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Background: Globo H (GH) is highly expressed in a variety of epithelial tumors with a limited expression in normal tissues rendering it a novel therapeutic target. OBI-999 is an antibody drug conjugate (ADC) consisting of a GH-specific monoclonal antibody conjugated with monomethyl auristatin E (MMAE) through a cathepsin B cleavable linker. MMAE is known to induce immunogenic cell death (ICD) which involves in the activation of cytotoxic T lymphocyte-driven adaptive immunity with long-term immunological memory. Aim: The aim of this study was to evaluate the synergistic effects of OBI-999 + pembrolizumab on tumor growth suppression in several xenograft tumor models. Methods: OBI-999-induced ICD was examined in vitro by the detection of damaging-associated molecular patterns (DAMPs) such as calreticulin (CRT), HMGB1, and ATP in incubated Globo H expressing cells. The synergistic effects with the combination of OBI-999 and pembrolizumab as well as the ICD-related immunity were assessed in vivo using advanced severe immunodeficient mice that were reconstituted with human peripheral blood mononuclear cells (PBMCs). Results: Incubation of OBI-999 with high GH expression cancer cell lines (HCC-1428, NCI-N87 and NCI-H526) and mid GH expression cancer cell line (SW-480) induced the release of DAMPs including CRT, HMGB1, and ATP, in a dose- and time-dependent manner, indicating that OBI-999 is capable of inducing ICD in vitro. In the high GH expression human breast cancer cell line HCC-1428 xenograft model, OBI-999+pembrolizumab exhibited significantly stronger inhibition of tumor growth compared to either OBI-999 or pembrolizumab alone. Similar synergistic effects were observed in other xenograft tumor models including gastric (NCI-N87), small cell lung (NCI-H526), and colorectal (SW-480) cancers. Analysis of tumor-infiltrating lymphocytes (TILs) in HCC-1428 humanized mice showed that OBI-999 + pembrolizumab induced populations of activated cytotoxic CD8 T-cells and mature dendritic cells. Pembrolizumab decreased PD-1 expression on CD8 and CD4 cells, and OBI-999 decreased PD-L1 expression on tumor cells, which reversed the exhausted status of immune cells and alleviated immunosuppression in the tumor microenvironment. Conclusion: We demonstrated significant synergistic effects of OBI-999 and pembrolizumab in several animal models. These synergistic effects may be attributed to the ability of OBI-999 to induce ICD, as demonstrated by the release of DAMPs in vitro and tumor-specific immunity in vivo, suggesting that OBI-999 can create a tumor microenvironment that enhances the function of pembrolizumab. The results suggest that combination therapy with OBI-999 and immune checkpoint inhibitors is warranted in human clinical studies. OBI-999 is currently in Phase 1/2 clinical trials for the treatment of advanced solid tumors with high GH expression (NCT04084366). Citation Format: Chun-Chung Wang, Chi-Huan Lu, Jhih-Jie Yang, Wan-Fen Li, Ming-Tain Lai. OBI-999, an anti-Globo H antibody drug conjugate, exhibits synergistic anti-tumor effect in combination with pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5946.

