specific interactions. Afterwards, the Na(+)-taurocholate 2.2.co-transporting Viral Entry polypeptide (NTCP) functions as a high affinity receptor for the recognition and attachment of the pre-S1 domain of L-HBsAg. NTCP is a liver-specific peptide that mediatesThe the HBV uptake virion of bile salts binds into hepatocytes, to the heparan and it is also sulfate an entry receptor proteoglycans for (HSPGs) cell-surface re- Hepatitis D virus (HDV) [17,25]. Interactions between HBV and NTCP are responsible for ceptors,the viral endocytosis via low (Figure-affinity3). According and tonon recent-specific studies, a complex interactions. formed between Afterwards, the Na(+)-taurocholate co-transporting polypeptide (NTCP) functions as a high affinity receptor for the recognition and attachment of the pre-S1 domain of L-HBsAg. NTCP is a liver-specific peptide that mediates the uptake of bile salts into hepatocytes, and it is also an entry receptor for Hepatitis D virus (HDV) [17,25]. Interactions between HBV and NTCP are responsible for the viral endocytosis (Figure 3). According to recent studies, a complex formed between NTCP and the epidermal growth factor receptor (EGFR) contributes to the HBV entry [26]. Due to its complicated structure, NTCP can be oligomerized, and this process seems to affect the viral internalization into the cell. Following entry, the nucleocapsid is released into the cytoplasm, followed by uncoating, and then the rcDNA is transported to the nucleus via the nuclear pore complexes [17,19,27].

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HBV replicates by reverse transcription. The RT activity of the P protein primes DNA synthesis using a tyrosine in the TP domain, covalently linking the enzyme to the product DNA. P then catalyzes the synthesis of the (−) DNA strand, which is the pattern for the (+) DNA strand synthesis, to form double-stranded rcDNA and mature DNA nucleocapsids [19,60]. During the (−) DNA strand synthesis, the RNaseH domain degrades the pgRNA template inside the capsids after it is copied into the minus-polarity DNA [61,62]. Either the newly formed mature nucleocapsids are surrounded by HBsAg and secreted non-cytolytically as virions that can infect new hepatocytes or they can re-enter the nucleus to maintain the cccDNA reservoir (Figure 3). This intracellular cccDNA recycling is likely one factor that makes the complete elimination of the HBV infection in a patient so diffi- cult. Smaller, non-infectious subviral particles (∼22 nm in diameter) are also released from the hepatocytes in vast excess over infectious virions [17]. These include empty envelopes of HBsAg (subviral particles), virions containing RNA, or a defective DNA genome, as well as naked nucleocapsids (lacking envelope) (Figure 1) [63]. These defective particles Pharmaceuticals 2021, 14, 417 do not participate in viral replication, although the subviral HBsAg particles4 of 27 help suppress antiviral immunity [18]. The dslDNA is an aberrant reverse transcription product that is able to integrate into the cellularNTCP and genome the epidermal early growthafter the factor initial receptor HBV (EGFR) infection contributes, and to it the has HBV been entry associated [26]. with promotingDue to itsthe complicated development structure, of HCC. NTCP The can integrated be oligomerized, HBV and DNA this processdoes not seems replicate, to but it affect the viral internalization into the cell. Following entry, the nucleocapsid is released contributesinto the to cytoplasm, HBsAg followedexpression, by uncoating, which contributes and then the to rcDNA HBV pathogenesis is transported toand the modulates the immunenucleus via response the nuclear [64]. pore complexes [17,19,27].

Figure 3. MainFigure features 3. Main features of the ofhepatitis the hepatitis B virus B virus replication replication cycle cycle andand potential potential therapeutic therapeutic targets. targets. (1) HBV (1) entry HBV inhibitors. entry inhibitors. Lipopeptides mimicking the pre-S1 region of HBV, monoclonal antibodies, and other small molecules under evaluation. Lipopeptides mimicking the pre-S1 region of HBV, monoclonal antibodies, and other small molecules under evaluation. (2) Targeting cccDNA. Damage and destruction of cccDNA via sequence-speciﬁc nucleases. Direct targeting of the HBx (2) Targeting cccDNA. Damage and destruction of cccDNA via sequence-specific nucleases. Direct targeting of the HBx protein. (3) RNA interference. Small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs). (4) HBV polymerase protein. (3)inhibitors. RNA interference. Reverse transcriptase Small interfering inhibitors (nucleos(t)ide RNAs (siRNAs), analogues) antisense are part of oligonucleotides the current treatment. (ASOs). RNaseH (4) inhibitors HBV polymerase are in preclinical evaluation. (5) Nucleocapsid assembly inhibitors or modulators can affect HBV capsid formation, reverse transcription, and pgRNA encapsidation. NTCP; Na(+) taurocholate co-transporting polypeptide, HSPG; heparan sulfate proteoglycan, rc-DNA; relaxed circular DNA, PF-rcDNA; protein-free rcDNA, cccDNA; covalently closed circular DNA, pgRNA; pregenomic RNA, preC; precore, mRNA; messenger RNA, P; polymerase, L-HBs; large hepatitis B surface protein, M-HBs; middle hepatitis B surface protein, S-HBs; small hepatitis B surface protein, HBx; hepatitis B X protein, HBsAg; hepatitis B surface antigen, HBeAg; hepatitis B e antigen, dslDNA; double-stranded linear DNA. Pharmaceuticals 2021, 14, 417 5 of 27

2.3. cccDNA Formation/Maintenance Multiple cellular factors repair the HBV rcDNA to form the episomal covalently closed circular DNA (cccDNA) that is located in the nucleus (Figure3). Both the viral P protein which is bound to the 50-end of the minus-polarity DNA strand [28] and the RNA primer attached to the 50-end of the plus DNA strand are removed to leave a protein-free rcDNA (PF-rcDNA). The gaps in both strands are filled and circularized to form the cccDNA. Cellular factors believed to be involved in this process include the DNA repair enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) by presumably breaking the phosphodiesterase bond between the HBV P and rcDNA [29,30]. Another enzyme that breaks down the RNA primer at the 50-end of the minus strand is the flap endonuclease 1 (FEN1) [31]. After removal of the proteins, the fill-in of the strands, DNA ligation, and DNA repair are conducted by other host enzymes such as DNA polymerases (κ and α), DNA ligases (LIG1 and LIG3), and topoisomerases I and II (TOP1 and TOP2) [17]. However, there is some functional redundancy among these factors, and it is not fully clear which of them function in an infected liver. The cccDNA is the template for transcription of viral RNAs. The stability of cccDNA is regulated by several cellular factors, such as the APOBEC3 protein family, that triggers cccDNA degradation [32,33]. The cccDNA is rather stable during antiviral therapy, declining by only ~1 log10 after more than a year of nucleos(t)ide analogue therapy [34]. However, the half-life of cccDNA has been measured at only approximately 40 days in HepG2 cells [17,35], and studies of cccDNA replacement during the reversion of nucleoside analogue resistance following the cessation of the therapy indicate that the cccDNA half-life in the liver is 16–28 weeks [36].

2.4. Transcription-Translation-Reverse Transcription-Nucleocapsid Assembly Using the cccDNA as a template, the host RNA polymerase II transcribes five RNAs of different lengths: three subgenomic mRNAs of 0.7, 2.1, and 2.4 kb and two longer than genomic mRNAs of 3.5 kb (Figure3). All of them are heterogenous, positively orientated, and have a 50-cap and a 30-polyadenylated tail [21]. The 3.5 kb pregenomic RNA (pgRNA) has two functions: it is the template for reverse transcription to generate the minus DNA strand and also the mRNA for the translation of the core protein and HBV P. The preC mRNA is slightly longer than the pgRNA. It contains an open reading frame that starts about 90 nucleotides upstream of the HBc ORF on the 3.5 kb pre-C mRNA and encodes the precore protein, which is converted to HBeAg upon post-translational processing in the endoplasmic reticulum (ER). The 2.4 kb mRNA encodes the L-HBs, whilst both M-HBs and S-HBs are encoded by the 2.1 kb transcript. The shortest transcript encodes the HBx protein [37–39]. The transcription process is regulated by four promoters (precore/core, pre-S1, pre-S2, and X) and two enhancers (Enh1 and Enh2), as well as several cis-acting negative regulatory elements [19,40–43]. HBx is the only purely regulatory protein encoded by HBV and has a multifunctional role. HBx promotes the degradation of the Smc5/6 complex (structural maintenance of chromosomes) host factor, thus enhancing the transcription of cccDNA [44–47]. Moreover, HBx represses development of the immune response to HBV infection, protecting the infected hepatocytes from immune-mediated apoptosis, and interferes with the host gene expression, facilitating the development of HCC [48–50]. HBV P contains four domains: the terminal protein (TP), the spacer, the reverse transcriptase (RT) domain, and the ribonuclease H (RNaseH) domain [51,52]. The pgRNA binds to P via the ε-stem loop located close to its 50-end with specific motifs in the TP, spacer, RT, and RNaseH domains to form a pgRNA-P ribonucleoprotein (RNP) complex [53–57]. This interaction is of great importance, as it is essential for the RNA packaging into nucleocapsids and initiation of reverse transcription [58,59]. Specifically, the RNP complex is packaged within HBcAg to form immature nucleocapsids, where reverse transcription occurs, producing either rcDNA, or less often, double-stranded linear DNA (dslDNA) forms (Figure3). Pharmaceuticals 2021, 14, 417 6 of 27

HBV replicates by reverse transcription. The RT activity of the P protein primes DNA synthesis using a tyrosine in the TP domain, covalently linking the enzyme to the product DNA. P then catalyzes the synthesis of the (−) DNA strand, which is the pattern for the (+) DNA strand synthesis, to form double-stranded rcDNA and mature DNA nucleocapsids [19,60]. During the (−) DNA strand synthesis, the RNaseH domain degrades the pgRNA template inside the capsids after it is copied into the minus-polarity DNA [61,62]. Either the newly formed mature nucleocapsids are surrounded by HBsAg and secreted non-cytolytically as virions that can infect new hepatocytes or they can re- enter the nucleus to maintain the cccDNA reservoir (Figure3). This intracellular cccDNA recycling is likely one factor that makes the complete elimination of the HBV infection in a patient so difﬁcult. Smaller, non-infectious subviral particles (∼22 nm in diameter) are also released from the hepatocytes in vast excess over infectious virions [17]. These include empty envelopes of HBsAg (subviral particles), virions containing RNA, or a defective DNA genome, as well as naked nucleocapsids (lacking envelope) (Figure1)[ 63]. These defective particles do not participate in viral replication, although the subviral HBsAg particles help suppress antiviral immunity [18]. The dslDNA is an aberrant reverse transcription product that is able to integrate into the cellular genome early after the initial HBV infection, and it has been associated with promoting the development of HCC. The integrated HBV DNA does not replicate, but it contributes to HBsAg expression, which contributes to HBV pathogenesis and modulates the immune response [64].

