Dysregulation of Hepatitis B Virus Nucleocapsid Assembly with RNA-directed Small Ligands

Abstract

RNA sequences/motifs dispersed across the pre-genomic copy of the Hepatitis B Virus genome regulate formation of nucleocapsids in vitro in an epsilon/polymerase independent fashion. These multiple RNA Packaging Signals (PSs) form stem-loops presenting in each loop a core protein recognition motif, -RGAG-. Small, drug-like molecules binding these motifs were identified by screening an immobilized library with a fluorescently-labelled RNA oligonucleotide encompassing the most conserved of these sites. This identified 66 "hits", with affinities ranging from low nanomolar to high micromolar in SPR assays. High affinity ligand binding is dependent on the presence of the -RGAG-motif, which also appears to be the common element in cross-binding to other PS sites. Some of these compounds are potent inhibitors of in vitro core protein assembly around the HBV pre-genome. Mathematical modelling confirms the potential of these novel anti-viral drug targets for disrupting replication of this major human pathogen. Preliminary structure-activity relationships of the highest affinity compound reveal critical functional groups for PS-binding. PS-regulated assembly is easily adapted to high-throughput screening allowing future development of pharmacologically active compounds.