Published papers

Chemical biology is not a new discipline, as research at the interface of chemistry and biology has been growing for decades. However, the field still needs regional and global support in emerging countries. The meeting organized by Asian Chemical Biology Initiative in Bangkok, Thailand in January 2013, brought together regional communities of young and established researchers to nurture chemical biology research programs and highlight some of the newer developments. Here, we report on the meeting and some of the key research topics discussed.

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF-1β and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications.

This report describes the development of a target-protein-oriented natural-products-isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5'- O-methyl-3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via the TPO-NAPI method developed using GLI1 protein-immobilized beads. 5'- O-Methyl-3-hydroxyflemingin A (3) inhibited Hh signaling (IC50 7.3 μM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5'- O-Methyl-3-hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5'- O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 ( Kd = 7.7 μM). Moreover, 5'- O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cell characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM. These results suggest that Hh inhibitors can efficiently suppress the activity of cancer stem cells.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50values (for infection of 293TA cells) of 19.7 and 9.7 μM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50concentrations.

Calnexin (CNX) and its soluable 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. The paper describes the synthesis of the non-natural undecsaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality-control system. Each possesses the key tetrasaccharide element (Glc(1)Man(3)) 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 colorimetry (ITC). As expected, each of them had a significant affinity towards CRT In addition, these compounds were shown to be resistant to glucosidose II digestion. Their activities in blocking the chaperone function of CRT wet I e next measured by using malate dehydrogenose (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.

The absolute stereochemistry of melleumin A (1) and B (2), novel peptide compounds isolated from the myxomycete Physarum melleum, was determined by synthesis of their segments and by a modified Mosher's method. Total synthesis of melleumin B (2) was achieved by a stereoselective method, which provided further evidence for all the absolute stereochemistries of melleumin B (2). The Wnt signal inhibitory activities of 2 and its 10R-epimer 19 were evaluated. Compound 19 showed moderate inhibition of Wnt signal transcription, which suggests that melleumin analogues might be useful as Wnt signal inhibitors. (C) 2007 Elsevier Ltd. All rights reserved.

This account focuses our works on the development of chiral spiro ligands bearing N-heterocycles as metal-coordinating units and their applications in the metal-catalyzed asymmetric reactions. The spiro bis(isoxazoline) ligands (SPRIXs), spiro bis(isoxazole) ligands, and spiro (isoxazole-isoxazoline) ligands were readily synthesized through intramolecular double nitrile oxide cycloaddition of the corresponding dioximes as a key step. An unprecedented activation of olefins was displayed by the Pd-11 complexes of these chiral spiro ligands in the enantioselective oxidative cyclizations, for example; the asymmetric tandem cyclization of dialkenyl alcohol via oxy-palladation produced a bicyclic ether in excellent enantioselectivity and enantioselective version of the aminocarbonylation proceeded for the first time. Other hitherto known chiral ligands we examined failed to promote these reactions.

Chromones and flavonoids are important bioactive compounds. We envisioned that new heterocyclic-substituted chromones or flavonoids might act as new bioactive compounds. To obtain diverse molecules, we developed an efficient one-pot synthesis by Michael aldol reaction of chromone and flavonoid derivatives bearing heterocyclic units. The 2,3-heterocyclic-substituted chromones were obtained in one step. Moreover, the use of substituted benzaldehydes and subsequent addition of heterocyclic aldehydes gave 3-pyridyl-substituted flavones. We also examined these one-pot reactions in the solid phase. To introduce an additional point of diversity into the molecules, Suzuki-Miyaura coupling was performed. Furthermore, we identified the cytotoxicity of the synthesized compounds against cancer cells (PANC1 and HeLa cells). Several compounds were cytotoxic to these cancer cells.

The hedgehog (Hh) signaling pathway performs important roles in embryonic development and cellular proliferation and differentiation. However, in many cancer cells Hh signaling is aberrantly activated, which has provided a strong impetus for the development of Hh pathway inhibitors. To address this, we synthesized a series of heterocyclic flavonoids and evaluated their Hh signaling inhibitory activity on cancer cell lines using our cell-based assay system. Of the synthetic flavonoids, compounds 4a and g showed good inhibitory activity (IC50 was 16.8 and 21.8 mu m, respectively), and were cytotoxic toward human pancreatic (PANC1) and prostate (DU145) cancer cells in which Hh signaling was activated. Compounds 4a and g had moderate selectivity against PANC1 cells. Western blotting analyses revealed that PTCH and GLI1 expression was reduced after treatment with these compounds. Overall, these synthetic flavonoids represent promising new additions to our expanding panel of Hh pathway inhibitors, and with further development these molecules may ultimately be considered for clinical use.

