Computational Methods Reveal a Series of Cyclic and Linear Lichenysins and Surfactins from the Vietnamese Marine Sediment-Derived Streptomyces Strain G222.

The Streptomyces strain G222, isolated from a Vietnamese marine sediment, was confidently identified by 16S rRNA gene sequencing. Its AcOEt crude extract was successfully analyzed using non-targeted LC-MS/MS analysis, and molecular networking, leading to a putative annotation of its chemical diversity thanks to spectral libraries from GNPS and in silico metabolite structure prediction obtained from SIRIUS combined with the bioinformatics tool conCISE (Consensus Annotation Propagation of in silico Elucidations). This dereplication strategy allowed the identification of an interesting cluster of a series of putative cyclic and linear lipopeptides of the lichenysin and surfactin families. Lichenysins (3-7) were isolated from the sub-fraction, which showed significant anti-biofilm activity against Pseudomonas aeruginosa MUC-N1. Their structures were confirmed by detailed 1D and 2D NMR spectroscopy (COSY, HSQC, HMBC, TOCSY, ROESY) recorded in CD3OH, and their absolute configurations were determined using the modified Marfey's method. The isolated lichenysins showed anti-biofilm activity at a minimum concentration of 100 µM. When evaluated for antibacterial activity against a panel of Gram-positive and Gram-negative strains, two isolated lichenysins exhibited selective activity against the MRSA strain without affecting its growth curve and without membranotropic activity. This study highlights the power of the MS/MS spectral similarity strategy using computational methods to obtain a cross-validation of the annotated molecules from the complex metabolic profile of a marine sediment-derived Streptomyces extract. This work provides the first report from a Streptomyces strain of combined cyclic and linear lichenysins and surfactins, known to be characteristic compounds of the genus Bacillus.

α,ω-Diacyl-Substituted Analogues of Natural and Unnatural Polyamines: Identification of Potent Bactericides That Selectively Target Bacterial Membranes.

In this study, α-ω-disubstituted polyamines exhibit a range of potentially useful biological activities, including antimicrobial and antibiotic potentiation properties. We have prepared an expanded set of diarylbis(thioureido)polyamines that vary in central polyamine core length, identifying analogues with potent methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Acinetobacter baumannii and Candida albicans growth inhibition properties, in addition to the ability to enhance action of doxycycline towards Gram-negative bacterium Pseudomonas aeruginosa. The observation of associated cytotoxicity/hemolytic properties prompted synthesis of an alternative series of diacylpolyamines that explored aromatic head groups of varying lipophilicity. Examples bearing terminal groups each containing two phenyl rings (15a-f, 16a-f) were found to have optimal intrinsic antimicrobial properties, with MRSA being the most susceptible organism. A lack of observed cytotoxicity or hemolytic properties for all but the longest polyamine chain variants identified these as non-toxic Gram-positive antimicrobials worthy of further study. Analogues bearing either one or three aromatic-ring-containing head groups were either generally devoid of antimicrobial properties (one ring) or cytotoxic/hemolytic (three rings), defining a rather narrow range of head group lipophilicity that affords selectivity for Gram-positive bacterial membranes versus mammalian. Analogue 15d is bactericidal and targets the Gram-positive bacterial membrane.

The potential role of dietary plant ingredients against mammary cancer: a comprehensive review.

Breast cancer is known as the most devastating cancer in the global female community and is considered as one of the severe health care burdens in both developed and developing countries. In many cases, breast cancer has shown resistance to chemotherapy, radiotherapy and hormonal therapy. Keeping in view these limitations, there is an urgent need to develop safe, readily available and effective breast anticancer treatments. Therefore, the scientists are keen in the extraction of plant-based phytochemicals (organosulfur compounds, betalains, capsaicinoids, terpenes, terpenoids, polyphenols, and flavonoids) and using them as breast anticancer agents. Results of numerous epidemiological investigations have revealed the promising role of phytochemicals in the prevention and treatment of breast cancer. The diverse classes of plant bioactive metabolites regulate different metabolic and molecular processes, which can delay the proliferation of cancers. These phytochemicals possess chemo-preventive properties as they down-regulate the expression of estrogen receptor-α, inhibit the proliferation of cancer cells, and cause cell cycle arrest by inducing apoptotic conditions in tumor cells. This review article discusses the potent role of various plant-based phytochemicals as potential therapeutic agents in the treatment or prevention of breast cancer along with the proposed mechanisms of action.

