Molecular design, synthesis, and biological evaluation of bisamide derivatives as cyclophilin A inhibitors for HCV treatment.

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. Direct-acting antivirals (DAAs), including inhibitors of nonstructural proteins (NS3/4A protease, NS5A, and NS5B polymerase), represent key components of anti-HCV treatment. However, some DAAs are associated with increased drug resistance and undesired side effects. Previous reports have shown that bisamides could be a novel class of cyclophilin A (CypA) inhibitors for treating HCV as a member of combinational therapies. To fully elucidate structure-activity relationships of bisamide derivatives and find a better hit compound with diverse binding modes, 16 biamides were designed with the help of docking program. They were then synthesized using one-pot four-component Ugi reaction. 7e with selectivity index of more than 18.9 (50% effective concentration of 5.3 µM, but no cytotoxicity at 100 µM) and unique binding mode that could be dived into gatekeeper pocket was selected as a new hit compound. Surface plasmon resonance experiments revealed that 7e is able to bind to CypA with a KD of 3.66 µM. Taken together, these results suggest that 7e as a CypA inhibitor could be used as an alternative anti-HCV agent in combinational therapy in the future.

Inhibition of protein disulfide isomerase in glioblastoma causes marked downregulation of DNA repair and DNA damage response genes.

Aberrant overexpression of endoplasmic reticulum (ER)-resident oxidoreductase protein disulfide isomerase (PDI) plays an important role in cancer progression. In this study, we demonstrate that PDI promotes glioblastoma (GBM) cell growth and describe a class of allosteric PDI inhibitors that are selective for PDI over other PDI family members. Methods: We performed a phenotypic screening triage campaign of over 20,000 diverse compounds to identify PDI inhibitors cytotoxic to cancer cells. From this screen, BAP2 emerged as a lead compound, and we assessed BAP2-PDI interactions with gel filtration, thiol-competition assays, and site-directed mutagenesis studies. To assess selectivity, we compared BAP2 activity across several PDI family members in the PDI reductase assay. Finally, we performed in vivo studies with a mouse xenograft model of GBM combining BAP2 and the standard of care (temozolomide and radiation), and identified affected gene pathways with nascent RNA sequencing (Bru-seq). Results: BAP2 and related analogs are novel PDI inhibitors that selectively inhibit PDIA1 and PDIp. Though BAP2 contains a weak Michael acceptor, interaction with PDI relies on Histidine 256 in the b' domain of PDI, suggesting allosteric binding. Furthermore, both in vitro and in vivo, BAP2 reduces cell and tumor growth. BAP2 alters the transcription of genes involved in the unfolded protein response, ER stress, apoptosis and DNA repair response. Conclusion: These results indicate that BAP2 has anti-tumor activity and the suppressive effect on DNA repair gene expression warrants combination with DNA damaging agents to treat GBM.

Ellagic Acid, the Active Compound of Phyllanthus urinaria, Exerts In Vivo Anti-Angiogenic Effect and Inhibits MMP-2 Activity.

This study aimed to assess the potential anti-angiogenic mechanism of Phyllanthus urinaria (P. urinaria) and characterize the major compound in P. urinaria that exerts anti-angiogenic effect. The water extract of P. urinaria and Ellagic Acid were used to evaluate the anti-angiogenic effect in chorioallantoic membrane (CAM) in chicken embryo and human vascular endothelial cells (HUVECs). The matrix metalloproteinase-2 (MMP-2) activity was determined by gelatin zymography. The mRNA expressions of MMP-2, MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Level of MMP-2 proteins in conditioned medium or cytosol was determined by western blot analysis. We confirmed that P. urinaria's in vivo anti-angiogenic effect was associated with a reduction in MMP-2 activity. Ellagic acid, one of the major polyphenolic components as identified in P. urinaria by high performance liquid chromatography mass spectrometry (HPLC/MS), exhibited the same anti-angiogenic effect in vivo. Both P. urinaria and Ellagic Acid inhibited MMP-2 activity in HUVECs with unchanged mRNA level. The mRNA expression levels of MMP-14 and TIMP-2 were not altered either. Results from comparing the change of MMP-2 protein levels in conditioned medium and cytosol of HUVECs after the P. urinaria or Ellagic Acid treatment revealed an inhibitory effect on the secretion of MMP-2 protein. This study concluded that Ellagic Acid is the active compound in P. urinaria to exhibit anti-angiogenic activity and to inhibit the secretion of MMP-2 protein from HUVECs.