Immunogenic cell death (ICD) involves the activation of cytotoxic T lymphocyte-driven adaptive immunity with long-term immunological memory. OBI-3424 is a highly selective prodrug that is activated by AKR1C3 to release a potent bis-alkylating agent. This study aims to investigate whether OBI-3424 can induce ICD and create a tumor microenvironment that benefits the combination therapy of OBI-3424 with immune checkpoint inhibitors. OBI-3424 induced ICD was examined in vitro by incubation of the prodrug with AKR1C3 positive cells followed by the detection of damaging-associated molecular patterns (DAMPs). The ICD-related immunity was assessed in vivo using advanced severe immunodeficient mice that were engrafted with human peripheral blood mononuclear cells (PBMCs). Anti-tumor effect of OBI-3424 in combination with pembrolizumab was evaluated in a xenograft model using PBMC-humanized mice. Incubation of OBI-3424 with AKR1C3 positive cells H460 and HepG2 induced the release of DAMPs including calreticulin, HMGB1, and ATP, in dose- and time-dependent manners. The detection of the DAMPs indicated that OBI-3424 induced ICD in vitro. The OBI-3424-induced ICD and its related immunity were also assessed in vivo. PBMC-humanized mice were immunized with OBI-3424- or PBS-treated HepG2 cells and then challenged with live HepG2 cells. No tumor growth was noted in mice that were immunized with OBI-3424-treated cells, indicating that the dying HepG2 cells induced by OBI-3424 elicited an adaptive, tumor-specific immune response. Furthermore, OBI-3424 showed a synergistic anti-tumor effect in combination with pembrolizumab in HepG2 xenograft model using PBMC-humanized mice. OBI-3424 plus pembrolizumab exhibited significantly stronger inhibition on tumor growth (TGI 77.2%) when compared with the treatment of OBI-3424 (TGI 27.8%) or pembrolizumab (TGI -15.3%) alone. Analysis of tumor-infiltrating lymphocytes (TILs) showed that OBI-3424 treatment induced the populations of activated cytotoxic CD8 T-cells (CD45+/CD8+/CD69+ and CD45+/CD8+/Granzyme), activated helper CD4 T-cells (CD45+/CD4+/CD69+), and mature dendritic cells (CD11b+/CD86+). In addition, OBI-3424 treatment also increased PD-1 expression on CD8 and CD4 cells, which in turn potentiated the anti-tumor effect of pembrolizumab. We demonstrated that OBI-3424 was able to induce ICD as shown by the release of DAMPs in vitro and tumor-specific immunity in vivo. OBI-3424 also created a tumor microenvironment that enhances the function of pembrolizumab, supported by the synergistic effect in animals with the cotreatment of the two drugs. The results suggest that a combination therapy of OBI-3424 and anti-PD-1 in human clinical study is warranted. OBI-3424 is currently in Phase 1/2 clinical trials for solid tumor and acute lymphoblastic leukemia (NCT03592264 and NCT04315324). Citation Format: Chun-Chung Wang, Wan-Fen Li, Chih-Chan Lee, Lu-Tzu Chen, Jhih-Jie Yang, Jiann-Shiun Lai, Ming-Tain Lai. OBI-3424, an AKR1C3-activated prodrug, exhibits in vivo synergistic anti-tumor effect in combination with pembrolizumab by induction of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6111.

AST-3424/OBI-3424 (denoted by 3424) is a novel prodrug bis-alkylating agent activated by AKR1C3. AKR1C3 is overexpressed in many types of cancer, particularly in liver, non-small cell lung, gastric, renal and CRPC cancer. Currently 3424 is being studied in phase 1/2 clinical trials for the treatment of solid and hematologic cancers, and it represents potentially a novel, selective anti-cancer agent for multiple indications. In this study, AKR1C3-dependent activation of 3424 was investigated in vitro using recombinant human AKR1C3. AKR1C3-dependent cytotoxicity of 3424 was determined in a wide range of human cancer cell lines with different AKR1C3 expression levels. In addition, anti-tumor activity of 3424 was also investigated in a broad panel of CDX and PDX models. AKR1C3-dependent activation of prodrug 3424 was evident by monitoring the decrease of 3424 and generation of the active form, 2660. Kinetic analysis indicated that AKR1C3 exhibited higher catalytic efficiency towards 3424 compared to the physiological substrates. There was a strong correlation between 3424 cytotoxic potency and AKR1C3 expression. The racemic mixture induced DNA cross-linking in a concentration dependent manner. Tumor growth inhibition of 3424 was shown to be better than or comparable to the standard of care chemotherapy at clinically achievable doses as a single agent in various CDX models with high expression of AKR1C3, including liver HepG2, lung H460, castration-resistant prostate VCaP, gastric SNU-16, and kidney A498 cancer cell lines. The excellent anti-tumor efficacy of 3424 was further demonstrated in PDX models which have high level of AKR1C3 expression, but not in a model with low level of AKR1C3 expression. In the combination therapy, we showed that 3424 could enhance the efficacy of the standard care of chemotherapy in the CDX models. The results described here highlight that 3424 exhibits AKR1C3-dependent cytotoxicity in vitro and anti-tumor activity in vivo in a wide range of human cancer types, which support further development of 3424 as an anti-cancer agent for treating different types of cancers and the use of AKR1C3 as a biomarker to profile cancer patients and further guide patient selection for therapy with 3424.