Valorisation of the diterpene podocarpic acid - Antibiotic and antibiotic enhancing activities of polyamine conjugates.

As part of our search for new antimicrobials and antibiotic adjuvants, a series of podocarpic acid-polyamine conjugates have been synthesized. The library of compounds made use of the phenolic and carboxylic acid moieties of the diterpene allowing attachment of polyamines (PA) of different lengths to afford a structurally-diverse set of analogues. Evaluation of the conjugates for intrinsic antimicrobial properties identified two derivatives of interest: a PA3-4-3 (spermine) amide-bonded variant 7a that was a non-cytotoxic, non-hemolytic potent growth inhibitor of Gram-positive Staphylococcus aureus (MRSA) and 9d, a PA3-8-3 carbamate derivative that was a non-toxic selective antifungal towards Cryptococcus neoformans. Of the compound set, only one example exhibited activity towards Gram-negative bacteria. However, in the presence of sub-therapeutic amounts of either doxycycline (4.5 µM) or erythromycin (2.7 µM) several analogues were observed to exhibit weak to modest antibiotic adjuvant properties against Pseudomonas aeruginosa and/or Escherichia coli. The observation of strong cytotoxicity and/or hemolytic properties for subsets of the library, in particular those analogues bearing methyl ester or n-pentylamide functionality, highlighted the fine balance of structural requirements and lipophilicity for antimicrobial activity as opposed to mammalian cell toxicity.

Metabolomics with multi-block modelling of mass spectrometry and nuclear magnetic resonance in order to discriminate Haplosclerida marine sponges.

A comprehensive metabolomic strategy, integrating 1H NMR and MS-based multi-block modelling in conjunction with multi-informational molecular networking, has been developed to discriminate sponges of the order Haplosclerida, well known for being taxonomically contentious. An in-house collection of 33 marine sponge samples belonging to three families (Callyspongiidae, Chalinidae, Petrosiidae) and four different genera (Callyspongia, Haliclona, Petrosia, Xestospongia) was investigated using LC-MS/MS, molecular networking, and the annotations processes combined with NMR data and multivariate statistical modelling. The combination of MS and NMR data into supervised multivariate models led to the discrimination of, out of the four genera, three groups based on the presence of metabolites, not necessarily previously described in the Haplosclerida order. Although these metabolomic methods have already been applied separately, it is the first time that a multi-block untargeted approach using MS and NMR has been combined with molecular networking and statistically analyzed, pointing out the pros and cons of this strategy.

Computational Metabolomics Tools Reveal Subarmigerides, Unprecedented Linear Peptides from the Marine Sponge Holobiont Callyspongia subarmigera.

A detailed examination of a unique molecular family, restricted to the Callyspongia genus, in a molecular network obtained from an in-house Haplosclerida marine sponge collection (including Haliclona, Callyspongia, Xestospongia, and Petrosia species) led to the discovery of subarmigerides, a series of rare linear peptides from Callyspongia subarmigera, a genus mainly known for polyacetylenes and lipids. The structure of the sole isolated peptide, subarmigeride A (1) was elucidated through extensive 1D and 2D NMR spectroscopy, HRMS/MS, and Marfey's method to assign its absolute configuration. The putative structures of seven additional linear peptides were proposed by an analysis of their respective MS/MS spectra and a comparison of their fragmentation patterns with the heptapeptide 1. Surprisingly, several structurally related analogues of subarmigeride A (1) occurred in one distinct cluster from the molecular network of the cyanobacteria strains of the Guadeloupe mangroves, suggesting that the true producer of this peptide family might be the microbial sponge-associated community, i.e., the sponge-associated cyanobacteria.

Total Synthesis of the Four Stereoisomers of Cyclo(l-Trp-l-Arg) Raises Uncertainty of the Structures of the Natural Products and Invalidates Their Promising Antimicrobial Activities.

