Jun-Goo Jee

Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13.

Abstract Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.

A Group of Novel HIF-1α Inhibitors, Glyceollins, Blocks HIF-1α Synthesis and Decreases Its Stability via Inhibition of the PI3K/AKT/mTOR Pathway and Hsp90 Binding

Glyceollins, a group of phytoalexins isolated from soybean, are known to exhibit anticancer, antiestrogenic, and antiangiogenic activities. However, whether glyceollins regulate tumor growth through regulation of hypoxia‐inducible factor (HIF)‐1α has not been investigated. We determined whether and how glyceollins regulate the synthesis and stability of HIF‐1α. Quantitative real‐time PCR revealed that glyceollins inhibited the expression of HIF‐1‐induced genes such as vascular endothelial growth factor (VEGF) in cancer cells. Enzyme‐linked immunosorbent assay and reporter luciferase assay showed that glyceollins decreased VEGF secretion and its promoter activity, respectively. Treatment of various cancer cells with 0.5–100 µM glyceollins under hypoxic conditions reduced the expression of HIF‐1α. Glyceollins blocked translation of HIF‐1α by inhibiting the PI3K/AKT/mTOR pathway under hypoxic conditions. Glyceollins decreased the stability of HIF‐1α after treatment with cycloheximide, a protein synthesis inhibitor, and increased the ubiquitination of HIF‐1α after treatment with MG132, a proteasome inhibitor. Glyceollins blocked the interaction of Hsp90 with HIF‐1α, as shown by immunoprecipitation assay. Chemical binding of Hsp90 with glyceollins, as confirmed by computational docking analysis, was stronger than that with geldanamycin at the HSP90 ATP‐binding pocket. We found that glyceollins decreased microvessel density, as well as expression of phosphorylated AKT/mTOR and the Hsp90 client protein CDK4, in solid tumor tissues. Glyceollins potently inhibited HIF‐1α synthesis and decreased its stability by blocking the PI3K/AKT/mTOR pathway and HSP90 binding activity, respectively. These results may provide new perspectives into potential therapeutic application of glyceollins for the prevention and treatment of hypervascularized diseases and into the mechanism of their anticancer activity. J. Cell. Physiol. 230: 853–862, 2015.

Barrier protective effects of piperlonguminine in LPS-induced inflammation in vitro and in vivo

Piperlonguminine (PL), an important component of Piper longum fruits, is well known to possess potent anti-hyperlipidemic, anti-platelet and anti-melanogenesis activities. In this study, we first investigated the possible barrier protective effects of piperlonguminine against proinflammatory responses induced by lipopolysaccharide (LPS) and the associated signaling pathways in vitro and in vivo. The barrier protective activities of PL were determined by measuring permeability, monocytes adhesion and migration, and activation of proinflammatory proteins in LPS-activated human umbilical vein endothelial cells (HUVECs) and in mice. We found that PL inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs) and adhesion/transendothelial migration of monocytes to human endothelial cells. PL also suppressed LPS-induced hyperpermeability and leukocytes migration in vivo. Further studies revealed that PL suppressed the production of tumor necrosis factor-α (TNF-α) or Interleukin (IL)-6 and activation of nuclear factor-κB (NF-κB) or extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, treatment with PL resulted in reduced LPS-induced septic mortality. Collectively, these results suggest that PL protects vascular barrier integrity by inhibiting hyperpermeability, expression of CAMs, adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.

Synthesis, antitumor activity, and structure-activity relationship study of trihydroxylated 2,4,6-triphenyl pyridines as potent and selective topoisomerase II inhibitors.

A series of eighteen trihydroxylated 2,4,6-triphenyl pyridines were designed and synthesized which contain hydroxyl groups at ortho, meta or para position of each phenyl rings attached to the central pyridine. They were evaluated for topoisomerase I and II inhibitory activity, and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Most of the compounds exhibited strong and selective topoisomerase II inhibitory activity compared to the positive control, etoposide, and also displayed significant cytotoxicity in low micromolar range. Trihydroxylated 2,4,6-triphenyl pyridines were more potent than mono- and di-hydroxylated 2,4,6-triphenyl pyridines, which have been previously studied in our research group. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was observed for the most compounds. Molecular docking study shows qualitatively consistent with the results of biological assays.