Novel chiral spiro bis(isoxazoline) ligands 3 and 4 are synthesized from an optically active olefin. The Cu(II)-spiro bis(isoxazoline) complex prepared in situ from Cu(OTf)(2) and 3 promoted the glyoxylate-ene reaction in high yield and moderate enantioselectivity (up to 70%). (c) 2007 Published by Elsevier Ltd.

Two authors (Midori A. Arai and Itsuki Ebihara) and one affiliation (Faculty of Science and Technology, Keio University) were added to the contribution list. Page 4998, Abstract, and p 5000, Figure 3: The peaks for compounds 1 and 2 are reversed. (Figure Presented).

In a screening program for natural products that can overcome TRAIL resistance, a new eudesmane-type sesquiterpenoid (1), three new guaianolides, mehirugins A-C (2-4), and two known guaianolides (5 and 6) were isolated from a Me OH extract of Kandelia candel leaves. Compounds 1 and 3-6 in combination with TRAIL showed cytotoxic activity in sensitizing TRAIL resistant human gastric adenocarcinoma cells.

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1α,25(OH)2D3 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 3H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1α,25(OH)2D3 analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2α- or 2β-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1α,25(OH)2D3. The 3H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1α,25(OH)2D3, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 or 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)2D3 for 7 days. 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 and 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)2D3 were also shown to be about 10-fold more active than 1α,25(OH)2D3 in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1α,25(OH)2D3 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. © 2006 Elsevier Ltd. All rights reserved.

TRAIL selectively kills cancer cells without harming most normal cells. However, TRAIL resistance is a major problem for its therapeutic use. Developing a strategy to overcome resistance is crucial for the successful use of TRAIL as an antitumor agent. In our screening program of natural products that can abrogate TRAIL resistance, four germacranolides (1-4) were isolated from the aerial parts of Enhydra fluctuans. All compounds exhibited potent TRAIL resistance overcoming activity against TRAIL-resistant human gastric adenocarcinoma (AGS) cells.

A screening study using a luciferase assay to identify natural products which inhibit Wnt signaling was carried out. Bioassay-guided fractionation of the culture broth and mycelia of actinomycetes CKK748 and CKK784, led to the isolation of griseoviridin (1), and four cyclic hydroxamates (4-7) as active compounds. 1, 4, and 5 inhibited TCF/beta-catenin transcriptional activity with IC50 values of 8.2-14 mu M.

The hedgehog (Hh) signaling pathway plays crucial roles in cell maintenance and proliferation during embryonic development. Naturally occurring Hh inhibitors were isolated from Artocarpus communis and Hyptis suaveolens using our previously constructed cell-based assay system. Bioactivity guided fractionation led to the isolation of 15 compounds, including seven new compounds (4, 5, 6, 7, and 9-11). The isolated compounds showed cytotoxicity against a cancer cell line (PANC1) in which Hh signaling was abnormally activated. Several compounds (12-14; GLI1 transcriptional inhibition IC50 = 7.6, 4.7, and 4.0 mu M, respectively) inhibited Hh related protein (BCL2) expression. Moreover, compounds 1, 12, and 13 disrupted GLI1 and DNA complex formation. (C) 2015 Elsevier Ltd. All rights reserved.

The hedgehog (Hh) signaling pathway performs an important role in embryonic development and in cellular proliferation and differentiation. However, aberrant activation of the Hh signaling pathway is associated with tumorigenesis. Hh signal inhibition was evaluated using a cell-based assay system that targets GLI1-mediated transcription. Activity-guided isolation of the Withania somnifera MeOH extract led to the isolation of six compounds: withaferin A (1) and its derivatives (2-6). Compounds 1 and 2 showed strong inhibition of Hh/GLI1-mediated transcriptional activity with IC50 values of 0.5 and 0.6 mu M, respectively. Compounds 1, 2, 3, and 6 were cytotoxic toward human pancreatic (PANC-1), prostate (DU145) and breast (MCF7) cancer cells. Furthermore, 1 also inhibited GLI1-DNA complex formation in EMSA. (C) 2015 Elsevier Ltd. All rights reserved.