Effects of Sponge-Derived Alkaloids on Activities of the Bacterial α-D-Galactosidase and Human Cancer Cell α-N-Acetylgalactosaminidase.

During a search for glycosidase inhibitors among marine natural products, we applied an integrated in vitro and in silico approach to evaluate the potency of some aaptamines and makaluvamines isolated from marine sponges on the hydrolyzing activity of α-N-acetylgalactosaminidase (α-NaGalase) from human cancer cells and the recombinant α-D-galactosidase (α-PsGal) from a marine bacterium Pseudoalteromonas sp. KMM 701. These alkaloids showed no direct inhibitory effect on the cancer α-NaGalase; but isoaaptamine (2), 9-demethylaaptamine (3), damirone B (6), and makaluvamine H (7) reduced the expression of the enzyme in the human colorectal adenocarcinoma cell line DLD-1 at 5 µM. Isoaaptamine (2), 9-demethylaaptamine (3), makaluvamine G (6), and zyzzyanone A (7) are slow-binding irreversible inhibitors of the bacterial α-PsGal with the inactivation rate constants (kinact) 0.12 min-1, 0.092 min-1, 0.079 min-1, and 0.037 min-1, as well as equilibrium inhibition constants (Ki) 2.70 µM, 300 µM, 411 µM, and 105 µM, respectively. Docking analysis revealed that these alkaloids bind in a pocket close to the catalytic amino acid residues Asp451 and Asp516 and form complexes, due to π-π interactions with the Trp308 residue and hydrogen bonds with the Lys449 residue. None of the studied alkaloids formed complexes with the active site of the human α-NaGalase.

Sponge-derived polybrominated diphenyl ethers and dibenzo-p-dioxins, irreversible inhibitors of the bacterial α-d-galactosidase.

An integrated in vitro and in silico approach was applied to evaluate the potency of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and spongiadioxins (OH-PBDDs) isolated from Dysidea sponges on the activity of the recombinant α-d-galactosidase of the GH36 family. It was revealed for the first time that all compounds rapidly and apparently irreversibly inhibited the bacterial α-d-galactosidase. The structure-activity relationship study in the series of OH-PBDEs showed that the presence of an additional hydroxyl group in 5 significantly enhanced the potency (IC50 4.26 µM); the increase of bromination in compounds from 1 to 3 increased their potency (IC50 41.8, 36.0, and 16.0 µM, respectively); the presence of a methoxy group decreased the potency (4, IC50 60.5 µM). Spongiadioxins 6, 7, and 8 (IC50 16.6, 33.1, and 28.6 µM, respectively) exhibited inhibitory action comparable to that of monohydroxylated diphenyl ethers 1-3. Docking analysis revealed that all compounds bind in a pocket close to the catalytic amino acid residues. Molecular docking detected significant compound-enzyme interactions in the binding sites of α-d-galactosidase. Superimposition of the enzyme-substrate and the enzyme-inhibitor complexes showed that their binding sites overlap.

Synthesis of Phenoxyundecyl Diphosphate Disaccharides for Studies of the Biosynthesis of O Antigenic Polysaccharides in Enteric Bacteria.

The biosynthesis of O antigenic polysaccharides in enteric bacteria from nucleoside diphosphate sugars (donor substrates) is catalyzed by the corresponding glycosyltransferases and proceeds through the intermediate formation of undecaprenyl diphosphate sugars (acceptor substrates). To study this process, a chemical synthesis of the compounds having the natural structure or their modified analogs is necessary. The phosphoroimidazolidate method is a universal method for synthesis of lipid diphosphate disaccharides containing 2-acetamido-2-deoxyglycosyl residue at the reducing end of the disaccharide moiety and 11-phenoxyundecyl residue as lipid fragment of the molecule. We report here protocols to synthesize the disaccharides P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate [D-Rha(α1-3)-D-GlcNAcα-PP-PhU, Compound 1] and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate [D-Gal(ß1-3)-D-GalNAcα-PP-PhU, Compound 6]. We describe the procedures for identification and structure estimation of compounds by TLC, NMR, and MS. We also include the biochemical testing of Compound 6 with α2,3-sialyltransferase WbwA from Escherichia coli O104.

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104.

Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P1-(11-phenoxyundecyl)-P2- (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively.

Identification and biochemical characterization of a novel α-1,3-mannosyltransferase WfcD from Escherichia coli O141.

