Apoptosis-inducing effect of diketopiperazine disulfides produced by Aspergillus sp. KMD 901 isolated from marine sediment on HCT116 colon cancer cell lines.

AIMS: Research is to identify the bioactive secondary metabolites produced by Aspergillus sp. KMD 901 isolated from marine sediment and to assess their apoptosis-inducing effects. METHODS AND RESULTS: Aspergillus sp. KMD 901 was isolated from marine sediment obtained from the East Sea of Korea. An ethyl acetate extract of KMD 901 exhibited potent cytotoxic activity towards five cancer cell lines (HCT116, AGS, A549, MCF-7 and HepG2). Sequencing of the internal transcribed spacer (ITS) region in this strain allowed us to identify KMD 901 as a strain of Aspergillus versicolor. The cytotoxic compounds from Aspergillus sp. KMD 901 were purified by reversed-phase high-performance liquid chromatography and identified as diketopiperazine disulfides through spectroscopic analyses including extensive 2D NMR and mass spectrometry. The diketopiperazine disulfides were found to induce apoptosis in HCT116 cells based on cell morphology, DNA fragmentation observed by agarose gel electrophoresis, Annexin-V/PI staining using a flow cytometer and cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-8, caspase-9 and Bcl-2 family proteins (Bcl-2, Bcl-xL and Bax) using Western blotting analysis. Further study using an in vivo xenograft model showed inhibitory effects of acetylapoaranotin (2) on tumour proliferation. CONCLUSION: A new diketopiperazine disulfide, deoxyapoaranotin (3), along with previously described acetylaranotin (1) and acetylapoaranotin (2) was separated from Aspergillus sp. KMD 901 and found to have direct cytotoxic and apoptosis-inducing effects towards HCT116 colon cancer cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the diketopiperazine disulfides produced from Aspergillus sp., KMD 901, could be candidates for the development of apoptosis-inducing antitumour agents. Also, this study indicates that marine natural products as potential source of pharmaceuticals.

Preparation of 1,4-oxaselenin from agNO(3)/LDA-assisted reaction of 3-selena-4-pentyn-1-one as potential antitumor agents.

1,4-Oxaselenins were synthesized from 3-selena-4-pentyn-1-ones by the use of AgNO(3) and LDA. One of the obtained oxaselenins, 2-(4-chlorophenyl)-6-phenyl-1,4-oxaselenin 5c, showed an inhibitory effect against the proliferation of human cancer cells and inducing effects on the early stage of apoptosis.

Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis.

The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area. The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.

Preparation and structural determination of dermatan sulfate-derived oligosaccharides.

Eight oligosaccharides were prepared from dermatan sulfate (DS) and their structures were elucidated. Porcine intestinal mucosal DS was subjected to controlled depolymerization using chondroitin ABC lyase (chondroitinase ABC). The oligosaccharide mixture formed was fractionated by low-pressure gel permeation chromatography (GPC). Size uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, and dodecasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange (SAX) high-performance liquid chromatography (HPLC). This approach has led to the isolation of six homogeneous oligosaccharides. The size of the oligosaccharides were determined using GPC-HPLC. Treatment of tetrasaccharide and hexasaccharide fragments with Hg(OAc)2 afforded trisaccharide and pentasaccharide products, respectively. The purity of the oligosaccharides obtained was confirmed by analytical SAX-HPLC, and capillary electrophoresis (CE). The molecular mass and degree of sulfation of the eight purified oligosaccharides were elucidated using electrospray ionization (ESI) mass spectrometry and their structures were established with high field nuclear magnetic resonance (NMR) spectroscopy. These DS-oligosaccharides are currently being used to study for interaction of the DS with biologically important proteins.

Chemoenzymatic preparation of dermatan sulfate oligosaccharides as arylsulfatase B and alpha-L-iduronidase substrates.

Dermatan sulfate was partially depolymerized with chondroitin ABC lyase to obtain an oligosaccharide mixture from which an unsaturated disulfated tetrasaccharide was purified and characterized using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Chemical removal of the unsaturated uronate residue with mercuric acetate, followed by de-4-O-sulfation with arylsulfatase B (N-acetylgalactosamine 4-sulfatase) and N- acetylhexosaminidase catalyzed removal of the 2-acetamido-2-deoxy-D-galactospyranosyl residue at the non-reducing end afforded a monosulfated disaccharide of the structure alpha-L-idopyranosyluronic acid (1-->3)-alpha,beta-D-2-acetamido-2-deoxy-4-O-sulfo galactopyranose. This monosulfated disaccharide serves as a substrate for mammalian alpha-L-iduronidase as demonstrated using fluorophore assisted carbohydrate electrophoresis.

In vitro platelet-activating factor receptor binding inhibitory activity of pinusolide derivatives: a structure-activity study.

Pinusolide, a labdane-type diterpene lactone isolated from Biota orientalis, was found to be a potent platelet-activating factor (PAF) receptor binding antagonist. To investigate the structure-activity relationship and find derivatives with improved pharmacological profiles, 17 pinusolide derivatives were prepared and tested for their ability to inhibit the PAF receptor binding. The results demonstrated that the carboxymethyl ester group at C-19, the integrity of the alpha,beta-unsaturated butenolide ring, and the exocyclic olefinic function of pinusolide are all necessary for its maximum PAF receptor binding inhibitory activity. Among the derivatives, the 17-nor-8-oxo derivative 8 was found to be as potent as pinusolide. The results also suggested that several derivatives warrant further pharmaceutical and pharmacological studies due to their improved water solubility (8 and 11) and apparent lack of susceptibility to Michael-type nucleophilic addition (13 and 18).

