Nomenclature of HBV Core Protein Targeting Antivirals

Introduction

A greater understanding of the Hepatitis B Virus (HBV) replication cycle has led to the discovery of drugs with novel targets, including drugs interfering with nucleocapsid assembly (formation of genomic nucleic acid containing capsids) and disassembly processes. In its role as a viral structural protein, the core protein (HBc) forms the viral capsid which packages the viral genome. Assembly agonists favor formation of aberrant capsids or morphologically normal capsids devoid of genetic material¹. A number of such “Capsid Assembly Modulator” (CAM) compounds are in clinical development.

Lack of consistent nomenclature for these drugs has generated confusion across the HBV community. Compounds targeting HBc have been described by various names, including capsid assembly modulators (CAMs), core protein allosteric modulators (CpAMs), core or capsid inhibitors, core targeting agents, and as subclasses (class 1 and class 2 agents with inconsistent use on what is class 1 or 2, CAM-A (aberrant), CAM-E (empty), and CAM-N (normal))². As HBc targeting compounds are undergoing clinical trials, an agreed upon classification would improve clarity in future discussions.

To address the need for a naming convention that appropriately categorizes these types of molecules with a uniform terminology, reduces confusion, and provides clear and precise language to document their development pathway, the HBV Forum and ICE-HBV jointly convened a working group to develop a standardized nomenclature that accommodates mechanisms of action while being simple, intuitive, and accessible to all members of the HBV community and beyond.

CAM: Mechanism of Action

Current CAMs are agonists that accelerate HBc assembly or disrupt HBc complexes, i.e. capsids. Both mechanisms take advantage of biological HBc activities, assembly and disassembly. An understanding of the molecular interaction between CAMs and HBc explains these disparate activities and the potential for CAMs to contribute to an HBV cure.

Late in the HBV replication cycle, the first stage of virus morphogenesis is the assembly of an RNA-filled virus capsid^1,2. In this process, 120 copies of the HBc homodimer form an icosahedral shell, or capsid, around a complex of viral polymerase and pregenomic RNA (pgRNA, an mRNA transcript of the HBV genome). Inside the RNA-filled virus capsid, the viral polymerase reverse transcribes the linear single-stranded pgRNA into the relaxed circular, partly double-stranded DNA (rcDNA) genome, which is then found in the mature HBV virion. HBc can also nucleate assembly spontaneously to form empty capsids devoid of genetic material. Remarkably, ≥90% of HBV capsids are empty, whether isolated from serum or culture medium, indicating that spontaneous assembly is a thermodynamically favorable reaction. CAMs can take advantage of this tendency.

CAMs interfere with assembly of new viral nucleocapsids probably by enhancing spontaneous nucleation leading to formation of empty and/or aberrant capsids (Figure 1). In vitro studies suggest that CAMs speed up nucleation and accelerate capsid formation^2-4 Structural studies show that CAMs bind in a hydrophobic pocket at the dimer:dimer interface, increasing interactions between HBc proteins and potentially promoting an assembly active state. While some CAMs favor formation of empty capsid particles that resemble normal HBV capsids, other CAMs can induce aberrant assembly, e.g. hexagonal sheets of HBc, large and incomplete balloon-like structures, and large cylinders (Figure 1). Aberrant assembly, however, is not general to all CAMs.

Figure 1: mode of action of CAMs supporting the proposed nomenclature.

PDB file is 6BVF. EM is from Kondylis, Schlicksup, et (2020)

Early in the replication cycle, DNA-filled capsids are delivered to nuclear pores and release viral rcDNA into the nucleus, where the rcDNA is converted into an episomal covalently closed circular (ccc) DNA, the viral persistence reservoir. rcDNA-filled capsids can also cycle back to the nucleus of infected cells. At this point in replication, the capsid must be disassembled to facilitate release of HBV DNA into the nucleus of hepatocytes. Because the double-stranded DNA (dsDNA) within HBV capsids carries approximately twice the negative charge of its single-stranded RNA template (neutralized by positive charges in the nucleic acid binding C-terminal domain of HBc) and the dsDNA is a stiff polymer compared to the diameter of an HBV capsid, the mature capsid can be described as “spring loaded”.

A subset of CAMs has been shown to prevent HBV from infecting cells when the cells are treated with the drug before, or shortly after, infection⁵. Mechanistically, this may be due to destruction of the DNA-filled capsid or by preventing newly formed capsids from cycling back to the nucleus and depositing additional copies of rcDNA to form cccDNA. Several labs have shown that a subset of CAMs can lead to capsid dissolution. A suspected mechanism of action (MOA) is that these CAMs favor an HBc-HBc interaction geometry that is incompatible with icosahedral geometry. A third alternative is that capsid-stabilizing CAMs that favor icosahedral geometry may inhibit uncoating at the proper time and place, although that mechanism has not been experimentally demonstrated.

