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. Compounds 1 and 2 exhibit strong inhibitory activity against HBV replication in both 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 induced the formation of morphologically intact capsids and accelerated the dynamics of capsid assembly, suggesting that both 1 and 2 were type I capsid assembly modulators (CAMs).