From anti-fouling to biofilm inhibition: New cytotoxic secondary metabolites from two Indonesian Agelas sponges

Chemical investigation of Indonesian marine sponges Agelas linnaei and A. nakamurai afforded 24 alkaloid derivatives representing either bromopyrrole or diterpene alkaloids. A. linnaei yielded 16 bromopyrrole alkaloids including 11 new natural products with the latter exhibiting unusual functionalities. The new compounds include the first iodinated tyramine-unit bearing pyrrole alkaloids, agelanesins A–D. These compounds exhibited cytotoxic activity against L5178Y mouse lymphoma cells with IC₅₀ values between 9.25 and 16.76 μM. Further new compounds include taurine acid substituted bromopyrrole alkaloids and a new dibromophakellin derivative. A. nakamurai yielded eight alkaloids among them are three new natural products. The latter include the diterpene alkaloids (?)-agelasine D and its oxime derivative and the new bromopyrrole alkaloid longamide C. (?)-Agelasine D and its oxime derivative exhibited cytotoxicity against L5178Y mouse lymphoma cells (IC₅₀ 4.03 and 12.5 μM, respectively). Furthermore, both agelasine derivatives inhibited settling of larvae of Balanus improvisus in an anti-fouling bioassay and proved to be toxic to the larvae. (?)-Agelasine D inhibited the growth of planktonic forms of biofilm forming bacteria S. epidermidis (MIC < 0.0877 μM) but did not inhibit biofilm formation whereas the oxime derivative showed the opposite activity profile and inhibited only biofilm formation but not bacterial growth. The structures of the isolated secondary metabolites were elucidated based on extensive spectroscopic analysis involving one- and two-dimensional NMR as well as mass spectrometry and comparison with literature data.     

Isolation of recombinant proteins from culture broth by co‐precipitation with an amino acid carrier to form stable dry powders

Protein‐coated microcrystals can be generated by co‐precipitation of protein and a water‐soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co‐precipitation with a L ‐glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co‐elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co‐precipitation process. Scale‐up of the process was readily achieved through in‐line mixing and subsequent reconstitution of the dried protein‐coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L ‐glutamine facilitated further purification of the recombinant protein immobilized on the L ‐glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing. Biotechnol. Bioeng. 2010;106: 764–773. © 2010 Wiley Periodicals, Inc.     

Cytosporones,coumarins, and an alkaloid from the endophytic fungus Pestalotiopsis sp. isolated from the Chinese mangrove plant Rhizophora mucronata

Chemical examination of the endophytic fungus Pestalotiopsis sp., isolated from the leaves of the Chinese mangrove Rhizophora mucronata, yielded 11 new compounds including cytosporones J–N (1–3, 5–6), five new coumarins pestalasins A–E (8–12), and a new alkaloid named pestalotiopsoid A (14), along with the known compounds cytosporone C (4), dothiorelone B (7), and 3-hydroxymethyl-6,8-dimethoxycoumarin (13). The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic data analysis.     

NF kappa B inhibitors and antitrypanosomal metabolites from endophytic fungus Penicillium sp. isolated from Limonium tubiflorum

Chemical investigation of the endophytic fungus Penicillium sp. isolated from Limonium tubiflorum growing in Egypt afforded four new compounds of polyketide origin, including two macrolides, penilactone (1) and 10,11-epoxycurvularin (2), a dianthrone, neobulgarone G (7), and a sulfinylcoumarin, sulfimarin (14), along with twelve known metabolites (3-6, 8-13, 15 and 16). The structures of all compounds were assigned by comprehensive spectral analysis (1D and 2D NMR) and mass spectrometry. Compounds 3, 4, 13 and 16 showed pronounced antitrypanosomal activity with mean MIC values ranging from 4.96 to 9.75 ??M. Moreover, when tested against a panel of three human tumor cell lines compounds 3, 4, 6 and 12 showed selective growth inhibition against Jurkat and U937 cell lines with IC₅₀ values ranging from 1.8 to 13.3 ??M. The latter compounds also inhibited TNF??-induced NF-??B activity in K562 cells with IC₅₀ values ranging from 1.6 to 10.1 ??M, respectively.     

