Antitumor activity of a pexidartinib bioisostere inhibiting CSF1 production and CSF1R kinase activity in human hepatocellular carcinoma.

Macrophage colony-stimulating factor (M-CSF, also known as CSF1) in tumor tissues stimulates tumor growth and tumor-induced angiogenesis through an autocrine and paracrine action on CSF1 receptor (CSF1R). In the present study, novel bioisosteres of pexidartinib (1) were synthesized and evaluated their inhibitory activities against CSF1R kinase and tumor growth. Among newly synthesized bioisosteres, compound 3 showed the highest inhibition (95.1%) against CSF1R tyrosine kinase at a fixed concentration (1 µM). The half maximal inhibitory concentration (IC50) of pexidartinib (1) and compound 3 was 2.7 and 57.8 nM, respectively. Unlike pexidartinib (1), which cross-reacts to three targets with structural homology, such as CSF1R, c-KIT, and FLT3, compound 3 inhibited CSF1R, c-KIT, but not FLT3, indicating compound 3 may be a more selective CSF1R inhibitor than pexidartinib (1). The inhibitory effect of compound 3 on the proliferation of various cancer cell lines was the strongest in U937 cells followed by THP-1 cells. In the case of cancer cell lines derived from solid tumors, the anti-proliferative activity of compound 3 was weaker than pexidartinib (1), except for Hep3B. However, compound 3 was safer than pexidartinib (1) in terminally differentiated normal cells such as macrophages. Pexidartinib (1) and compound 3 suppressed the production of CSF1 in Hep3B liver cancer cells as well as in the co-culture of Hep3B cells and macrophages. Also, pexidartinib (1) and compound 3 decreased the population ratio of the M2/M1 phenotype and inhibited their migration. Importantly, compound 3 preferentially inhibited M2 phenotype over M1, and the effect was about 4 times greater than that of pexidartinib (1). In addition, compound 3 inhibited maintenance of cancer stem cell population. In a chick chorioallantoic membrane (CAM) tumor model implanted with Hep3B cells, tumor growth and tumor-induced angiogenesis were significantly blocked by compound 3 to a similar extent as pexidartinib (1). Overall, compound 3, a bioisostere of pexidartinib, is an effective dual inhibitor to block CSF1R kinase and CSF1 production, resulting in significant inhibition of tumor growth.

6-Amino-2,4,5-trimethylpyridin-3-ol and 2-amino-4,6-dimethylpyrimidin-5-ol derivatives as selective fibroblast growth factor receptor 4 inhibitors: design, synthesis, molecular docking, and anti-hepatocellular carcinoma efficacy evaluation.

A novel series of aminotrimethylpyridinol and aminodimethylpyrimidinol derivatives were designed and synthesised for FGFR4 inhibitors. Structure-activity relationship on the FGFR4 inhibitory activity of the new compounds was clearly elucidated by an intensive molecular docking study. Anti-cancer activity of the compounds was evaluated using hepatocellular carcinoma (HCC) cell lines and a chick chorioallantoic membrane (CAM) tumour model. Compound 6O showed FGFR4 inhibitory activity over FGFR1 - 3. Compared to the positive control BLU9931, compound 6O exhibited at least 8 times higher FGFR4 selectivity. Strong anti-proliferative activity of compound 6O was observed against Hep3B, an HCC cell line which was a much more sensitive cell line to BLU9931. In vivo anti-tumour activity of compound 6O against Hep3B-xenografted CAM tumour model was almost similar to BLU9931. Overall, compound 6O, a novel derivative of aminodimethylpyrimidinol, was a selective FGFR4 kinase inhibitor blocking HCC tumour growth.

TPH1 and 5-HT7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma.

