Dual mechanism of actions of novel HBV core protein allosteric modifiers (CpAMs): inhibiting viral replication and blocking cccDNA formation
Qi Huang1, Dawei Cai1, Pao-Chen Li1, Alex Mercier1, Renuka Kumar1, Emily Connelly1, Yuhua Zong1, Ran Yan1, Xiulan Zhou1, Yi Zhou1, Lida Guo1, Ariel Tang1, Geoffrey Chen1,Esteban Carabajal1, Katherine Nabel1, Lichun Li1,Steve Dunkelbarger1, Sarah Katen1, Jason Deer1, Earl May1, Uri Lopatin1, Adam Zlotnick1,2, Richard Colonno1
1Assembly Biosciences, Carmel, USA; 2Indiana University,Bloomington, USA
Background: HBV core protein (Cp) is involved in multiple steps of the HBV life cycle, including the formation, amplification and maintenance of cccDNA. A novel class of direct acting HBV antivirals, Core Protein Allosteric Modifiers (CpAMs), have been discovered to target HBV core protein and reduce HBV replication and cccDNA levels. Here we report on the mechanism of actions of two prototype CpAMs (GLS4 and AT-130) and Assembly Biosciences proprietary CpAMs using a variety of biochemical and cellular assays.
Methods: In vitro capsid assembly assay: BOPIPY-FL-labeled Cp150 were used in the fluorescence quenching Cpasid assembly assay to test potential CpAMs. Molecular profiling of CpAMs: HepAD38 cells were treated with inhibitors and induced simultaneously. HBV total RNA/encapsidated pgRNA, Cp, capsid, and capsid associated core DNA were detected by Northern Blot, Western Blot, Enzyme Immunoassay (EIA), and Southern Blot, respectively. HBV DNA/pgRNA were quantified using bDNA probes (Affimetrix). HBe and HBs antigens were quantified using ELISA assays. HBc were visualized using Standard Immunostaining method. cccDNA Southern Blots: cells were harvested and extrachromosomal DNA extracted by a Hirt-modified method.
Result: In contrast to earlier published data suggesting different class of CpAMs function differently, either inhibits capsid assembly (HAP)or leads to mis-assembly (AT-130), our data demonstrated that all CpAMs induced empty capsids, either as normal or aberrant size in vitro (assembly assay and EM) and in vivo (EIA assays in HepAD38). Treatment of CpAMs in HepAD38 cells also re-distribute of Core Protein, shown as punctate or large aggregates by Cp immunostaining. Western blot analysis indicated that CpAMs may alter the phosphorylation of Cp and cause accumulation of insoluble Cp in cells. HAPs also induce accelerated Cp degradation. While all CpAMs effectively block pgRNA encapsidation and HBV DNA replication, only certain classes of CpAM prevent HBV infection and cccDNA formation during de novo infection. The effect of cccDNA formation is time-dependent but sustainable, resulting in dose-dependent reduction of intracellular HBV RNA transcription and secretion of HBsAg and HBeAg.
Conclusion: CpAMs represent novel HBV DAAs that induce inappropriat oligomerization of HBV core protein, resulting in empty capsid formation. Some CpAMs also prevent delivery of rcDNA to the nucleus and establishment of cccDNA during HBV de novo infection, based on surrogate markers HBeAg, HBsAg and pgRNA levels, and direct cccDNA detection. In conclusion, we demonstrate that certain class of CpAMs can interfere with multiple steps in viral life cycle, perturb the normal maintenance of cccDNA longevity (key deficiency of current nucleos(t)ide therapy). Therefore, CpAMs have the potential to significantly increase cure rates in HBV patients when used in combination with current therapies.