3. Current Therapies Two types of treatment are currently available against hepatitis B viral infection, interferon α derivatives (IFNs), and nucleos(t)ide analogues (NAs). Interferon α (IFN-α) was first approved for the treatment of HBV infection in 1991 [65]. However, the addition of a polyethylene glycol chain to IFN-α led to significantly improved pharmacological properties. Thus, IFN-α was replaced by its pegylated counterpart, PEG- IFN-α, in 2005. There are two forms of PEG-IFN-α available today, PEG-IFN-α2a (Pegasys©, Roche) and PEG-IFN-α2b (Pegintron©, Merck). They have improved pharmacokinetics and allowed for a longer half-life, enabling a weekly administration [66]. PEG-IFN-α is administered subcutaneously and has direct antiviral as well as immunomodulatory activity [67–70]. One year of PEG-IFN-α treatment in HbeAg-positive patients led to HbeAg seroconversion in 29–32% of the patients and sustainable reduced HbsAg levels in 3–7% of the patients, 24 weeks after the end of the treatment, highlighting the effectiveness of PEG-IFN-α against HBV [2,71]. Nevertheless, the PEG-IFN-α treatment causes adverse reactions including flu-like symptoms, bone marrow suppression, fatigue, and depression, and is contraindicated for patients suffering from hepatic failure or cirrhosis [2,72]. Patient compliance is also low due to the subcutaneous administration. Nucleoside analogues (Figure4) inhibit the HBV reverse transcriptase activity and therefore block HBV DNA replication. The active form of most of these drugs is the triphosphate that results from their phosphorylation by hepatocyte kinases. Nucleoside triphosphate analogues are substrates for the RT. During reverse transcription, they act as immediate or delayed transcriptional terminators and prevent the synthesis of both (−) and (+) HBV DNA strands. They are administered per os, having acceptable pharmacokinetics and limited drug-drug interactions. NAs suppress viremia at clinically undetectable levels in up to 76% of HBeAg (+) and 93% of HBeAg (−) patients after one year of treatment. Efficacy can vary in patients with different HBV genotypes [73,74]. Although some HBeAg (−) patients can discontinue treatment with NAs, their use is essentially life-long for the large majority of patients. However, virological relapse almost always occurs. Eight NAs have been approved against the HBV, of which the current recommended ones are entecavir and the two tenofovir prodrugs, disoproxil and alafenamide [73]. Pharmaceuticals 2021, 14, 417 7 of 27 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 27

(i) Lamivudine (ii) Adefovir Dipivoxil (iii) Telbivudine

(vi) Tenofovir Disoproxil (iv) Entecavir (v) Clevudine Fumarate

(ix) Besifovir Dipivoxil Male- (vii) Tenofovir Exalidex (viii) Tenofovir Alafenamide Fumarate ate

Figure 4. Nucleos(t)ide Analogues Analogues (NAs) (NAs) approved approved fo forr the the treatment treatment of of hepatitis hepatitis BB [28,73]. [28,73].

4. NovelThe Therapeutic first approved Strategies NA which was effective against HBV was lamivudine (3TC, LMV, © © © © EpivirMajor, Zeffix scientific, Heptodin breakthroughs,, Hepitec such). It as was the approved identification in the of United the NTCP States cell of surface America receptor,in 1998 [73 detailed], and it knowledge is administered gained once about daily, cccD withNA fewformation, side effects. regulation It is no and longer its epige- widely neticused control, because the it is mechanism less potent thanof cccDNA newer drugsand pgRNA and most degradation, patients develop and the resistance determination within ofone the to HBx five protein’s years [65 role]. Data in viral from transcription, a randomized have controlled enabled an trial in-depth showed understanding that treatment withof the LMV HBV forlife acycle. median In addition, duration innovative of approximately cell and 32 animal months models reduced have the improved frequency the of inHCC vitro occurrence and in vivo [75]. assessment As shown inof anotherthe antiviral study, activity receiving and LMV potential reduced toxicity the risk of ofnovel HCC compounds.even in patients All of with the liverabove cirrhosis have paved [76]. the The way long-term for investigating use of LMV multiple is limited new thera- by the peuticdevelopment targets ofthat resistance will lead associated to substantial with progress mutations toward in the achieving YMDD (tyrosine-methionine- a functional HBV cureaspartic [7,95]. acid-aspartic acid) motif in the viral RT active site. A study carried out by Kwon et al. in 2013 [77] suggests that treatment in patients without mutations in this region may 4.1.be continued HBV Entry for Inhibitors more than five years until the complete loss of HBsAg is achieved [77]. However,The discovery the sustained of NTCP viral responseas the entry obtained receptor with for LMV HBV for provided more than key five knowledge years showed on theno furtherviral entry decrease mechanism, in the incidence thus facilitating of HCC the [75 identification]. In the opposite of a direction,variety of Euncompounds et al. [78 ] found that long-term LMV administration and subsequent prolonged viral suppression had a beneficial impact on the risk of HCC [75]. Lamivudine therapy has been confirmed to reduce liver-related mortality in patients with HBV and even in patients with co-infection

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with human immunodeficiency virus (HIV), especially along with other NA as combination therapy [75,79]. The next approved NA was adefovir dipivoxil (bis(POM) PMEA, ADV, Hepsera© or Preveon©) in 2002 [73]. Given once daily, it has shown only few side effects; however, the renal function should be monitored to avoid the development of renal impairment. It is considered a second-line treatment option, except for the case of LMV resistance, where it is used as the drug of choice [65]. Although ADV monotherapy is effective in HBV patients and its long-term use reduced the rate of liver fibrosis, resistant mutations conferred decreased susceptibility to ADV [75]. Entecavir (BMS-200475-01, ETV, Baraclude©) was approved in 2005 [73]. It is taken once daily and causes few side effects. It is a first-line treatment with exceptional resistance profile [65], and it has been proved that it reduces the incidence of HCC [80]. The monitoring of serum alanine aminotransferase (ALT), an enzyme released by dead hepatocytes, is recommended at 6 and 12 months of treatment with ETV, since normal ALT levels are related to a reduced risk of developing HCC. Furthermore, the follow-up monitoring of serum alpha-fetoprotein as a biomarker for HCC is suggested [75]. Telbivudine (LdT, TBV, Tyzeka© or Sebivo©) was approved in 2006 as a second- line treatment option [65]. Randomized clinical trials revealed that TBV is superior to lamivudine and adefovir in the treatment of patients with chronic HBV, regardless of the HBeAg detection [81–83]. In 2013, Tsai et al. [84] found that the cumulative incidence of HCC in patients who had received telbivudine was 2.5% and 4.1% at two and three years, respectively, rates similar to that of entecavir administration (3.1% and 7.5% in two and three years, respectively). TBV is associated with few side effects, including muscle toxicity and peripheral neuropathy [85,86]. Renal function should be considered when choosing between NAs, and it is worth noting that TBV can prevent nephrotoxicity [75]. At the same year, clevudine (L-FMAU, CLV, Levovir© or Revovir©) was approved in South Korea and the Philippines. It was soon recalled due to the induction of skeletal myopathy caused by mitochondrial dysfunction [73]. Tenofovir Disoproxil Fumarate (bis(POC) PMPA Fumarate, TDF, Viread©) was first released in 2008. It is taken once daily and has few serious adverse effects, including dose-limiting renal toxicity. Although being a first-line treatment, TDF is also effective as a second-line rescue treatment after therapy with other nucleos(t)ide analogues has failed due to resistance evolution [65,75]. To a great extent, TDF is not susceptible to resistance development, and thus its use provides sufficient virological suppression [87]. Some studies demonstrate that patients receiving TDF have a lower incidence of HCC compared with patients receiving entecavir [75,88–90]. Contrarily, other studies indicate that both tenofovir and entecavir monotherapies display a comparable risk for HCC [91,92]. Tenofovir alafenamide fumarate (GS-7340-03, TAF, Vemlidy©) was first released in 2016, and it was developed to tackle the dose-limiting renal toxicity of TDF. The primary purpose of another analogue, tenofovir exalidex (a prodrug which is in early clinical development) is to improve the safety compared to formulations of TDF [93]. In 2017 another analogue was discovered, besifovir dipivoxil maleate (ANA-380/LB80380 maleate, BSV dipivoxil maleate, Besivo©), showing significantly reduced bone and kidney toxicity, compared to tenofovir [73,93]. Overall, NAs which are administered per os, require long-term duration of therapy, achieve better control of HBV replication, and show many fewer side effects compared to PEG-IFN-α. Treatment with PEG-IFN-α is shorter in duration and can lead to stable, off-treatment multi-log10 reductions in viral titers in about 30% of patients [2], but it is not well tolerated in many patients because of severe side effects [7,94]. Pharmaceuticals 2021, 14, 417 9 of 27

4. Novel Therapeutic Strategies Major scientiﬁc breakthroughs, such as the identiﬁcation of the NTCP cell surface receptor, detailed knowledge gained about cccDNA formation, regulation and its epigenetic control, the mechanism of cccDNA and pgRNA degradation, and the determination of the HBx protein’s role in viral transcription, have enabled an in-depth understanding of the HBV life cycle. In addition, innovative cell and animal models have improved the in vitro and in vivo assessment of the antiviral activity and potential toxicity of novel compounds. All of the above have paved the way for investigating multiple new therapeutic targets that will lead to substantial progress toward achieving a functional HBV cure [7,95].

4.1. HBV Entry Inhibitors The discovery of NTCP as the entry receptor for HBV provided key knowledge on the viral entry mechanism, thus facilitating the identification of a variety of compounds that block the viral entry into the host hepatocytes [96]. As mentioned in Section 2.2, interactions between the pre-S1 domain of L-HBsAg and NTCP are the key process for viral entry [97]. Various strategies have been proposed for the inhibition of HBV entry into hepatocytes. Small, acetylated peptides derived from the pre-S1 domain of L-HBs can effectively inhibit viral entry, exhibiting promising results both in vitro and in vivo [98–100]. In a recent report, novel cyclic peptides led to a significant HBsAg loss in vivo, with IC50 values between 0.66 and 2.54 µM, without affecting the physiological function of the NTCP receptor [101]. Another study revealed that peptide 4B10 was able to inhibit HBV infection in a human hepatocyte culture, with IC50 values in the nM range and no observed cytotoxicity [102]. The most important compound of this category is Myrcludex B (also known as Bulevirtide). Myrcludex B is a synthetic myristoylated lipopeptide consisting of 47 amino acids of the pre-S1 region. It strongly inhibits the HBV entry in the cell culture (IC50 = 80 pM) and in a uPA/SCID humanized mouse model of HBV infection [103–105]. Moreover, Myrcludex B inhibits bile salt uptake only at much higher concentrations (IC50 = 52.5 nM) [106]. The safety and efficacy results from clinical trials IIb are also excellent [107,108]. A liposomal formulation of Myrcludex B is under development for per os administration with an excellent pharmacological drug-drug interaction profile [109,110]. Several FDA-approved compounds have recently been identified as efficient inhibitors of the NTCP-L-HBs interaction. Those include the immunosuppressant cyclosporin A and its derivatives [106,111,112], the antihyperlipidemic ezetimibe [113], the angiotensin II receptor antagonist irbesartan [114], and the immunosuppressant rapamycin [115], among many drugs already in clinical use [116–118]. Another study identified that the green tea flavonoid epigallocatechin-3-gallate can efficiently block the NTCP-mediated viral entry [119]. Other recently identified HBV entry inhibitors include zafirlukast, vanitaracin A, proanthocyanidin, and its analogues, betulin derivatives, and novel synthetic compounds like B7 (Table1)[117,120–124]. Pharmaceuticals 2021, 14, 417 10 of 27 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 11 of 27 PharmaceuticalsPharmaceuticals 20212021,, 1414,, xx FORFOR PEERPEER REVIEWREVIEW 1111 ofof 2727 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 11 of 27

Table 1. Compounds that inhibit HBV entry in hepatocytes and their in vitro IC50 values for HBV infection, measured in Table 1. Compounds that inhibit HBV entry in hepatocytes and their in vitro IC50 values for HBV Table 1. Compounds that inhibit HBV entry in hepatocytes and their in vitro IC50 values for HBV HepG2 cell cultures. infection,Table 1. Compounds measured in that HepG2 inhibit cel lHBV cultures. entry in hepatocytes and their in vitro IC50 values for HBV Tableinfection, 1. Compounds measured in that HepG2 inhibit cel HBVl cultures. entry in hepatocytes and their in vitro IC50 values for HBV infection,infection, measuredmeasured inin HepG2HepG2 celcelll cultures.cultures. infection, measured in HepG2 cell cultures. 1 Name Structure Class IC 50Ref.1 Name Structure Class 50 IC50 1 Ref. Name Structure Class IC50 1 Ref. Name Structure Class IC50 1 Ref. Name Structure Class IC50 1 Ref.

