Red Sea Sponge Negombata Magnifica Research Articles

Differential Binding of Latrunculins to G-Actin: A Molecular Dynamics Study

Latrunculins are unique macrolides containing a thiazolidinone moiety. Latrunculin A (1), latrunculin B (2), 16-epi-latrunculin B (3), and latrunculin T (4) were isolated from the Red Sea sponge Negombata magnifica. In the present study, after testing compounds 2-4 for cytotoxic activity, they were docked into the crystal structure of G-actin and subjected to binding energy calculation and a 20 ns MD simulation. The modeling study shows that latrunculins binding depends on both hydrophobic interaction of the macrocycle as well as H bonding of the thiazolidinone ring with Asp157 and Thr186. It was noticed that epimerization at C16 of latrunculin B was well tolerated as it could form an alternative H bonding network. However, opening of the macrocyclic ring deteriorates the actin binding due to reduced hydrophobicity. MD simulation showed that latrunculin B (2) possesses a more significant stabilizing effect on G-actin than latrunculin T (4) and could efficiently hinder the flattening transition of G-actin into F-actin. These findings could explain, at the molecular level, the impact of epimerization and macrolide ring-opening on latrunculins activity, an issue that has not been addressed before. Also, the study gives insights into the mechanism of cytotoxicity of diverse latrunculins and provides direction for future lead optimization studies.

Antifungal Potential of Marine Sponge Extract against Plant and Fish Pathogenic Fungi

The need for discovery of new antibiotics is imperative because previous evidence suggest that development of resistance to any new antimicrobial agents is inevitable. In addition, compounds not tested for antimicrobial activity also gives ample scope for rediscovering compounds with antimicrobial activity. This study screens extracts (Diethyl Ether and Butanol) of a marine red-Sea sponge Negombata magnifica for in vitro fungicidal activity against 10 plant and 3 fish pathogens. Fungicidal activity of the crude diethylether fraction of the sponge against test strains showed exceptionally high activity against three virulent fish pathogens Exophiala salmonis, Branchiomyces demigrans and Saprolegnia sp. Furthermore, Minimal Inhibitory Concentration (MIC) of the compound was evaluated with respect to Exophiala salmonis (1.5 μg/ml) Branchiomyces demigrans (0.75 μg/ml) and Saprolegnia sp. (3 μg/ml) respectively. Finally, bioassay guided isolation and purification led to the identification of the active compound as latrunculin B on the basis of spectroscopic data. This finding suggests that latrunculin B, could be considered as a lead molecule for the design of new antifungals for managing common fungal diseases in aquaculture. Toxicity testing of this compound on shrimps indicated that the compound was non toxic up to a concentration as high as 5 mg/ml suggesting its suitability for use in aquaculture.

3D-QSAR studies of latrunculin-based actin polymerization inhibitors using CoMFA and CoMSIA approaches

The marine-derived macrolide latrunculins A and B, from the Red Sea sponge Negombata magnifica, are known to reversibly bind actin monomers, forming 1:1 complex with G-actin, disrupting its polymerization. Latrunculins have remarkable physiological properties and widely used as biochemical markers. Nevertheless, no QSAR studies have been developed for any kind of actin disruptors. In the present study, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) implemented in the SYBYL software packages were used to develop predictive 3D-QSAR models. Two alignment strategies were used to fit analyzed molecules to a suitable conformational template. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA results presented cross-validated correlation coefficient values ( q 2) of 0.621 and 0.659, and non-cross-validated values ( r 2) of 0.938and 0.965, respectively. Comparable to multifit-derived models, CoMFA and CoMSIA 3D-QSAR models were also derived using a molecular alignment obtained by docking latrunculin derivatives into the ATP active site of actin. In addition to q 2, the predictive ability was validated using external test set of five compounds. The results of this study suggest that the established model has a strong predictive ability and can be prospectively used in the molecular design and action mechanism analysis of this kind of cytotoxic compounds.

