Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles

Capsid assembly modulators (CAMs) are a novel class of therapeutic small molecules with the potential to address the continued global challenge posed by chronic hepatitis B (CHB). Class A CAMs (CAM-As) are particularly attractive because they induce loss of hepatitis B virus (HBV)-infected hepatocytes in animal models. All CAM-As described to date are heteroaryldihydropyrimidines (HAPs) which come with several drawbacks. Here, we report on the first non-HAP CAM-As ALG-005398 and ALG-005863 and provide a detailed in vitro intracellular characterization. These non-HAP CAM-As are potent inhibitors of HBV DNA production and also block the establishment of cccDNA. Non-HAP CAM-As can be classified into two distinct profiles: CAM-Ai and CAM-At, which are in turn differentiated from the HAP CAM-Ah profile. CAM-Ai molecules induce larger and more irregular capsids in electron microscopy and cellular HBV core protein (HBc) staining, whereas CAM-At-induced capsids and aggregates are smaller but more numerous. CAM-Ai and CAM-At also induce a different subnuclear localization (no co-localization with promyelocytic leukemia bodies) and show a lower propensity to CAM-A-induced HBc-dependent cell death compared to CAM-Ah. CAM-Ats ALG-005398 and ALG-006162 showed steady reductions of circulating HBsAg and HBeAg with minimal alanine aminotransferase (ALT) elevation in the adeno-associated virus (AAV)-HBV mouse model, accompanied by loss of the AAV-HBV episome and infected hepatocytes. Moreover, these effects were sustained for at least 13 weeks after end of treatment. The differentiated mechanism of action and sustained in vivo response make non-HAP CAM-As a promising potential component of future functional cure regimens for CHB patients.