| Name | Valeria |
| Surname | Costantino |
| Institution | University of Naples – Federico II |
| valeria.costantino@unina.it | |
| Address | Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, via Domenico Montesano 49, 80131 Napoli, Italy |
| Resume | Download |
The 6-epimer of the plakortide H acid (1), along with the endoperoxides plakortide E (2), plakortin (3), and dihydroplakortin (4) have been isolated from a sample of the Caribbean sponge Plakortis halichondrioides. To perform a comparative study on the cytotoxicity towards the drug-sensitive leukemia CCRF-CEM cell line and its multi-drug resistant subline CEM/ADR5000, the acid of plakortin, namely plakortic acid (5), as well as the esters plakortide E methyl ester (6) and 6-epi-plakortide H (7) were synthesized by hydrolysis and Steglich esterification, respectively. The data obtained showed that the acids (1, 2, 5) exhibited potent cytotoxicity towards both cell lines, whereas the esters showed no activity (6, 7) or weaker activity (3, 4) compared to their corresponding acids. Plakortic acid (5) was the most promising derivative with half maximal inhibitory concentration (IC50) values of ca. 0.20 µM for both cell lines.
There is an urgent need for novel strategies to fight drug resistance and multi-drug resistance. As an alternative to the classic antibiotic therapy, attenuation of the bacteria virulence affecting their Quorum sensing (QS) system is a promising approach. Quorum sensing (QS) is a genetic regulation system that allows bacteria to communicate with each other and coordinate group behaviors. A new γ-lactone that is capable of inhibiting the LasI/R QS system, plakofuranolactone (1), was discovered in the extract of the marine sponge Plakortis cf. lita, and its structure, including absolute configuration, was determined by NMR spectroscopy, MS spectrometry, and quantum-mechanical prediction of optical rotation. The quorum quenching activity of plakofuranolactone was evaluated using reporter gene assays for long- and short-chain signals (E. coli pSB1075, E. coli pSB401, and C. violeaceum CV026) and was confirmed by measuring the total protease activity (a virulence factor which is under control of the LasI/R system) of the wild-type P. aeruginosa PAO1. Further research will be pursued to assess the potential of plakofuranolactone as a new antivirulence lead compound and a chemical tool to increase the knowledge in this field.
Marine natural products with antibiotic activity have been a rich source of drug discovery; however, the emergence of antibiotic-resistant bacterial strains has turned attention towards the discovery of alternative innovative strategies to combat pathogens. In many pathogenic bacteria, the expression of virulence factors is under the regulation of quorum sensing (QS). QS inhibitors (QSIs) present a promising alternative or potential synergistic treatment since they disrupt the signaling pathway used for intra- and interspecies coordination of expression of virulence factors. This review covers the set of molecules showing QSI activity that were isolated from marine organisms, including plants (algae), animals (sponges, cnidarians, and bryozoans), and microorganisms (bacteria, fungi, and cyanobacteria). The compounds found and the methods used for their isolation are the emphasis of this review.
The halogenated alkaloid chloromethylhalicyclamine B (1), together with the known natural compound halicyclamine B (2), was isolated from the extract of the sponge Acanthostrongylophora ingens. The structure of compound 1 was determined by spectroscopic means, and it was shown that 1 is produced by reaction of 2 with CH2Cl2 used for extraction. Compound 1 was a selective CK1δ/ε inhibitor with an IC50 of 6 μM, while the natural compound 2 was inactive. The absolute configuration of 1 was determined by quantum mechanical calculation of its ECD spectrum, and this also determined the previously unknown absolute configuration of the parent halicyclamine B (2). Computational studies, validated by NOESY data, showed that compound 1 can efficiently interact with the ATP-binding site of CK1δ in spite of its globular structure, very different from the planar structure of known inhibitors of CK1δ. This opens the way to the design of a new structural type of CK1δ/ε inhibitors.
