Bioactivity-directed fractionation of a hot H2O extract from a blue-green alga Spirulina platensis led to the isolation of a novel sulfated polysaccharide named calcium spirulan (Ca-SP) as an antiviral principle. This polysaccharide was composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate, and calcium. Ca-SP was found to inhibit the replication of several enveloped viruses, including Herpes simplex virus type 1, human cytomegalovirus, measles virus, mumps virus, influenza A virus, and HIV-1. It was revealed that Ca-SP selectively inhibited the penetration of virus into host cells. Retention of molecular conformation by chelation of calcium ion with sulfate groups was suggested to be indispensable to its antiviral effect.

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Blown algae like wakame (Undaria pinnatifida) and kombu (Laminaria sp.) are popular foods in East Asia. These edible algae have been used as rich sources of minerals, vitamins and dietary fibers. They have been attracted recently as multifunctional foods for maintaining our health.Fucoidan (fucan sulfate) is a fucose-containing sulfated polysaccharide from brown algae such as Fucus vesiculosus, 1) Ecklonia kurome, 2) and Cladosiphon okamuranus.3) Fucoidans showed various biological activities like anticoagulant 4) and antivirus. 5) In addition, fucoidan prevents microvascular thrombus formation induced by endothelial damage in arterioles and venules in vivo.6) It is suggested that this activity is not attributable to inhibition of P-and L-selectin but instead to the anticoagulative capacity of fucoidan. Thus, fucoidan might be useful for anticoagulant, antiviral, and anti-thrombus formation agent.Mekabu is a sporophyll of U. pinnatifida and it is also used as a safe, inexpensive and savory food in Japan. It has been reported that Mekabu has potential for chemoprevention of human breast cancer since Mekabu extract shows strong suppressive effect on rat mammary carcinogenesis.7) In addition, Mekabu fucoidan showed antitumor effect in vivo and its mechanism was mediated by IFN-g activated NK cells.8) Although Mekabu fucoidan has been subjected to different kinds of biological evaluation, its structural feature is still unknown. This paper describes the structural characterization of Mekabu fucoidan as well as evaluation for its antiviral effects. Results and DiscussionAfter washing and crushing the dried alga, it was treated with EtOH to remove lipophyllic compounds. The defatted alga was then extracted with 0.15 N HCl to prevent extraction of alginates. After neutralization and precipitation with 4 vol. of EtOH, crude fucoidan (CF) was obtained. CF was successively fractionated by ion exchange column chromatographies on DEAE Toyopearl 650M and Q-Sepharose FF and gel filtration on Sephacryl S-300 HR. As indicated in Fig. 1, CF was separated to four fractions. The most abundant fraction (CF-4) was further fractionated by Q-Sepharose (Fig. 2), and the second eluate (CF-4B) was collected. Then CF-4B was applied to a gel filtration on Sephacryl S-300 HR (Fig. 3) and the most abundant fraction was collected as purified fucoidan.The fucoidan was eluted as a single peak, and its apparent molecular weight was estimated to be 9000 by HPLC on TSK gel GMPW XL . In addition, the electrophoretic pattern indicated that the electric charge of fucoidan was homogeneous (Fig. 4). These results revealed that the fucoidan might be a homogeneous polysaccharide on the basis of molecular Medicine, Toyama Medical and Pharmaceutical University; 2630 Sugitani, Toyama 930-0194, Japan: and c Riken Vitamin Co., Ltd.; 3 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-8370, Japan. Received April 8, 2004; accepted June 9, 2004 Structural characterization and antiviral activities of fucoidan from sporophyll of Undaria pinnatifida (Mekabu) was exa...

A sulfated polysaccharide named calcium spirulan (Ca-SP) has been isolated from a sea alga, Spirulina platensis, as an antiviral component. The anti-human immunodeficiency virus type 1 (HIV-1) and anti-herpes simplex virus type 1 (HSV-1) activities of Ca-SP were compared with those of dextran sulfate (DS) as a representative sulfated polysaccharide. Anti-HIV-1 activities of these agents were measured by three different assays: viability of acutely infected CD4-positive cells, or a cytopathology assay; determination of HIV-1 p24 antigen released into culture supernatants; and inhibition of HIV-induced syncytium formation. Anti-HSV-1 activity was assessed by plaque yield reduction. In addition, their effects on the blood coagulation processes and stability in the blood were evaluated. These data indicate that Ca-SP is a potent antiviral agent against both HIV-1 and HSV-1. Furthermore, Ca-SP is quite promising as an anti-HIV agent because even at low concentrations of Ca-SP an enhancement of virus-induced syncytium formation was not observed, as was observed in DS-treated cultures, Ca-SP had very low anticoagulant activity, and showed a much longer half-life in the blood of mice when compared with that of DS. Thus, Ca-SP can be a candidate agent for an anti-HIV therapeutic drug that might overcome the disadvantages observed in many sulfated polysaccharides. When the role of chelation of calcium ion with sulfate groups was examined by removing calcium or its replacement by sodium, the presence of calcium ion in the molecule was shown to be essential for the dose-dependent inhibition of cytopathic effect and syncytium formation induced by HIV-1.

As a first step in structure-based design of highly selective and potent Cdk4 inhibitors, we performed structure-based generation of a novel series of Cdk4 inhibitors. A Cdk4 homology model was constructed according to X-ray analysis of an activated form of Cdk2. Using this model, we applied a new de novo design strategy which combined the de novo design program LEGEND with our in-house structure selection supporting system SEEDS to generate new scaffold candidates. In this way, four classes of scaffold candidates including diarylurea were identified. By constructing diarylurea informer libraries based on the structural requirements of Cdk inhibitors in the ATP binding pocket of the Cdk4 model, we were able to identify a potent Cdk4 inhibitor N-(9-oxo-9H-fluoren-4-yl)-N'-pyridin-2-ylurea 15 (IC(50) = 0.10 microM), together with preliminary SAR. We performed a docking study between 15 and the Cdk4 model and selected a reasonable binding mode which is consistent with the SAR. Further modification based on the proposed binding mode provided a more potent compound, N-[(9bR)-5-oxo-2,3,5,9b-tetrahydro-1H-pyrrolo[2,1-a]isoindol-9-yl]-N'-pyridin-2-ylurea 26a (IC(50) = 0.042 microM), X-ray analysis of which was accomplished by the soaking method. The predicted binding mode of 15 in Cdk4 was validated by X-ray analysis of the Cdk2-26a complex.