Cholesterol absorption 2 2 EzetimibeEzetimibe Cholesterol absorption inhibitor18 µM 18 μΜ[113 2 ] [113] EzetimibeEzetimibe CholesterolCholesterolinhibitor absorptionabsorption inhibitorinhibitor 1818 μΜμΜ 2 [113][113] Ezetimibe Cholesterol absorption inhibitor 18 μΜ 2 [113]

(−(−)-epigallocatechin-3-)-epigallocatechin- Flavonoid in green tea ((−−))--epigallocatechinepigallocatechin-- Flavonoid in green tea extract≈10 µM[ ≈10 μ119M ] [119] (−)-epigallocatechingallate3-gallate - FlavonoidFlavonoidextract inin greengreen teatea extractextract ≈10≈10 μμMM [119][119] 33--gallategallate Flavonoid in green tea extract ≈10 μM [119] 3-gallate

OligomericOligomeric ﬂavonoid, flavonoid, derived Proanthocyanidin OligomericOligomeric flavonoid,flavonoid, derivedderived 7.8 μM [122] ProanthocyanidinProanthocyanidinProanthocyanidin derivedOligomericfrom from extract extract flavonoid, of of grape derived seed7.8 µ M[ 7.87.8 μμM122M ] [122][122] Proanthocyanidin fromfromgrape extractextract seed ofof grapegrape seedseed 7.8 μM [122] from extract of grape seed

Oolonghomobisfla- Oolonghomobisfla-Oolonghomobisfla- ProanthocyanidinProanthocyanidin analogue 4.3 μM [122] OolonghomobisﬂavanOolonghomobisfla-van C C ProanthocyanidinProanthocyanidin analogueanalogue4.3 µ M[ 4.34.3 μμM122M ] [122][122] vanvan CC Proanthocyanidinanalogue analogue 4.3 μM [122] van C

Rapamycin derivative and pro- Temsirolimus RapamycinRapamycin derivativederivative andand pro-pro- 7.86 μM [115] TemsirolimusTemsirolimus RapamycinRapamycin derivative derivativedrug and pro- 7.867.86 μμMM [115][115] TemsirolimusTemsirolimus drugdrug 7.86 µM[7.86 μ115M ] [115] and prodrugdrug

Compound 2 Betulin derivative 4 μM [123] CompoundCompound 22 BetulinBetulin derivativederivative 44 μμMM [123][123] CompoundCompound 2 2 BetulinBetulin derivative derivative 4 µM[4 μM123 ] [123]

Pharmaceuticals 2021, 14, 417 11 of 27

Pharmaceuticals 2021, 14, x FOR PEER REVIEW 12 of 27 Pharmaceuticals 2021,, 14,, xx FORFOR PEERPEER REVIEWREVIEW Table 1. Cont. 12 of 27 PharmaceuticalsPharmaceuticals 2021 2021, ,14 14, ,x x FOR FOR PEER PEER REVIEW REVIEW 1212 of of 27 27 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 12 of 27 Pharmaceuticals Pharmaceuticals 2021 2021, 14, 14, x, FORx FOR PEER PEER REVIEW REVIEW 1 1212 of of27 27 Name Structure Class IC50 Ref.

CyclosporinCyclosporin A A ImmunosuppressantImmunosuppressant 1.17 µM[1.17 μ111M ] [111] CyclosporinCyclosporin AA ImmunosuppressantImmunosuppressant 1.171.17 μμMM [111][111] CyclosporinCyclosporin A A ImmunosuppressantImmunosuppressant 1.171.17 μ μMM [111][111] CyclosporinCyclosporin A A ImmunosuppressantImmunosuppressant 1.171.17 μ MμM [111][111]

ThiazolidinedioneThiazolidinedione (PPAR-γ ago- RosiglitazoneRosiglitazone ThiazolidinedioneThiazolidinedione (PPAR(PPAR--5.1γγ ago-ago-µM[5.1 μM117 ] [117] Rosiglitazone (PPAR-Thiazolidinedioneγ agonist)nist) (PPAR-γ ago- 5.1 μM [117] RosiglitazoneRosiglitazone ThiazolidinedioneThiazolidinedionenist) (PPAR (PPAR-γ- γago- ago- 5.15.1 μ μMM [117][117] RosiglitazoneRosiglitazone Thiazolidinedionenist)nist) (PPAR -γ ago- 5.15.1 μ MμM [117][117] Rosiglitazone nist)nist) 5.1 μM [117] nist) Thiazolidinedione (PPAR-γ ago- Ciglitazone ThiazolidinedioneThiazolidinedione (PPAR-γ ago- 4.7 μM [116] CiglitazoneCiglitazone ThiazolidinedioneThiazolidinedionenist) (PPAR (PPAR-4.7-γγ ago- µago-M[4.7 μM116 ] [116] CiglitazoneCiglitazone Thiazolidinedione(PPAR-Thiazolidinedioneγ agonist)nist) (PPAR (PPAR-γ- γago- ago- 4.74.7 μ μMM [116][116] CiglitazoneCiglitazone Thiazolidinedionenist)nist) (PPAR -γ ago- 4.74.7 μ MμM [116][116] Ciglitazone nist)nist) 4.7 μM [116] nist)

Irbesartan AngiotensinAngiotensin II receptorII receptor antagonist 3.3 μM [114] IrbesartanIrbesartanIrbesartan AngiotensinAngiotensin IIII receptorreceptor antagonistantagonist3.3 µM[ 3.33.3 μμM114M ] [114][114] IrbesartanIrbesartan AngiotensinAngiotensinantagonist II II receptor receptor antagonist antagonist 3.33.3 μ μMM [114][114] IrbesartanIrbesartan AngiotensinAngiotensin II IIreceptor receptor antagonist antagonist 3.33.3 μ MμM [114][114]

Zafirlukast Leukotriene receptor antagonist 6.5 μM [117] Zafirlukast LeukotrieneLeukotriene receptor receptor antagonist 6.5 μM [117] ZaﬁrlukastZafirlukastZafirlukast LeukotrieneLeukotriene receptor receptor antagonist antagonist6.5 µM[ 6.56.5 μ μ117MM ] [117][117] ZafirlukastZafirlukast LeukotrieneLeukotrieneantagonist receptor receptor antagonist antagonist 6.56.5 μ MμM [117][117] Zafirlukast Leukotriene receptor antagonist 6.5 μM [117]

TRIAC Thyroid hormone analogue 6.9 μM [117] TRIAC Thyroid hormone analogue 6.9 μM [117] TRIAC ThyroidThyroid hormone hormone analogue 6.9 μM [117] TRIACTRIACTRIAC ThyroidThyroid hormone hormone analogue analogue6.9 µ M[ 6.96.9 μ μM117M ] [117][117] TRIACTRIAC ThyroidThyroidanalogue hormone hormone analogue analogue 6.96.9 μ MμM [117][117]

B7 Novel synthetic compound 7.36 μM [124] B7B7 NovelNovel syntheticsynthetic compoundcompound 7.367.36 μμMM [124][124] B7B7 NovelNovelNovel synthetic synthetic synthetic compound compound 7.367.36 μ μMM [124][124] B7B7B7 NovelNovel synthetic synthetic compound compound7.36 µ M[ 7.367.36 μ124 MμM ] [124][124] compound

Vanitaracin A Isolated from Talaromyces sp. 0.61 μM [121] Vanitaracin A Isolated from Talaromyces sp. 0.61 μM [121] VanitaracinVanitaracin A A IsolatedIsolated from from Talaromyces Talaromyces sp. sp. 0.610.61 μ μMM [121][121] Vanitaracin A IsolatedIsolated fromfrom Talaromyces sp. 0.61 μM [121] VanitaracinVanitaracinVanitaracin A A A IsolatedIsolated from from Talaromyces Talaromyces0.61 sp. µsp. M[ 0.610.61 μ121 MμM ] [121][121] Talaromyces sp. 1 2 1 2 50 1 Inhibitor concentration required for 50% inhibition; 2 EC50 value (half-maximal effective concentration). 1 Inhibitor concentration required for 50% inhibition; 2 EC50 value (half-maximal effective concentration). 1Inhibitor concentration required for 50% inhibition; 2EC 50 value (half-maximal effective concentration). 1 Inhibitor concentration required for 50% inhibition;2 EC50 value (half-maximal effective concentration). Inhibitor1 concentration required for 50% inhibition; EC2 50 value (half-maximal effective concentration). 11 InhibitorInhibitor concentration concentration4.2. Directly required required Targeting for for 50% 50% inhibition; cccDNA inhibition;2 2 EC EC50 value50 value (half (half-maximal-maximal effective effective concentration). concentration). Inhibitor concentration4.2. required Directly for Targeting 50% inhibition; cccDNAEC50 value (half-maximal effective concentration). 4.2.4.2. Directly Directly Targeting Targeting cccDNA cccDNA 4.2. DirectlyThe ability Targeting to eliminate cccDNA or inactivate HBV cccDNA has been considered as the “holy 4.2.4.2. DirectlyTheThe Directly abilityability Targeting Targeting toto eliminateeliminate cccDNA cccDNA oror inactivate inactivate HBVHBV cccDNAcccDNA hashas beenbeen consideredconsidered asas thethe “holy“holy grail”TheThe of HBVability ability treatments to to eliminate eliminate [93] or or b inactivateecause inactivate achieving HBV HBV cccDNA cccDNA a “functional has has been been cure considered considered” for chronic as as the theHBV “holy “holy in- grail”grail”The ofTheof HBVabilityHBV ability treatmentstreatments to to eliminate eliminate [93][93] or orbb inactivateecauseecause inactivate achievingachieving HBV HBV cccDNA cccDNA aa ““functionalfunctional has has been been curecure considered considered”” forfor chronicchronic as as the HBV HBVthe “holy “holy in-in- grail”fectionsgrail” of of requiresHBV HBV treatments treatments the permanent [93] [93] b because ecauseinactivation achieving achieving or degradationa a “ “functionalfunctional of cure curethe” ”cccDNA for for chronic chronic [131]. HBV HBV Steps inin- fectionsgrail”fectionsgrail” of of requiresrequiresHBV HBV treatments treatments thethe permanentpermanent [93] [93] b ecauseb inactivationecauseinactivation achieving achieving oror degradationdegradationa a“ functional“functional ofof cure thethecure” cccDNA cccDNA”for for chronic chronic [131].[131]. HBV HBV StepsSteps inin- fectionstowardfections disruptingrequires requires the the cccDNA permanent permanent have inactivation inactivation been enabled or or degradation bydegradation understanding of of the the thecccDNA cccDNA roles [131]. of[131]. host Steps Steps en- towardfectionstowardfections disrupting disruptingrequires requires the the cccDNA cccDNA permanent permanent have have inactivation beeninactivation been enabled enabled or or degradation by by degradation understanding understanding of of the the thecccDNA the cccDNA roles roles [131]. of of [131]. hosthost Steps Steps en- en- towardzymestoward such disrupting disrupting as TDP2, cccDNA cccDNA FEN1, and have have Pol been been-K in enabled enabledcccDNA by byformation. understanding understanding Similarly, the the the roles roles identification of of host host en- en- zymestowardzymestoward suchsuch disrupting disrupting asas TDP2,TDP2, cccDNA cccDNAFEN1FEN1,, andand have have PolPol been - been-KK inin enabled cccDNAcccDNA enabled by formation. byformation. understanding understanding Similarly,Similarly, the the the rolesthe roles identificationidentification of of host host en- en- zymesofzymes host suchcell such nuclear as as TDP2, TDP2, histones FEN1 FEN1, and,and and chromatinPol Pol-K-K in in cccDNA cccDNA-modifying formation. formation. enzymes Similarly, Similarly, that are essentialthe the identification identification for viral ofzymesofzymes hosthost such cellcell such nuclearnuclear as as TDP2, TDP2, histoneshistones FEN1 FEN1, andand, and chromatinchromatinPol Pol-K- Kin in cccDNA- -cccDNAmodifyingmodifying formation. formation. enzymesenzymes Similarly, Similarly, thatthat areare essentialtheessential the identification identification forfor viravirall ofminichromosomeof host host cell cell nuclear nuclear formation histones histones and andand chromatin functionchromatin has-modifying-modifying also enabled enzymes enzymes novel that drugthat are are discovery essential essential avenues. for for vira viral l minichromosomeofminichromosomeof host host cell cell nuclear nuclear formationformation histones histones and andand and chromatin functionfunction chromatin hashas-modifying-modifying alsoalso enabledenabled enzymes enzymes novelnovel that drugdrug that are arediscoverydiscovery essential essential avenues. avenues.for for vira viral l minichromosomeminichromosome formation formation and and function function has has also also enabled enabled novel novel drug drug discovery discovery avenues. avenues. minichromosomeminichromosome formation formation and and function function has has also also enabled enabled novel novel drug drug discovery discovery avenues. avenues.