New therapeutic options to combat the growing incidence of antimicrobial resistance are urgently needed. A 2015 publication reported the isolation and biological evaluation of two diketopiperazine natural products, cyclo(l-Trp-l-Arg) (CDP 2) and cyclo(d-Trp-d-Arg) (CDP 3), from an Achromobacter sp. bacterium, finding that the latter metabolite in particular exhibited strong antibacterial activity towards a range of wound-related microorganisms and could synergize the action of ampicillin. Intrigued by these biological activities and noting inconsistencies in the structural characterization of the natural products, we synthesized the four diastereomers of cyclo(Trp-Arg) and evaluated them for antimicrobial and antibiotic enhancement properties. The detailed comparison of spectroscopic data raises uncertainty regarding the structure of CDP 2 and disproves the structure of CDP 3. In our hands, none of the four stereoisomers of cyclo(Trp-Arg) exhibited detectable intrinsic antimicrobial properties towards a range of Gram-positive and Gram-negative bacteria or fungi nor could they potentiate the action of antibiotics. These discrepancies in biological properties, compared with the activities reported in the literature, reveal that these specific cyclic dipeptides do not represent viable templates for the development of new treatments for microbial infections.

Repurposing primaquine as a polyamine conjugate to become an antibiotic adjuvant.

In our search for new antibiotic adjuvants as a novel strategy to deal with the emergence of multi-drug resistant (MDR) bacteria, a series of succinylprimaquine-polyamine (SPQ-PA) conjugates and derivatives of a cationic amphiphilic nature have been prepared. Evaluation of these primaquine conjugates for intrinsic antimicrobial properties and the ability to restore the antibiotic activity of doxycycline identified two derivatives, SPQ-PA3-8-3 and SPQ-PA3-10-3 that exhibited intrinsic activity against the Gram-positive bacteria Staphylococcus aureus and the yeast Cryptococcus neoformans. None of the analogues were active against the Gram-negative bacterium Pseudomonas aeruginosa. However, in the presence of a sub-therapeutic amount of doxycycline (4.5 µM), both SPQ-PA3-4-3 and SPQ-PA3-10-3 compounds displayed potent antibiotic adjuvant properties against P. aeruginosa, with MIC's of 6.25 µM. A series of derivatives were prepared to investigate the structure-activity relationship that explored the influence of both a simplified aryl lipophilic substituent and variation of the length of the polyamine scaffold on observed intrinsic antimicrobial properties and the ability to potentiate the action of doxycycline against P. aeruginosa.

Functional diversity of microboring Ostreobium algae isolated from corals.

The filamentous chlorophyte Ostreobium sp. dominates shallow marine carbonate microboring communities, and is one of the major agents of reef bioerosion. While its large genetic diversity has emerged, its physiology remains little known, with unexplored relationship between genotypes and phenotypes (endolithic versus free-living growth forms). Here, we isolated nine strains affiliated to two lineages of Ostreobium (>8% sequence divergence of the plastid gene rbcL), one of which was assigned to the family Odoaceae, from the fast-growing coral host Pocillopora acuta Lamarck 1816. Free-living isolates maintained their bioerosive potential, colonizing pre-bleached coral carbonate skeletons. We compared phenotypes, highlighting shifts in pigment and fatty acid compositions, carbon to nitrogen ratios and stable isotope compositions (δ13 C and δ15 N). Our data show a pattern of higher chlorophyll b and lower arachidonic acid (20:4ω6) content in endolithic versus free-living Ostreobium. Photosynthetic carbon fixation and nitrate uptake, quantified via 8 h pulse-labeling with 13 C-bicarbonate and 15 N-nitrate, showed lower isotopic enrichment in endolithic compared to free-living filaments. Our results highlight the functional plasticity of Ostreobium phenotypes. The isotope tracer approach opens the way to further study the biogeochemical cycling and trophic ecology of these cryptic algae at coral holobiont and reef scales.

6-Bromoindolglyoxylamido derivatives as antimicrobial agents and antibiotic enhancers.

The combination of increased incidence of drug-resistant strains of bacteria and a lack of novel drugs in development creates an urgency for the search for new antimicrobials. Initial screening of compounds from an in-house library identified two 6-bromoindolglyoxylamide polyamine derivatives (3 and 4) that exhibited intrinsic antimicrobial activity towards Gram-positive bacteria, Staphylococcus aureus and S. intermedius with polyamine 3 also displaying in vitro antibiotic enhancing properties against the resistant Gram-negative bacterium Pseudomonas aeruginosa. A series of 6-bromo derivatives (5-15) were prepared and biologically evaluated, identifying analogues with enhanced antibacterial activity towards Escherichia coli and with moderate to excellent antifungal properties. Polyamine 3, which includes a spermine chain, was the most potent of the series - its mechanism of action was attributed to rapid membrane permeabilization and depolarization in both Gram-positive and Gram-negative bacteria.

