Disrupting TSLP-TSLP receptor interactions via putative small molecule inhibitors yields a novel and efficient treatment option for atopic diseases.

Thymic stromal lymphopoietin (TSLP) is a key player in atopic diseases, which has sparked great interest in therapeutically targeting TSLP. Yet, no small-molecule TSLP inhibitors exist due to the challenges of disrupting the protein-protein interaction between TSLP and its receptor. Here, we report the development of small-molecule TSLP receptor inhibitors using virtual screening and docking of >1,000,000 compounds followed by iterative chemical synthesis. BP79 emerged as our lead compound that effectively abrogates TSLP-triggered cytokines at low micromolar concentrations. For in-depth analysis, we developed a human atopic disease drug discovery platform using multi-organ chips. Here, topical application of BP79 onto atopic skin models that were co-cultivated with lung models and Th2 cells effectively suppressed immune cell infiltration and IL-13, IL-4, TSLP, and periostin secretion, while upregulating skin barrier proteins. RNA-Seq analysis corroborate these findings and indicate protective downstream effects on the lungs. To the best of our knowledge, this represents the first report of a potent putative small molecule TSLPR inhibitor which has the potential to expand the therapeutic and preventive options in atopic diseases.

G Protein-Coupled Estrogen Receptor-Mediated Anti-Inflammatory and Mucosal Healing Activity of a Trimethylpyridinol Analogue in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by abnormal immune responses, including elevated proinflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) in the gastrointestinal (GI) tract. This study presents the synthesis and anti-inflammatory evaluation of 2,4,5-trimethylpyridin-3-ol analogues, which exhibit dual inhibition of TNFα- and IL-6-induced inflammation. Analysis using in silico methods, including 3D shape-based target identification, modeling, and docking, identified G protein-coupled estrogen receptor 1 (GPER) as the molecular target for the most effective analogue, 6-26, which exhibits remarkable efficacy in ameliorating inflammation and restoring colonic mucosal integrity. This was further validated by surface plasmon resonance (SPR) assay results, which showed direct binding to GPER, and by the results showing that GPER knockdown abolished the inhibitory effects of 6-26 on TNFα and IL-6 actions. Notably, 6-26 displayed no cytotoxicity, unlike G1 and G15, a well-known GPER agonist and an antagonist, respectively, which induced necroptosis independently of GPER. These findings suggest that the GPER-selective compound 6-26 holds promise as a therapeutic candidate for IBD.

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.

Synthesis of Pyridoxine-Derived Dimethylpyridinols Fused with Aminooxazole, Aminoimidazole, and Aminopyrrole.

Described in this paper are studies on the preparation of three classes of dimethylpyridinols derived from pyridoxine fused with aminooxazole, aminoimidazole, and aminopyrrole. The key feature of this synthetic strategy is the manipulation of hydroxymethyl moiety of C(5)-position of the pyridoxine starting material along with the installation of an amino group at C(6)-position. Efficient and practical synthesis for the oxazole- and imidazole-fused targets was accomplished, while the instability of the pyrrole-fused one was observed.

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.

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.

Synthesis and activity of N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)acetamide analogues as anticolitis agents via dual inhibition of TNF-α- and IL-6-induced cell adhesions.

Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are the critical pro-inflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Inhibition of these cytokines and related signaling pathways has been a target for the development of IBD therapeutics. In the current study, 6-acetamido-2,4,5-trimethylpyridin-3-ol (1) and various analogues with the amido scaffold were synthesized and examined for their inhibitory activities in in vitro and in vivo IBD models. The parent compound 1 (1 µM) showed an inhibitory activity against TNF-α- and IL-6-induced adhesion of monocytes to colon epithelial cells, which was similar to tofacitinib (1 µM), a JAK inhibitor, but much better than mesalazine (1,000 µM). All the analogues showed a positive relationship (R2 = 0.8943 in a linear regression model) between the inhibitory activities against TNF-α-induced and those against IL-6-induced adhesion. Compound 2-19 turned out to be the best analogue and showed much better inhibitory activity against TNF-α- and IL-6-induced adhesion of the cells than tofacitinib. In addition, oral administration of compound 1 and 2-19 resulted in a significant suppression of clinical signs of TNBS-induced rat colitis, including weight loss, colon tissue edema, and myeloperoxidase activity, a marker for inflammatory cell infiltration in colon tissues. More importantly, compound 2-19 (1 mg/kg) was more efficacious in ameliorating colitis than compound 1 and sulfasalazine (300 mg/kg), the commonly prescribed oral IBD drug. Taken together, the results suggest that compound 2-19 can be a novel platform for dual-acting IBD drug discovery targeting both TNF-α and IL-6 signaling.

Synthesis and anticancer evaluation of 6-azacyclonol-2,4,6-trimethylpyridin-3-ol derivatives: M3 muscarinic acetylcholine receptor-mediated anticancer activity of a cyclohexyl derivative in androgen-refractory prostate cancer.

We recently reported 2,4,5-trimethylpyridin-3-ol with C(6)-azacyclonol, whose code name is BJ-1207, showing a promising anticancer activity by inhibiting NOX-derived ROS in A549 human lung cancer cells. The present study was focused on structural modification of the azacyclonol moiety of BJ-1207 to find a compound with better anticancer activity. Ten new compounds (3A-3J) were prepared and evaluated their inhibitory actions against proliferation of eighteen cancer cell lines as a primary screening. Among the ten derivatives of BJ-1207, the effects of compounds 3A and 3J on DU145 and PC-3, androgen-refractory cancer cell lines (ARPC), were greater than the parent compound, and compound 3A showed better activity than 3J. Antitumor activity of compound 3A was also observed in DU145-xenografted chorioallantoic membrane (CAM) tumor model. In addition, the ligand-based target prediction and molecular docking study using DeepZema® server showed compound 3A was a ligand to M3 muscarinic acetylcholine receptor (M3R) which is overexpressed in ARPC. Carbachol, a muscarinic receptor agonist, concentration dependently increased proliferation of DU145 in the absence of serum, and it also activated NADPH oxidase (NOX). The carbachol-induced proliferation and NOX activity was significantly blocked by compounds 3A in a concentration-dependent manner. This finding might become a new milestone in the development of pyridinol-based anti-cancer agents against ARPC.