Globo H (GH), a hexasaccharide, is expressed at low levels in normal tissues but is highly expressed in multiple cancer types, rendering it a promising target for cancer immunotherapy. OBI-999, a novel antibody–drug conjugate, is derived from a conjugation of a GH-specific mAb with a monomethyl auristatin E (MMAE) payload through a site-specific ThioBridge and a cleavable linker. OBI-999 high homogeneity with a drug-to-antibody ratio of 4 (>95%) was achieved using ThioBridge. OBI-999 displayed GH-dependent cellular internalization and trafficked to endosome and lysosome within 1 and 5 hours, respectively. Furthermore, OBI-999 showed low nanomolar cytotoxicity in the assay with high GH expression on tumor cells and exhibited a bystander killing effect on tumor cells with minimal GH expression. Tissue distribution indicated that OBI-999 and free MMAE gradually accumulated in the tumor, reaching maximum level at 168 hours after treatment, whereas OBI-999 and free MMAE decreased quickly at 4 hours after treatment in normal organs. Maximum MMAE level in the tumor was 16-fold higher than in serum, suggesting that OBI-999 is stable during circulation and MMAE is selectively released in the tumor. Excellent tumor growth inhibition of OBI-999 was demonstrated in breast, gastric, and pancreatic cancer xenograft or lung patient–derived xenograft models in a dose-dependent manner. The highest nonseverely toxic dose in cynomolgus monkeys is 10 mg/kg determined by a 3-week repeated-dose toxicology study demonstrating an acceptable safety margin. Taken together, these results support further clinical development of OBI-999, which is currently in a phase I/II clinical study in multiple solid tumors (NCT04084366). OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibition in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers, warranting further investigation in the treatment of solid tumors.

Terminal RNA elements of the dengue virus (DENV) genome are necessary for balanced stability of linear and circular conformations during replication. We examined the small hairpin (sHP) and co-existing and mutually-exclusive terminal RNA elements by mutagenesis analysis, compensatory mutation screening, and by probing with RNA fragments to explore localized RNA folding and long-range RNA interactions. We found that the first base pair of the sHP and the stability of SLB and the 3′SL bottom stem affected circularization; sHPgc/C10631G+G10644C prohibited circularization, sHPuG accelerated and stabilized 5′-to-3′ RNA hybridization, while C94A and A97G and C10649 mutations loosened SLB and 3′SL, respectively, for circularization. sHPuG+C10649G induced circularization and impeded replication, whereas point mutations that loosened the UAR or DAR ds region, strengthened the sHP, or reinforced the 3′SL bottom stem, rescued the replication deficiency. Overall, we reveal structural and sequence features and interplay of DENV genome terminal RNA elements essential to viral replication.