Pharmaceuticals 2021, 14, 417 12 of 27

Monoclonal antibodies are also efﬁcient HBV entry inhibitors [125–128]. Studies have proven that monoclonal antibodies not only inhibit viral entry but also block the secretion of new infectious virions from hepatocytes [129]. The combination of two monoclonal antibodies, HBV-Ab17 and HBV-Ab19, has been evaluated in phase I clinical trials, demonstrating great safety and efﬁcacy against HBV infection [130].

4.2. Directly Targeting cccDNA The ability to eliminate or inactivate HBV cccDNA has been considered as the “holy grail” of HBV treatments [93] because achieving a “functional cure” for chronic HBV infections requires the permanent inactivation or degradation of the cccDNA [131]. Steps toward disrupting cccDNA have been enabled by understanding the roles of host enzymes such as TDP2, FEN1, and Pol-K in cccDNA formation. Similarly, the identification of host cell nuclear histones and chromatin-modifying enzymes that are essential for viral minichromosome formation and function has also enabled novel drug discovery avenues. Thus, targeting the cccDNA formation has led to many novel candidates in the pipeline of HBV chemotherapy [132,133]. As referred in Section 2.3, APOBEC3 proteins can trigger cccDNA degradation. Several studies have proven that IFN-α administration induces APOBEC3 expression, resulting in the elimination of cccDNA in infected hepatocytes [134]. A potentially useful tool for the complete inactivation of cccDNA is the CRISPR-Cas9 endonuclease system to perform RNA-guided disruption and mutagenesis of cccDNA. The CRISPR-Cas9 endonuclease is complexed with a synthetic guide RNA (gRNA) that perfectly matches with the target sequence of cccDNA, resulting in the cleavage of the selected region. Therefore, to inactivate cccDNA, several gRNAs, targeting multiple different sites in the HBV genome, are required [133,135,136]. Integrated HBV DNA is also sensitive in CRISPR-Cas9-mediated inactivation, which could cause alterations in the host genome and subsequent gene malfunction [132]. Before using this genome editing approach in clinical practice, a number of serious potential issues need to be addressed. These include the incomplete cccDNA degradation, the need of a delivery system that will transfer the CRISPR-Cas9 system to all infected hepatic and extrahepatic cells, potential off- target effects, an immune response induced against the bacterial enzyme that could provoke serious toxicity, and the unpredictable effects of editing the integrated chromosomal HBV DNA [133,137]. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TAL- ENs) are other methods that can destroy HBV cccDNA [133,138]. This treatment could slow down the growth of resistant HBV strains and increase the probability of a prolonged viral response [139–141], yet off-target activity, limited efficacy, effects on integrated HBV DNAs, and the potential induction of immune responses remain serious obstacles [140]. TALENs are newly developed nucleases that cleave selected DNA sequences, thus leading to gene disruptions. Several types of TALENs have been developed that target conserved regions of viral DNA among different HBV genotypes. Overall, TALENs can target and inactivate the HBV genome with a higher specificity than ZFNs and ameliorate the antiviral activity in synergy with IFN-α. Thus, a potential therapeutic strategy for the treatment of chronic hepatitis B infection is provided [140–142]. Another method that could contribute to the transcriptional control of cccDNA is direct targeting of the HBV X protein. HBx induces the proteasomal degradation of the Smc5/6 complex, that normally suppresses cccDNA transcription. Consequently, HBx protein inhibition will prevent the expression of all HBV transcripts from existing cccDNA molecules and suppress the formation of new cccDNA molecules [132,143]. cccDNA formation can also be directly targeted with substituted sulfonamides that interfere with the conversion of rcDNA to cccDNA (Figure5)[144,145]. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 13 of 27

Thus, targeting the cccDNA formation has led to many novel candidates in the pipeline of HBV chemotherapy [132,133]. As referred in Section 2.3, APOBEC3 proteins can trigger cccDNA degradation. Several studies have proven that IFN-α administration induces APOBEC3 expression, resulting in the elimination of cccDNA in infected hepatocytes [134]. A potentially useful tool for the complete inactivation of cccDNA is the CRISPR-Cas9 endonuclease system to perform RNA-guided disruption and mutagenesis of cccDNA. The CRISPR-Cas9 endonuclease is complexed with a synthetic guide RNA (gRNA) that perfectly matches with the target sequence of cccDNA, resulting in the cleavage of the selected region. Therefore, to inactivate cccDNA, several gRNAs, targeting multiple different sites in the HBV genome, are required [133,135,136]. Integrated HBV DNA is also sensitive in CRISPR-Cas9-mediated inactivation, which could cause alterations in the host genome and subsequent gene malfunction [132]. Before using this genome editing approach in clinical practice, a number of serious potential issues need to be addressed. These include the incomplete cccDNA degradation, the need of a delivery system that will transfer the CRISPR-Cas9 system to all infected hepatic and extrahepatic cells, potential off-target effects, an immune response induced against the bacterial enzyme that could provoke serious toxicity, and the unpredictable effects of editing the integrated chromosomal HBV DNA [133,137]. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are other methods that can destroy HBV cccDNA [133,138]. This treatment could slow down the growth of resistant HBV strains and increase the probability of a prolonged viral response [139–141], yet off-target activity, limited efficacy, effects on integrated HBV DNAs, and the potential induction of immune responses remain serious obstacles [140]. TALENs are newly developed nucleases that cleave selected DNA sequences, thus leading to gene disruptions. Several types of TALENs have been developed that target conserved regions of viral DNA among different HBV genotypes. Overall, TALENs can target and inactivate the HBV genome with a higher specificity than ZFNs and ameliorate the antiviral activity in synergy with IFN-α. Thus, a potential therapeutic strategy for the treatment of chronic hepatitis B infection is provided [140–142]. Another method that could contribute to the transcriptional control of cccDNA is direct targeting of the HBV X protein. HBx induces the proteasomal degradation of the Smc5/6 complex, that normally suppresses cccDNA transcription. Consequently, HBx protein inhibition will prevent the expression of all HBV transcripts from existing cccDNA molecules and suppress the formation of new cccDNA molecules [132,143]. cccDNA for- Pharmaceuticals 2021, 14, 417 mation can also be directly targeted with substituted sulfonamides13 ofthat 27 interfere with the conversion of rcDNA to cccDNA (Figure 5) [144,145].

Pharmaceuticals 2021, 14, x FOR PEER REVIEW 14 of 27

PRRs interact with specific components, essential for pathogens’ survival, called patho- (i) CCC-0975 (ii) CCC-0346 gen-associated molecular patterns (PAMPs), and trigger the production of pro-inflammatory factors, like cytokinesFigureFigure 5.5.Disubstituted Disubstituted from immune Sulfonamides Sulfonamides cells (DSS) [146 [144 (DSS)].–149] [144].. Thus, TLR, and RIG-I agonists can stimulate the immune4.3. Immune response Therapy against HBV infection and contribute significantly to its “functional cure4.3.”4.3.1.. Several Immune Targeting TLR7, Therapy Innate TLR8 Immunity , and TLR9 agonists are being evaluated in clin- 4.3.1.The Targeting innate immune Innate responses Immunity constitute the first line of defense against pathogens. ical trials [150–153]These. Phase systems I include clinical membrane trials and for cytoplasmic TLR7pattern agonists recognition RO7020531, receptors (PRRs). RG7795 (ANA773), and RG7854PRRsThe interact (Roche innate with© specific)immune are components, currently responses essential underway. constitute for pathogens’ TLR7the survival,first agonist line called of JNJdefense pathogen--64794964 against pathogens. © Theseassociated systems molecular include patterns membrane (PAMPs), and and trigger cytoplasmic the production pattern of pro-inflammatory recognition receptors (PRRs). (Janssen ) demonstratedfactors, an like excellent cytokines from safety immune and cells tolerability [146–149]. Thus, profile TLR, andin healthy RIG-I agonists adults can during a double-blinded,stimulate randomized the immune phase response I trial against [154]. HBV infectionPhase II and clinical contribute trial significantly results tofor its TLR7 agonist GS-9620 (also“functional known cure”. as vesatolimod) Several TLR7, TLR8, revealed and TLR9 that agonists it is are safe being an evaluatedd well in-tolerated clinical in trials [150–153]. Phase I clinical trials for TLR7 agonists RO7020531, RG7795 (ANA773), chronic hepatitis B patientsand RG7854 receiving (Roche©) are NAs, currently although underway. no TLR7 significant agonist JNJ-64794964 HBsAg (Janssenloss was©) observed after 24 weeksdemonstrated of treatment an excellent [155] safety. The and same tolerability compound profile in healthy also adults caused during no a double-significant blinded, randomized phase I trial [154]. Phase II clinical trial results for TLR7 agonist HBsAg decline in combinationGS-9620 (also known with as vesatolimod)tenofovir revealedin treatment that it is safe-naïve and well-toleratedpatients [156] in chronic. Pyrimi- dine analogues werehepatitis recently B patients identified receiving NAs,as potent although nodual significant TLR7/8 HBsAg modulators loss was observed (Figure after 6ii) 24 weeks of treatment [155]. The same compound also caused no significant HBsAg decline [157]. Structural modificationsin combination led with to tenofovir novel in 2,4 treatment-naïve-diaminoquinazoline patients [156]. Pyrimidinedual TLR7/8 analogues agonists with increased potencywere recentlyand proved identified that as potent changing dual TLR7/8 the stereoch modulatorsemistry (Figure6ii) in [ 157 one]. Structural single stereocenter leads to TLR8modifications selectivity led to(Figure novel 2,4-diaminoquinazoline 6iii) [158]. TLR8 dual agonist TLR7/8 GS agonists-9688 with (also increased known as potency and proved that changing the stereochemistry in one single stereocenter leads to selgantolimod) is underTLR8 selectivityphase II (Figure clinical6iii) [ 158 trial]. TLR8 evaluation agonist GS-9688 [150,159] (also known. Finally as selgantolimod), RIG-I agonist is SB-9200 (also knownunder as phaseInarigrivir) II clinical trialshowed evaluation promising [150,159]. Finally, results RIG-I in agonist a woodchuck SB-9200 (also known model of as Inarigrivir) showed promising results in a woodchuck model of HBV infection [160,161], HBV infection [160,161]and phase, and II clinicalphase trials II clinical demonstrated trials the increaseddemonstrated benefit of combining the increased classic antiviral benefit of combining classic antiviraltreatment withtreatment immune therapywith immune [162]. therapy [162].

