Small molecule inhibitors of HBx that decrease hepatitis B virus replication

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Chronic infection with hepatitis B virus (HBV) is the 7th leading cause of death worldwide and the 5th leading cause of cancer. The key to HBV chronic infection is the nuclear localized HBV episomal covalently closed circular DNA (cccDNA) that drives HBV transcription and replication. The risk of HCC is directly related to the level of replication from the cccDNA. An HBV cure requires eliminating cccDNA, which is not feasible at present. However, achieving a functional cure, defined as a sustained loss of hepatitis B surface antigen (HBsAg), may be possible by silencing the cccDNA. The current therapies, treatment with interferon alpha or life-long maintenance on nucleos(t)ide analogs, have low functional cure rates highlighting the critical need for new HBV therapeutics. The HBV HBx protein is an excellent candidate for the development of antivirals due to its critical roles in the virus life cycle – regulation of viral transcription, degradation of viral restriction factors that silence cccDNA, and interference with many host cellular processes. Despite its central role, HBx has yet to be the target of antiviral therapy. Our central hypothesis is that targeting HBx will inhibit viral replication, silence cccDNA, and facilitate an HBV functional cure. Our proposed studies build on several recent advances. First, we have shown HBx interacts with cellular DDB1 to mediate the degradation of cellular proteins known to silence cccDNA. Second, we have developed an authentic HBV infection model using human liver stem cell-derived organoids (HLOs). Finally, we have established a collaboration with the Center for Drug Discovery (CDD) at Baylor College of Medicine that has developed 50 libraries containing over 5 billion novel DNA-bar coded small drug-like molecules (DEC-Tec). We now propose a highly innovative project to ultimately identify new HBV antivirals that target HBx. In Aim 1, purified HBx protein will be screened by affinity selection against the DEC- Tec libraries. Binders will be identified by DNA sequencing and validated with fresh target protein. Considering that HBx interacts with over 100 cellular proteins, we expect to identify multiple HBx binders. Structure-activity relationship (SAR) will be determined using computational software, and the binder's affinity for HBx measured. Compounds with SAR and high affinity will be pursued. In Aim 2, high affinity HBx binders will be screened for the ability to inhibit the critical HBx-DDB1 interaction as measured in a split luciferase assay. Compounds that inhibit HBx-DDB1 will then be tested for the ability to inhibit HBV replication in the HLO replication model and compared against the known nucleos(t)ide analogue entecavir. Successful HBx binders will serve as leads for future in vivo evaluation that includes toxicity, drug metabolism, and pharmacokinetics. Other high affinity binders can be incorporated into proteolysis targeting chimeric molecules (PROTACs) for proteasome-mediated degradation. Worldwide, over 257 million people are chronically infected with HBV and approximately 1 million die each year of liver cirrhosis or cancer. The proposed studies hold tremendous potential for the discovery of novel approaches to treat chronic HBV, prevent HBV-associated cancer, and lead to a functional cure.