Midori A. Arai

Naturally occurring small-molecule inhibitors of hedgehog/GLI-mediated transcription.

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. To search for Hh/GLI inhibitors, we screened for naturally occurring inhibitors of the transcriptional activator GLI1 by using a cell‐based assay. We identified zerumbone (1), zerumbone epoxide (2), staurosporinone (9), 6‐hydroxystaurosporinone (10), arcyriaflavin C (11) and 5,6‐dihydroxyarcyriaflavin A (12) as inhibitors of GLI‐mediated transcription. In addition, we isolated physalins F (17) and B (18) from Physalis minima, which are also potent inhibitors. These compounds also inhibited GLI2‐mediated transactivation. Semiquantitative RT‐PCR and Western blotting analysis further revealed that 1, 9, 17, and 18 decreased Hh‐related component expressions. We also show that inhibitors of GLI‐mediated transactivation reduce the level of the antiapoptosis Bcl2 expression. Finally, these identified compounds were cytotoxic to PANC1 pancreatic cancer cells, which express Hh/GLI components. These results strongly suggest that the cytotoxicity of the compounds to PANC1 cells correlates with their inhibition of GLI‐mediated transcription.

The first enantioselective intramolecular aminocarbonylation of alkenes promoted by Pd(II)-spiro bis(isoxazoline) catalyst

Abstract The highly ligand acceleration effect of spiro bis(isoxazoline) ligand (SPRIX) on the Pd(II)-catalyzed intramolecular aminocarbonylation of alkenyl amine derivatives was realized. Furthermore, the chiral Pd(II)–SPRIX catalyst accomplished the first enantioselective intramolecular aminocarbonylation. The reaction of N -(2,2-dimethyl-pent-4-enyl)- p -toluenesulfonamide in the presence of Pd(II)–SPRIX catalyst and p -benzoquinone in methanol under a carbon monoxide atmosphere afforded [4,4-dimethyl-1-( p -toluene-sulfonyl)-pyrrolidin-2-yl]-acetic acid methyl ester in good yield with moderate enantioselectivity.

Terpenoids and a flavonoid glycoside from Acacia pennata leaves as Hedgehog/GLI-mediated transcriptional inhibitors.

Overexpression of glioma-associated oncogene 1 (GLI1), which has been characterized as a terminal effector and a target gene of the Hedgehog (Hh) signaling pathway, is associated with the development of cancer. A cellular screen was applied utilizing of a GLI-dependent luciferase reporter in human keratinocyte cells (HaCaT) and identified two terpenoids (1 and 2) and a flavonoid glycoside (5) from Acacia pennata as Hh/GLI inhibitors. Compounds 1, 2, and 5 exhibited selective cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells with no toxic effect on normal cells. This result was consistent with a dose-dependent reduction of the protein levels of antiapoptotic BCL-2 and the tumor suppressor patched 1 protein (PTCH). Additionally, treatment of 1 downregulated mRNA expression of Ptch in PANC1, suggesting that the compound has an inhibitory effect on the transcription of Hh/GLI.

Hedgehog/GLI-mediated transcriptional inhibitors from Zizyphus cambodiana.

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. By screening tropical plant extracts by using our screening system, Zizyphus cambodiana was found to include Hh/GLI signaling inhibitors. Bioassay-guided fractionation of this plant extract led to the isolation of three active pentacyclic triterpenes, colubrinic acid (1), betulinic acid (2) and alphitolic acid (3), as potent inhibitors. The inhibition of GLI-related protein expression with 1 or 2 was observed in HaCaT cells with exogenous GLI1, or human pancreatic cancer cells (PANC1), which express Hh/GLI components aberrantly. The expressions of GLI-related proteins PTCH and BCL2 were clearly inhibited by 1 or 2. We also examined the cytotoxicity of these active compounds against PANC1, human prostate cancer cells (DU145) and mouse embryo fibroblast cells (C3H10T1/2). The cytotoxicity against cancer cells (PANC1 and DU145) by 1 or 2 would be caused by inhibition of the expression of the anti-apoptosis protein BCL2. These pentacyclic triterpene inhibitors showed an important relationship between Hh/GLI signaling inhibition, the decrease of BCL2, and cytotoxicity against cancer cells.

