PROJECT SUMMARY/ABSTRACT Despite the availability of an effective vaccine, epidemiologic data estimates about 2 billion people globally are infected with hepatitis B virus (HBV). Approximately 350 million people are chronic HBV carriers and at high risk for the development of hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Current anti-HBV treatment options suppress the virus but do not eliminate the virus, requiring costly lifetime therapy. All FDA approved therapeutic approaches fail to target the HBV covalently closed-circular DNA (cccDNA; associated with viral persistence) or the virus capsid which is essential for virus proliferation. Our approach in this application is to target capsid assembly, which is essential for replication, as DNA synthesis from cccDNA occurs exclusively within the capsid encoded particle. HBV core proteins (Cp) constitute the subunits in viral capsid assembly and Capsid Assembly Modulators (CAM) accelerate the kinetics of capsid assembly whereby they prevent pol- pgRNA complex encapsidation and block HBV replication. CAMs also interfere with cccDNA transcription/de novo formation during early steps of infection. As part of our ongoing HBV CAM discovery program NIH- supported over the last 4 years, we have been successful in developing several highly potent (sub-micromolar) class II CAMs with one of our lead compounds entering phase 1 clinical trials in October 2020. However, there are numerous hurdles that could derail our efforts towards FDA approval. Here, we describe for the first time a novel class of homo or hetero-dimer CAM displaying selective anti-HBV activity in culture in the picomolar range. Because a dimeric structure linking two CAM moieties can interact with two distinct sites of one capsid or eventually connect two (or more) capsids together, we hypothesized that these compounds would have a more profound impact on HBV capsid assembly than known class I or II CAMs. Based on the potency and the unique mode of action (MoA) we termed our new compounds as “class III” CAMs. We propose to evaluate these class III CAMs by pursuing three specific aims: 1) To chemically optimize and characterize a unique series of CAM homo/heterodimers made from novel monomers; 2) To characterize (structurally, biochemically, and biologically) novel CAM homo/heterodimer binding interaction with HBV capsid; 3) To determine pharmacokinetics (PK) and in vivo efficacy of novel CAM homo and heterodimers. Novel homo and heterodimers will be synthesized and evaluated to reach maximum potency and drug-like properties. To differentiate our compounds from existing class I and II CAMs, we will characterize structural and dynamical effects of our new CAMs by determining a) their effect on the morphology of HBV capsids and their localization within cells, b) binding to HBV wild-type and known mutant Cp, c) resistance profile and activity against major CAM resistant HBV strains and d) intra- or inter-capsid connections. Results fr...