(i) GS-9620 (ii) 50d (iii) 31 (iv) GS-9688 FigureFigure 6. InnateInnate immunity immunity modulators; modulators; (i) Selective ( TLR7i) Selective agonist [163 TLR7](ii) TLR7/8 agonist dual [163] agonist ( [ii157) TLR7/8](iii) Dual dual TLR7/8 agonist [157]agonist. (iii (R)) Dual isomer TLR7/8 results in selectiveagonist. TLR8 (R) agonist isomer [158 ](resultsiv) Selective in selective TLR8 agonist TLR8 [159]. agonist [158] (iv) Selective TLR8 agonist [159].

4.3.2. Targeting Adaptive Immunity The PD-1 (programmed death-1) receptor is expressed on HBV-specific T cells, and compounds that block the interactions with its physiological ligand, PD-L1, can increase the number and response of HBV-specific T cells, resulting in increased cytotoxic T cell activity against HBV-infected cells’ anti-HBV-antigen production by B cells [164–166]. Ex vivo studies have shown that blocking PD-1/PD-L1 interactions in chronically infected patients can partially restore the HBV-specific T and B cells’ function [167–169]. PD-1 antagonists have been associated with a high risk of hepatic failure [170]. On the other hand, the anti-PD-1:PD-L1 monoclonal antibody nivolumab has already been evaluated in phase I and II clinical trials in over 100 patients with advanced HCC and no hepatotoxicity incidents were observed [171,172].

Pharmaceuticals 2021, 14, 417 14 of 27

4.3.2. Targeting Adaptive Immunity The PD-1 (programmed death-1) receptor is expressed on HBV-specific T cells, and compounds that block the interactions with its physiological ligand, PD-L1, can increase the number and response of HBV-specific T cells, resulting in increased cytotoxic T cell activity against HBV-infected cells’ anti-HBV-antigen production by B cells [164–166]. Ex vivo studies have shown that blocking PD-1/PD-L1 interactions in chronically infected patients can partially restore the HBV-specific T and B cells’ function [167–169]. PD-1 antagonists have been associated with a high risk of hepatic failure [170]. On the other hand, the anti-PD-1:PD-L1 monoclonal antibody nivolumab has already been evaluated in phase I and II clinical trials in over 100 patients with advanced HCC and no hepatotoxicity incidents were observed [171,172].

4.4. RNA Interference—Post-Transcriptional Control The inhibition of HBV replication by targeting mRNA production and stability is an innovative method for the therapy of chronic hepatitis B, whilst several inhibitors have made it into phase II clinical trials [7,28,93,173]. Inhibitors should bind to HBV mRNA with high specificity and therefore disrupt HBV protein expression by suppressing mRNA translation or inducing mRNA degradation. Such compounds are either small RNA interference (RNAi) molecules, antisense oligonucleotides (ASOs), or possibly even specific ribonucleic acid enzymes (riboenzymes) [133]. RNA interference is mediated by a sequence of 20–30 nucleotides, known as small interfering RNAs (siRNAs) [174]. An advantage stemming from HBV’s transcriptional profile is that multiple mRNA copies of HBV can be targeted selectively at the same time by selecting siRNAs that bind within the overlapping coding regions [133]. To date, three types of siRNAs with different modes of administration are under preclinical evaluation and/or in early-phase clinical trials [7]. An early RNAi drug against HBV, tested in human clinical trials, is ARC-520. The injection consists of two cholesterol-conjugated siRNAs, along with N-acetylgalactosamine (NAG) to achieve hepatocyte-specific delivery via the asialoglycoprotein receptor [39,93]. Potential use limitations are the intravenous administration, the contingent hepatotoxicity, and the off-target binding, as well as the risk of immune activation by PRRs [7]. Despite the barriers mentioned, ARC-520 seems to be very efficient in reducing HBV DNA, HBeAg, and HBsAg levels after experiments on chimpanzees [39,93]. Having passed through phase I with only few hypersensitivity reactions, it proceeded to phase II trials [28]. The co-administration of antihistamines is also recommended [28]. The next step in siRNA evolution came with JNJ-3989 (formerly ARO-HBV, phase II clinical trials), designed to target different HBV genome sites. It is administered subcutaneously, affecting transcripts from both cccDNA and integrated HBV DNA, is safe, and has not shown serious drug-drug interactions [39,93,175]. The HBV inhibitor RG7834 (Figure7), has been studied for its complicity in hepatitis B and has been confirmed not to act as an RNAi molecule, but as an HBV transcription sup- pressor, in a specific, unknown manner [28,176]. Furthermore, the combination of RG7834, entecavir, and PEG-IFN-α significantly decreases HBV DNA and HBsAg levels [133]. AB- 729 is also an siRNA molecule which is administered subcutaneously conjugated with NAG. This compound showed an important suppression of HBsAg in mice models infected by HBV [93]. Pharmaceuticals 2021, 14, 417 15 of 27

Figure 7. HBV transcription inhibitor [176].

Antisense oligonucleotides are short, single-stranded fragments of nucleic acids, either DNA or RNA, that bind to the complementary sequence of viral mRNAs through base pairing. As a result, when binding to RNA, they form hybrids of DNA:RNA (antisense DNA) and duplexes of RNA:RNA (antisense RNA), respectively. The subsequent degradation of the transcribed RNA and the silencing of protein expression occur via a host RNase H-dependent mechanism [7,177,178].

4.5. Ribonuclease H Inhibitors Ribonucleases H are endonuclease enzymes that catalyze cleavage of RNA sequences in DNA:RNA hybrids [179,180]. The HBV RNaseH degrades the viral pgRNA during minus-polarity DNA strand synthesis by reverse transcriptase within immature nucleocapsids [181]. Inhibiting HBV RNaseH activity results in the accumulation of long DNA:RNA hybrids and halts the reverse transcription process [62]. Consequently, newly synthesized virions are non-infectious since they contain a defective genome [182]. Thus, compounds that inhibit RNaseH are promising antiviral candidates against HBV infection. The RNaseH catalytic site includes a ‘DEDD’ (aspartic acid-glutamic acid-aspartic acid-aspartic acid) motif that coordinates two Mg2+ ions. Both Mg2+ are essential during the RNA hydrolysis process [183,184]. All known HBV RNaseH inhibitors contain three electron donors (O or N) that chelate these two cations [185]. RNaseH inhibitors primarily belong to two chemical classes: α-hydroxytropolones (α-HTs) and N-hydroxyimides. The latter include N-hydroxyisoquinolinediones (HIDs), N-hydroxynapthyrydinones (HNOs), N-hydroxypyridinediones (HPDs), and N-hydroxypyrimidinediones [185–188]. One of the first identified HBV RNaseH inhibitors was β-thujaplicinol (compound 46, Table2), an α-HT isolated from the heartwood of western red cedar. β-thujaplicinol blocks the RNaseH of HBV genotypes D and H with EC50 values of 5.9 and 2.3 µM, respectively [189]. This finding led to the design and synthesis of several novel hydroxylated tropolone analogues that suppress HBV replication in EC50 values as low as 0.34 µM (compound 110) [185,190,191], and with CC50 values up to 100 µM. Therapeutic index values were up to 200 [191]. Compound 110 has also been found to inhibit the HBV RNaseH activity, in a molecular beacon assay, and to suppress viremia in animal models [192]. Further structure-activity relationship studies on the hydroxylated tropolone ring revealed that the α-OH substitution is essential for the HBV RNaseH inhibition. Bulky substitution in positions R1,R2 and R3 leads to a decreased inhibitory activity, indicating that sulfonyl- or lactone substituents can increase the efficacy [190,191,193]. These findings have been val- idated by recent studies, which also highlighted the increased efficacy of amide-substituted α-HTs [194–196]. PharmaceuticalsPharmaceuticalsPharmaceuticalsPharmaceuticals 2021 2021, 14 ,2021, x14 FOR ,2021 ,x 14 FOR PEER, ,x 14 FOR PEER, x REVIEW FOR PEER REVIEW PEER REVIEW REVIEW 16 of16 27 of 16 27 of 16 27 of 27

