Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine-1-Phosphate (S1P) Receptor Agonist Ceralifimod (ONO-4641). A S1P1 and S1P5 Selective Agonist for the Treatment of Autoimmune Diseases.

The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, is described. While it has been revealed that the modulation of the S1P1 receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P3 receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P1 over S1P3 and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing).

p120-Catenin is essential for N-cadherin-mediated formation of proper junctional structure, thereby establishing cell polarity in epithelial cells.

The role of p120-catenin in the function of classical cadherins is still enigmatic despite various studies. To elucidate its role, we examined the effect of p120-catenin on the N-cadherin-mediated localization of junctional proteins in epithelial cells in this study. Cadherin-deficient MIA PaCa-2 epithelial cells did not show linear localization of tight junction proteins ZO-1 and occludin. When N-cadherin was expressed in these cells, however, the resultant transfectant cells revealed strong cell adhesion activity and linear localization of ZO-1, occludin, and N-cadherin in the lateral membrane. When the p120-catenin-binding site of N-cadherin was disrupted, the linear localization of ZO-1 and occludin disappeared, and the mutant N-cadherin became localized more diffusely in the transfectant, although the cell adhesion activity did not change much. Knockdown of p120-catenin also resulted in the very weak localization of ZO-1 and occludin. A similar effect of p120-catenin on the localization of junctional proteins was obtained under more dynamic conditions in a wound healing assay. Moreover, p120-catenin was essential for the regulation of centrosome orientation in this healing assay. Taken together, the present data indicate that p120-catenin is essential for N-cadherin-mediated formation of proper junctional structures and thereby the establishment of the cell polarity. Similar results were obtained when E-cadherin mutants comparable to those of N-cadherin were used, suggesting that p120-catenin plays the same role in the function of other classical cadherins.

The extracellular domains of E- and N-cadherin determine the scattered punctate localization in epithelial cells and the cytoplasmic domains modulate the localization.

The accumulation of classical cadherins is essential for their function, but the mechanism is poorly understood. Hence, we investigated the accumulation of E- and N-cadherin and the formation of cell junctions in epithelial cells. Immunostaining revealed a scattered dot-like accumulation of E- and N-cadherin throughout the lateral membrane in MDCK II and other epithelial cells. Mutant E-cadherin lacking the beta-catenin binding site accumulated granularly at cell-cell contact sites and showed weak cell aggregation activity in cadherin-deficient epithelial cells, MIA PaCa2 cells. Mutant E-cadherin lacking the p120-catenin binding site exhibited scattered punctate accumulation and strong cell adhesion activity in MIA PaCa2 cells. Electron microscopy demonstrated that MIA PaCa2 transfectants of E-cadherin containing beta-catenin binding site formed adherens junction, whereas E-cadherin lacking the binding site did not. Mutant N-cadherins showed accumulation properties similar to those of corresponding mutant E-cadherins. Moreover, wild type and mutant N-cadherin lacking the p120-catenin binding site showed subapical accumulation in polarized DLD-1 cells, whereas mutant N-cadherin lacking beta-catenin binding site did not. These results indicate that the extracellular domains of E- and N-cadherin determines the basic localization pattern, whereas the cytoplasmic domains modulate it thereby affects the cell adhesion activity, subapical accumulation, and the formation of adherens junction.

Negative regulation of adipogenesis from human mesenchymal stem cells by Jun N-terminal kinase.

Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions. We found that SP600125, an inhibitor of Jun N-terminal kinase (JNK), promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors, CCAAT/enhancer-binding proteins alpha and beta as well as peroxisome proliferator-activated receptor gamma2, which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus, JNK represses adipogenesis from hMSCs probably by, at least in part, inhibiting the transactivating function of CRE-binding protein. Another action of JNK, phosphorylation at Ser(307) of insulin receptor substrate-1, was also predicted to contribute to the repression of adipogenesis.

SKIP modifies gene expression by affecting both transcription and splicing.

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a beta-thalassemia mutation in the human beta-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.

Growth hormone has dual stage-specific effects on the differentiation of 3T3-L1 preadipocytes.

Reports vary on the role of growth hormone (GH) in adipocyte differentiation. In this study, we showed that GH exerted dual effects depending on the stage of differentiation, using a serum-free culture of 3T3-L1 preadipocytes. GH promoted the differentiation when added to the medium during differentiation-inducing treatment with a hormone cocktail, but apparently suppressed it when added after the treatment. Only the suppressive effect was observed in the presence of 10% fetal bovine serum (FBS). Immunodepletion study showed that GH contributes to the differentiation-promoting activity of FBS. Insulin-like growth factor-1 could not replicate either the stimulative or the suppressive effect of GH. Stimulation of differentiation by GH involved the enhanced expression of mRNA of middle to late adipocyte markers. Among the key regulators of adipogenesis, peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha, but not C/EBPbeta, were stimulated for mRNA expression by GH added during the treatment with hormone cocktail. The stimulation of adipogenesis by GH was indeed due to the increase in the ratio of differentiated cells, though GH also promoted cell growth.