Direct Inhibition of Hepatitis B e Antigen by Core Protein Allosteric Modulator

Abstract

Hepatitis B e antigen (HBeAg) is an important immunomodulator for promoting host immune tolerance during chronic hepatitis B (CHB) infection. In patients with CHB, HBeAg loss and seroconversion represent partial immune control of CHB infection and are regarded as valuable endpoints. However, the current approved treatments have only a limited efficacy in achieving HBeAg seroconversion in HBeAg‐positive patients. Hepatitis B virus (HBV) core protein has been recognized as an attractive antiviral target, and two classes of core protein allosteric modulator (CpAM) have been discovered: the phenylpropenamides (PPAs) and the heteroaryldihydropyrimidines (HAPs). However, their differentiation and potential therapeutic benefit beyond HBV DNA inhibition remain to be seen. Here, we show that in contrast to PPA series compound AT‐130, a HAP CpAM, HAP_R01, reduced HBeAg levels in multiple in vitro and in vivo HBV experimental models. Mechanistically, we found that HAP_R01 treatment caused the misassembly of capsids formed by purified HBeAg in vitro. In addition, HAP_R01 directly reduces HBeAg levels by inducing intracellular precore protein misassembly and aggregation. Using a HAP_R01‐resistant mutant, we found that HAP_R01‐mediated HBeAg and core protein reductions were mediated through the same mechanism. Furthermore, HAP_R01 treatment substantially reduced serum HBeAg levels in an HBV mouse model. Conclusion: Unlike PPA series compound AT‐130, HAP_R01 not only inhibits HBV DNA levels but also directly reduces HBeAg through induction of its misassembly. HAP_R01, as well as other similar CpAMs, has the potential to achieve higher anti‐HBeAg seroconversion rates than currently approved therapies for patients with CHB. Our findings also provide guidance for dose selection when designing clinical trials with molecules from HAP series.

Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation

“…Cell fractionation and pSTAT1/2 detection in cytoplasmic and nuclear fractions were conducted as described for panel C. p22 shares the exact same amino acid sequence as the core protein except for a 10-aa N-terminal extension, it fails to form a capsid-like structure and support HBV DNA replication ( Fig. 2A), which is consistent with previous studies showing that precore is not required for HBV infection in vivo or replication in vitro (58,59), though it can form heterocapsid with core protein (60,61). It has been shown that the presence of a cysteine at the Ϫ7 position in the N-terminal precore domain of HBeAg causes a preference for the formation of an intramolecular disulfide bond between the Ϫ7 and 61 cysteines over an intermolecular disulfide bond, as seen in the case of the core protein, resulting in HBeAg dimerization in an inverted orientation relative to the core dimer and, thus, precluding capsid formation from HBeAg.…”

New Approaches to the Treatment of Chronic Hepatitis B

“…Interestingly, studies in multiple in vitro and in vivo HBV experimental models have found that the type I core inhibitor HAP_R01 reduced HBV DNA and HBeAg levels through causing misassembly of its 22 kDa precore protein precursor [ 61 ]. It seems that HAP_R01, as well as other similar CpAMs, have the potential to achieve higher anti-HBe seroconversion rates than the currently approved therapies for patients with CHB.…”

A New Role for Capsid Assembly Modulators To Target Mature Hepatitis B Virus Capsids and Prevent Virus Infection

“…HAP_R01 is a novel HAP-type CpAM (Fig. 1A) that binds to the core protein dimer-dimer interface and effectively inhibits HBV replication and HBeAg biosynthesis in HBVreplicating hepatoma cells (14)(15)(16).…”