Novel mouse model for evaluating in vivo efficacy of xCT inhibitor.

xCT, a well-known cystine transporter, is reported to be involved in the proliferation of various cells, such as cancer cells, immune cells, and fibroblasts. xCT inhibitor is expected to be a promising drug for cancer or immune diseases. However, there are little studies reporting that xCT inhibitors improve disease progression in vivo. To invent potent xCT inhibitors in vivo, we established a new in vivo model for assessing efficacy of xCT inhibition. dl-propargylglycine (PPG) was administered intraperitoneally to wild-type C57BL/6J mice. Concentration of cystathionine, another substrate of xCT, in the thymus and spleen was measured by LC-MS/MS. PPG increased cystathionine amounts in the thymus and spleen in a dose- and time-dependent manner. At 7 h after PPG administration, the efficacy of erastin, a representative xCT inhibitor, was clearly shown. We synthesized a new compound, Compound A, which had much higher inhibitory effect on xCT than erastin both in vitro and in vivo. We established a mouse model of PPG-induced cystathionine accumulation for assessing xCT inhibition in vivo. By using this model, we discovered that Compound A was approximately 15 times more effective in vivo than erastin.

Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.

Protein tyrosine phosphatase receptor-type Z (PTPRZ) is aberrantly over-expressed in glioblastoma and a causative factor for its malignancy. However, small molecules that selectively inhibit the catalytic activity of PTPRZ have not been discovered. We herein performed an in vitro screening of a chemical library, and identified SCB4380 as the first potent inhibitor for PTPRZ. The stoichiometric binding of SCB4380 to the catalytic pocket was demonstrated by biochemical and mass spectrometric analyses. We determined the crystal structure of the catalytic domain of PTPRZ, and the structural basis of the binding of SCB4380 elucidated by a molecular docking method was validated by site-directed mutagenesis studies. The intracellular delivery of SCB4380 by liposome carriers inhibited PTPRZ activity in C6 glioblastoma cells, and thereby suppressed their migration and proliferation in vitro and tumor growth in a rat allograft model. Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.

Orally active ghrelin receptor inverse agonists and their actions on a rat obesity model.

A series of 2-alkylamino nicotinamide analogs was prepared as orally active ghrelin receptor (ghrelinR) inverse agonists. Starting from compound 1, oral bioavailability was improved by modifying metabolically unstable sites and reducing molecular weight. Brain-permeable compound 33 and compound 24 with low brain permeability were tested in rat models of obesity; 30 mg/kg of compound 33 suppressed weight gain. PK/PD analysis revealed that the anti-obesity effect of ghrelinR inverse agonists depends on their brain concentrations.

2-Aminoalkyl nicotinamide derivatives as pure inverse agonists of the ghrelin receptor.

New inverse agonists of the ghrelin receptor (ghrelinR) were obtained through high-throughput screening and subsequent structural modification of 2-aminoalkyl nicotinamide derivatives. The key structural feature to improve in vitro activity was the introduction of a diazabicyclo ring at the 5-position of the pyridine ring. The final product showed potent inverse agonist activity and, despite its low brain permeability, reduced food intake in both normal and obese mice. These results implied that peripheral ghrelinR activity is important for appetite control and that a peripheral ghrelinR inverse agonist could be an anti-obesity drug with reduced risk of central nervous system (CNS)-related side effects.

Quantitative analysis of cadherin-11 and ß-catenin signalling during proliferation of rheumatoid arthritis-derived synovial fibroblast cells.

