Evgeny A. Pislyagin

Urupocidin A: A New, Inducing iNOS Expression Bicyclic Guanidine Alkaloid from the Marine Sponge Monanchora pulchra

Urupocidins A and B (1 and 2), bisguanidine alkaloids with an unprecedented skeleton system, derived from polyketide precursors and containing an unusual N-alkyl-N-hydroxyguanidine moiety, have been isolated from the sponge Monanhora pulchra. The structures of 1 and 2, including absolute configuration, were established using the detailed analysis of 1D and 2D NMR, CD, and mass spectra as well as chemical transformations. Compound 1 increases nitric oxide production in murine macrophages via inducing iNOS expression.

Cyclic Steroid Glycosides from the Starfish Echinaster luzonicus: Structures and Immunomodulatory Activities

Five new steroid glycosides, luzonicosides B-E (2-5), belonging to a rare structure group of marine glycosides, containing carbohydrate moieties incorporated into a macrocycle, and a related open carbohydrate chain steroid glycoside, luzonicoside F (6), were isolated from the starfish Echinaster luzonicus along with the previously known cyclic steroid glycoside luzonicoside A (1). The structures of compounds 2-6 were established by extensive NMR and ESIMS techniques as well as chemical transformations. Luzonicoside A (1) at concentrations of 0.01-0.1 μM was shown to be potent in lysosomal activity stimulation, intracellular ROS level elevation, and NO synthesis up-regulation in RAW 264.7 murine macrophages. Luzonicoside D (4) was less active in these biotests.

Chapter 3 - Immunomodulatory and Anticancer Activity of Sea Cucumber Triterpene Glycosides

Abstract The triterpene glycosides are composed of a carbohydrate chain and triterpene aglycone and are widely distributed in sea cucumbers ( Holothurioidea , Echinodermata ). Most aglycones have 18(20)-lactones and belong to the holostane type. Carbohydrate chains of sea cucumber glycosides have from two to six monosaccharide residues including xylose, quinovose, glucose, and 3- O -methylglucose and sometimes 3- O -methylxylose, 3- O -methylquinovose, 3- O -methylglucuronic acid, and 6- O -acetylglucose. They may contain one, two, or three sulfate groups. At the milli- and micromolar concentrations, sea cucumber glycosides show hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. The basis of membranotropic action of the glycosides is their ability to attach to cell biomembranes and form nonselective ion-conducting complexes with 5(6)-nonsaturated sterol components of cell membranes, preferably with cholesterol. Such sterol/saponin interactions result in an efflux of some ions, nucleotides, and peptides, disrupting ion homeostasis and osmolarity followed by lysis and cell death. Some sea cucumber glycosides show an immunostimulatory effect at subtoxic nanomolar concentrations. Incubation of immune cells with the glycosides induces their activation resulting in an increase of immune cell adhesion on an extracellular matrix, enhancement of cell spreading and motility, increase of macrophage lysosomal activity, ROS formation, and phagocytic activity. The most effective immunostimulants are monosulfated glycosides, whereas di- and trisulfated glycosides are immunodepressants. Injection of subtoxic doses of some glycosides induces an increase in the number of antibody-producing plaque-forming cells in mouse spleens, an increase in the number, size and acidity of lysosomes of peritoneal macrophages, and increase of phagocytic index, resulting in heightened resistance by host animals against bacterial infections. Proteomic methods have demonstrated that the mechanism of immunomodulatory action of some sea cucumber glycosides on mouse splenocytes includes regulation of the expression of some proteins involved in the formation of cellular immune response. These glycosides regulate the expression of proteins associated with lysosome maturation, activation and merging, phagocytosis, cytoskeletal reorganization, cell adhesion, mobility, and proliferation of immune cells. It was shown that glycosides moderately induce production of some cytokines, restore the level of some CD markers of lymphocytes, increase bactericidal activity of leukocytes, and induce a significant increase in mouse resistance to lethal doses of some pathogenic microorganisms and radiation. Cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. These studies have shown the triterpene glycosides block egg cleavage and development of sea urchin embryos, suppress the proliferation of various human tumor cell lines in vitro , possess antiangiogenic effect, and cause cancer cell cycle arrest. Several sea cucumber glycosides are reported to induce tumor cell apoptosis in vitro and more importantly, IP administration in rodents of solutions of some sea cucumber triterpene glycosides show significant reduction of both tumor burden and metastasis. Recently, it was found that the new immunomodulatory lead compound, Cumaside, based on the holothurian triterpene glycoside, cucumarioside A 2 -2, demonstrates inhibition of tumor initiation and proliferation, in vivo and exhibits significant synergy with 5-fluorouracil.

Polyoxygenated steroids from the gorgonian Menella woodin with capabilities to modulate ROS levels in macrophages at response to LPS.

Four new polyoxygenated sterol derivatives (1-4) along with the compounds (5-7) previously known from other biological sources were isolated from the gorgonian Menella woodin, collected from the Vietnamese waters. Structures of 1-4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as comparison with those reported in literature data. Compounds 1, 4, and 6 decrease the production of reactive oxygen species (ROS) by the murine macrophages of RAW 264.7 line at induction by endotoxic lipopolysaccharide (LPS) from Escherichia coli.

[Anti-Inflammatory Activity of the Polypeptide of the Sea Anemone, Heteractis crispa].

The anti-inflammatory activity of the HCGS 1.20 recombinant polypeptide (a Kunitz-type serine protease inhibitor from the Heteractis crispa sea anemone) was investigated. The polypeptide was shown to inhibit the histamine-induced increase in the concentration of calcium ions and the lipopolysaccharidestimulated increase in the concentration of nitric oxide (II) in macrophages. A possible mechanism of this anti-inflammatory activity of the polypeptide was discussed.