TheThe RNaseHThe RNaseHThe RNaseH catalyticRNaseH catalytic catalytic sitecatalytic site includes siteincludes site includes aincludes ‘DEDD’ a ‘DEDD’ a ‘DEDD’ a(aspartic ‘DEDD’ (aspartic (aspartic acid(aspartic acid-glutamic acid-glutamic acid-glutamic -acidglutamic acid-aspartic acid-aspartic acid-aspartic -aspartic acidacid-asparticacid-asparticacid-aspartic acid)-aspartic acid) motif acid) motif acid) thatmotif thatmotif coordinates that coordinates that coordinates coordinates two two Mg two Mg2+ twoions. 2+Mg ions. Mg2+ Both ions.2+ Both ions. Mg Both Mg2+ Both are 2+Mg are essentialMg2+ areessential2+ areessential duringessential during during during the theRNA theRNA hy theRNA drolysishy RNAdrolysis hydrolysis hy processdrolysis process process [183,184]. process [183,184]. [183,184]. [183,184].All Allknown Allknown Allknown HBV known HBV RNaseH HBV RNaseH HBV RNaseH inhibitors RNaseH inhibitors inhibitors inhibitorscontain contain contain three contain three three three electronelectronelectron donorselectron donors donors (O donors or(O N) or(O thatN) or(O thatN) orchelate thatN) chelate that chelate these chelate these two these two thesecations two cations two cations [185]. cations [185]. RNaseH [185]. RNaseH [185]. RNaseH inhibitors RNaseH inhibitors inhibitors inhibitorsprimarily primarily primarily primarily belongbelongbelong to twobelong to two tochemical two tochemical two chemical classes:chemical classes: classes: α- classes:hydroxytropolones α-hydroxytropolones α-hydroxytropolones α-hydroxytropolones (α- HTs)(α- HTs)(α and- HTs)(α and- NHTs) -andhydroxyimides. N- andhydroxyimides. N-hydroxyimides. N-hydroxyimides. The The The The latterlatter latterinclude latterinclude include includeN -hydroxyisoquinolinedionesN -hydroxyisoquinolinedionesN -hydroxyisoquinolinedionesN-hydroxyisoquinolinediones (HIDs), (HIDs), (HIDs), (HIDs),N -hydroxynapthyrydinonesN -hydroxynapthyrydinonesN -hydroxynapthyrydinonesN-hydroxynapthyrydinones (HNOs),(HNOs),(HNOs), N(HNOs),-hydroxypyridinediones N-hydroxypyridinediones N-hydroxypyridinediones N-hydroxypyridinediones (HPDs) (HPDs) (HPDs), and (HPDs), and N, -andhydroxypyrimidinediones N,- andhydroxypyrimidinediones N-hydroxypyrimidinediones N-hydroxypyrimidinediones [185 [185–188]. [185–188]. [185 –188]. –188]. OneOne ofOne t ofhe One t firstheof t firstofhe identified tfirsthe identified first identified identifiedHBV HBV RNaseH HBV RNaseH HBV RNaseH inhibitor RNaseH inhibitor inhibitor was inhibitor was β- thujaplicinolwas β- thujaplicinolwas β-thujaplicinol β-thujaplicinol (compound (compound (compound (compound 46, 46, 46, 46, TableTable 2),Table an2),Table αan 2),-HT α an2),-HT iso αan- latedHTiso α-latedHT iso from latediso fromlated the from theheartwood from theheartwood theheartwood heartwood of western of western of western of red western red cedar. redcedar. red βcedar.-thujaplicinol βcedar.-thujaplicinol β-thujaplicinol β-thujaplicinol blocks blocks blocks blocks the theRNaseH theRNaseH theRNaseH of RNaseH HBVof HBV of genotypes HBV of genotypes HBV genotypes genotypes D and D and DH andwith DH andwith H EC with H 50EC withvalues 50EC values 50EC valuesof50 5.9valuesof 5.9and of and5.9 of2.3 5.9and 2.3μΜ and μΜ2.3, respectively 2.3μΜ, respectively μΜ, respectively, respectively [189].[189]. This[189]. This[189]. finding This finding This finding led finding ledto the ledto the design ledto thedesign to thedesign and designand synthesis and synthesis and synthesis ofsynthesis several of several of several ofnovel several novel hydroxylated novel hydroxylated novel hydroxylated hydroxylated tropolone tropolone tropolone tropolone analoguesanaloguesanaloguesanalogues that that suppress that suppress that suppress HBVsuppress HBV replication HBV replication HBV replication replication in ECin 50 ECin values 50 ECin values 50EC valuesas50 valueslowas lowas as low as0.34as low 0.34as μ M 0.34as μ (compoundM0.34 μ (compoundM μ (compoundM (compound 110)110) [185,190,191]110) [185,190,191]110) [185,190,191] [185,190,191], and, and with, and with, CCand with CC50 withvalues 50CC values 50CC valuesup50 valuesupto 100 toup 100 μuptoM. 100 μto M.Therapeutic 100 μ TherapeuticM. μ M.Therapeutic Therapeutic index index valuesindex values index valueswere valueswere up were up were up up to 200to 200 [191].to 200[191].to 200Compound [191]. Compound [191]. Compound Compound 110 110 has 110has also 110 hasalso been has also been foundalso been found been tofound inhibit tofound inhibit to inhibit theto inhibit theHBV theHBV RNaseH theHBV RNaseH HBV RNaseH activity, RNaseH activity, activity, in activity, a in a in a in a molecumolecumolecularmolecu larbeacon beaconlar larbeacon assay, beacon assay, assay,and andassay, to and suppressto andsuppress to suppressto viremiasuppress viremia viremia in viremia animalin animal in animal inmodels animal models models [192]. models [192]. Further[192]. Further[192]. Further struc- Further struc- struc- struc- tureture-activityture-activityture-activity relationship-activity relationship relationship relationship studies studies studies on studies theon theonhydroxylated theonhydroxylated thehydroxylated hydroxylated tropolone tropolone tropolone tropolonering ring revealed ring revealed ring revealed that revealed that the that theα -that theα- theα- α- OHOH substitutionOH substitutionOH substitution substitution is essential is essential is essential is foressential forthe for theHBV fortheHBV RNaseH theHBV RNaseH HBV RNaseH inhibition. RNaseH inhibition. inhibition. inhibition. Bulky Bulky substitutionBulky substitutionBulky substitution substitution in posi-in posi- in posi-in posi- tionstions Rt1ions, RRt1ions2, andR12, andRR R12, 3and RleadsR2 3and leadsR3 to leadsR 3a toleads decreased ato decreased ato decreased a decreased inhibitory inhibitory inhibitory inhibitory activity, activity, activity, indicating activity, indicating indicating indicating that that sulfonyl that sulfonyl that sulfonyl- orsulfonyl- or- or- or lactonelactonelactone substituentslactone substituents substituents substituents can can increase canincrease can increase the increase theefficacy theefficacy theefficacy [190,191,193] efficacy [190,191,193] [190,191,193] [190,191,193]. These. These. findingsThese. findingsThese findings have findings have been have been have val- been val- been val- val- idatedidated idatedby idatedbyrecent recentby byrecentstudies, recentstudies, studies, which studies, which whichalso whichalso highlighted also highlighted also highlighted highlighted the theincreased theincreased theincreased increasedefficacy efficacy efficacy of efficacy aofmide aofmide - asubsti-ofmide -asubsti-mide-substi--substi- tutedtuted αtuted-HTs αtuted-HTs α[194-HTs α[194–-HTs196] [194–196] .[194 –196]. –196]. . All Allfour Allfour mentioned Allfour mentioned four mentioned mentioned classes classes classes of N classesof-hydroxyimides N of-hydroxyimides N of-hydroxyimides N-hydroxyimides contain contain contain N containor N Oor N atoms O or N atoms O or inatoms O suitable inatoms suitable in suitable in po- suitable po- po- po- sitionssitions sitionsin ordersitionsin order in to orderin chelate toorder chelate to chelate tothe chelate thetwo thetwo Mg thetwo Mg2+ twoions 2+Mg ions Mg2+and ions 2+and inhibitions and inhibit and inhibitRNaseH inhibitRNaseH RNaseH in RNaseH thein the samein thesamein way thesame way same as way theas way theas theas the α-HTsα-HTs α[185].-HTs α[185].-HTs Several[185]. Several[185]. Several analogues Several analogues analogues analogues have have been have been have synthesized been synthesized been synthesized synthesized and and assessed and assessed and assessed pharmacologicallyassessed pharmacologically pharmacologically pharmacologically for fortheir fortheir HBV fortheir HBV their RNaseH HBV RNaseH HBV RNaseH inhibitory RNaseH inhibitory inhibitory inhibitory activity, activity, activity, exhibiting activity, exhibiting exhibiting exhibiting low low EC low 50EC valueslow 50EC values 50EC values(as50 values (aslow low(as as (as low 1as1 0 low1 asnM),10 1nM),as1 0 1 nM),1 0 nM), limitedlimitedlimited cytotoxicitylimited cytotoxicity cytotoxicity cytotoxicity (most (most (mostCC CC(most50 values50 CC values 50 CC values50 between values between between 25 between 25and and25 100 25and 100 μΜ and 100μΜ), 100 andμΜ), and μΜ) TI, and ) TIvalues, and values TI values TI >300 values >300 >300 >300 [187,188,197,198][187,188,197,198][187,188,197,198][187,188,197,198]. One. One compound. One compound. One compound compound from from this from this classfrom this class hasthis class has al classso hasal beenso has al beenso evaluatedal beenso evaluated been evaluated evaluatedin vivo in vivo in, and vivo in, and vivothe, and the, and the the resultsresultsresults verified resultsverified verified N verified-hydroxyimides N-hydroxyimides N-hydroxyimides N-hydroxyimides as being as being as effective being as effective being effective HBV effective HBV RNaseH HBV RNaseH HBV RNaseH inhibitors RNaseH inhibitors inhibitors inhibitors[192]. [192]. Finally, [192]. Finally, [192]. Finally, Finally, RNaseHRNaseHRNaseH inhibitionRNaseH inhibition inhibition inhibitionis unlikely is unlikely is unlikely is to unlikely be to affectedbe to affected be to affected be by affected HBV’sby HBV’s by HBV’slargeby HBV’slarge genetic large genetic large genetic diversity, genetic diversity, diversity, diversity,and and RNaseH and RNaseH and RNaseH RNaseH inhibitorsinhibitorsinhibitors inhibitorshave have demonstrated have demonstrated have demonstrated demonstrated great great synerg great synerg great synergistic synergistic activityistic activityistic activity with activity with antiviral with antiviral with antiviral compounds antiviral compounds compounds compounds with with a with a with a a Pharmaceuticals 2021, 14, 417differentdifferentdifferent mechanismdifferent mechanism mechanism mechanism of action,of action,of action, ofindicating action,indicating indicating indicating that that they that they canthat they can potentiallythey canpotentially can potentially potentially be usedbe usedbe in usedbe effective,in used effective,in effective,in effective,16 of 27 combinationcombinationcombinationcombination therapeutic therapeutic therapeutic therapeutic schemes schemes schemes against schemes against against HBV against HBV infection HBV infection HBV infection [199,200].infection [199,200]. [199,200]. [199,200].The The structures The structures The structures s oftructures sev-of sev- of sev-of sev- eraleral HBVeral HBV RNaseHeral HBV RNaseH HBV RNaseH inhibitors RNaseH inhibitors inhibitors inhibitorsidenti identified identified identiupfied upto fieddate toup date upto are date to areshown date areshown areshown in Table shownin Table in 2Table .in 2 Table. 2. 2. Table 2. HBV Ribonuclease inhibitors. The highlighted atoms chelate the two Mg2+ ions in the enzyme’s catalytic site. TableTable 2.Table HBV 2.Table HBV 2.Ribonuclease HBV 2.Ribonuclease HBV Ribonuclease Ribonuclease inhibitors. inhibitors. inhibitors. Theinhibitors. The highlighted Thehighlighted The highlighted highlighted atoms atoms chelate atoms chelate atoms chelatethe thechelatetwo twothe Mg twothe2+Mg ions two 2+Mg ions in 2+Mg theions in2+ theenzyme’sions in enzyme’sthe in enzyme’sthe catalytic enzyme’s catalytic catalytic site. catalytic site. HID; site. HID; site. HID; HID; HID; N-hydroxyisoquinolinedione, HNO; N-hydroxynaphthyridinone, HPD; N-hydroxypyridinedione, EC50; half-maximal N-hydroxyisoquinolinedione,N-hydroxyisoquinolinedione,N-hydroxyisoquinolinedione,N-hydroxyisoquinolinedione, HNO; HNO; N HNO;-hydroxynaphthyridinone, N HNO;-hydroxynaphthyridinone, N-hydroxynaphthyridinone, N-hydroxynaphthyridinone, HPD; HPD; N HPD;-hydroxypyridinedione, N HPD;-hydroxypyridinedione, N-hydroxypyridinedione, N-hydroxypyridinedione, EC 50EC; h50 alfEC; h-maximalalf50 EC; -hmaximal50alf; h-maximalalf ef--maximal ef- ef- ef- effective concentration, CC50; 50% cytotoxic concentration, TI; therapeutic index (TI = CC50/EC50). fectivefective concentration,fective concentration,fective concentration, concentration, CC 50CC; 50%50 CC; 50% cytotoxic50CC; 50% cytotoxic50; 50% cytotoxic concentration, cytotoxic concentration, concentration, concentration, TI; therapeuticTI; therapeutic TI; therapeuticTI; therapeutic index index (TI index =(TI indexCC =(TI 50CC/EC =(TI50 CC/EC50 =). 50CC 50/EC).50 /EC50). 50). α-hydroxytropolonesα-hydroxytropolonesα-hydroxytropolonesαα-hydroxytropolones-hydroxytropolones