In the present study, we investigated the regulatory mechanisms underlying overexpression of EZH2, tryptophan hydroxylase 1 (TPH1), and 5-HT7, in relation to gemcitabine resistance and CSC survival in PDAC cells. In aggressive PANC-1 and MIA PaCa-2 cells, knock-down (KD) of EZH2, TPH1, or HTR7 induced a decrease in CSCs and recovery from gemcitabine resistance, while preconditioning of less aggressive Capan-1 cells with 5-HT induced gemcitabine resistance with increased expression of EZH2, TPH1, and 5-HT7. Such effects of the gene KD and 5-HT treatment were mediated through PI3K/Akt and JAK2/STAT3 signaling pathways. EZH2 KD or GSK-126 (an EZH2 inhibitor) inhibited activities of these signaling pathways which altered nuclear level of NF-kB, Sp1, and p-STAT3, accompanied by downregulation of TPH1 and 5-HT7. Co-immunoprecipation with EZH2 and pan-methyl lysine antibodies revealed that auto-methylated EZH2 served as a scaffold for binding with methylated NF-kB and Sp1 as well as unmethylated p-STAT3. Furthermore, the inhibitor of EZH2, TPH1, or 5-HT7 effectively regressed pancreatic tumor growth in a xenografted mouse tumor model. Overall, the results revealed that long-term exposure to 5-HT upregulated EZH2, and the noncanonical action of EZH2 allowed the expression of TPH1-5-HT7 axis leading to gemcitabine resistance and CSC population in PDAC.

Novel Pyridine Bioisostere of Cabozantinib as a Potent c-Met Kinase Inhibitor: Synthesis and Anti-Tumor Activity against Hepatocellular Carcinoma.

Two novel bioisosteres of cabozantinib, 3 and 4, were designed and synthesized. The benzene ring in the center of the cabozantinib structure was replaced by trimethylpyridine (3) and pyridine (4), respectively. Surprisingly, the two compounds showed extremely contrasting mesenchymal-epithelial transition factor (c-Met) inhibitory activities at 1 µM concentration (4% inhibition of 3 vs. 94% inhibition of 4). The IC50 value of compound 4 was 4.9 nM, similar to that of cabozantinib (5.4 nM). A ligand-based docking study suggested that 4 includes the preferred conformation for the binding to c-Met in the conformational ensemble, but 3 does not. The anti-proliferative activity of compound 4 against hepatocellular carcinoma (Hep3B and Huh7) and non-small-cell lung cancer (A549 and H1299) cell lines was better than that of cabozantinib, whereas 3 did not show a significant anti-proliferative activity. Moreover, the tumor selectivity of compound 4 toward hepatocellular carcinoma cell lines was higher than that of cabozantinib. In the xenograft chick tumor model, compound 4 inhibited Hep3B tumor growth to a much greater extent than cabozantinib. The present study suggests that compound 4 may be a good therapeutic candidate against hepatocellular carcinoma.

Synthesis and anti-hepatocellular carcinoma activity of aminopyridinol-sorafenib hybrids.

Sorafenib is recommended as the primary therapeutic drug for patients with hepatocellular carcinoma. To discover a new compound that avoids low response rates and toxic side effects that occur in sorafenib therapy, we designed and synthesized new hybrid compounds of sorafenib and 2,4,5-trimethylpyridin-3-ols. Compound 6 was selected as the best of 24 hybrids that inhibit each of the four Raf kinases. The anti-proliferative activity of 6 in HepG2, Hep3B, and Huh7 cell lines was slightly lower than that of sorafenib. However, in H6c7 and CCD841 normal epithelial cell lines, the cytotoxicity of 6 was much lower than that of sorafenib. In addition, similar to sorafenib, compound 6 inhibited spheroid forming ability of Hep3B cells in vitro and tumour growth in a xenograft tumour model of the chick chorioallantoic membrane implanted with Huh7 cells. Compound 6 may be a promising candidate targeting hepatocellular carcinoma with low toxic side effects on normal cells.

Potent Inhibitory Effect of BJ-3105, a 6-Alkoxypyridin-3-ol Derivative, on Murine Colitis Is Mediated by Activating AMPK and Inhibiting NOX.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation in the gastrointestinal tract. Biological therapeutics and orally available small molecules like tofacitinib (a JAK inhibitor) have been developed to treat IBD, but half of the patients treated with these drugs fail to achieve sustained remission. In the present study, we compared the therapeutic effects of BJ-3105 (a 6-alkoxypyridin-3-ol derivative) and tofacitinib in IBD. BJ-3105 induced activation of AMP-activated protein kinase (AMPK) in the kinase activity measurement and recovery from cytokine-induced AMPK deactivation in HT-29 human colonic epithelial cells. Similar to tofacitinib and D942 (an AMPK activator), BJ-3105 inhibited IL-6-induced JAK2/STAT3 phosphorylation and TNF-α-stimulated activation of IKK/NF-κB, and consequently, stimulus-induced upregulations of inflammatory cytokines and inflammasome components. In addition, unlike tofacitinib or D942, BJ-3105 inhibited NADPH oxidase (NOX) activation and consequent superoxide production induced by activators (mevalonate and geranylgeranyl pyrophosphate) of the NOX cytosolic component Rac. In mice, oral administration with BJ-3105 ameliorated dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS-induced colitis-associated tumor formation (CAT) much more potently than that with tofacitinib. Moreover, BJ-3105 suppressed the more severe form of colitis and CAT formation in mice with AMPK knocked-out in macrophages (AMPKαfl/fl-Lyz2-Cre mice) with much greater efficacy than tofacitinib. Taken together, our findings suggest BJ-3105, which exerted a much better anti-colitis effect than tofacitinib through AMPK activation and NOX inhibition, is a promising candidate for the treatment of IBD.

