Teng-Yuan Chang

Design and Synthesis of Tetrahydropyridothieno[2,3-d]pyrimidine Scaffold Based Epidermal Growth Factor Receptor (EGFR) Kinase Inhibitors: The Role of Side Chain Chirality and Michael Acceptor Group for Maximal Potency

HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11 and 19 showed over 3 orders of magnitude enhanced HCC827 antiproliferative activity compared to HTS hit 7 and also inhibited gefitinib-resistant double mutant (DM, T790M/L858R) EGFR kinase at nanomolar concentration. Moreover, treatment with 19 shrinked tumor in nude mice xenograft model.

Diosgenin, a Plant-Derived Sapogenin, Exhibits Antiviral Activity in Vitro against Hepatitis C Virus

Diosgenin (3β-hydroxy-5-spirostene, 1), a plant-derived sapogenin, is used as a dietary supplement. However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 μM. No cellular toxicity was observed at this concentration. Diosgenin (1) also significantly reduced the levels of viral RNA and viral proteins as evaluated by quantitative real-time reverse transcriptase PCR and Western blot analysis, respectively. In addition, in an alternative HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 μM. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1 and interferon-α exerted an additive effect on the resultant anti-HCV activity.

Identification, SAR studies, and X-ray co-crystallographic analysis of a novel furanopyrimidine aurora kinase A inhibitor

Herein we reveal a simple method for the identification of novel Aurora kinase A inhibitors through substructure searching of an in‐house compound library to select compounds for testing. A hydrazone fragment conferring Aurora kinase activity and heterocyclic rings most frequently reported in kinase inhibitors were used as substructure queries to filter the in‐house compound library collection prior to testing. Five new series of Aurora kinase inhibitors were identified through this strategy, with IC50 values ranging from ∼300 nM to ∼15 μM, by testing only 133 compounds from a database of ∼125 000 compounds. Structure–activity relationship studies and X‐ray co‐crystallographic analysis of the most potent compound, a furanopyrimidine derivative with an IC50 value of 309 nM toward Aurora kinase A, were carried out. The knowledge gained through these studies could help in the future design of potent Aurora kinase inhibitors.

Aurora kinase A inhibitors : Identification, SAR exploration and molecular modeling of 6,7-dihydro-4H-pyrazolo-[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione scaffold

Tricyclic 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione was identified as a novel scaffold for Aurora kinase A inhibition through virtual screening. SAR exploration coupled with molecular modeling of 8a reveals the minimum pharmacophore requirements for Aurora kinase A inhibition.

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.

Bispecific antibody conjugated manganese-based magnetic engineered iron oxide for imaging of HER2/neu- and EGFR-expressing tumors

The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

BackgroundThere are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated.ResultsIn the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.ConclusionThe data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.

EGFR and myosin II inhibitors cooperate to suppress EGFR-T790M-mutant NSCLC cells

An acquired mutation (T790M) in the epidermal growth factor receptor (EGFR) accounts for half of all relapses in non‐small cell lung cancer (NSCLC) patients who initially respond to EGFR kinase inhibitors. In this study, we demonstrated for the first time that EGFR‐T790M interacts with the cytoskeletal components, myosin heavy chain 9 (MYH9) and β‐actin, in the nucleus of H1975 cells carrying the T790M‐mutant EGFR. The interactions of EGFR with MYH9 and β‐actin were reduced in the presence of blebbistatin, a specific inhibitor for the MYH9‐β‐actin interaction, suggesting that the EGFR interaction with MYH9 and β‐actin is affected by the integrity of the cytoskeleton. These physical interactions among MYH9, β‐actin, and EGFR were also impaired by CL‐387,785, a kinase inhibitor for EGFR‐T790M. Furthermore, CL‐387,785 and blebbistatin interacted in a synergistic fashion to suppress cell proliferation and induce apoptosis in H1975 cells. The combination of CL‐387,785 and blebbistatin enhanced the down‐regulation of cyclooxygenase‐2 (COX‐2), a transcriptional target of nuclear EGFR. Overall, our findings demonstrate that disrupting EGFR interactions with the cytoskeletal components enhanced the anti‐cancer effects of CL‐387,785 against H1975 cells, suggesting a novel therapeutic approach for NSCLC cells that express the drug‐resistant EGFR‐T790M.

The Combination Effects of LiCl and the Active Leflunomide Metabolite, A771726, on Viral-Induced Interleukin 6 Production and EV-A71 Replication

Enterovirus 71 (EV-A71) is a neurotropic virus that can cause severe complications involving the central nervous system. No effective antiviral therapeutics are available for treating EV-A71 infection and drug discovery efforts are rarely focused to target this disease. Thus, the main goal of this study was to discover existing drugs with novel indications that may effectively inhibit EV-A71 replication and the inflammatory cytokines elevation. In this study, we showed that LiCl, a GSK3β inhibitor, effectively suppressed EV-A71 replication, apoptosis and inflammatory cytokines production (Interleukin 6, Interleukin-1β) in infected cells. Furthermore, LiCl and an immunomodular agent were shown to strongly synergize with each other in suppressing EV-A71 replication. The results highlighted potential new treatment regimens in suppressing sequelae caused by EV-A71 replication.

Abstract B211: Augmentation of Fas (CD95) signaling pathway sensitizes non-small cell lung cancer (NSCLC) cells.

Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a subset of NSCLC harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR kinase inhibitors, suggesting that other factors may play a role in determining survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) functions as a transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been described in numbers of cancers. Our previous report showed that suppression of Stat3 activity by a pharmacological approach, WP1066, sensitized NSCLC cells. Along with Stat3 inhibition, we also found that WP1066 treatment up-regulated the expression of Fas, a death receptor, regulating numbers of physiological and pathological process of cell death. Although Fas-mediated apoptosis pathway has been studied mostly in the immune system, the identification of Fas mutations in non-lymphoid malignancies, such as NSCLC, suggests its implication in the pathogenesis of non-lymphoid malignancies as well. Results from this study showed that levels of Fas were increased in cells with down-regulated Stat3 by small interfering RNA (siRNA), suggesting that Stat3 may play an inhibitory role to regulate the expression of Fas. To examine whether the Fas pathway is responsible for the WP1066-mediated cytotoxicity, Fas apoptosis signaling was induced by anti-Fas antibody and Fas ligand. Our results revealed that NSCLC cells showed different degrees of sensitivity to anti-Fas antibody but resistance to Fas ligand. However, augmentation of Fas expression by WP1066 remarkably increased the sensitivity of NSCLC cells to both anti-Fas antibody and Fas ligand. Taken together, our findings suggest that regulation of the Fas system may be a potential strategy to treat NSCLC and the combined use of a Fas inducer, such as WP1066, may further strengthen its efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B211.