The role of thioredoxin reductase and glutathione reductase in plumbagin-induced, reactive oxygen species-mediated apoptosis in cancer cell lines.

Plumbagin is a secondary metabolite that was first identified in the Plumbago genus of plants. It is a naphthoquinone compound with anti-atherosclerosis, anticancer, anti-inflammatory, antimicrobial, contraceptive, cardiotonic, immunosuppressive, and neuroprotective activities. However, the mechanisms of plumbagins activities are largely unknown. In this study, we examined the effect of plumbagin on HepG2 hepatocellular carcinoma cells as well as LLC lung cancer cells, SiHa cervical carcinoma cells. Plumbagin significantly decreased HepG2 cell viability in a dose-dependent manner. Additionally, treatment with plumbagin significantly increased the Bax/Bcl-2 ratio and caspase-3/7 activity. Using the similarity ensemble approach (SEA)-a state-of-the-art cheminformatic technique-we identified two previously unknown cellular targets of plumbagin: thioredoxin reductase (TrxR) and glutathione reductase (GR). This was then confirmed using protein- and cell-based assays. We found that plumbagin was directly reduced by TrxR, and that this reduction was inhibited by the TrxR inhibitor, sodium aurothiomalate (ATM). Plumbagin also decreased the activity of GR. Plumbagin, and the GR inhibitor sodium arsenite all increased intracellular reactive oxygen species (ROS) levels and this increase was significantly attenuated by pretreatment with the ROS scavenger N-acetyl-cysteine (NAC) in HepG2 cells. Plumbagin increased TrxR-1 and heme oxygenase (HO)-1 expression and pretreatment with NAC significantly attenuated the plumbagin-induced increase of TrxR-1 and HO-1 expression in HepG2 cells, LLC cells and SiHa cells. Pretreatment with NAC significantly prevented the plumbagin-induced decrease in cell viability in these cell types. In conclusion, plumbagin exerted its anticancer effect by directly interacting with TrxR and GR, and thus increasing intracellular ROS levels.

Selective inhibition of the cytochrome P450 isoform by hyperoside and its potent inhibition of CYP2D6

Hyperoside, quercetin-3-O-galactoside, is a flavonoid isolated from Oenanthe javanica. In the present study, we investigated potential herb-drug inhibitory effects of hyperoside on nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) and human recombinant cDNA expressed CYP using a cocktail probe assay. Hyperoside strongly inhibited CYP2D6-catalyzed dextromethorphan O-demethylation, with IC₅₀ values of 1.2 and 0.81 μM after 0 and 15 min of preincubation, and a Ki value of 2.01 μM in HLMs, respectively. Hyperoside strongly decreased CYP2D6 activity dose-, but not time-, dependently in HLMs. In addition, the Lineweaver-Burk and Secondary plots for the inhibition of CYP2D6 in HLMs fitted a competitive inhibition mode. Furthermore, hyperoside decreased CYP2D6-catalyzed dextromethorphan O-demethylation activity of human recombinant cDNA-expressed CYP2D6, with an IC₅₀ value of 3.87 μM. However, other CYPs were not inhibited significantly by hyperoside. In conclusion, our data demonstrate that hyperoside is a potent selective CYP2D6 inhibitor in HLMs, and suggest that hyperoside might cause herb-drug interactions when co-administrated with CYP2D substrates.

Ensemble-Based Virtual Screening Led to the Discovery of New Classes of Potent Tyrosinase Inhibitors.