Glycosyltransferases (GTs) catalyze the formation of regio- and stereospecific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the wfcD gene from the Escherichia coli O141 O-antigen gene cluster encoding an α-1,3-mannosyltransferase that catalyzed the formation of the linkage Man(α1-3)-GlcNAc was biochemically characterized. WfcD was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn) and glycosidase digestion using the donor substrate GDP-Man and the synthetic acceptor substrate decyl diphosphate 2-acetamido-2-deoxy-α-D-glucopyranose (GlcNAc-PP-De). The kinetic and physiochemical properties and the substrate specificity of WfcD were investigated. WfcD is the first characterized bacterial mannosyltransferase that acts on the Man(α1-3)-GlcNAc linkage. This study enhances our knowledge of the diverse functions of GTs.

Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3.

Glycosyltransferases (GTs) catalyze the formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the gene wclR from the Escherichia coli O3 O-antigen gene cluster that encodes an α 1, 3-galactosyltransferase (GalT) that acts on the linkage Gal α 1, 3-GlcNAc was biochemically characterized. WclR was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)) and galactosidase digestion, using UDP-Gal as the donor substrate and the synthetic acceptor substrate GlcNAc-PP-De (decyl diphosphate N-acetylglucosamine). The physiochemical properties and the substrate specificity of WclR were investigated. WclR is the first bacterial GalT characterized that acts on the linkage Gal α 1, 3-GlcNAc. This study enhanced our knowledge of the diversified functions of GTs and provided a novel enzyme source for possible pharmaceutical application.

Mass spectrometric characterization of a two-glycosyltransferase tandem reaction for assembly of tetrasaccharide repeating unit of Escherichia coli O77 O-antigen.

The wbaD gene and wbaC gene from Escherichia coli O77 O-antigen gene cluster encoding mannosyltransferases were functionally characterized in vitro. A synthetic acceptor P(1)-(11-phenoxyundecyl)-P(2)-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate (GlcNAc-PP-PhU) was used as an acceptor and GDP-Man as a donor substrate; the activities of WbaD and WbaC were confirmed by detailed structural characterization of their lipooligosacharide enzyme products using high-sensitivity negative-ion electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (CID) MS-MS. The extensive fragmentation unequivocally demonstrated that the Man(1-3)-GlcNAc linkage in WbaD catalyzed reaction product and two Man(1-2)-Man linkages in tandem WbaD/WbaC catalyzed reaction product are present, respectively. This study provided valuable information for the understanding of diversified glycosyltransferase (GT) functions and the two GTs characterized can serve as additional enzyme sources for possible pharmaceutical related applications.

Influence of Merosesquiterpenoids from Marine Sponges on Seedling Root Growth of Agricultural Plants.

The impact of the merosesquiterpenoids avarol (1), avarone (2), 18-methylaminoavarone (3), melemeleone A (4), isospongiaquinone (5), ilimaquinone (6), and smenoquinone (7), isolated from marine sponges of the Dictyoceratida order, was studied on the root growth of seedlings of buckwheat (Fagopyrumesculentum Moench), wheat (Triticumaestivum L.), soy (Glycine max (L.) Merr.), and barley (Hordeumvulgare L.). Compounds 2and 6 were effective for the root growth of wheat seedlings, compound 3 stimulated the root growth of seedlings of buckwheat and soy, compound 4 affected the roots of barley seedlings, and compound 5 stimulated the root growth of seedlings of buckwheat and barley. Compounds 1 and 7 showed no activity on the root growth of the seedlings of any of the studied plants. The stimulatory effect depends on the chemical structure of the compounds and the type of crop plant.

N-Demethylaaptanone, A new Congener of Aaptamine Alkaloids from the Vietnamese Marine Sponge Aaptos aaptos.

A new compound, N-demethylaaptanone (5), having an oxygenated 1,6-naphthyridine core, has been isolated from the Vietnamese marine sponge Aaptos aaptos, along with the known metabolites, aaptamine (1), isoaaptamine (2), 9-demethylaaptamine (3), and aaptanone (4). The structure of N-demethylaaptanone was determined as 9-hydroxy-8-methoxy-4H-benzo[de][1,6]-naphthyridine-5,6-dione from spectroscopic data.

Sagitol D, a New Thiazole Containing Pyridoacridine Alkaloid from a Vietnamese Ascidian.

A new thiazole containing pyridoacridine alkaloid, named sagitol D (1), and five known alkaloids kuanoniaminesA (2), C (3), D (4), E (5), and F (6), have been isolated from an unidentified Vietnamese ascidian. The structure of the new compound was established from NMR spectroscopic data. Kuanoniamines C, D, E, and F showed moderate antioxidant activity in the DPPH (IC50 36 µM) and ABTS assays (TE = 0.5), while sagitol D showed weak activity (IC50 92 M;TE = 0.10), and kuanoniamine A was inactive.

