Toshihiro Chikanishi

Objective— Serine protease inhibitors (serpin) play a central role in various pathological processes including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serpins may prove therapeutic. As yet, however, only very few small molecule serpin inhibitors have been reported. For the first time, we apply a new approach of virtual screening to discover novel, orally active, small molecule serpin inhibitors and report their effectiveness. Methods and Results— We focused on a clinically important serpin, plasminogen activator inhibitor-1 (PAI-1), whose crystal structure has been described. We identify novel, orally active molecules able to enter into the strand 4 position (s4A) of the A β-sheet of PAI-I as a mock compound. In vitro they specifically inhibit the PAI-1 activity and enhance fibrinolysis activity. In vivo the most effective molecule (TM5007) inhibits coagulation in 2 models: a rat arteriovenous (AV) shunt model and a mouse model of ferric chloride–induc...

Procede de selection d'un accelerateur de thermogenese contenant un compose ayant un effet d'activation de PPARδ, medicaments contenant ledit compose et medicaments ayant un effet antidiabetique, contre l'obesite et des effets cumulatifs visceraux de reduction des graisses. Des medicaments contenant un compose ayant un effet d'activation d'un recepteur PPARδ active par le proliferateur de peroxisome et des effets d'acceleration de la thermogenese sans tremblement (nTS), et specifiquement d'acceleration de la respiration decouplante des mitochondries dans les cellules telles que les adipocytes ou de la fuite de protons dans la membrane intramitochondriale et d'elevation de la dose d'expression de UPC1 sont egalement decrits. La presente invention concerne en outre des antidiabetiques, des medicaments contre l'obesite et des agents de reduction des graisses cumulatifs visceraux contenant ledit compose. Elle concerne encore un procede de selectio...

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.

Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or...

Spontaneous herniated disc resorption occurs via inflammatory reactions involving abundant neovascularization and macrophage phagocytotic activity. Nonthermal low-intensity pulsed ultrasound (LIPUS) treatment might be effective in shortening the duration of disc resorption. We developed a rat in vitro resorption model in which a coccygeal intervertebral disc and peritoneal macrophages were cocultured. Secretion of tumor necrosis factor-alpha (TNF-alpha) from macrophages was promoted by LIPUS, and the process of disc degeneration was thus accelerated. In this study, we further examined the effects of LIPUS using this in vitro model focusing on whether LIPUS affects cyclooxygenase-2 (COX-2) signaling pathways. We found that the levels of COX-2 and prostaglandin E2 (PGE2) secreted from macrophages were increased by LIPUS. However, these phenomena were not caused by LIPUS directly, as the levels of these substances were reduced by neutralizing TNF-alpha activity. Moreover, the wet weights of the disc samples were not changed by addition of PGE2, but were reduced by recombinant TNF-alpha. Our results suggest that the effects of LIPUS in enhancing the process of herniated disc resorption are caused mainly by TNF-alpha.

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1alpha and -1beta genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1alpha gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1alpha gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.