In this study, we report new classes of potent tyrosinase inhibitors identified by enhanced structure-based virtual screening prediction; the enzyme and melanin content assays were also confirmed. Tyrosinase, a type-3 copper protein, participates in two distinct reactions, hydroxylation of tyrosine to DOPA and conversion of DOPA to dopaquinone, in melanin biosynthesis. Although numerous inhibitors of this reaction have been reported, there is a lag in the discovery of the new functional moieties. In order to improve the performance of virtual screening, we first produced an ensemble of 10,000 structures using molecular dynamics simulation. Quantum mechanical calculation was used to determine the partial charges of catalytic copper ions based on the met and deoxy states. Second, we selected a structure showing an optimal receiver operating characteristic (ROC) curve with known direct binders and their physicochemically matched decoys. The structure revealed more than 10-fold higher enrichment at 1% of the ROC curve than those observed in X-ray structures. Third, high-throughput virtual screening with DOCK 3.6 was performed using a library consisting of approximately 400,000 small molecules derived from the ZINC database. Fourth, we obtained the top 60 molecules and tested their inhibition of mushroom tyrosinase. The extended assays included 21 analogs of the 21 initial hits to test their inhibition properties. Here, the moieties of tetrazole and triazole were identified as new binding cores interacting with the dicopper catalytic center. All 42 inhibitors showed inhibitory constant, Ki, values ranging from 11.1 nM and 33.4 μM, with a tetrazole compound exhibiting the strongest activity. Among the 42 molecules, five displayed more than 30% reduction in melanin production when treated in B16F10 melanoma cells; cell viability was >90% at 20 μM. Particularly, a thiosemicarbazone-containing compound reduced melanin content by 55%.

Bisdemethoxycurcumin Induces apoptosis in activated hepatic stellate cells via cannabinoid receptor 2.

Activated Hepatic Stellate Cells (HSCs), major fibrogenic cells in the liver, undergo apoptosis when liver injuries cease, which may contribute to the resolution of fibrosis. Bisdemethoxycurcumin (BDMC) is a natural derivative of curcumin with anti-inflammatory and anti-cancer activities. The therapeutic potential of BDMC in hepatic fibrosis has not been studied thus far in the context of the apoptosis in activated HSCs. In the current study, we compared the activities of BDMC and curcumin in the HSC-T6 cell line and demonstrated that BDMC relatively induced a potent apoptosis. BDMC-induced apoptosis was mediated by a combinatory inhibition of cytoprotective proteins, such as Bcl2 and heme oxygenase-1 and increased generation of reactive oxygen species. Intriguingly, BDMC-induced apoptosis was reversed with co-treatment of sr144528, a cannabinoid receptor (CBR) 2 antagonist, which was confirmed with genetic downregulation of the receptor using siCBR2. Additionally, incubation with BDMC increased the formation of death-induced signaling complex in HSC-T6 cells. Treatment with BDMC significantly diminished total intracellular ATP levels and upregulated ATP inhibitory factor-1. Collectively, the results demonstrate that BDMC induces apoptosis in activated HSCs, but not in hepatocytes, by impairing cellular energetics and causing a downregulation of cytoprotective proteins, likely through a mechanism that involves CBR2.

Analogues of ethionamide, a drug used for multidrug-resistant tuberculosis, exhibit potent inhibition of tyrosinase.

Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: the hydroxylation of tyrosine to DOPA followed by the oxidation of DOPA to dopaquinone. The central roles of melanin in living species have motivated researchers to maintain constant efforts to discover new agents that modulate tyrosinase activity. In this study, we report on the inhibition of tyrosinase by ethionamide and its analogues. Ethionamide, 2-ethylpyridine-4-carbothioamide, is a second-line antituberculosis drug used for the treatment of multidrug-resistant tuberculosis. The chemical similarity of ethionamide to phenylthiourea, a well-known tyrosinase inhibitor, led us to investigate its inhibitory effects on mushroom tyrosinase and the IC50 was calculated as 4 μM. Five analogues of ethionamide, including another antituberculosis drug, prothionamide, were also inhibitory, with values for IC50 in the range of 3-43 μM. Fluorescence quenching experiments supported a mechanism of direct binding. In contrast, isoniazid, a structural analogue and first-line antituberculosis drug, was a poor inhibitor of tyrosinase. We also tested the effects of ethionamide and its analogues on melanin content in B16F10 cells. At a concentration of 50 μM, the molecules, pyridine-2-carbothioamide and thiobenzamide substantially decreased the melanin content by 44% and 37%, respectively. In addition to identifying other interactions, docking simulations showed that the carbothioamide groups of the molecules make essential contacts with the catalytic di-copper atoms. Our results suggest that carbothioamide can be a central moiety for the development of new and potent tyrosinase inhibitors.

Thymosin Beta4 Regulates Cardiac Valve Formation Via Endothelial-Mesenchymal Transformation in Zebrafish Embryos

Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor β (TGFβ) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-β-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT.