The Wnt/β-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/β-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated "31" that was found to be an inhibitor of the Wnt/β-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/β-catenin signaling pathway.

In this study, the effects of side-chain configurations of D-Ile residues of a retro–inverso (RI)-type inhibitor on the human T-cell leukemia virus type 1 (HTLV-1) protease containing a hydroxyethylamine dipeptide isostere were clarified. Prior to evaluation using the RI-type inhibitor, the effects of side-chain configurations of Ile residues of the substrate peptide on the HTLV-1 protease were examined to estimate the influence of side-chain configurations on enzyme activity. Based on the estimation of the influence of side-chain configurations on protease affinity, the RI-type inhibitors containing a D-allo-Ile residue in the corresponding substrate sequence, instead of a D-Ile residue, were synthesized via 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis. Refolded recombinant HTLV-1 protease (1-116, L40I) was used for the simple and short evaluation of the inhibitory activities of the synthesized RI-type inhibitors. The results clearly indicated that mimicking the whole topology, comprising both the main- and side-chain structures of the parent inhibitor, is effective for the design of potent RI-modified protease inhibitors.

Background/Aim: Aberrant activation of the Wnt/β-catenin signaling pathway contributes to the pathogenesis of acute myelogenous leukemia (AML). Thus, targeting this pathway offers a promising therapeutic strategy against AML. Here, we synthesized a novel dipeptide-type inhibitor of the Wnt/β-catenin signaling pathway, compound #41, and explored its anti-tumor effects on AML cells. Materials and Methods: We evaluated the inhibitory effect of compound #41 on T cell factor (TCF)/β-catenin transcriptional activity using a luciferase (Luc) reporter assay. The anti-tumor effects were assessed on KG1a and MV4;11 human AML cells using RT-qPCR, western blotting, and WST-8, cell cycle, and apoptosis assays. Differentially expressed genes were analyzed by RNA-sequencing (RNA-seq). Additionally, we investigated the in vivo effects of compound #41 using KG1a-Luc/GFP cells in an orthotopic mouse model. Results: The Luc reporter assay showed that compound #41 decreased the TCF/β-catenin transcriptional activity. Compound #41 blocked the cell cycle progression, inhibited cell proliferation, and induced apoptosis in AML cells. Treatment with compound #41 down-regulated the expression of β-catenin, Survivin, and β-catenin-specific target genes, as demonstrated by RNA-seq. In vivo analysis showed that compound #41 blocked the expansion of KG1a-Luc/GFP cells in the bone marrow and prolonged the overall survival of KG1a-Luc/GFP-transplanted mice. Conclusion: Compound #41 suppressed the Wnt/β-catenin signaling pathway by reducing CTNNB1 levels and induced apoptosis in AML cells. Furthermore, compound #41 inhibited the proliferation of KG1a-Luc/GFP cells in the bone marrow and extended the overall survival of mice. Thus, compound #41 is an attractive Wnt/β-catenin signaling pathway inhibitor of AML.

The plant alkaloids, iso-6-spectaline and spectaline, isolated from the Cassia or Senna genera contain a characteristic 2,6-disubstituted piperidin-3-ol scaffold. Although both natural products are reported to exhibit a variety of interesting biological activities, few stereo-selective schemes for the construction of the 2,6-disubstituted scaffold have been reported. Following our previous studies regarding the synthesis of (+)-spectaline, herein we report the first convergent synthesis of (−)-iso-6-spectaline using a cross-metathesis under thermal conditions where the cis-2,6-disubstituted piperidin-3-ol scaffold is condensed with a long alkyl chain containing a terminal olefin. The cis-2,6-disubstituted piperidin-3-ol used in the synthesis was prepared simply via Pd(II)-catalyzed diastereoselective cyclization. It was confirmed that (+)-spectaline, an epimer of (−)-iso-6-spectaline, was selectively synthesized by the cross-metathesis reaction under less intense thermal conditions starting from the same cis-2,6-disubstituted piperidin-3-ol derivative.

The inhibition mode of a retro-inverso (RI) inhibitor containing a hydroxyethylamine dipeptide isostere against the human T-cell leukemia virus type-1 (HTLV-1) protease was examined. Enzymatic evaluation of the RI-modified inhibitor containing a D-allo-Ile residue revealed that HTLV-1 was competitively inhibited. IC50 values of the RI-modified inhibitor and pepstatin A, a standard inhibitor of aspartic proteases, were nearly equivalent.

We previously demonstrated that a novel coumarin-based compound, 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC), suppresses metastasis of LM8 osteosarcoma cells both in vitro and in vivo. However, the molecular target of DHC remains unclear. The aim of this study was to develop novel DHC-derived compounds and to identify their target molecule. Novel coumarin-based compounds were designed and evaluated for their effects on LM8 osteosarcoma cell invasion and migration. To identify a candidate target molecule of the newly synthesized compound SSKP-0076, liquid chromatography-mass spectrometry analysis was performed. The identified candidate molecule was further validated using RNA interference. Three novel coumarin-based compounds, SSKP-0076, SSKP-0081, and SSKP-0082, were synthesized. All compounds significantly inhibited invasion and migration of LM8 cells. Liquid chromatography-mass spectrometry combined with Mascot database searching identified Y-box binding protein 1 (Ybx1) as a candidate target molecule of SSKP-0076. Knockdown of Ybx1 by specific siRNAs significantly suppressed invasion and migration of LM8 cells and reduced the protein expression of the small GTP-binding proteins RhoA and Rac1, without affecting their mRNA expression levels. These effects were consistent with those observed following DHC treatment. Ybx1 is a potential target molecule of the novel anti-metastatic coumarin-based compound SSKP-0076.