Pinusolidic acid: a platelet-activating factor inhibitor from Biota orientalis.

The water extract of Biota orientalis showed a potent inhibitory effect on platelet activating factor (PAF) binding to rabbit platelets using [3H]PAF as a ligand in our previous screening studies for Korean medicinal antagonists by the activity-guided purification studies. Another active compound, compound 1 (IC50 = 2.3 x 10(-5) M, 7.48 +/- 2.11 micrograms/ml, n = 4) was isolated from the title plant. The chemical structure of compound 1 was elucidated as pinusolidic acid by chemical and spectrometric analyses.

Pinusolidic acid: a platelet-activating factor inhibitor from Biota orientalis.

The water extract of Biota orientalis showed a potent inhibitory effect on platelet activating factor (PAF) binding to rabbit platelets using [ (3)H]PAF as a ligand in our previous screening studies for Korean medicinal antagonists by the activity-guided purification studies. Another active compound, compound 1 (IC (50) = 2.3 x 10 (-5)M, 7.48 +/- 2.11 microg/ml, n = 4) was isolated from the title plant. The chemical structure of compound 1 was elucidated as pinusolidic acid by chemical and spectrometric analyses.

Biological and pharmacological effects of pinusolide, a novel platelet activating factor antagonist.

Pinusolide, a potent platelet activating factor (PAF) receptor binding antagonist, inhibited PAF-induced aggregation of rabbit platelets with an IC50 value of 5 microM, whereas no inhibitory effects on the aggregation induced by ADP, thrombin, and collagen were observed. Pinusolide protected the mice from PAF-induced lethality with ED50 values of 1.1 mg/kg, i.v. and 69 mg/kg, p.o. Topically given pinusolide at 2 mg/ear effectively inhibited croton oil induced mouse ear edema. All the pinusolide treated ears recovered to normal healthy appearance in a sharp contrast to totally necrotized untreated ears. These results indicated that pinusolide is a potent and specific PAF antagonist in all experimental models as shown in vitro, in vivo, and in animal tests.

Metabolic fate of the new antithrombotic agent aspalatone in rats. In vivo and in vitro study.

In order to determine whether aspalatone ([3-(2-methyl-4-pyronyl)]-2- acetyloxybenzoate, CAS 147249-33-0), a new antiplatelet agent, behaves as a prodrug of acetylsalicylic acid (ASA), metabolism studies in rats were carried out in vivo and in vitro using the whole animal and tissue homogenates. The ASA molecule was not detected in the plasma samples taken after oral administration of aspalatone at 80 mg/kg. Instead, salicylic acid maltol ester (SM), the deacetylated metabolite of aspalatone, was detected in the plasma and it was rapidly hydrolyzed to salicylic acid. In in vitro experiments with rat serum, intestinal fluid, liver and gastric mucosal homogenates, SM was the only compound formed after 4 min incubation at 37 degrees C. From these results it was concluded that aspalatone does not behave as a prodrug of ASA in rats. In addition, the results of experiments using certain esterase inhibitors, suggested the major contribution of B-esterase(s) in the metabolism of aspalatone to SM particularly in serum and intestinal fluid.

Isolation and characterization of platelet-activating factor receptor binding antagonists from Biota orientalis.

The leaf extract of Biota orientalis showed potent PAF receptor binding antagonistic activity in our previous screening studies on 234 Korean medicinal plants using rabbit platelet receptor binding tests. The activity-guided purification of the plant extract resulted in the isolation of six compounds, including two active substances. The chemical structures of the compounds isolated were established by chemical and spectrometric analyses as dotriacontane, totarol, 8 beta-hydroxy-3-oxopimar-15-ene, cedrol (IC50 = 1.3 x 10(-5) M), pinusolide (IC50 = 2.52 x 10(-7) M), and 5-hydroxy-7,4'-dimethoxyflavone.

Synthesis and antiplatelet effects of the new antithrombotic agent aspalatone with low ulcerogenicity.

A new compound, aspalatone (acetylsalicylic acid maltol ester), was synthesized by esterification of acetylsalicylic acid (ASA) and maltol, an antioxidant, and studied for its bleeding time prolongation effect in rats, for its antiplatelet aggregation activity in vitro and ex vivo in rats, and for its antithrombotic activity in vivo using the mouse thromboembolism test. Aspalatone treatment (15 mg/kg p.o.) for 10 days prolonged bleeding time by 57% (p < 0.005) in Sprague-Dawley rats vs control, while ASA treatment (15 mg/kg p.o.) prolonged by 44%. At the low dose of 15 mg/kg p.o. at least 8 days of treatment were necessary for aspalatone and ASA to prolong the bleeding time significantly. On the other hand, salicylic acid maltol ester which lacks the acetyl group did not significantly affect bleeding time at a dose of 15 mg/kg. Aspalatone produced a potent inhibition of collagen-induced platelet aggregation in vitro with IC50 of 1.8 x 10(-4) mol/l, but, similar to ASA, did not significantly inhibit ADP-induced aggregation. The ability of oral aspalatone to inhibit platelet aggregation in rats ex vivo was compared with other reference antiplatelet drugs. Relative potency was ASA > dipyridamole approximately equal to aspalatone > ticlopidine. A single dose of aspalatone potently prevented death due to collagen-induced platelet aggregation in mice in vivo with ED50 value of 32 mg/kg p.o., but failed to prevent death due to ADP-induced platelet aggregation. When given for 10 days, aspalatone prevented collagen-induced death by 90% (p < 0.001) at 20 mg/kg, and this antithrombotic effect lasted after 4 days of wash-out period.(ABSTRACT TRUNCATED AT 250 WORDS)