AMPK is essential for IL-10 expression and for maintaining balance between inflammatory and cytoprotective signaling.

BACKGROUND: AMP-activated protein kinase (AMPK) exerts its anti-inflammatory effects by suppressing redox-sensitive nuclear factor kappa B (NF-κB) and pro-inflammatory cytokines including TNF-α. However, it is unclear whether AMPK regulates anti-inflammatory cytokine expressions in the presence of oxidative stress-induced inflammation. We sought to elucidate the mechanisms whereby AMPK regulates inflammatory cytokine expressions under NADPH oxidase (NOX)-induced oxidative stress. METHODS: HT-29 human colonic epithelial cells transfected with AMPKα shRNA and mouse models with AMPKα knocked out in epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) were used to examine the effects of AMPK and NOX on signaling pathways and cytokine expressions. RESULTS: In HT-29 cells, 5-hydroxytryptamine (5-HT)-induced NOX activity was enhanced by AMPKα silencing, and resulted in inflammatory cell death. AMPKα deletion specific for colon epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) intensified 5-HT- or dextran sulfate sodium (DSS)-induced upregulations of NOX2, TNF-α, and IL-6, but completely abolished basal and 5-HT- or DSS-induced upregulation of IL-10 in colon epithelium. Furthermore, 5-HT- and DSS-induced changes were accompanied by marked upregulations of increased inflammatory signaling pathways linked to NF-κB, AP-1, and STAT3 transcription factors, and to GATA, a cell fate-directing signaling. In addition, AMPKα deletion significantly fortified 5-HT- or DSS-induced downregulations of cytoprotective signaling pathways (Nrf2, HIF-1α, and KLF4). CONCLUSION: Basal AMPKα maintains an anti-inflammatory state by inhibiting NOX, balancing pro-/anti-inflammatory signaling pathways, and directing IL-10 production. When these regulatory roles of AMPK are diminished by oxidative stress, colon epithelium undergoes inflammation despite IL-10 production.

Artesunate and chloroquine induce cytotoxic activity on cholangiocarcinoma cells via different cell death mechanisms.

Chemotherapy for cholangiocarcinoma (CCA) is not quite successful. In this study, we revisited the possibility of artesunate (ART) and chloroquine (CQ), the antimalarial drugs, as therapeutic agents against CCA. The possible mechanisms of these drugs to exert cytotoxicity on CCA cells were also explored. The effects of ART and CQ on proliferation and death patterns of two CCA cell lines, KKU-214 and its highly metastatic subtype KKU-214L5, were examined using water soluble tetrazolium (WST) assay and time-lapse photometry, respectively. To differentiate and verify the death patterns between necrosis and apoptosis, lactate dehydrogenase (LDH) release, and caspase 3 activity were measured. CellROXTM green reagent staining method was used to assess reactive oxygen species (ROS) production in ART- and CQ-treated cells. ART and CQ significantly inhibited proliferation of CCA cells. Both drugs kill malarial parasites via similar mechanism depending on ROS formation, however, ART induced necrotic cell death and CQ induced apoptotic cell death in CCA cells. ART induced LDH release, whereas CQ activated caspase 3, confirming induction of necrotic and apoptotic cell deaths by ART and CQ, respectively. ART treatment induced higher ROS production than CQ. ART and CQ induce CCA cells death via different death pathways. ART should be suitable for necrosis-sensitive CCA, whereas CQ is more suitable for apoptosis-sensitive CCA.