Owing to the extensive development of drug resistance in pathogens against the available antibiotic arsenal, antimicrobial resistance is now an emerging major threat to public healthcare. Anti-virulence drugs are a new type of therapeutic agent aiming at virulence factors rather than killing the pathogen, thus providing less selective pressure for evolution of resistance. One promising example of this therapeutic concept targets bacterial quorum sensing (QS), because QS controls many virulence factors responsible for bacterial infections. Marine sponges and their associated bacteria are considered a still untapped source for unique chemical leads with a wide range of biological activities. In the present study, we screened extracts of 14 sponge species collected from the Red and Mediterranean Sea for their quorum-quenching (QQ) potential. Half of the species showed QQ activity in at least 2 out of 3 replicates. Six out of the 14 species were selected for bacteria isolation, to test for QQ activity also in isolates, which, once cultured, represent an unlimited source of compounds. We show that ≈20% of the isolates showed QQ activity based on a Chromobacterium violaceum CV026 screen, and that the presence or absence of QQ activity in a sponge extract did not correlate with the abundance of isolates with the same activity from the same sponge species. This can be explained by the unknown source of QQ compounds in sponge-holobionts (host or symbionts), and further by the possible non-symbiotic nature of bacteria isolated from sponges. The potential symbiotic nature of the isolates showing QQ activity was tested according to the distribution and abundance of taxonomically close bacterial Operational Taxonomic Units (OTUs) in a dataset including 97 sponge species and 178 environmental samples (i.e., seawater, freshwater, and marine sediments). Most isolates were found not to be enriched in sponges and may simply have been trapped in the filtration channels of the sponge at the time of collection. Our results highlight potential for QQ-bioactive lead molecules for anti-virulence therapy both from sponges and the bacteria isolated thereof, independently on the symbiotic nature of the latter.
Cyclic N (1)-pentylinosine monophosphate (cpIMP), a novel simplified inosine derivative of cyclic ADP-ribose (cADPR) in which the N (1)-pentyl chain and the monophosphate group replace the northern ribose and the pyrophosphate moieties, respectively, was synthesized. The role played by the position of the phosphate group in the key cyclization step, which consists in the formation of a phosphodiester bond, was thoroughly investigated. We have also examined the influence of the phosphate bridge on the ability of cpIMP to mobilize Ca(2+) in PC12 neuronal cells in comparison with the pyrophosphate bridge present in the cyclic N (1)-pentylinosine diphosphate analogue (cpIDP) previously synthesized in our laboratories. The preliminary biological tests indicated that cpIMP and cpIDP induce a rapid increase of intracellular Ca(2+) concentration in PC12 neuronal cells.
Cyanotoxins obtained from a freshwater cyanobacterial collection at Green Lake, Seattle during a cyanobacterial harmful algal bloom in the summer of 2014 were studied using a new approach based on molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data. This MS networking approach is particularly well-suited for the detection of new cyanotoxin variants and resulted in the discovery of three new cyclic peptides, namely microcystin-MhtyR (6), which comprised about half of the total microcystin content in the bloom, and ferintoic acids C (12) and D (13). Structure elucidation of 6 was aided by a new microscale methylation procedure. Metagenomic analysis of the bloom using the 16S-ITS rRNA region identified Microcystis aeruginosa as the predominant cyanobacterium in the sample. Fragments of the putative biosynthetic genes for the new cyanotoxins were also identified, and their sequences correlated to the structure of the isolated cyanotoxins.
Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N-acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei. The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales, here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli, produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium.
The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.
Sponge-associated microorganisms are able to assemble the complex machinery for the production of secondary metabolites such as polyketides, the most important class of marine natural products from a drug discovery perspective. A comprehensive overview of polyketide biosynthetic genes of the sponge Plakortis halichondrioides and its symbionts was obtained in the present study by massively parallel 454 pyrosequencing of complex and heterogeneous PCR (Polymerase Chain Reaction) products amplified from the metagenomic DNA of a specimen of P. halichondrioides collected in the Caribbean Sea. This was accompanied by a survey of the bacterial diversity within the sponge. In line with previous studies, sequences belonging to supA and swfA, two widespread sponge-specific groups of polyketide synthase (PKS) genes were dominant. While they have been previously reported as belonging to Poribacteria (a novel bacterial phylum found exclusively in sponges), re-examination of current genomic sequencing data showed supA and swfA not to be present in the poribacterial genome. Several non-supA, non-swfA type-I PKS fragments were also identified. A significant portion of these fragments resembled type-I PKSs from protists, suggesting that bacteria may not be the only source of polyketides from P. halichondrioides, and that protistan PKSs should receive further investigation as a source of novel polyketides.