4646 [190][46190 [190]46] [190]46 [190] 110110 [190] 110110[190] 110 [190][190 [190]] 280280 [191] 280[191] 280 280[191] [191][191 ] µ α-hydroxytropolonesα-hydroxytropolonesα-hydroxytropolonesα-hydroxytropolonesα- ECEC50EC == 501.0EC 1.0 = 501.0ECμΜµ M= 50μΜ1.0 = 1.0μΜ μΜ EC50EC = 500.34ECEC = 500.34EC μΜ== 500.34 0.34 μΜ = 0.34 µμΜ M μΜ EC50EC = 500.5EC = 500.5ECμΜEC = 50μΜ0.5 == 0.5μΜ 0.5 μΜ M PharmaceuticalsPharmaceuticals 2021 2021, ,14 14, ,x x FOR FOR PEER PEER REVIEW REVIEW µ µ CC = >771717 of of M 27 27 Pharmaceuticals Pharmaceuticalshydroxytropolones 2021 ,2021 14, x, 14FOR, x FORPEER PEER REVIEW REVIEWCC CC50 =50 25 = 25M μΜ CC50CC = 5032= μΜ 32 M CC50 = >77 50μΜ 17 of 1727 of 27 CC50 = CC25 μΜ50CC = 50 25 = μΜ25 μΜ CC50 = CC32 μΜ50CC = 50 32 = μΜ32 μΜ CC50 = CC>7750CC μΜ = 50>77 = >77 μΜ μΜ Pharmaceuticals 2021, 14, x FOR PEER REVIEWTI = 25 TI = 94 TI = >15417 of 27 ΤΙ =ΤΙ 25 =ΤΙ 25 = ΤΙ 25 = 25 ΤΙ =ΤΙ 94 =ΤΙ 94 = ΤΙ 94 = 94 ΤΙ =ΤΙ >154 =ΤΙ >154 =ΤΙ >154 = >154 N-hydroxyimides N-hydroxyimidesN-hydroxyimidesN-hydroxyimidesN-hydroxyimides

17 [185] 1717 [185][185] 86 [188] 1212 [185][185] N-hydroxypyrimidinedione17 [185]17 [185] 86 [188] 1212 [185][18512 ][185] NN--hydroxypyrimidinedionehydroxypyrimidinedione 86 [188]8686 [188][188 ] HNO N-hydroxypyrimidinedioneEC1750 =[185] 5.5 µ M HIDHID HNO12 [185]HNO N-hydroxypyrimidinedione 86HID [188]HID HNO ECECCC505050 == = 5.55.5 >100 μμMMµ M ECECEC5050 == 3.43.4 3.4 μµμMM N-hydroxypyrimidinedioneEC50EC = 5.550 = μ 5.5M μM ECEC5050 EC == 1.41.450 = μΜμΜ 1.4 µM EC50EC =HNO 3.450 = μ 3.4 M μM TI = >18 EC50EC =HID 1.450 = μΜ 1.4 μΜ CCCC5050 == >100>100 μΜμΜ CC = 99 µM CCCCCC5050 == 7.17.1 μµμMM CCEC50CC =50 >10050= 5.5= >100 μΜμM μΜ CCCC5050 == 999950 μΜμΜ CCEC50CC50 = =7.150 3.4 = μ 7.1 μMM μ M CCEC50CC50 = = 9950 1.4 =μΜ 99μΜ μΜ TI = 2 TITI == >18>18 TI = 71 TITI == 22 CCTI50 = TI>100>18 = >18 μΜ ΤΙΤΙ == 7171 CCTI50 == TI7.12 = μ 2M CCΤΙ50 == ΤΙ 7199 = μΜ 71 TI = >18 ΤΙ = 71 TI = 2

A23A23A23 [187][187][187] A24A24A24 [187][187][187] A23A23 [187] [187] A24A24 [187] [187] HPDHPD HPDHPD A23HPD [187]HPD A24HPD [187]HPD EC50HPD= 0.11 µM EC50HPD= 0.29 µM ECEC5050 == 0.110.11 μμMM ECEC5050 == 0.290.29 μμMM ECCC50EC50 = HPD=0.115033 = 0.11µμ MM μM ECCC5050EC == HPD0.2950 102 = 0.29μµ M μM CCCC5050 == 3333 μμMM CCCC5050 == 102102 μΜμΜ CCEC50TI50CC == 33500.11 300 =μ 33M μM μM CCEC50TICC50 = = 10250 0.29 352 = μΜ102 μM μΜ TITI == 300300 ΤΙΤΙ == 352352 CCTI50 = = TI300 33 = μ300M CCΤΙ50 == ΤΙ 352102 = 352μΜ TI = 300 ΤΙ = 352 4.6.4.6. NucleocapsidNucleocapsid AssemblyAssembly InhibitorsInhibitors oror ModulatorsModulators 4.6. Nucleocapsid4.6. Nucleocapsid Assembly Assembly Inhibitors Inhibitors or Modulators or Modulators TheThe HBV HBV core core particle particle is is actively actively involved involved in in the the HBV HBV replication replication cycle. cycle. It It is is required required 4.6. TheNucleocapsid TheHBV HBV core Assemblycore particle particle isInhibitors actively is actively or involved Modulators involved in the in HBVthe HBV replication replication cycle. cycle. It is Itrequired is required forfor thethe transfertransfer ofof thethe viralviral genomegenome toto andand fromfrom thethe nucleusnucleus ofof thethe infectedinfected hepatocyte,hepatocyte, asas for theforThe transferthe HBV transfer ofcore the of particle viralthe viral genome is activelygenome to and involvedto andfrom from the in the nucleusthe HBV nucleus ofreplication the of infectedthe infected cycle. hepatocyte, It hepatocyte, is required as as wellwell as as for for a a successful successful reverse reverse transcription transcription [39]. [39]. Thus, Thus, it it is is a a promising promising target target for for antiviral antiviral wellforwell asthe for transferas a for successful a successful of the reverseviral reverse genome transcription transcription to and [39].from [39]. Thus, the Thus,nucleus it is ait promisingis of a thepromising infected target target hepatocyte, for antiviral for antiviral as drugsdrugs [11]. [11]. New New regulators regulators or or inhibitors inhibitors of of nucleocapsid nucleocapsid assembly assembly can can affect affect various various drugswelldrugs as [11]. for [11]. a New successful New regulators regulators reverse or transcription inhibitors or inhibitors of [39]. nucleocapsid of Thus, nucleocapsid it is a assembly promising assembly can target can affect for affect variousantiviral various stagesstages of of the the HBV HBV replication replication cycle, cycle, including including capsid capsid formation, formation, reverse reverse transcription transcription,, and and stagesdrugsstages of [11]. the of HBVthe New HBV replication regulators replication cycle, or cycle, inhibitors including including of capsid nucleocapsid capsid formation, formation, assembly reverse reverse transcription can transcription affect various, and, and pgRNApgRNA encapsidation encapsidation [28]. [28]. Based Based on on the the three three--dimensionaldimensional structurestructure of of capsids capsids when when pgRNAstagespgRNA of encapsidation the encapsidation HBV replication [28]. [28]. Based cycle, Based on including the on threethe capsid three-dimensional-dimensional formation, structure reverse structure oftranscription capsids of capsids when, and when theythey interactinteract withwith aa ligand,ligand, twotwo categoriescategories ofof analoguesanalogues havehave beenbeen developeddeveloped [28].[28]. theypgRNAthey interact interact encapsidation with with a ligand, a ligand, [28]. two Based twocategories categories on the of threeanalogues of analogues-dimensional have have been structure been developed developed of capsids [28]. [28]. when TheThe firstfirst categorycategory isis thethe ClassClass II corecore proteinprotein allostericallosteric modulatorsmodulators (CpAMs),(CpAMs), repre-repre- theyThe interact Thefirst first categorywith category a ligand, is the is two Classthe categoriesClass I core I core protein of proteinanalogues allosteric allosteric have modulators been modulators developed (CpAMs), (CpAMs), [28]. repre- repre- sentedsented byby heteroaryldihydropyrimidinesheteroaryldihydropyrimidines (HAPs)(HAPs) suchsuch asas GLS4,GLS4, RO7RO7049389049389,, andand Bay41Bay41-- sentedsentedThe by heteroaryldihydropyrimidines firstby heteroaryldihydropyrimidines category is the Class I core (HAPs) protein (HAPs) such allosteric such as GLS4, as modulators GLS4, RO7 RO7049389 049389(CpAMs),, and, andBay41 repre- Bay41- - 41094109 [28,201]. [28,201]. Class Class I I CpAMs CpAMs induce induce thethe formationformation of of deformed deformed nucleocapsids nucleocapsids 4109sented4109 [28,201]. by [28,201]. heteroaryldihydropyrimidines Class Class I CpAMs I CpAMs induce induce (HAPs)the theformation suchformation as GLS4, of deformed of RO7 deformed049389 nucleocapsids , nucleocapsidsand Bay41 - [11,39,133,145][11,39,133,145].. The The other other category category is is that that of of Class Class II II CpAMs, CpAMs, such such as as phenylpropenamides phenylpropenamides [11,39,133,145]4109[11,39,133,145] [28,201].. The Class. Theother other I category CpAMs category is inducethat is thatof Class theof Class IIformation CpAMs, II CpAMs, such of such deformedas phenylpropenamides as phenylpropenamides nucleocapsids (PP)(PP) oror sulfamoylbenzamidessulfamoylbenzamides (SBA),(SBA), itsits mainmain representativesrepresentatives being:being: ATAT--13130,0, NVRNVR--3778,3778, (PP)[11,39,133,145](PP) or sulfamoylbenzamides or sulfamoylbenzamides. The other category (SBA), (SBA), is itsthat main its of Classmain representatives IIrepresentatives CpAMs, such being: asbeing: phenylpropenamidesAT- 13AT0,- 13NVR0, NVR-3778,-3778, JNJ6379,JNJ6379, JNJ0440,JNJ0440, JNJJNJ--632,632, JNJ56136379,JNJ56136379, ABAB--423.423. TheseThese compoundscompounds accelerateaccelerate thethe assem-assem- JNJ6379,(PP)JNJ6379, or JNJ0440,sulfamoylbenzamides JNJ0440, JNJ- JNJ632,- 632,JNJ56136379, JNJ56136379, (SBA), its AB main- 423.AB -representatives 423.These These compounds compounds being: accelerate AT accelerate-130, the NVR assem-the-3778, assem- blybly ofof morphologicallymorphologically normalnormal HBVHBV capsidscapsids thatthat lacklack thethe viralviral genomegenome [28,133,201,202][28,133,201,202].. blyJNJ6379, ofbly morphologically of JNJ0440,morphologically JNJ- 632,normal normalJNJ56136379, HBV HBV capsids capsidsAB -that423. that lackThese lack the compounds viralthe viral genome genome accelerate [28,133,201,202] [28,133,201,202] the assem-. . NVRNVR 33--778778 isis thethe firstfirst SBASBA derivativederivative developeddeveloped inin thethe USA,USA, administeredadministered perper osos,, andand isis NVRblyNVR of3- 778morphologically 3- 778is the is firstthe firstSBA normal SBA derivative derivative HBV developedcapsids developed that in lack the in USA,the viralUSA, administered genomeadministered [28,133,201,202] per osper, and os, andis . is underunder pphasehase IaIa clinicalclinical trialtrial (NCT02112799,(NCT02112799, NCT02401737),NCT02401737), exhibitingexhibiting synergisticsynergistic effectseffects underNVRunder p3hase-778 phase isIa theclinical Ia first clinical trialSBA trial (NCT02112799,derivative (NCT02112799, developed NCT02401737), NCT02401737), in the USA, exhibiting administered exhibiting synergistic synergistic per os effects, and effects is inin combination combination with with PEG PEG--IFNIFN--α,α, after after evaluation evaluation in in HBV HBV--infectedinfected mice mice with with a a humanized humanized inunder combinationin combination phase Ia with clinical with PEG PEGtrial-IFN- -(NCT02112799,IFNα, after-α, after evaluation evaluation NCT02401737), in HBV in HBV-infected- infectedexhibiting mice mice with synergistic with a humanized a humanized effects liverliver [7,28,39,175] [7,28,39,175].. Recent Recent in in vitro vitro studies studies in in primary primary human human hepatocytes hepatocytes have have shown shown that that liverin combinationliver [7,28,39,175] [7,28,39,175] with. Recent. PEG Recent in-IFN vitro in- α,vitro studies after studies evaluation in primary in primary in human HBV human-infected hepatocytes hepatocytes mice withhave have ashown humanized shown that that JNJJNJ--632632 (SBA)(SBA) andand Bay41Bay41--41094109 (HAP)(HAP) inhibitinhibit cccDNAcccDNA formationformation andand decreasedecrease bothboth intra-intra- JNJliver-JNJ632 [7,28,39,175]- (SBA)632 (SBA) and and.Bay41 Recent Bay41-4109 in- vitro4109 (HAP) studies(HAP) inhibit inhibit in primarycccDNA cccDNA humanformation formation hepatocytes and anddecrease decrease have both shown both intra- that intra- cellularcellular HBV HBV RNA RNA and and HBeAg HBeAg and and HBsAg HBsAg levels levels [28]. [28]. Further Further in in vitro vitro studies studies have have cellularJNJcellular-632 HBV (SBA) HBV RNA and RNA Bay41 and and HBeAg-4109 HBeAg (HAP) and and HBsAg inhibit HBsAg cccDNA levels levels [28]. formation [28]. Further Further and in vitrodecrease in vitro studies both studies haveintra- have provedproved that that phenylpropenamide phenylpropenamide derivatives derivatives demonstrate demonstrate improved improved an antiviraltiviral activity activity provedcellularproved that HBV that phenylpropenamide RNA phenylpropenamide and HBeAg derivativesand derivatives HBsAg demonstrate levels demonstrate [28]. Further improved improved in vitro antiviral an studiestiviral activity haveactivity whenwhen combinedcombined withwith NAsNAs [145]. [145]. JNJ JNJ--63796379 bindsbinds toto thethe HBVHBV corecore proteinprotein andand disruptsdisrupts thethe whenprovedwhen combined thatcombined phenylpropenamide with with NAs NAs [145]. [145]. JNJ derivatives-JNJ6379-6379 binds binds to demonstrate the to HBVthe HBV core improved core protein protein and antiviral anddisrupts disrupts activity the the encapsidationencapsidation of of pgRNA. pgRNA. It It also also blocks blocks the the cccDNA cccDNA formation. formation. This This drugdrug seems seems to to have have a a encapsidationwhenencapsidation combined of pgRNA. withof pgRNA. NAs It also[145]. It alsoblocks JNJ blocks-6379 the cccDNAthebinds cccDNA to formation.the HBVformation. core This protein This drug drug seems and seems disrupts to have to have the a a veryvery longlong halfhalf--lifelife,, ofof approximatelyapproximately 120120––140140 hh [175,203,204][175,203,204].. ABIABI--H0731H0731 markedmarked thethe be-be- veryencapsidationvery long long half -halflife of ,-pgRNA. lifeof approximately, of approximately It also blocks 120 –the120140 cccDNA– 140h [175,203,204] h [175,203,204] formation.. ABI This. -ABIH0731 drug-H0731 marked seems marked to the have be-the a be- ginningginning of of a a new new category category of of compounds. compounds. It It is is a a dibenzo dibenzo--thiazepinethiazepine--22--carboxamidecarboxamide deriv- deriv- ginningveryginning long of a half ofnew a- lifenew category, of category approximately of compounds. of compounds. 120 It–140 is Ita hdibenzois [175,203,204] a dibenzo-thiazepine-thiazepine. ABI--2H0731-carboxamide-2-carboxamide marked deriv-the deriv-be- ative,ative, and and it it has has been been shown shown to to cause cause a a significant significant reduction reduction in in HBV HBV DNA DNA and and RNA RNA levels levels ative,ginningative, and of andit hasa new it beenhas category been shown shown of to compounds. cause to cause a significant a significant It is a dibenzo reduction reduction-thiazepine in HBV in HBV DNA-2-carboxamide DNA and andRNA RNA levels deriv- levels inin pphasehase II clinicalclinical trials,trials, asas aa corecore proteinprotein modulatormodulator [39,20[39,205].5]. inative, phasein p andhase I clinical it I has clinical been trials, trials, shown as a ascore to a causecore protein protein a significant modulator modulator reduction [39,20 [39,205]. in5]. HBV DNA and RNA levels in phase I clinical trials, as a core protein modulator [39,205].