Anti-Inflammatory Activity of Lauraceae Plant Species and Prediction Models Based on Their Metabolomics Profiling Data

The Lauraceae is a botanical family known for its anti-inflammatory potential. However, several species have not yet been studied. Thus, this work aimed to screen the anti-inflammatory activity of this plant family and to build statistical prediction models. The methodology was based on the statistical analysis of high-resolution liquid chromatography coupled with mass spectrometry data and the ex vivo anti-inflammatory activity of plant extracts. The ex vivo results demonstrated significant anti-inflammatory activity for several of these plants for the first time. The sample data were applied to build anti-inflammatory activity prediction models, including the partial least square acquired, artificial neural network, and stochastic gradient descent, which showed adequate fitting and predictive performance. Key anti-inflammatory markers, such as aporphine and benzylisoquinoline alkaloids were annotated with confidence level 2. Additionally, the validated prediction models proved to be useful for predicting active extracts using metabolomics data and studying their most bioactive metabolites.     

Chemical synthesis and biological activities of 3-alkyl pyridinium polymeric analogues of marine toxins

Two new large poly-1,3-dodecylpyridinium salts, APS12 and APS12-2 of 12.5- and 14.7-kDa size, respectively, were synthesised and tested for their pore-forming and transfection capabilities in HEK 293 and undifferentiated mouse ES cells using patch-clamp recording, Ca(2+) imaging and flow cytometry. Polymerisation reactions were enhanced by microwaves, and the product sizes were controlled by altering the irradiation time. This method can also be applied to obtain polymers with variable linking chains as shown by the preparation of poly-(1,3-octylpyridinium) salt of 11.9-kDa size. Molecular weights of the final products were determined using ESIMS analysis, which also indicated the products to be amongst the largest macro-cycles ever recorded, up to a 900-membered ring. Anti-bacterial, haemolytic and anti-acetylcholinesterase activities were also reported for the two dodecyl pyridinium polymers. These biological activities are characteristic to the structurally related marine toxin, poly-APS. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-010-0036-4) contains supplementary material, which is available to authorized users.     

Callyaerins A–F and H,new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa

Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new cytotoxic cyclic peptides callyaerins A–F (1–6) and H (8). Their structures were determined using extensive 1D (¹H, ¹³C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5–9 amino acids and side chains of 2–5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the template for ring closure and afforded the linkage to the peptidic side chain which was always initiated with a proline moiety. All peptides contained three or more proline residues and the remaining residues were predominantly hydrophobic residues with all amino acids present in the l form. Callyaerins A–F (1–6) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited strong activity against the L5178Y cell line with ED₅₀ values of 0.39 and 0.48 μM, respectively. On the other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans.     

Metabolomic Tools to Assess the Chemistry and Bioactivity of Endophytic Aspergillus Strain

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology that may find applications as pharmaceutical and agrochemical drugs. In this study, a combination of metabolomics and bioactivity‐guided approaches were employed to isolate secondary metabolites with cytotoxicity against cancer cells from an endophytic Aspergillus aculeatus. The endophyte was isolated from the Egyptian medicinal plant Terminalia laxiflora and identified using molecular biological methods. Metabolomics and dereplication studies were accomplished by utilizing the MZmine software coupled with the universal Dictionary of Natural Products database. Metabolic profiling, with aid of multivariate data analysis, was performed at different stages of the growth curve to choose the optimized method suitable for up‐scaling. The optimized culture method yielded a crude extract abundant with biologically‐active secondary metabolites. Crude extracts were fractionated using different high‐throughput chromatographic techniques. Purified compounds were identified by HR‐ESI‐MS, 1D‐ and 2D‐NMR. This study introduced a new method of dereplication utilizing both high‐resolution mass spectrometry and NMR spectroscopy. The metabolites were putatively identified by applying a chemotaxonomic filter. We also present a short review on the diverse chemistry of terrestrial endophytic strains of Aspergillus, which has become a part of our dereplication work and this will be of wide interest to those working in this field.