Discovery of New Hepatitis B Virus Capsid Assembly Modulators by an Optimal High-throughput Cell-based Assay.

In this article, a simple and effective high-throughput screening (HTS) assay was developed to identify anti-HBV compounds by using a HepAD38 luciferase reporter (HepAD38-luc) cell line that can effectively exclude the false positive hit compounds targeted on the tetracycline off (tet-off) regulation system. Through screening in-house chemical libraries, N-phenylpiperidine-3-carboxamide derivatives, represented by 1 and 2 were identified, while the other false positive hits, i. e. quinoxaline (3) and benzothiazin (4) derivatives were simultaneously excluded. Compound 1 and 2 exhibit strong inhibitory activity against HBV replication both in HepAD38 and HepG2.2.15 cells. Further studies revealed that 1 and 2 reduced extracellular HBV DNA, HBeAg and intracellular HBV intermediates, including total DNA, RNA and precore RNA of HBV. Size exclusion chromatography (SEC) and electron microscopy (EM) investigations demonstrated that 1 and 2 remarkably induce the formation of morphologically intact capsids and accelerate the dynamics of capsid assembly，suggesting that both 1 and 2 were type I capsid assembly modulators (CAMs).