Aligos Belgium presented research on ALG-005398, a first representative of a series of non-heteroaryldihydropyrimidines (HAPs) class I capsid-assembly modulators, that was identified using structure-based drug design and scaffold hopping.

To treat chronic hepatitis B, class I CAMs induce hepatitis B virus (HBV) core protein (HBc) aggregation and sustained HBsAg reduction in AAV-HBV mice, in addition to inhibiting HBV RNA encapsidation and formation of infectious HBV particles. Antiviral activity on HBV DNA was determined in HepG2.117 cells using quantitative PCR. Both the primary effect on HBV DNA and the secondary effect on covalently closed circular DNA (cccDNA) establishment were studied in HBV-infected primary human hepatocytes (PHHs). Inhibition over several orders of magnitude was assessed in an optimized log drop assay.

Using this assay, the EC50, EC90, EC99 and EC99.9 values were determined to be 1.70, 4.80, 8.12 and 13.7 nM, respectively, for ALG-005398 (n = 3) vs. 59.6, 204, 458 and 989 nM for RG-7907 (n = 2), another class I CAM. In addition, ALG-005398 potently inhibited RNA encapsidation and cccDNA establishment in HBV-infected PHHs. The EC50/EC90 values for HBV DNA, HBsAg, HBeAg and HBV RNA were 9.29/83.6, 167/650, 97.3/353 and 128/446 nM, respectively, for ALG-005398 (n = 3) vs. 5.41/55.3, 848/> 10000, 372/2730 and 294/> 10000 nM for RG-7907 (n = 2), respectively. Furthermore, ALG-005398 induced rapid formation of small aberrant capsids in vitro. Compared with RG-7907, binding of ALG-005398 to HBc produced rapid oligomerization, with slightly lower maximum quenching but a steeper slope (3.4 vs. 1.9).

In the log drop assay, a steeper slope was also observed for ALG-005398 compared with RG-7907 (2.3 vs. 1.5). Both compounds sharply reduced the amount of detectable HBc in Western blot, but ALG-005398 was approximately 10-fold more potent than RG-7907 in inducing HBc spots. The respective EC50 and EC90 values were 34.7 and 192 nM for ALG-005398 (n = 4) vs. 529 and 1970 nM for RG-7907 (n = 4). Regarding the cellular compartments in which the nuclear HBc exist, colocalization of HAP-induced HBc spots with PML bodies was confirmed.

It was concluded that the nuclear localization of HBc spots induced by ALG-005398 may be different. A hypothesis for possible future evaluation is that localization of the HBc spots to structures such as PML bodies might be related to HBsAg loss, perhaps through post-translational modification (for example, small ubiquitin-like modifier) or the activation of interferon responses. The delayed antigen reductions and lack of clear ALT elevation may suggest a slightly different mechanism of action for ALG-005398 vs. HAPs, which is being currently investigated by Aligos. In vivo, ALG-005398 (30 mg/kg b.i.d.) resulted in a multiphasic reduction in HBV DNA, with a 6.4 log10 IU/mL reduction after 95 days. A significant decrease in serum HBsAg levels was observed with a 2.24 log10 IU/mL reduction with paced kinetics: the main decline was observed between day 49 and 95 of ALG-005398 treatment, with only a minimal change in ALT.

Among other findings, significant decreases in HBsAg levels were noted, but with a slight delay compared with HBsAg decline; pgRNA levels were significantly reduced; intrahepatic HBsAg showed a 'dramatic disappearance' in immunohistochemically stained liver sections at day 95; and a 10-fold reduction occurred in the amount of AAV-HBV episome in the livers of class I CAM-treated animals