OBJECTIVES: Cadherin-11 (CDH11) is an adhesion molecule that anchors ß-catenin and is involved with various functions of synovial fibroblast cells (SFCs) during the development of rheumatoid arthritis (RA). However, the mechanism of CDH11 during RA-SFC proliferation is unclear. The aim of our study was to clarify the involvement of CDH11 and ß-catenin signalling during proliferation. METHODS: IL-1ß-induced and tumour necrosis factor-α (TNF-α)-induced cell proliferation, with CDH11 siRNAs, ß-catenin-specific siRNAs and a CDH11-neutralizing antibody, were assessed by 5-Bromo-2'-deoxy-uridine ELISA. KEY FINDINGS: Using CDH11 siRNAs, there were a 42% reduction in IL-1ß-induced proliferation and a 64% reduction in ß-catenin protein. When ß-catenin siRNAs were applied, there was a 63% reduction in IL-1ß-induced proliferation. The median effective concentration (EC50 ) values for IL-1ß-induced proliferation via CDH11-mediated ß-catenin-dependent, total ß-catenin-dependent and ß-catenin-independent signalling were 0.0015, 0.016 and 0.18 ng/ml, respectively. Blocking CDH11 ligation with a CDH11-neutralizing antibody did not decrease IL-1ß-induced proliferation. CONCLUSIONS: CDH11-mediated ß-catenin signalling was 42% involved in IL-1ß-induced proliferation and had the highest susceptibility to IL-1ß among the proliferative signallings analysed in this study. The mode of action for CDH11 during the cell proliferation was likely associated with a pool of ß-catenin protein. In contrast, CDH11 and ß-catenin were not involved in TNF-α-induced RA-SFC proliferation.

Inhibition of phosphodiesterase 7A ameliorates Concanavalin A-induced hepatitis in mice.

An intravenous injection of Concanavalin A (Con A) elevated the serum level of alanine aminotransferase (ALT) activity, a marker for liver damage, and an oral administration of PDE7A inhibitor SUN11817 suppressed the increase of ALT activity in a dose-dependent manner. Histological analysis revealed that Con A injection caused extensive liver damage, and that the SUN11817 treatment improved the degenerative change in the liver. In addition, SUN11817 inhibited not only the production of IL-4 and TNF-alpha in the Con A-induced hepatitis model but also that in vitro by murine splenocytes stimulated with alpha-galactosylceramide, an activator specific for NKT cells. The Con A injection to mice also induced expression of Fas ligand (FasL) on NKT cells, which was significantly prevented by SUN11817. As NKT cells are known to contribute to the pathogenesis in Con A-induced hepatitis by producing cytokines such as IL-4 and TNF-alpha and inducing FasL-mediated hepatocyte injury, it is thought that PDE7A inhibitor SUN11817 improves liver injury in the Con A model by blocking cytokine production and FasL expression in NKT cells. PDE7A might be a novel pharmaceutical target for hepatitis.

ASB16165, a novel inhibitor for phosphodiesterase 7A (PDE7A), suppresses IL-12-induced IFN-gamma production by mouse activated T lymphocytes.

Phosphodiesterase 7A (PDE7A) has been suggested to be involved in activation of T lymphocytes. In the present study, a possible involvement of PDE7A in function of preactivated T cells (i.e. T lymphoblasts) was investigated using ASB16165, an inhibitor for PDE7A. ASB16165, which has an IC50 value of 15 nM for human PDE7A, suppressed IL-12-induced IFN-gamma production by T lymphoblasts which have been prepared by stimulating mouse T cells with anti-CD3 antibody. In the same experiment, rolipram, a PDE4-specific inhibitor, showed similar effect, while calcineurin antagonist FK506 did not. Forskolin (an adenylyl cyclase activator) and dibutyryl cAMP also inhibited the IL-12-induced IFN-gamma synthesis. Rp-8-Br-cAMPS, an inhibitor of protein kinase A (PKA), reduced the suppressive effect of ASB16165 on the IFN-gamma production by T lymphoblasts. The rescue of IFN-gamma production by Rp-8-Br-cAMPS was also observed in the inhibition by rolipram and forskolin. These findings suggest that PDE7A may regulate function of activated T cells in a cAMP/PKA-dependent manner, and that PDE4 might share the role. The data in our study also indicate that PDE7 inhibitors such as ASB16165 will be beneficial for the patients with immunological disorders.