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Epidermal growth factor receptor (EGFR)-targeted therapy in non-small cell lung cancer represents a breakthrough in the field of precision medicine. Previously, we have identified a lead compound, furanopyrimidine 2, which contains a (S)-2-phenylglycinol structure as a key fragment to inhibit EGFR. However, compound 2 showed high clearance and poor oral bioavailability in its pharmacokinetics studies. In this work, we optimized compound 2 by scaffold hopping and exploiting the potent inhibitory activity of various warhead groups to obtain a clinical candidate, 78 (DBPR112), which not only displayed a potent inhibitory activity against EGFRL858R/T790M double mutations but also exhibited tenfold potency better than the third-generation inhibitor, osimertinib, against EGFR and HER2 exon 20 insertion mutations. Overall, pharmacokinetic improvement through lead-to-candidate optimization yielded fourfold oral AUC better that afatinib along with F = 41.5%, an encouraging safety profile, and significant antitumor efficacy in in vivo xenograft models. DBPR112 is currently undergoing phase 1 clinical trial in Taiwan.

Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a small subset of non small cell lung cancer (NSCLC) harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR inhibitors, suggesting that other factors are implicated in survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) function as transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been discovered in a number of cancers. In this study, we found that a small molecule, reactivation of p53 and induction of tumor cell apoptosis (RITA), showed anti-cancer activity against gefitinib-resistant H1650 cells through a p53-independent pathway. Stat3 suppression by RITA attracted our attention to investigate the role of Stat3 in sustaining survival of H1650 cells. Pharmacological and genetic approaches were employed to down-regulate Stat3 in H1650 cells. WP1066, a known Stat3 inhibitor, was shown to exhibit inhibitory effect on the growth of H1650 cells. Meanwhile, apoptosis activation by siRNA-mediated down-regulation of Stat3 in H1650 cells provides more direct evidence for the involvement of Stat3 in viability maintenance of H1650 cells. Moreover, as a novel identified Stat3 inhibitor, RITA increased doxorubicin sensitivity of H1650 cells in vitro and in vivo, suggesting that doxorubicin accompanied with Stat3 inhibitors may be considered as an alternative strategy to treat NSCLC patients who have inherent resistance to doxorubicin. Overall, our observations reveal that targeting Stat3 may be an effective treatment for certain NSCLC cells with oncogenic addition to Stat3.

Designed from a high throughput screened hit compound, novel 2-amino-1-thiazolyl imidazoles were synthesized and demonstrated cytotoxicity against human cancer cells. 1-(4-Phenylthiazol-2-yl)-4-(thiophen-2-yl)-1H-imidazol-2-amine (compound 2), a 2-amino-1-thiazolyl imidazole, inhibited tubulin polymerization, interacted with the colchicine-binding sites of tubulins, and caused cell cycle arrest at the G(2)/M phase in human gastric cancer cells. Disruption of the microtubule structure in cancer cells by compound 2 was also observed. Compound 2 concentration-dependently inhibited the proliferation of cancer cells in histocultured human gastric and colorectal tumors. Given orally, compound 2 prolonged the lifespans of leukemia mice intraperitoneally inoculated with the murine P388 leukemic cells. We report 2-amino-1-thiazolyl imidazoles as a novel class of orally active microtubule-destabilizing anticancer agents.

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.

Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly. In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.

Mortalities of white shrimp Litopenaeus vannamei juveniles exceeding 80% have occurred in Taiwan since late 1998. It has been determined that Taura syndrome virus (TSV) is responsible for the epizootic outbreaks. In order to clarify the origin of these epizootics, a 3288 base pair fragment that encodes a TSV coat protein was amplified by RT-PCR. Analysis showed that the Taiwan isolate had 97% and 98% identity to the sequences in Genebank originated from isolates obtained from Mexico and Hawaii, respectively, both in cDNA and deduced amino acid sequences. These results suggest that TSV that infected the white shrimp in Taiwan in late 1998 and early 1999 is similar to the Western hemisphere isolates. We conclude that the Taiwan isolate originated in the Western hemisphere. Urgent and strict quarantine is advised to prevent Taura syndrome dissemination to other areas in Asia.

Background Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells. Principal Findings BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats. Conclusions and Significance BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.

A focused library of furanopyrimidine (350 compounds) was rapidly synthesized in parallel reactors and in situ screened for Aurora and epidermal growth factor receptor (EGFR) kinase activity, leading to the identification of some interesting hits. On the basis of structural biology observations, the hit 1a was modified to better fit the back pocket, producing the potent Aurora inhibitor 3 with submicromolar antiproliferative activity in HCT-116 colon cancer cell line. On the basis of docking studies with EGFR hit 1s, introduction of acrylamide Michael acceptor group led to 8, which inhibited both the wild and mutant EGFR kinase and also showed antiproliferative activity in HCC827 lung cancer cell line. Furthermore, the X-ray cocrystal study of 3 and 8 in complex with Aurora and EGFR, respectively, confirmed their hypothesized binding modes. Library construction, in situ screening, and structure-based drug design (SBDD) strategy described here could be applied for the lead identification of other kinases.

This study isolated agonists of peroxisome proliferator activated receptors (PPARs) from the green algae Chlorella sorokiniana, using a bioassay-guided purification strategy. PPARs are widely recognized as the molecular drug targets for many diseases including hyperglycemia, diabetes, obesity and cancer. Two independent bioassays were developed. The first is the scintillation proximity assay, a ligand binding assay. The other is the cell-based transcriptional activation assay which uses the Dual-Luciferase reporter system as the reporter gene under the control of the PPAR response element. Using these two assays, a PPARgamma-active fraction, CE 3-3, was obtained from C. sorokiniana extracts, which was also able to activate PPARalphamediated gene expression. To elucidate the active ingredients in the CE 3-3 fraction, GC-MS analysis was employed. The results showed that the CE 3-3 fraction consisted of at least ten fatty acids (FAs). The bioactivities of several of the individual FAs were evaluated for their PPARgamma activity and the results showed that linolenic acid and linoleic acid were the most potent FAs tested. Our studies indicate that Chlorella sorokiniana could have potential health benefits through the dual activation of PPARalpha/gamma via its unique FA constituents.

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.

Chronic hepatitis C virus (HCV) infection is a worldwide health problem causing serious complications, such as liver cirrhosis and hepatoma. Alpha interferon (IFN-α) or its polyethylene glycol-modified form combined with ribavirin is the only recommended therapy. However, an alternative therapy is needed due to the unsatisfactory cure rate of the IFN-based therapy. Using a modified reporter assay based on the HCV subgenomic-replicon system, we found that sodium stibogluconate (SSG), a compound used for leishmania treatment, suppressed HCV replication. We have previously reported that SSG is effective at inhibiting HCV replication in a cell line permissive for HCV infection/replication and in an ex vivo assay using fresh human liver slices obtained from patients infected with HCV (26). In this study, we show that the SSG 50% inhibitory dose for HCV replication is 0.2 to 0.3 mg/ml (equivalent to 345 to 517 μM of Sb) in the HCV subgenomic-replicon system. We also found that SSG and IFN-α exert a strong synergistic anti-HCV effect in both the traditional isobologram analysis and the median effect principle (CalcuSyn analysis). The combination of SSG and IFN-α could sustain the antiviral response better than SSG or IFN-α alone. The results suggest that SSG may be a good drug candidate for use in combination with other therapeutics, such as IFN-α and ribavirin, to treat HCV infection.