AKR1C3 is overexpressed at both levels of protein and RNA in many types of cancer, particularly in liver, gastric, renal, CRPC and non-small cell lung cancer. High AKR1C3 expression is directly related to treatment resistance and poor prognosis. AST-3424/OBI-3424 (denoted by 3424) is a novel prodrug activated by AKR1C3. Currently 3424 is being studied in phase I clinical trials for the treatment of solid and hematologic cancers. Here we characterize 3424 on its specificity against AKR1C3 in biochemical and cell-based assays. In addition, its anti-tumor activity as a single agent or in combination therapy in a broad panel of CDX and PDX models was also investigated. AKR1C3-dependent activation of prodrug 3424 was evident by monitoring the decrease of 3424 and generation of the active form, 2660, based on LC/MS-MS analysis. Kinetic analysis indicated that AKR1C3 exhibited higher catalytic efficiency towards 3424 compared to the physiological substrates. Cytotoxicity of 3424 was found to be dependent on the level of AKR1C3 expression in a wide range of human cancer cell lines. There was a strong correlation between 3424 cytotoxic potency and AKR1C3 expression at both protein and RNA levels across all the cell lines examined. Cytotoxicity of 3424, its enantiomer and racemic mixture were also greatly inhibited by the AKR1C3-specific inhibitor 3021. The racemic mixture induced DNA cross-linking in a concentration dependent manner. Tumor growth inhibition of 3424 was shown to be better than or comparable to the standard of care chemotherapy at clinically achievable doses as a single agent in various CDX models with high expression of AKR1C3 at both levels of protein and RNA, including gastric SNU-16, kidney A498, and castration-resistant prostate VCaP, liver HepG2 and lung H460 cancers. The excellent anti-tumor efficacy of 3424 was further demonstrated in PDX models that have high level of AKR1C3 expression (pancreatic PA1280, gastric GA6201, Lung cancer LU2505) but not in model with low level of AKR1C3 expression (lung cancer LU2057). A significant correlation was found between AKR1C3 expression level and 3424 anti-tumor activity in two lung LU2505 and LU2057 PDX xenograft models confirming that 3424 in vivo activity is AKR1C3-dependent, which is consistent with its AKR1C3-dependent cytotoxicity in vitro. In the combination therapy, we have showed that 3424 could enhance the efficacy of the standard care of chemotherapy in the CDX models of VCaP CRPC, SNU-16 gastric, and A498 renal cell carcinoma. In conclusion, the results described here highlight that 3424 exhibits AKR1C3-dependent cytotoxicity in vitro and anti-tumor activity in vivo in a wide range of human cancer types, which support further development of 3424 as an anti-cancer agent for treating different types of cancer and the use of AKR1C3 as a biomarker to profile cancer patients and further guide patient selection for therapy with 3424. Citation Format: Fanying Meng, Wan-Fen Li, Donald Jung, Chun-Chung Wang, Tianyang Qi, Chi-Sheng Shia, Ren-Yu Hsu, Yin-Cheng Hsieh, Jianxin Duan. Selective and broad anti-tumor activity of AKR1C3-activated prodrug AST-3424/OBI-3424 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1220.