Enantioselective synthesis of α-methylene-γ-butyrolactones using chiral Pd(II)-SPRIX catalyst

Combination of Pd(OCOCF3)2 and (M,S,S)-iPr-SPRIX ligand promoted the intramolecular cyclization of 2-alkynoates very effectively to form α-methylene-γ-butyrolactones in good yields with up to 92% ee.

Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1alpha,25(OH)(2)D(3) can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized (3)H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1alpha,25(OH)(2)D(3) analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2alpha- or 2beta-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1alpha,25(OH)(2)D(3). The (3)H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1alpha,25(OH)(2)D(3), 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) or 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) for 7 days. 19-Nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) and 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) were also shown to be about 10-fold more active than 1alpha,25(OH)(2)D(3) in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1alpha,25(OH)(2)D(3) molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.

Design and Synthesis of Oligosaccharides that Interfere with Glycoprotein Quality‐control systems

Calnexin (CNX) and its soluble homologue calreticulin (CRT) are lectin‐like molecular chaperones that help newly synthesized glycoproteins to fold correctly in the rough endoplasmic reticulum (ER). To investigate the mechanism of glycoprotein‐quality control, we have synthesized structurally defined high‐mannose‐type oligosaccharides related to this system. This paper describes the synthesis of the non‐natural undecasaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality‐control system. Each possesses the key tetrasaccharide element (Glc1Man3) critical for the CNX/CRT binding, while lacking the pentamannosyl branch required for glucosidase II recognition. These oligosaccharides were evaluated for their ability to bind CRT by isothermal titration calorimetry (ITC). As expected, each of them had a significant affinity towards CRT. In addition, these compounds were shown to be resistant to glucosidase II digestion. Their activities in blocking the chaperone function of CRT were next measured by using malate dehydrogenase (MDH) as a substrate. Their inhibitory effects were shown to correlate well with their CRT‐binding affinities, both being critically dependent upon the presence of the terminal glucose (Glc) residue.

COMPOUND ANALYSIS VIA GRAPH KERNELS INCORPORATING CHIRALITY

High accuracy is paramount when predicting biochemical characteristics using Quantitative Structural-Property Relationships (QSPRs). Although existing graph-theoretic kernel methods combined with machine learning techniques are efficient for QSPR model construction, they cannot distinguish topologically identical chiral compounds which often exhibit different biological characteristics. In this paper, we propose a new method that extends the recently developed tree pattern graph kernel to accommodate stereoisomers. We show that Support Vector Regression (SVR) with a chiral graph kernel is useful for target property prediction by demonstrating its application to a set of human vitamin D receptor ligands currently under consideration for their potential anti-cancer effects.

New Hedgehog/GLI signaling inhibitors from Excoecaria agallocha.

The inhibition of the Hedgehog (Hh) signaling pathway has emerged as an anti-cancer strategy. Three flavonoid glycosides including 2 new compounds (1-2) were isolated from Excoecaria agallocha as Hedgehog/GLI1-mediated transcriptional inhibitors and exhibited cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells. Our data revealed that compound 1 clearly inhibited the expression of GLI-related proteins (PTCH and BCL-2) and blocked the translocation of GLI1 transcription factors into the nucleus in PANC1. Deleting the Smoothened (Smo) function in PANC1 treated with 1 led to downregulation of the mRNA expression of Ptch. This study describes the first Hh signaling inhibitor which blocks GLI1 movement into the nucleus without interfering with Smo.

Synthesis of fluorine substituted oligosaccharide analogues of monoglucosylated glycan chain, a proposed ligand of lectin-chaperone calreticulin and calnexin

As a part of a exploring the N-glycan-mediated glycoprotein quality control in endoplasmic reticulum, 2-fluorinated derivative Glcα1 → 3Man(F) 1, Glcα1 → 3Man(F)α1 → 2Man2, and Glcα1 → 3Man(F)α1 → 2Manα1 → 2Man 3 were synthesized in a concise manner. These oligosaccharides were subjected to binding studies with calreticulin by using isothermal titration calorimetry. It was revealed that disaccharide 1 was a poor ligand, while tri- (2) and tetrasaccharide (3) had observable affinity. Published in 2003.