Semisynthetic Latrunculin Derivatives as Inhibitors of Metastatic Breast Cancer: Biological Evaluations, Preliminary Structure–Activity Relationship and Molecular Modeling Studies

The microfilament cytoskeleton protein actin plays an important role in cell biology and affects cytokinesis, morphogenesis, and cell migration. These functions usually fail and become abnormal in cancer cells. The marine-derived macrolides latrunculins A and B, from the Red Sea sponge Negombata magnifica, are known to reversibly bind actin monomers, forming 1:1 stoichiometric complexes with G-actin, disrupting its polymerization. To identify novel therapeutic agents for effective treatment of metastatic breast cancer, several semisynthetic derivatives of latrunculin A with diverse steric, electrostatic, and hydrogen bond donor and acceptor properties were rationally prepared. Analogues were designed to modulate the binding affinity toward G-actin. Examples of these reactions are esterification, acetylation, and N-alkylation. Semisynthetic latrunculins were then tested for their ability to inhibit pyrene-conjugated actin polymerization, and subsequently assayed for their antiproliferative and anti-invasive properties against MCF7 and MDA-MB-231 cells using MTT and invasion assays, respectively.

Particulate organic matter as a food source for a coral reef sponge

The ability of sponges to feed in diverse (including oligotrophic) ecosystems significantly contributes to their ubiquitous aquatic distribution. It was hypothesized that sponges that harbour small amounts of symbiotic bacteria in their mass feed mainly on particulate organic matter (POM). We examined the nearly symbiont-free (by microscopic observation) filter-feeding Red Sea sponge Negombata magnifica in order to: (a) study removal efficiency of naturally occurring organic particles, (b) measure the total amount of absorbed particulate organic carbon (POC) and nitrogen (PON), and (c) estimate organic carbon and nitrogen flux in this sponge. Total amount of organic carbon and nitrogen in the Gulf of Aqaba was found to be 48.46+/-5.69 microg l(-1) and 6.45+/-0.7 microg l(-1), respectively. While detritus contributed 54% of POC, most PON (84%) came from planktonic microorganisms, mainly prokaryotes. Particle removal efficiency ranged from 99% (the cyanobacterium Synechococcus sp.) to 37% (for eukaryotic cells >8 microm). On average, N. magnifica ingested 480 microg C day(-1) g(-1) (wet mass, WM) sponge and 76.6 microg N day(-1) g(-1) sponge. Ingested POC balanced 85% of the sponge's energetic demand but more is needed for biomass production because it cannot digest all of the carbon. 54.4+/-16.1 microg N day(-1) g(-1) (WM) nitrogen was excreted as total ammonia nitrogen (TAN); however, nitrogen allowance should be higher because more nitrogen is deposited for sponge biomass during growth. It is hypothesized that the discrepancy in the nutritional requirements should be covered by the sponge absorbing carbon and nitrogen from sources that are not dealt with in the present research, such as dissolved organic carbon and nitrogen. This study highlights the significance of detritus as a carbon source, and prokaryotes as a PON source in sponge feeding.

Semisynthetic latrunculin B analogs: Studies of actin docking support a proposed mechanism for latrunculin bioactivity

Latrunculins are unique macrolides containing a thiazolidinone moiety. Latrunculins A, B and T and 16-epi-latrunculin B were isolated from the Red Sea sponge Negombata magnifica. N-Alkylated, O-methylated analogs of latrunculin B were synthesized and biological evaluation was performed for antifungal and antiprotozoal activity. The natural latrunculins showed significant bioactivity, while the semisynthetic analogs did not. Docking studies of these analogs into the X-ray crystal structure of G-actin showed that, in comparison with latrunculins A and B, N-alkylated latrunculins did not dock satisfactorily. This suggests that the analogs do not fit well into the active site of G-actin due to steric clashes and provides an explanation for the absence of bioactivity.