Stage-specific embryonic antigen-4 (SSEA-4) ceramide, a globo-series hexasaccharide glycosphingolipid, has been reported to be a tumor-associated antigen. Here, we present a novel antibody-drug conjugate (ADC) targeting SSEA-4 to evaluate its potential as a therapeutic agent for cancer treatment. OBI-998 is an ADC comprising the humanized anti-SSEA-4 antibody (OBI-898) that is conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE) through maleimide and PEGylated cleavable linkers. We demonstrated the specificity of the naked antibody to SSEA-4 by screening against a panel of 21 related carbohydrate antigens using ELISA. OBI-998 displayed potent cytotoxic activity against cells (SKOV3) with a high level of SSEA-4 expression at sub-nanomolar potency but no effect on the viability of cells (SKBR3) with negligible SSEA-4 expression. The bystander killing effect of OBI-998 was shown by the transferring of conditioned medium from SKOV3 cells treated with 1 or 5 nM of OBI-998 to SKBR3 cells. Furthermore, significant bystander effects were observed by co-culturing high (NCI-N87) and low (PANC-1/GFP) SSEA-4-expressing cell lines at different ratios in the presence of 5 or 10 nM of OBI-998. OBI-998 was found to be rapidly internalized into cancer cells within 5 minutes upon binding to its target on the cell surface by confocal microscopy. OBI-998 showed significant anti-tumor efficacy in multiple cancer cell–derived xenograft models at doses of 1, 3, and 10 mg/kg in a dose-dependent manner. More importantly, OBI-998 at a dose of 10 mg/kg showed complete tumor regression in an EGFR-triple mutation non–small cell lung cancer xenograft model, which is resistant to the current last-line tyrosine kinase inhibitor, osimertinib. Pharmacokinetic analysis of OBI-998 revealed that total antibody and the conjugated antibody exhibited similar pharmacokinetic profiles, suggesting OBI-998 is highly stable in vivo. The biodistribution study in HCC1428 tumor-bearing mice indicated that MMAE accumulated in the tumor site at a higher level compared with other blood-rich organs. In addition, the MMAE levels in tumors reached peak level at 24 hours post-treatment, which was much higher than the maximum levels in other organs, and the level was sustained for 168 hours with tumor-to-muscle ratio of 329. OBI-998 is a novel ADC targeting SSEA-4 that possesses desired properties such as high target specificity, rapid internalization, potent cytotoxicity, and significant bystander effects. Furthermore, OBI-998 showed a high level of deposition and a persistent presence of MMAE in tumors and significant anti-tumor efficacy in a variety of animal models. Taken together, these results support the further development of OBI-998 as a therapeutic agent for SSEA-4-targeting cancer therapy. Citation Format: I-Ju Chen, Chun-Chung Wang, Chi-Sheng Shia, Chung-Chen Su, Chi-Huan Lu, Hui-Wen Chang, Ping-Tzu Chiu, Yueh-Chin Wu, Ming-Tain Lai, Wei-Chien Tang, Hsin-Yi Tung, Ren-Yu Hsu. Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 955.

An unrecorded disease of persimmon (Diospyros kaki) with black spot symptoms on its mature fruit sporadically occurred in central Taiwan in August 2014. The causal agent was isolated and re-inoculated to show its pathogenicity on persimmon. The pathogen was identified as Phyllosticta capitalensis based on morphology of its ascospores and conidia, and molecular barcodes from ex-type sequences on GenBank of the internal transcribed spacer region (ITS), the actin gene (ACT) and the translation elongation factor 1-alpha gene (TEF1). This is the first report of persimmon disease caused by P. capitalensis in Taiwan.

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified.

Garcinia multiflora is an evergreen arbor, which belongs to the family of Guttiferae. It grows mainly at the lower altitudes of the mountains in the Hengchun Peninsula, which is in southern Taiwan. In this study, bioactivity-guided screening was conducted to evaluate the anti-hepatocellular carcinoma bioactivity of the fruit's methanol extract of G. multiflora. The methanol extract residue was partitioned with various solvents. It was then separated and purified using flash chromatography. The active component was confirmed to be guttiferone F. In our present study, cytotoxicity assay revealed that the IC50 of guttiferone F was 14.15 ± 2.36 μM in PLC/PRF/5 cells, 10.17 ± 0.51 μM in HepG2 cells and 9.71 ± 1.28 μM in Hep3B cells. In the in vitro anticancer study, we found guttiferone F induced apoptosis by up-regulating cleaved pro-caspase-3 and PARP expressions in PLC/PRF/5 cells. The antitumor potency is similar to sorafenib, a therapeutic drug for liver cancer. Further in vivo study showed that guttiferone F inhibits minor tumor growth on s.c. xenograft tumor of human liver cancer cell line in female SCID mice. Although antitumor efficiency of guttiferone F is not superior to sorafenib, the dose (i.p., 25 mg/kg × 5 days) is proved safe to animal. According to our studies, guttiferone F can be a promising lead for liver cancer therapy and become a new liberator for liver cancer patients after modification of the drug.

Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a, which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress.

