Shiow-Ju Lee

Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay

ABSTRACT Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC50) of 15.43 ± 2.12 μM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC50) of 0.17 ± 0.01 μM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

ABSTRACT Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 μM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

Tylophorine arrests carcinoma cells at G1 phase by downregulating cyclin A2 expression.

Tylophorine, a representative phenanthroindolizidine alkaloid from Tylophoraindica plants, exhibits anti-inflammatory and anti-cancerous growth activities. However, the underlying mechanisms of its anti-cancer activity have not been elucidated and its effects on cell cycle remain ambiguous. Here, we reveal by asynchronizing and synchronizing approaches that tylophorine not only retards the S-phase progression but also dominantly arrests the cells at G1 phase in HepG2, HONE-1, and NUGC-3 carcinoma cells. Moreover, tylophorine treatment results in down regulated cyclin A2 expression and overexpressed cyclin A2 rescues the G1 arrest by tylophorine. Thus, we are the first to report that the downregulated cyclin A2 plays a vital role in G1 arrest by tylophorine in carcinoma cells.

c-Jun-mediated anticancer mechanisms of tylophorine

Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal constituent of herb Tylophora indica. Tylophorine treatment increased the accumulation of c-Jun protein, a component of activator protein 1 (AP1), in carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun phosphorylation was primarily mediated via activated c-Jun N-terminal protein kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed c-Jun in conjunction with tylophorine significantly increased the number of carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated downregulation of cyclin A2 protein levels is known to be involved in the primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter activity upon increased binding of c-Jun to the deregulation AP1 site and decreased binding to the upregulation activating transcription factor (ATF) site in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further, biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein level occurs primarily via two discrete prolonged signaling pathways: (i) the NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its stability by slowing its ubiquitination, and (ii) the PI3K_PDK1_PP2A_eEF2 cascade, which sustains eukaryotic elongation factor 2 (eEF2) activity and thus c-Jun protein translation. To the best of our knowledge, this report is the first to demonstrate the involvement of c-Jun in the anticancer activity of tylophorine and the release of c-Jun translation from a global translational blockade via the PI3K_PDK1_eEF2 signaling cascade.

Cytotoxic flavonoids from the leaves of Cryptocarya chinensis.

Bioassay-guided fractionation led to the isolation of six new tetrahydroflavanones, cryptochinones A-F (1-6), from the neutral CHCl(3) fraction of Cryptocarya chinensis leaves, together with 14 known compounds (7-20). The structures of these new compounds were determined through spectroscopic analyses, including 2D-NMR, MS, CD, and X-ray crystallographic analysis. Among the isolates, infectocaryone (7) showed cytotoxic activities with IC(50) values of 11.0 and 3.7 μM against NCI-H460 and SF-268 cell lines, respectively, and cryptocaryanone A (9) showed cytotoxic activities with IC(50) values of 5.1, 4.3, and 5.0 μM against MCF-7, NCI-H460, and SF-268 cell lines, respectively.

Synthesis and Biological Evaluation of Tylophorine-Derived Dibenzoquinolines as Orally Active Agents-Exploration of the Role of Tylophorine E Ring on Biological Activity.

A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further development into orally active agents.

Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus

Abstract The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses still remains an important mission. Here, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV). The potent compounds showed 50% maximal effective concentration (EC50) values ranging from 8 to 1468nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure–activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC50 values ranging from less than 5 to 340nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection.

Isolation and biological activities of phenanthroindolizidine and septicine alkaloids from the Formosan Tylophora ovata.

An investigation of alkaloids present in the leaves and stems of Tylophora ovata led to the isolation of two new septicine alkaloids and one new phenanthroindolizidine alkaloid, tylophovatines A, B, C (1, 2, and 5), respectively, together with two known septicine and six known phenanthroindolizidine alkaloids. The structures of the new alkaloids 1, 2, and 5 were established by means of spectroscopic analyses. These eleven alkaloids show in vitro anti-inflammatory activities with IC₅₀ values ranging from 84 nM to 20.6 μM through their suppression of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide and interferon-γ. Moreover, these substances display growth inhibition in HONE-1, NUGC-3, HepG2, SF-268, MCF-7, and NCI-H460 cancer cell lines, with GI₅₀ values ranging from 4 nM to 24.2 μM. In addition, tylophovatine C (5) and 13a(S)-(+)-tylophorine (7) were found to exhibit potent in vivo anti-inflammation activities in a rat paw edema model. Finally, structure–activity relationships were probed by using the isolated phenanthroindolizidines and septicines. Phenanthroindolizidines are suggested to be divided into cytotoxic agents (e.g., 10 and 11) and anti-inflammation based anticancer agents (e.g., 5–9).

Novel Small-Molecule Inhibitors of Transmissible Gastroenteritis Virus

ABSTRACT We used swine testicle (ST) cells infected with transmissible gastroenteritis virus (TGEV) and an indirect immunofluorescent assay with antibodies against TGEV spike and nucleocapsid proteins to screen small-molecule compounds that inhibit TGEV replication. Analogues of initial hits were collected and subjected to a 3CL protease (3CLpro) inhibition assay with recombinant 3CLpro and a fluorogenic peptide substrate. A series of benzothiazolium compounds were found to have inhibitory activity against TGEV 3CLpro and to exert anti-TGEV activities in terms of viral protein and RNA replication in TGEV-infected ST cells, with consequent protection of TGEV-infected ST cells from cytopathic effect by blocking the activation of caspase-3.

Cytotoxic cardenolide glycosides from the root of Reevesia formosana.

Bioassay-guided fractionation of the root tissue of Reevesia formosana led to isolation of 13 cardenolide glycosides, reevesiosides A-I and epi-reevesiosides F-I. Their structures were determined by means of spectroscopic analysis and single-crystal X-ray diffraction was performed using reevesioside A. Reevesioside A, reevesioside F, and epi-reevesioside F displayed especially potent cytotoxicity against the MCF-7 and NCI-H460 cancer cell lines, with IC(50) values of 63±2 and 19±1, 72±8 and 20±0, and 34±6 and 10±1 nM, respectively. Identification of the sugar constituents and unusual 18,20-epoxide cardenolide glycosides are described herein. Cardiac glycosides were previously unknown in the Sterculiaceae family.