Anti-tumor and anti-angiogenic effects of Phyllanthus urinaria in mice bearing Lewis lung carcinoma.

Phyllanthus urinaria, a widely used herb medicine in Asia, was tested for its anti-tumor effect in vivo for the first time. The anti-tumor activity in P. urinaria extract was evaluated by its effect on tumor developed in C57BL/6J mice with implantation of Lewis lung carcinoma cells. The oral administration of P. urinaria to mice caused significant inhibition of tumor development with lower occurrence rate and markedly reduced tumor size. Neither the total body weight of mouse nor the weights of organs including heart, lung, liver, spleen and kidney revealed any difference between two groups, suggesting limited in vivo cytotoxic effect of P. urinaria in mice. TUNEL assay demonstrated the increase of apoptosis in tumor sections prepared from P. urinaria-treated mice compared with control mice. It is worth of note that the neovascularization in tumor was inhibited in P. urinaria-treated mice, which implicated the potential anti-angiogenic effect of P. urinaria. Further study using an in vitro matrix-induced tube formation of HUVECs again confirmed the anti-angiogenic action of P. urinaria. P. urinaria exerted no inhibitory effect on the growth of HUVECs, however, the migration of HUVECs as analyzed using transwell assay was suppressed markedly by P. urinaria in a dose-dependent manner. All together, the present study indicated that P. urinaria extract is an anti-tumor and anti-angiogenic agent, which can be used safely in animals.

Protective effect of propylthiouracil independent of its hypothyroid effect on atherogenesis in cholesterol-fed rabbits: PTEN induction and inhibition of vascular smooth muscle cell proliferation and migration.

BACKGROUND: Propylthiouracil (PTU) is used to treat hyperthyroid patients by its hypothyroid effect. PTU also is found to have potent antioxidant and immunosuppressive effects. These findings suggest that PTU may play a role in the prevention of atherosclerosis. METHODS AND RESULTS: This study evaluates the effect of PTU on atherosclerotic change in rabbits fed a high-cholesterol diet. The pronounced atherosclerotic lesions in the aortas of rabbits fed a 2% cholesterol diet for 12 weeks were significantly attenuated by the concurrent addition of 0.1% PTU to the drinking water. However, exogenous supplementation of thyroid hormone in hypothyroid PTU-treated rabbits did not abrogate the protective effect of PTU on atherogenesis. Immunohistochemical analysis showed that PTU administration apparently reduced the intimal smooth muscle cell/macrophage ratio in the atherosclerotic plaques of rabbits fed a 2% cholesterol diet. In vitro, the addition of PTU to the medium of cultured rat vascular smooth muscle cells led to a dose-dependent inhibition of cell proliferation and migration. Furthermore, this study confirmed that PTU dose-dependently increased expression of PTEN, a tumor suppressor gene known to be involved in the coordinate inhibition of VSMC proliferation and migration. CONCLUSIONS: This study demonstrated that PTU inhibited the development of atherosclerosis through a thyroid-independent mechanism that may be explained, at least in part, by the ability of PTU to inhibit vascular smooth muscle cell proliferation and migration. Furthermore, PTEN induction, via disruption of the phosphatidylinsitol 3-kinase-mediated pathway, plays a crucial role in mediating the inhibitory action on vascular smooth muscle cell proliferation and migration.