Alkaloids from marine sponges as stimulators of initial stages of development of agricultural plants.

Damirone A (1), damirone B (2), makaluvamine G (3), debromohymenialdisine (4), and dibromoagelaspongin (5) were examined for their ability to stimulate growth of seedling roots of barley (Hordeum vulgare L.), buckwheat (Fagopyrum esculentum Moench), corn (Zea mays L.), soy (Glycine max (L.) Merr.}, and wheat (Triticum aestivum L.). It was shown that the stimulatory effects depend on the chemical structure of the alkaloids and on the plant species. Compounds 1, 3, and 4 are efficient for growth of seedling roots of barley, compounds 2-5, at different concentrations, stimulate growth of buckwheat roots, and compound 5 stimulates growth of wheat roots. These compounds can be recommended for field study as plant growth stimulators.

Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology.

For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([¹²5I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (K(i) 0.5-1.3 µM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [¹²5I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (K(i) ~ 1.5 µM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.

Biochemical characterization of a new ß-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit.

In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn(2+) ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a ß-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid ß-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts.

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.

Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5.

The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P¹-[11-(anthracen-9-ylmethoxy)undecyl]-P²-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E. coli) O157 and for galactosyltransferase WbwC from E. coli O5. This is the first report of the Gal-transferase activity of the wbwC gene product of E.coli O5. The presence of the fluorescent label in the acceptor molecule allows the detection of glycosyltransferase reaction products with high sensitivity, eliminating the need for radioactive nucleotide sugars.

Antioxidant activity of zyzzyanones and makaluvamines from the marine sponge Zyzzya fuliginosa.

Antioxidant activities of zyzzyanones A-D (1-4) and makaluvamines C (8), E (5), G (6), H (9), and L (7) isolated from the marine sponge Zyzzya fuliginosa (Carter, 1879) were evaluated using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) scavenging assay and AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) induced autoxidation of linoleic acid. Zyzzyanones A, B (1, 2; TE = 0.3), C, D (3, 4; TE = 0.24) and makaluvamines 5-7 (TE = 0.6) displayed moderate activities in the ABTS assay and in the autoxidation of linoleic acid (61-66% inhibition at concentrations of 0.1 mM). Makaluvamines C (8) and H (9) were essentially inactive in the both assays. Structure-activity relationships showed that antioxidant activities of tested compounds depended on the presence of a phenolic function in molecules. Makaluvamines 5-7 possessing a p-hydroxystyryl moiety were more active than zyzzyanones 1-4 possessing a p-hydroxyphenyl fragment. The presence of a charged side chain in 1 and 2 slightly increases their ABTS scavenging activity in comparison with compounds 3 and 4. Structural variations in a pyrroloquinoline skeleton of 5-7 and in a dipyrroloquinone core of 1-4 have no effect on activities.

Free radical scavenging activities of naturally occurring and synthetic analogues of sea urchin naphthazarin pigments.

Antioxidant activities of minor pigments of sea urchins (1-5) and synthetic naphthazarins (7-13) were evaluated and compared with echinochrome A (6) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) scavenging assays. Structure-activity relationships showed that the antioxidant activities of the tested compounds depended on the number and positions of hydroxyl groups. Compounds bearing 3 or 2 hydroxyl groups on a naphthazarin core (5,8-dihydroxy-1,4-naphthoquinone) were the most active in both assays. Echinochrome A (6) (IC50 7.0 microM) and its monomethyl ethers 7 (IC50 15.0 microM) and 8 (IC50 15.0 microM) displayed stronger activities than Trolox (IC50 16.0 microM) in the DPPH and ABTS assays (TE = 3.41, 2.35, and 2.35 mM, respectively). Compounds with either one or without hydroxyl groups on a naphthazarin core displayed activities significantly lower than Trolox in both assays. These results suggest that hydroxylated naphthazarin pigments of sea urchins have a potential use as natural antioxidants.

New meroterpenoids from the marine sponge Aka coralliphaga.

Three new sulfated meroterpenoids containing sesquiterpene and hydroquinone moieties, namely siphonodictyal A sulfate (1), akadisulfate A (2) and akadisulfate B (3), along with the known siphonodictyal B3 and bis(sulfato)-cyclosiphonodictyol A were isolated from the sponge Aka coralliphaga. Their structures were elucidated on the basis of spectroscopic data. Akadisulfate B and siphonodictyal B3 showed a radical-scavenging activity comparable with that of the known lipophylic antioxidant BHT.

Biochemical characterization of WbdN, a ß1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157.

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcß1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was ß1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid ß1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.