In summary, CAMs, are small molecules with two well-established MOAs against HBV. First, they stimulate HBc assembly to decrease proper formation of pgRNA-filled capsids, resulting in suppression of viral replication by inhibiting production of new infectious virus. CAMs can also bind mature HBV capsids and prevent them from properly releasing their contents, blocking cccDNA formation in newly infected cells (Supplementary Figure 1). By inhibiting formation of both new virus and new rounds of infection, CAMs offer at least two routes to suppress HBV infection. They may also inhibit cccDNA replenishment in already infected cells by preventing the recycling of capsids to the nucleus and release of rcDNA to form cccDNA.

Pre-clinical studies have shown that CAMs exhibit antiviral activity across a number of HBV genotypes, and against nucleos(t)ide analog (NUC) resistant mutants. Next generation CAMs have demonstrated an improved resistance profiles compared to first generation CAMs in cell culture models.

Clinical Studies

More than 20 CAMs are in preclinical or clinical development^6,7 (Supplementary Table 1). Several have completed short Phase 1b studies; limited data is available for CAMS given for longer duration.

In recent phase 2 studies, addition of a CAM to a NUC intensified the on-treatment suppression of viral replication, inhibited serum HBV DNA and HBV RNA by multiple logs, and prevented virologic breakthroughs from selection of CAM-resistant variants. Next generation CAMs have in vitro antiviral potency corresponding 2-3 log₁₀ against cccDNA formation than earlier CAMs.

In the phase II JADE study, two groups of chronic hepatitis B (CHB) patients, one not currently receving treatment and one virologically suppressed by NUC therapy, were randomized to receive bersacapavir (JNJ-379), a CAM, once daily or placebo with NUC or bersacapavir alone for ≥24 and ≤48 weeks⁸. Overall, bersacapavir with a NUC showed promising antiviral activity in CHB patients, with pronounced reductions in both HBV DNA and HBV RNA. However, the effects on hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) levels were small and mainly observed in previously untreated HBeAg-positive patients. This suggests a limited impact on the DNA template for HBsAg expression, i.e. cccDNA and integrated viral sequences, and for HBeAg expression, i.e. cccDNA.

A pilot sub-study of vebicorvir (ABI-H0731), plus NUC evaluated whether intensified virologic suppression could allow safe discontinuation of all therapy after 72 weeks with profound viral suppression, defined by HBV total nucleic acid (both HBV DNA and RNA) < 20 IU/mL and low or undetectable HBeAg^9,10. All patients relapsed, indicating that despite a profound viral suppression, the pool of cccDNA was not eradicated. Intensified on-treatment virologic suppression was not associated with significant reduction in HBsAg levels.

Several ongoing studies are assessing how CAMs can be combined with other antiviral (e.g. RNA interference) and immunomodulatory approaches. Long-term combination treatment with potent, next-generation CAMs may be required to engage its secondary MOA, and contribute to HBsAg loss and a finite therapy resulting in functional cure. Thus, agreeing on a consensus nomenclature is vital given the important role CAMs are expected to play in combination therapy for HBV Cure.

Discussion

The HBV Forum and ICE-HBV led the CAM working group, including scientific experts from regulatory agencies (U.S. Food & Drug Administration and European regulatory system National Competent Authorities), industry developing CAM-based therapeutics, and academic/clinical researchers. It was tasked with reviewing commonly used nomenclatures in the context of the molecule’s MOA and proposing a standardized nomenclature that is simple, easy to remember, scientifically accurate, and yet allows for future development of drugs involving interaction with core protein with other MOA. Through a process of evolving consensus, the HBV Forum and ICE-HBV propose CAM, with “C” standing for capsid, ”A” for assembly, and “M" for modulator. This nomenclature encompasses how the molecules function, and indicate that the capsid assembly (and disassembly) is being modulated, thus descriptive of the MOA (Figure 1). We propose “Core Protein Targeting Antivirals” as an overarching classification for drugs interacting with core proteins with other MOAs. While more information is needed to sub-categorize CAM drugs, we propose CAM-A (aberrant) and CAM-E (empty) as an interim subclassification (Figure 1).

Conclusion

Our proposed nomenclature emerged from the working group’s review of available information and will be updated when more information is available to further distinguish between the chemical classes. This work was undertaken to facilitate efficiency and clarity in HBV drug development through standardized nomenclature reflecting the science on which drug development is grounded.