Background To identify a novel therapeutic agent for hepatocellular carcinoma (HCC), for which no promising therapeutic agent exists, we screened a panel of plants and found that Juniperus chinensis exhibited potential antiangiogenic and anti-HCC activities. We further investigated the antiangiogenic and anti-HCC effects of the active ingredient of J. chinensis extract, CBT-143-S-F6F7, both in vitro and in vivo. Methods A tube formation assay conducted using human umbilical vein endothelial cells (HUVECs) was first performed to identify the active ingredient of CBT-143-S-F6F7. A series of angiogenesis studies, including HUVEC migration, Matrigel plug, and chorioallantoic membrane (CAM) assays, were then performed to confirm the effects of CBT-143-S-F6F7 on angiogenesis. The effects of CBT-143-S-F6F7 on tumor growth were investigated using a subcutaneous and orthotopic mouse model of HCC. In vitro studies were performed to investigate the effects of CBT-143-S-F6F7 on the cell cycle and apoptosis in HCC cells. Moreover, protein arrays for angiogenesis and apoptosis were used to discover biomarkers that may be influenced by CBT-143-S-F6F7. Finally, nuclear magnetic resonance analysis was conducted to identify the compounds of CBT-143-S-F6F7. Results CBT-143-S-F6F7 showed significantly antiangiogenic activity in various assays, including HUVEC tube formation and migration, CAM, and Matrigel plug assays. In in vivo studies, gavage with CBT-143-S-F6F7 significantly repressed subcutaneous Huh7 tumor growth in severe combined immunodeficient (SCID) mice, and prolonged the survival of orthotopic Huh7 tumor-bearing SCID mice (a 40 % increase in median survival duration compared with the vehicle-treated mice). Immunohistochemical staining of subcutaneous Huh7 tumors in CBT-143-S-F6F7-treated mice showed a significantly decrease in the cell cycle regulatory protein cyclin D1, cellular proliferation marker Ki-67, and endothelial marker CD31. CBT-143-S-F6F7 caused arrest of the G2/M phase and induced Huh7 cell apoptosis, possibly contributing to the inhibition of HCC tumors. Protein array analysis revealed that several angiogenic and antiapoptotic factors were suppressed in CBT-143-S-F6F7-treated Huh7 cells. Finally, five compounds from CBT-143-S-F6F7 were identified. Conclusions According to these results, we report for the first time the antiangiogenic and anti-HCC activities of CBT-143-S-F6F7, the active fractional extract of J. chinensis. We believe that CBT-143-S-F6F7 warrants further evaluation as a new anti-HCC drug. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1250-6) contains supplementary material, which is available to authorized users.

Dengue virus (DENV) is a mosquito-borne human pathogen that causes a serious public-health threat in tropical and subtropical regions of the world. Neither a vaccine to prevent nor an effective therapeutic agent to treat DENV infection is currently available. We established a stable cell line harboring a luciferase-reporting DENV subgenomic replicon to screen for inhibitors of DENV. A total of 14,400 small-molecule (MW

Angiogenesis and apoptosis array analysis of CBT-143-S-F6F7-treated Huh7 cells. (PPT 1587 kb)

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid-NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA-protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA-protein complex, reduce the nucleic acid chaperone activity of NdAg.

