Phenylpropenamide derivatives as inhibitors of hepatitis B virus replication.

A non-nucleoside class of compounds that inhibits the replication of hepatitis B virus (HBV) in cell culture has been discovered. A series of substituted analogues of phenylpropenamide 6 has been prepared and evaluated in the HepAD38 cellular assay. Structure-activity relationships of this series are discussed.

Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-L-2',3'-dideoxy-3'-thiacytidine.

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

COLO 205 utilizes E-selectin to adhere to human endothelium.

We have demonstrated that pretreatment of human umbilical vein endothelial cells (HUVEC) with tumor necrosis factor-alpha (TNF) or phorbol myristate acetate (PMA) augmented the binding of COLO 205, a human colon carcinoma cell line, in vitro. The increased adherence was both concentration and time dependent with a peak for tumor cell attachment at 4 hr. The increase in binding required both RNA and protein synthesis. This pattern is consistent with increased expression of the adhesion molecule E-selectin (ELAM-1) on the endothelium. Incubation of TNF- or PMA-stimulated HUVEC with BB11, an anti-E-selectin mAb, prior to the addition of COLO 205, led to almost complete inhibition of adherence of the tumor cells. Flow cytometric analysis confirmed that there was expression of E-selectin on HUVEC following both TNF and PMA stimulation.

Effect of inflammatory cytokines on the adherence of tumor cells to endothelium in a murine model.

We have demonstrated that pretreatment of mouse brain microvascular endothelial cells (MBE) with tumor necrosis factor-alpha (TNF), IL-1, or LPS augmented the binding of P815 mastocytoma cells in vitro. The effect of these agents was dose and time dependent. PMA was able to mimic the influence of these factors to a limited degree. The effect of TNF on endothelium was accompanied by the appearance of changes in the expression of proteins isolated from endothelial cell membranes. The adherence of tumor cells to endothelium was not inhibited by RGD-containing peptides but could be decreased by preincubation of endothelium with high concentrations of FCS. Our data suggest that cytokines regulate the synthesis of endothelial adhesion proteins which may be involved in tumor cell adherence leading to metastasis. These results raise the possibility that cytokines may exert paradoxical effects in vivo, i.e., a cytotoxic effect that reduces tumor mass accompanied by a metastasis-enhancing effect that actually promotes dissemination of the remaining tumor cells. Definition of the molecular events involved in tumor cell-endothelial cell interactions may lead to strategies for minimizing the latter effect in therapeutic settings.