Latrunculin A and Its C-17-O-Carbamates Inhibit Prostate Tumor Cell Invasion and HIF-1 Activation in Breast Tumor Cells

The marine-derived macrolides latrunculins A ( 1) and B, from the Red Sea sponge Negombata magnifica, have been found to reversibly bind actin monomers, forming a 1:1 complex with G-actin and disrupting its polymerization. The microfilament protein actin is responsible for several essential functions within the cell such as cytokinesis and cell migration. One of the main binding pharmacophores of 1 to G-actin was identified as the C-17 lactol hydroxyl moiety that binds arginine 210 NH. Latrunculin A-17- O-carbamates 2- 6 were prepared by reaction with the corresponding isocyanates. Latrunculin A ( 1) and carbamates 4- 6 displayed potent anti-invasive activity against the human highly metastatic human prostate cancer PC-3M cells in a Matrigel assay at a concentration range of 50 nM to 1 microM. Latrunculin A ( 1, 500 nM) decreased the disaggregation and cell migration of PC-3M-CT+ spheroids by 3-fold. Carbamates 4 and 5 were 2.5- and 5-fold more active than 1, respectively, in this assay with less actin binding affinity. Latrunculin A ( 1, IC 50 6.7 microM) and its 17- O-[ N-(benzyl)carbamate ( 6, IC 50 29 microM) suppress hypoxia-induced HIF-1 activation in T47D breast tumor cells.

Biocatalytic and semisynthetic optimization of the anti-invasive tobacco (1 S,2 E,4 R,6 R,7 E,11 E)-2,7,11-cembratriene-4,6-diol

Tobacco cembranoids were reported to inhibit tumorigenesis. Biocatalysis of (1 S,2 E,4 R,6 R,7 E,11 E)-2,7,11-cembratriene-4,6-diol ( 1) using the symbiotic Bacillus sp. NC5, Bacillus sp. NK8, and Bacillus sp. NK7, isolated from the Red Sea sponge Negombata magnifica, afforded two new and four known hydroxylated metabolites 3– 8. The use of symbiotic marine bacteria as biocatalysts for bioactive natural product scaffolds is very rare. Cembranoid 1 carbamate analogs 9– 11 were prepared by its reaction with corresponding isocyanates. Cembranoid 1 and its bioconversion and carabamate products show anti-invasive activity against the human highly metastatic prostate PC-3M cancer cell line at 10–50 nM doses in Matrigel assay.

Bioactive Natural and Semisynthetic Latrunculins

Marine-derived macrolides latrunculins A and B, of the Red Sea sponge Negombata magnifica, are the first marine natural products that have been found to reversibly bind to actin monomers and to disrupt its organization. Latrunculins are structurally related to many antimicrobial and antiangiogenic macrolides. Several grams of latrunculin B (1), together with a new latrunculin named latrunculin T (2), were isolated from a recent collection of N. magnifica. Semisynthetic modifications of 1, including acetylation, acetalization, and N-hydroxymethylation, afforded four new (4, 5, 7, 8) and two known (6 and 9) semisynthetic analogues. Specifically, 15-O-methyllatrunculin B (6) showed a promising antiangiogenic activity in a chick chorioallantoic membrane assay and antimigratory activity in Boyden's chamber assay. Moreover, latrunculin B (1) and the new N-acetyllatrunculin B (4) displayed potent antimigratory activity in a wound-healing assay. Natural and semisynthetic latrunculins showed potent antimicrobial activity against Candida albicans, Saccharomyces cerevisiae, Staphylococcus aureus, and Bacillus cereus. Latrunculins are potential leads that can be developed as anticancer and antimicrobial agents.

Quantitative determination of latrunculins A and B in the Red Sea sponge Negombata magnifica by high performance liquid chromatography.

An accurate, reproducible and sensitive method for the quantitative determination of latrunculins A and B in the organic extract of the Red Sea sponge Negombata magnifica was developed and validated. Latrunculin A and B concentrations were determined by RP-C18-HPLC and a mobile phase consisting of acetonitrile and water (60:40, v/v). The flow rate utilized was 1 mL min(-1) and the detector was set at 235 nm. The HPLC analysis of several N. magnifica samples collected from different locations in the Red Sea revealed that Ras Mohamed had the highest concentrations of latrunculin A, while Safaga had the highest levels of latrunculin B. Also, a comparison between latrunculin concentrations in the summer and winter revealed that the yield of latrunculins were generally higher in the winter.

