総説・解説等

During our studies on a search for natural products from myxomycetes, we have isolated novel compounds, fuligocandin A and B containing cycloanthranilylproline skeleton, from the extract of fruit bodies of Fuligo candida. We studied the total synthesis of fuligocandin A, B and its derivatives. Fuligocandin B consists of three residues, anthranilic acid, L-proline, and indole-3-carboxyaldehyde. Total synthesis of fuligocandin A, B and their enantiomers and fuligocandin B derivatives were achieved.TRAIL-resistance overcoming activities of fuligocandin A, B and their enantiomers and fuligocandin B derivatives AGS cells were evaluated. Fuligocandin B and its derivatives showed effects on the TRAIL-resistance AGS cells.

An asymmetric Heck reaction-carbanion capture process was realized for the first time, making possible the catalytic asymmetric synthesis of various functionalized bicyclo[3.3.0]octane derivatives 6 in up to 94% ee. Sodium bromide had interesting effects on this asymmetric Heck reaction-carbanion capture process, and these effects were useful for improving the enantiomeric excess. Furthermore, the catalytic asymmetric synthesis of (-)-Delta(9(12))-capnellene (7) was achieved for the first time, using 6b as a key intermediate and a radical cyclization as a key step.

1背景：ウィント（Wnt）シグナルは線虫からヒトに至るまで生物種を超えて高度に保存されたシグナル伝達経路で，初期発生における体軸形成から各種組織・器官の形態形成，出生後の細胞の増殖・分化・運動，組織幹細胞の維持などの様々な生命現象において多彩かつ重要な役割を果たしている．一方で，大腸がんを始めとする様々ながんにおいて異常亢進していることが知られているほか，糖尿病や精神疾患など各種疾患との関わりも示唆されている．したがってWntシグナルに作用する化合物は各種生命現象を研究する上での分子ツールや，医薬品リード化合物となることが期待される1．当研究室ではタイ・バングラデシュ産植物の抽出物ライブラリーを独自に構築しており，これらからのWntシグナル調節作用をもつ天然物の探索を進めてきた2-4．本会ではこれまでの研究で見出した強力なWntシグナル阻害作用をもつ天然物とその活性発現機構に関する解析について報告する．2スクリーニング試験：当研究室保有の独自に構築した南方アジア産植物の天然物抽出物ライブラリーを対象に，培養細胞を用いたルシフェラーゼアッセイシステムを用いてスクリーニング試験を行った．スクリーニング試験は，ヒト胎児腎細胞HEK293に野生型TCF結合領域をもつSuperTOP-Flashレポーター遺伝子を安定導入したSTF/293細胞を用い，試料添加によるルシフェラーゼ活性（TOP活性）の変化を測定することによりWntシグナルの最下流に位置するTCF/β-catenin転写活性の評価した2,5．スクリーニングの結果，良好な活性を示した以下の二種の植物について活性成分の探索を行った．3 Calotropis giganteaから得られたWntシグナル阻害成分6バングラデシュにて採取したガガイモ科植物Calotropis gigantea（和名：カイガンタバコ）滲出液メタノールエキスより，上記活性試験を指標として，分画を行い，6種のカルデノライド類（1-6）を単離した．これらは10位にアルデヒド基をもつ共通するカルデノライド骨格をもつが，3'位の置換基が異なっていた．これら化合物はnMオーダーでTOP活性を阻害し，そのIC50値は0.7-3.8 nMであった．また，これら化合物は1-10 nMの濃度範囲において，細胞増殖がWntに依存するヒト大腸がん細胞（SW480，DLD1，HCT116）に対して選択的に細胞毒性（IC50値：1.8-7.0 nM）を示したが，細胞増殖がWntに依存性しないヒトRKO細胞に対しては細胞毒性を示さなかった．このことから，これら化合物は，Wntシグナルを阻害することにより，Wntシグナル依存性細胞の細胞生存率を低下させると示唆された．得られた化合物のうち，calotropin（1）について，そのWntシグナル阻害作用のメカニズムを解明する目的でWntシグナル経路において重要な役割を担う転写活性化因子β-cateninレベルへの影響をウェスタンブロットにて調べた．その結果，Wntシグナルの亢進が認められているSW480細胞において，1は濃度依存的に核内および細胞質のβ-cateninを減少させた．また，1は本シグナルの標的遺伝子であるc-mycタンパクを濃度依存的に低下させた（図1A）．化合物1は核および細胞質の両方のβ-cateninを減少させてい(View PDFfor the rest of the abstract.)