Obesity, the excessive accumulation of lipids in the body, is closely associated with many prevalent human disorders. Continued efforts to identify plant extracts that exhibit anti-obesity effects have drawn much attention. This study investigated whether a Polygala tenuifolia extract (PTE) possesses anti-obesity activity and how PTE may affect liver gene expression and gut microbiota. We used 3T3-L1 adipocytes and a high-fat diet–induced obese mouse model to determine the effects of PTE on lipid accumulation. Next-generation sequencing analysis of liver gene expression and gut microbiota profiles following PTE treatment were conducted to elucidate possible mechanisms. We found that treatment of fully differentiated 3T3-L1 adipocytes with PTE inhibited lipid accumulation in the cells through reducing lipid formation and triglyceride content and by increasing lipase activity. No cytotoxicity was observed from the PTE treatment. After 5 weeks of treatment with PTE, the increased body weight, elevated serum triglyceride content, and liver steatosis in the high-fat diet–induced obese mice were each reduced. Liver transcriptomic analysis revealed that expression of genes involved in lipid and cholesterol metabolism was significantly altered. The low-grade chronic inflammation of obesity caused by a high-fat diet was also decreased after PTE treatment. In addition, treatment with PTE improved the relatively low Bacteroidetes/Firmicutes ratio in the gut of high-fat diet–fed mice through enrichment of the Proteobacteria population and reduction of the Deferribacteres population. In conclusion, treatment with PTE inhibited lipid accumulation by inducing the expression of the master transcription factor PPARα, attenuated the low-grade chronic inflammation of obesity, and also altered gut microbiota profiles. These results indicate that PTE has the potential to be developed into an anti-obesity food supplement and therapy. Abbreviations: Abcg5: ATP-binding cassette subfamily G member 5; ALT: alanine aminotransferase; AMPK: adenosine monophosphate-activated protein kinase; AST: aspartate aminotransferase; B/F: Bacteroidetes to Firmicutes [ratio]; C/EBPα: CCAAT/enhancer-binding protein alpha; CR: creatinine; Cyp51: cytochrome P450 family 51; DMEM: Dulbecco’s modified Eagle’s medium; Fabp5: fatty acid-binding protein 5; FBS: fetal bovine serum; Fdps: farnesyl diphosphate synthase; Glc: Glucose; HFD: high-fat diet; GO: gene ontology; HPRT: hypoxanthine guanine phosphoribosyl transferase; IBMS: 3-isobutyl-1-methylxanthine; Idi1: isopentenyl-diphosphate delta isomerase 1; IL-1β: interleukin-1-beta; Lpin1: phosphatidic acid phosphohydrolase; LPS: lipopolysaccharide; Mvd: mevalonate diphosphate decarboxylase; ND: normal diet; OTU: operational taxonomic units; Pcsk9: proprotein convertase subtilisin/kexin 9; Pctp: phosphatidylcholine transfer protein; PPARα: peroxisome proliferator-activated receptor alpha; PPARγ: peroxisome proliferator-activated receptor gamma; PTE: Polygala tenuifolia extract; Saa1: serum amyloid A1; SD: standard deviation; SEM: standard error of the mean; Serpina12: serpin family member 12; Sqle: squalene monooxygenase; SREBP1C: sterol regulatory element-binding protein 1C; TCHO: total cholesterol; TG: triglyceride

B-Raf is a member of the RAF family of serine/threonine kinases: it mediates cell division, differentiation, and apoptosis signals through the RAS-RAF-MAPK pathway. Thus, B-Raf is of keen interest in cancer therapy, such as melanoma. In this study, we propose the first combination approach to integrate the pharmacophore (PhModel), CoMFA, and CoMSIA models for B-Raf, and this approach could be used for screening and optimizing potential B-Raf inhibitors in silico. Ten PhModels were generated based on the HypoGen BEST algorithm with the flexible fit method and diverse inhibitor structures. Each PhModel was designated to the alignment rule and screening interface for CoMFA and CoMSIA models. Therefore, CoMFA and CoMSIA models could align and recognize diverse inhibitor structures. We used two quality validation methods to test the predication accuracy of these combination models. In the previously proposed combination approaches, they have a common factor in that the number of training set inhibitors is greater than that of testing set inhibitors. In our study, the 189 known diverse series B-Raf inhibitors, which are 7-fold the number of training set inhibitors, were used as a testing set in the partial least-squares validation. The best validation results were made by the CoMFA09 and CoMSIA09 models based on the Hypo09 alignment model. The predictive r(2)(pred) values of 0.56 and 0.56 were derived from the CoMFA09 and CoMSIA09 models, respectively. The CoMFA09 and CoMSIA09 models also had a satisfied predication accuracy of 77.78% and 80%, and the goodness of hit test score of 0.675 and 0.699, respectively. These results indicate that our combination approach could effectively identify diverse B-Raf inhibitors and predict the activity.