Sea ranching of the marine sponge Negombata magnifica (Demospongiae, Latrunculiidae) as a first step for latrunculin B mass production

Marine sponges are a rich source of bioactive metabolites that can potentially be used as lead compounds to treat various human diseases. Due to the difficulty in obtaining a steady supply of such metabolites, few of these compounds have reached the clinical trials stage. Sponge sea ranching is one of the most promising methods to overcome this “supply problem”. The Red Sea sponge Negombata magnifica is a potential source of the cytotoxin, latrunculin B (lat-B). The aim of the research was to compare the effect of different sea ranching culture methods on N. magnifica growth and survival rates and to identify factors that might enhance these rates. Since the ultimate goal of the project is to produce bioactive metabolites, we measured the lat-B concentration in the sponge tissues. Ninety-eight fragments were cut from nine different individuals and attached to PVC plates or threaded on fishing lines. The PVC-attached sponges were fastened to plastic nets and the threaded sponges were suspended on horizontal ropes. Each of these treatments was stocked at 10 or 20 m depth. Within 177 days, the sponges’ average size increased from 11.6±0.7 to 24.5±2 gr and the survival weighted mean of all treatments was 71.4%. The specific growth rate (SGR) of sponges that were suspended on nylon threads was significantly higher ( P<0.05) than the SGR of sponges that were attached to the net (0.9±0.08% day −1 and 0.5±0.05% day −1, respectively). No significant difference ( P>0.05) in growth rate was found between the two stocking depths. Fouling organisms significantly ( p<0.05) reduced the sponge SGR compared to that of sponges grown free of fouling organisms (0.26±0.07% day −1 and 0.55±0.09% day −1, respectively). The amount of latrunculin B found within body of sponges cultured for a year in the sea ranching system (1.2±0.16 mg lat-B gr dry matter sponge −1) was higher than the concentration previously reported for this species (0.35 mg lat-B gr dry matter sponge −1). The present results demonstrate that N. magnifica could be cultured in sea ranching systems for the production of lat-B with relatively good survival and growth rates.

A new dimension to the biosynthetic products isolated from the sponge Negombata magnifica.

Latrunculeic acid (3), a novel analogue of latrunculin B (2a), was isolated from the Red Sea sponge Negombata magnifica and characterized. Several known compounds were also isolated, including latrunculin B (2a), 15-methoxylatrunculin B (2b), 16-epi-latrunculin B (4), and latrunculin C (5). In contrast to the other members of the latrunculin family, the novel compound 3 is a polyketide devoid of the normal macrocyclic and thiazolidinone rings present in previously identified members of this family.

Immunolocalization of the Toxin Latrunculin B within the Red Sea Sponge Negombata magnifica (Demospongiae, Latrunculiidae).

The location of latrunculin B, the major toxin of the Red Sea sponge Negombata magnifica, was revealed using specific antibodies. Antibodies from rabbits immunized with a conjugate of latrunculin B with keyhole limpet hemocyanin (KLH) were purified over a latrunculin B-Sepharose affinity column. Analysis of immunohistochemical and immunogold-stained sponge sections, using light and transmission electron microscopy, revealed latrunculin B labeling mostly beneath the sponge cortex at the border between the external (ectosome) and internal (endosome) layers (ectosome-endosome border). The endosome was less labeled than the border. Immunogold localization revealed latrunculin B in the sponge cells but not in its prokaryotic symbionts. Archeocytes and choanocytes were significantly more labeled than other cells. The antibodies primarily labeled membrane-limited vacuoles within archeocytes and choanocytes that are perhaps latrunculin B secretory or storage vesicles. Peripheral latrunculin B may have a role in defense against external epibionts, predators, and competitors.