VHL-independent degradation of hepatitis B virus e antigen (HBeAg) by VHL-binding chimeric small molecules

Abstract

Hepatitis B virus (HBV) is a leading cause of liver cancer worldwide, with current treatment options unable to provide lasting efficacy against chronic infection. A key viral protein, HBV e antigen (HBeAg), plays an important role in suppressing the cellular and humoral immune response during infection and its loss is a precursor to clearance of chronic HBV infection. Its structural similarity to capsid forming HBV core protein antigen (HBcAg) makes it an intriguing, yet understudied target for pharmaceutical intervention. Recently, targeted protein degradation has been successfully applied against several viral proteins. This work investigates the targeting of HBeAg using heterobifunctional degraders derived from reported HBcAg ligands known to interact with HBeAg. Multiple compounds designed to recruit the VHL E3 ligase were found to be capable of reducing recombinant HBeAg protein levels in a HiBiT reporter assay system. Surprisingly, this decrease was found to be independent of VHL recruitment but driven by structural motifs of the VHL recruiting ligand, VH032. Virological assessment of these compounds against wildtype virus revealed an equipotent capability to reduce secreted HBeAg compared to the parental inhibitor, however increased efficacy was observed against an inhibitor resistant strain. Together, this work provides an initial description of the feasibility of converting HBV capsid-targeting ligands into degraders and provides evidence that such degraders may harbour improved activity against mutated forms of target which are resistant to parental compounds.

Fig. 1. A. Crystal structure of NVR-010-001-E2 bound to HBcAg N-domain (Y132A mutant, PDB: 5E0I), B. Structure of second-generation HAP, NVR-010-001-E2, C. Structure of fluorophore-linked HAP, HAP-ALEX.

Fig. 2. A. Chemical structure of degrader candidates, composed of a heteroaryldihydropyridine (HAP) HBeAg recruiting domain, varied linkers, and one of three E3L recruiters. B. HEK293T^{HiBiT-HBeAg-NS} cells were treated with candidates at 10 μM for 24 h, then luminescence detected and normalised to the DMSO control. Compounds with a statistically significant increase in potency compared to parental compound 2 are marked, where **** represents P < 0.0001, *** P ≤ 0.001, as determined by one-way ANOVA. C. A matched ViaLight™ assay was performed to determine the effect of treatment on cell viability.

Scheme 1. Synthesis of key HAP intermediates and initial series of HBeAg degrader candidates. a) N-Bromosuccinimide, DCM, r.t, 1 h, 64%, b) morpholine, MeOH, 3 h, r.t., 40%.

Fig. 3. A. HEK293T^{HiBiT-HBeAg-S} cells were titrated with compounds 2 and LH-3 for a 24 h treatment period. B. The HEK293T^{HiBiT-HBeAg-S} cell line was treated with compounds 2, LH-1, cisLH-1, LH-3 and cisLH-3 at 10 μM for 24 h. C. Regular or VHL KO HEK293T^{HiBiT-HBeAg-S} cell lines were treated with compounds 2, LH-1 or LH-3 at 10 μM for 24 h. Secreted HBeAg levels were reduced in all treatment groups, with minimal difference observed between cell lines. VHL KO was also unable to rescue intracellular HBeAg levels. **** represents P < 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns = P > 0.05 as determined by one-way ANOVA.

Fig. 4. A. A series of LH-3 analogues were prepared bearing truncated or altered E3L recruiting domains. B and C. HEK293T^{HiBiT-HBeAg-S} cells were treated with LH-3 derivatives at 10 μM for 24 h then levels of secreted (B) and intracellular (C) HBeAg levels were assessed via HiBiT assay. Varying levels of activity against secreted HBeAg were observed, while only LH-3-T1, LH-3-desHyd and LH-3-Pom retained activity against intracellular HBeAg. **** represents P < 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns = P > 0.05 as determined by one-way ANOVA. D. Consensus log P of LH-3 and derivatives were calculated using Swiss ADME, and compared to activity against intracellular HBeAg. Relative activity is colour coded as high (green), moderate (yellow), or low (red).

Fig. 5. A. Compounds were prepared based on LH-3 where the VH032 motif was replaced with reported hydrophobic tags. B and C. HEK293T^{HiBiT-HBeAg-S} cells were titrated with HyT deriviatives for 24 h and assessed for secreted (B) and intracellular HBeAg levels (C).

Fig. 6. Virological assessment of degrader candidates. A. HepAD38 cells treated with compounds at 10 μM for 6 days before removal of media and western blot analysis of cell lysate using anti-core protein antibody, B. Antiviral activity of LH-3/cisLH-3 was assessed in Huh7 cells transfected with either WT or T109I mutant strain of HBV. The cells were treated with compound at 10 μM for 24 h and media levels of HBeAg quantified. * P ≤ 0.05, ns = P > 0.05 as determined by two-way ANOVA.