Hepatocellular carcinoma (HCC) is one of the most common cancers and is also a leading cause of cancer-related deaths worldwide. Until now, treatments for HCC remain limited, especially for the shortage of promising systemic therapies to replace systemic chemotherapy. Sorafenib, the first drug applied in targeted therapy for HCC, was approved and drew considerable attention. However, the efficacy and side effects of sorafenib are still unsatisfied, which make sorafenib far from ideal for treatment of HCC. Thus, developing novel therapeutics for HCC is necessary. A novel compound, ITRI-2531, is developed to improve the efficacy from sorafenib. ITRI-2531 shows cytotoxicity in HCC cell lines (Huh-7 and PLC/PRF/5) and patient-derived HCC tissue cell lines. In studies of kinase activities, ITRI-2531 suppresses the phosphorylation of MEK and ERK in PLC/PRF/5 cells and shows inhibition of multiple kinases (FLT3, FLT4, PDGFRA, PGDFRB, and VEGFR2) in KINOMEscanTM study. In in vivo studies, oral treatment of ITRI-2531 repressed subcutaneous Huh-7 and PLC/PRF/5 tumor growth in severe combined immunodeficient (SCID) mice and shows better efficacy than sorafinib. Moreover, ITRI-2531 prolongs the survival of SCID mice with orthotopic patient-derived xenograft tumor and suppresses alpha-fetoprotein in serum. On the other hand, ITRI-2531 also has good pharmacokinetic profiles. According to these results, we believe that ITRI-2531 warrants further evaluation as a new anti-HCC drug. Citation Format: Tsung-Keng Kuo, On Lee, Mai-Wei Lin, Li-Zong Lin, Chun-Min Liu, Tai-ju Hsieh, Chun-Chung Wang, Shyh-Horng Lin, Chia-Ni Chang, Hui-Chun Hsu, Nien-Tzu Chou, Chin-Pen Lai, Chih-Hung Chen, Chia-Mu Tu, Shih-Ta Chen, Yuan-Jang Tsai, Chih-Peng Liu, Jenn-Tsang Hwang, Jui-Wen Huang, Yen-Chun Chen, Chrong-Shiong Hwang, Hsiang-Wen Tseng. ITRI-2531 - A novel kinase inhibitor for treating hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B186.

The helicase domain of dengue virus NS3 protein (DENV NS3H) contains RNA-stimulated nucleoside triphosphatase (NTPase), ATPase/helicase, and RNA 5′-triphosphatase (RTPase) activities that are essential for viral RNA replication and capping. Here, we show that DENV NS3H unwinds 3′-tailed duplex with an RNA but not a DNA loading strand, and the helicase activity is poorly processive. The substrate of the divalent cation-dependent RTPase activity is not restricted to viral RNA 5′-terminus, a protruding 5′-terminus made the RNA 5′-triphosphate readily accessible to DENV NS3H. DENV NS3H preferentially binds RNA to DNA, and the functional interaction with RNA is sensitive to ionic strength.

NS3H, the helicase domain of HCV NS3, possesses RNA-stimulated ATPase and ATP hydrolysis-dependent dsRNA unwinding activities. Here, the ability of NS3H to facilitate RNA structural rearrangement is studied using relatively long RNA strands as the model substrates. NS3H promotes intermolecular annealing, resolves three-stranded RNA duplexes, and assists dsRNA and ssRNA inter-conversions to establish a steady state among RNA structures. NS3H facilitates RNA structure conversions in a mode distinct from an ATP-independent RNA chaperone. These findings expand the known function of HCV NS3 helicase and reveal a role for viral helicase in assisting RNA structure conversions during virus life cycle.