Determination of cucumarioside A2-2 in mouse spleen by radiospectroscopy, MALDI-MS and MALDI-IMS

The distribution of triterpene glycoside cucumarioside A₂-2, the main compound of medical lead Cumaside in immunodeficiency diseases, in mouse spleen was determined. For this purpose the stability and dynamics of glycoside content changes over time in Balb/c mouse spleen tissue homogenate as well as the study of the cucumarioside A2-2 spatial distribution in tissue sections were investigated using radiospectroscopy, MALDI-MS and MALDI Imaging Mass Spectrometry (IMS), correspondingly. Cucumarioside A₂-2 is reliably detected by MALDI-MS in the mouse spleen tissue after single intraperitoneal (i.p.) injection at a dosage of 5 mg/kg. The glycoside is stable in the spleen and does not undergo metabolic transformation in either tissue homogenates or in the intact organ within 24 h after i.p. injection. The cucumarioside A₂-2 was absorbed fairly rapidly: the glycoside maximum concentration (Cmax) in tissue homogenate was observed in the first 30 min after injection; the minimum values were registered in 3 h. These results are in agreement with those obtained in the pharmacokinetic study of (3)H-cucumarioside A₂-2. It was established by MALDI-IMS that glycoside was mainly located in the tunica serosa part of the spleen and only a small amount was detected within the red and white pulp of the organ. MALDI MS images obtained 15-30 min post dosage clearly reflect high drug concentrations in the regions surrounding the organ followed by its decline in the surface part and a very slight redistribution to the internal part of the spleen.

Glycosides from edible sea cucumbers stimulate macrophages via purinergic receptors

Since ancient times, edible sea cucumbers have been considered a jewel of the seabed and used in Asian folk medicine for stimulation of resistance against different diseases. However, the power of this sea food has not been established on a molecular level. A particular group of triterpene glycosides was found to be characteristic metabolites of the animals, responsible for this biological action. Using one of them, cucumarioside A2-2 (CA2-2) from the edible Cucumaria japonica species as an example as well as inhibitory analysis, patch-clamp on single macrophages, small interfering RNA technique, immunoblotting, SPR analysis, computer modeling and other methods, we demonstrate low doses of CA2-2 specifically to interact with P2X receptors (predominantly P2X4) on membranes of mature macrophages, enhancing the reversible ATP-dependent Ca2+ intake and recovering Ca2+ transport at inactivation of these receptors. As result, interaction of glycosides of this type with P2X receptors leads to activation of cellular immunity.

Anthenosides L–U, Steroidal Glycosides with Unusual Structural Features from the Starfish Anthenea aspera

Ten new polyhydroxysteroidal glycosides, anthenosides L-U (1-10), with rare positions of carbohydrate fragment attachments, were isolated from the starfish Anthenea aspera. The structures of 1-10 were established by NMR and ESIMS techniques as well as by chemical transformations. The unoxidized Δ22-24-nor-cholestane (1), (24S)-Δ22-24-methylcholestane (2-5), and Δ22-cholestane (7) side chains of the steroidal aglycons, 3-O-methyl-β-d-galactofuranosyl residue (2, 8), and 5α-cholest-8(14)-ene-3α,7β,16α-trihydroxysteroidal nucleus (9, 10) have not been found previously in starfish polar steroidal compounds. The mixture of glycosides 9 and 10 showed hemolytic activity with an EC50 = 8 μM. Compound 4 at a dose of 10 μM exhibited a potential immunomodulatory action, decreasing by 24% the intracellular ROS content in RAW 264.7 murine macrophages, induced by pro-inflammatory endotoxic lipopolysaccharide from E. coli.

Pallidopenillines: Polyketides from the Alga-Derived Fungus Penicillium thomii Maire KMM 4675

Eleven new polyketides, pallidopenillines 1-11, were isolated from the alga-derived fungus Penicillium thomii. The structures of these compounds were established based on spectroscopic methods. The absolute configuration of pallidopenilline A (1) as 4R, 5S, 8S, 9R, 10R, 13R was established using a combination of the modified Moshers method, X-ray analysis, and NOESY data. The absolute configurations of 2-5 were determined by time-dependent density functional theory calculations of the ECD spectra and ECD and NOESY data. It was shown that 1-acetylpallidopenilline A (2) and pallidopenilline G (10) inhibit the growth of colonies of 22Rv1 cells by 40% at 2 and 1 μM, respectively.

Cucumarioside A2-2 causes changes in the morphology and proliferative activity in mouse spleen.

The immunomodulatory effect of triterpene glycoside cucumarioside A2-2 (CA2-2), isolated from the Far Eastern sea cucumber Cucumaria japonica, on the mouse spleen was investigated in comparison with lipopolysaccharide (LPS). It has been shown that the intraperitoneal (i.p.) glycoside administration did not influence on splenic weights, while the statistically significant increase in splenic weight was observed after LPS administration. Changes in the ratio of red to white pulp after CA2-2 or LPS administration were observed. The proportion of splenic white pulp after glycoside or LPS administration increased by up to 34% and 36%, respectively. A detailed study of the distribution of the РСNA (Proliferating Cell Nuclear Antigen) marker showed that the proliferative activity in the white pulp under CA2-2 and LPS influence increased 2.07 and 2.24 times, respectively. The localization of PCNA-positive nuclei in the white pulp region, as well as their dimensional characteristics, suggests that a large proportion of the proliferating cell population consisted of B cells. The mass spectrometry profiles of spleen peptide/protein homogenate were obtained using the MALDI-TOF-MS (Matrix -Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) approach. It was found that i.p. stimulation of animals with CA2-2 or LPS leads to marked changes in the intensity of revealed characteristic peaks of peptides/proteins after exposure to immunostimulants.