Highly efficient screening method of naturally occurring products that bind to a specific target protein was demonstrated by using hVDR-magnetic beads. The native ligand 1α,25(OH)_2VD_3 was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was proven to be applicable to the identification of natural products, which interact with a specific protein immoblized on the beads, from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, neturally occurring products that bind to specific target proteins. A high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor Hes1 was construcrted. Successful demerization of Hes1 immobilized on a microplate and fluorophore (Cy3)-labeled Hes1 was confirmed. Using this system, several natural products were identified as the first Hes1 dimer inhibitors. Of these, two compounds which were isolated from myxomycetes (true slime molds) inhibited Hes1 from N box-dependent suppression of the gene expression in C3H10T1/2 cells.

The hedgehog (Hh)/GLI signaling pathway has been implicated not only in a variety of developmental processes in wide range of organisms, but also in the formation and development of different tumors including skin, brain, prostate, upper gastrointestinal tract, pancreas and lung. Targeting Hh/GLI signaling has been expected as an effective cancer therapeutic strategy. To find the specific inhibitors of Hh/GLI signaling pathway from natural resources, a cell-based screening assay system targeting transcriptional activator GLI1, which constitutes the final step in the Hh signaling pathway, was constructed. A pGL4-Luc reporter vector inserted with 12×GLI binding sites was stably transfected into HaCaT cell line expressing GLI1 under tetracycline repressor control. A suitable protocol for the screening was then established. The screening of our natural products library with the above constructed system identified six active compounds; staurosporinone (1), 6-hydroxystaurosporinone (2), arcyriaflavin C (3), 5,6-dihydroxyarcyriaflavin A (4), zerumbone (5), and zerumbone epoxide (6). Their IC_<50> values of GLI1 transcriptional inhibitory activity were 1.8, 3.6, 11.3, 6.9, 3.0 and 55μM, respectively. The next screening of our natural plants extracts library gave 15 hit plants. Repeated chromatography separations of the MeOH extracts of Physalis minima and Zizyphus cambodiana gave active compounds. P minima gave two compounds; physalin F (10) and physalin B (11), and Z. combodiana a compound; colubrinic acid (13). Their IC_<50> values were 0.66, 0.62 and 29μM, respectively. It was reported that glil, ptchl, bcl2 are the Hh/GLI-mediated transcriptional targets. The influences of these inhibitors (1, 5, 10 and 11) on these protein expressions were investigated with western blotting analysis. These inhibitors decreased the expression of GLI1 and PTCH1, which are related Hh signaling pathway. Moreover, BCL2, which is related with apoptosis, was also decreased.

Natural products have their important meanings in biological systems. Isolation of new natural products and synthesis of small molecules based on their scaffolds would be of chemical relevance to living cells and organs. In this study, we focused on the Wnt signaling, which pathways show aberrant activation in many cancer cells. We synthesized natural products melleumin A (1) and B (2), and trans-dihydroarcyriarubin C, which were isolated by us from myxomycetes (true slime molds), and also synthesized their isomers. Melleumin A (1) and B (2) are novel peptide compounds isolated from the myxomycete Physarum melleum. Their syntheses have been achieved by coupling procedures of their components, L-threonine, glycine, and an unusual amino acid, a tyrosine-attached acetic acid. We found cis-dihydroarcyriarubin C (12), and isomers of melleumin B are moderate inhibitors of Wnt signal transcription. We also developed a high-throughput system for analyzing of inhibition of making a complex between TCF and β-catenin by using microplates. The regulation of transcription by TCF/β-catenin complex is a final step of the Wnt signal pathway. In this assay, cis-dihydroarcyriarubin C (12) showed inhibition, which indicates that a cell-based Wnt signal inhibition would be caused by disruption of TCF/β-catenin complex. We also found several natural products from our natural products library that show a cell-based Wnt signal inhibition and a protein-based TCF/β-catenin complex inhibition.