Pharmaceuticals 2021, 14, 417 17 of 27

All four mentioned classes of N-hydroxyimides contain N or O atoms in suitable positions in order to chelate the two Mg2+ ions and inhibit RNaseH in the same way as the α-HTs [185]. Several analogues have been synthesized and assessed pharmacologically for their HBV RNaseH inhibitory activity, exhibiting low EC50 values (as low as 110 nM), limited cytotoxicity (most CC50 values between 25 and 100 µM), and TI values >300 [187,188,197,198]. One compound from this class has also been evaluated in vivo, and the results veriﬁed N-hydroxyimides as being effective HBV RNaseH inhibitors [192]. Finally, RNaseH inhibition is unlikely to be affected by HBV’s large genetic diversity, and RNaseH inhibitors have demonstrated great synergistic activity with antiviral compounds with a different mechanism of action, indicating that they can potentially be used in effective, combination therapeutic schemes against HBV infection [199,200]. The structures of several HBV RNaseH inhibitors identiﬁed up to date are shown in Table2.

4.6. Nucleocapsid Assembly Inhibitors or Modulators The HBV core particle is actively involved in the HBV replication cycle. It is required for the transfer of the viral genome to and from the nucleus of the infected hepatocyte, as well as for a successful reverse transcription [39]. Thus, it is a promising target for antiviral drugs [11]. New regulators or inhibitors of nucleocapsid assembly can affect various stages of the HBV replication cycle, including capsid formation, reverse transcription, and pgRNA encapsidation [28]. Based on the three-dimensional structure of capsids when they interact with a ligand, two categories of analogues have been developed [28]. The first category is the Class I core protein allosteric modulators (CpAMs), repre- sented by heteroaryldihydropyrimidines (HAPs) such as GLS4, RO7049389, and Bay41- 4109 [28,201]. Class I CpAMs induce the formation of deformed nucleocapsids [11,39,133,145]. The other category is that of Class II CpAMs, such as phenylpropenamides (PP) or sulfamoylbenzamides (SBA), its main representatives being: AT-130, NVR-3778, JNJ6379, JNJ0440, JNJ-632, JNJ56136379, AB-423. These compounds accelerate the assembly of morphologically normal HBV capsids that lack the viral genome [28,133,201,202]. NVR 3-778 is the ﬁrst SBA derivative developed in the USA, administered per os, and is under phase Ia clinical trial (NCT02112799, NCT02401737), exhibiting synergistic effects in combination with PEG-IFN-α, after evaluation in HBV-infected mice with a humanized liver [7,28,39,175]. Recent in vitro studies in primary human hepatocytes have shown that JNJ-632 (SBA) and Bay41-4109 (HAP) inhibit cccDNA formation and decrease both intracellular HBV RNA and HBeAg and HBsAg levels [28]. Further in vitro studies have proved that phenylpropenamide derivatives demonstrate improved antiviral activity when combined with NAs [145]. JNJ-6379 binds to the HBV core protein and disrupts the encapsidation of pgRNA. It also blocks the cccDNA formation. This drug seems to have a very long half-life, of approximately 120–140 h [175,203,204]. ABI-H0731 marked the beginning of a new category of compounds. It is a dibenzo-thiazepine-2-carboxamide derivative, and it has been shown to cause a signiﬁcant reduction in HBV DNA and RNA levels in phase I clinical trials, as a core protein modulator [39,205]. Both classes of CpAMs inhibit the release of viral particles. Thus, the amount of HBV DNA and RNA leaving the hepatocyte is reduced. They also prevent de novo cccDNA formation due to blocking the formation of functional capsids, and hence viral replication [133]. The structures of the abovementioned compounds are shown in Figure8. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 18 of 27

Both classes of CpAMs inhibit the release of viral particles. Thus, the amount of HBV DNA and RNA leaving the hepatocyte is reduced. They also prevent de novo cccDNA Pharmaceuticals 2021, 14, 417 formation due to blocking the formation of functional capsids, and hence18 ofviral 27 replication [133]. The structures of the abovementioned compounds are shown in Figure 8.

(i) GLS4 (ii) Bay-41-4109

(iii) NVR 3-378 (iv) AT-130

(v) JNJ-632 (vi) AB-423

(vii) ABI-HO731

FigureFigure 8. Nucleocapsid assembly assembly modulators modulators or inhibitors or inhibitors [201,205– 208[201,205]. –208].

5.5. Perspectives Perspectives Hepatitis B vaccines are very effective in preventing infection, and antiviral drugs are partiallyHepatitis effective B invaccines reducing are disease very progression effective and in deathpreventing from the infection, infection. However,and antiviral drugs areaccess partially to both effective the vaccine in and reducing the drugs disease remains progression a challenge for and a large death percentage from the of infection the . How- ever,world’s access population to both because the vaccine the majority and of the chronically drugs remains infected patients a challenge live in developingfor a large percentage ofcountries, the world’s most oftenpopulation in sub-Saharan because Africa the ormajority southeast of Asia chronically [209], with infected varying degreespatients live in de- velopingof access tocountries, medical care. most Therefore, often in developing sub-Saharan an affordable Africa or and southeast readily deliverable Asia [209], cure with varying for chronic hepatitis B is urgent. It is widely believed that achieving a broadly applicable degrees“functional of cure”access for to chronic medical HBV care. infection Therefore, will require developi a combinationng an therapy affordable using agentsand readily deliv- erablethat target cure multiple for chronic different hepatitis viral targets B is plusurgent. immune It is modulators widely believed that harness that the achieving power a broadly applicableof the patients’ “functional defenses against cure” the for virus chronic [132,210 HBV]. Drugs infection to improve will controlrequire and a combination eliminate therapy usingHBV willagents have that to tackle target the multiple unique features different of this viral infection, targets particularly plus immune the durability modulators of that har- the cccDNA during current therapies, which is the reason why the existing drugs so rarely ness the power of the patients’ defenses against the virus [132,210]. Drugs to improve induce a “functional cure”. Fortunately, there is a very wide range of drugs in preclinical control and eliminate HBV will have to tackle the unique features of this infection, particularly the durability of the cccDNA during current therapies, which is the reason why the existing drugs so rarely induce a “functional cure”. Fortunately, there is a very wide range of drugs in preclinical and clinical development, so chances are high that combinations of these strategies may be found that substantially improve treatment for HBV patients.

Pharmaceuticals 2021, 14, 417 19 of 27

and clinical development, so chances are high that combinations of these strategies may be found that substantially improve treatment for HBV patients.

Author Contributions: G.-M.P. writing—original draft, D.M. writing—original draft, E.G. writing— review & editing and preparing the original manuscript figures, V.P. writing—review & editing, J.E.T. review & editing G.Z. conceptualization, methodology, review & editing, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: The APC was funded by the Special Account for Research Grants and the National and Kapodistrian University of Athens. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conflicts of Interest: The authors declare no conflict of interest.

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