New Benthic Cyanobacteria from Guadeloupe Mangroves as Producers of Antimicrobials.

Benthic cyanobacteria strains from Guadeloupe have been investigated for the first time by combining phylogenetic, chemical and biological studies in order to better understand the taxonomic and chemical diversity as well as the biological activities of these cyanobacteria through the effect of their specialized metabolites. Therefore, in addition to the construction of the phylogenetic tree, indicating the presence of 12 potentially new species, an LC-MS/MS data analysis workflow was applied to provide an overview on chemical diversity of 20 cyanobacterial extracts, which was linked to antimicrobial activities evaluation against human pathogenic and ichtyopathogenic environmental strains.

A Collection of Bioactive Nitrogen-Containing Molecules from the Marine Sponge Acanthostrongylophora ingens.

Thirteen nitrogen-containing molecules (1a/1b and 2-12) were isolated from the Indonesian sponge Acanthostrongylophora ingens, highlighting the richness of this organism as a source of alkaloids. Their structures were elucidated using one- and two-dimensional NMR spectroscopy and HR-ESI-MS, while the stereochemistry of the diketopiperazines was established using Marfey's method. All compounds were screened in our standard bioactivity assays, including antibacterial, antikinases, and amyloid ß-42 assays. The most interesting bioactivity result was obtained with the known acanthocyclamine A (3), which revealed for the first time a specific Escherichia coli antimicrobial activity and an inhibitory effect on amyloid ß-42 production induced by aftin-5 and no cytotoxicity at the dose of 26 µM. These results highlight the potentiality of a bipiperidine scaffold as a promising skeleton for preventing or reducing the production of amyloid ß-42, a key player in the initiation of Alzheimer's disease.

New 9α-Hydroxy-5α,6α-epoxyhydroxysterols from the Vietnamese Marine Sponge Ircinia echinata.

Chemical investigation of the methanol extract of the Vietnamese marine sponge Ircinia echinata led to the isolation of six new 9α-hydroxy-5α,6α-epoxysterols: 5α,6α-epoxycholesta-7,22(E)-dien-3ß,9α-diol (1), 5α,6α-epoxycholesta-7,24(28)-dien-3ß,9α-diol (2), (24R)-5α,6α-epoxy-24-ethyl-cholesta-7-en-3ß,9α-diol (3), 5α,6α-epoxycholesta-7-en-3ß,9α-diol (4), (24S)-5α,6α-epoxyergosta-7,22-dien-3ß,9α-diol (5), and (24R)-5α,6α-epoxy-24-methyl-cholesta-7-en-3ß,9α-diol (6) along with the known 5α-6α-epoxysterols: 5α,6α-epoxystigmasta-7-en-3ß-ol (7), 5α,6α-epoxystigmasta-7,22-dien-3ß-ol (8), and 5α,6α-epoxyergosta-7-en-3ß-ol (9). Their structures and their configurations were established on the basis of high resolution mass spectra and extensive 1D and 2D NMR spectroscopic data and by comparison with the literature. Their cytotoxic activity, evaluated against three human cancer cell lines, MCF-7, Hep-G2 and LU-1, revealed that only compounds 3 and 4 exhibited significant antiproliferative activity and compound 3 showed a selective inhibition towards the MCF-7 human breast cancer cells.

Major Antimicrobial Representatives from Marine Sponges and/or Their Associated Bacteria.

The rapid emergence of resistant bacteria during the last 20 years has stimulated research efforts in order to overcome this thorny problem. Marine sponges and their associated bacteria, which have been proven to be a source of bioactive natural products, have appeared as a promising opportunity to identify new antibiotic compounds. An overview of the major antibacterial compounds isolated from marine sponges and/or their associated bacteria is presented in this chapter, highlighting new potential antibiotics.

Synthesis and biological evaluation of the ascidian blood-pigment halocyamine A.

Synthesis of the antimicrobial marine natural product halocyamine A has been achieved utilizing a combination of Sonogashira coupling, ruthenium complex/ytterbium triflate catalyzed hydroamidation and solid-phase peptide synthesis (SPPS) chemistry. The synthetic natural product exhibited only modest levels of antibacterial activities but significant antioxidant activity.

Structure-activity relationship studies on thiaplidiaquinones A and B as novel inhibitors of Plasmodium falciparum and farnesyltransferase.

Marine meroterpenoids, thiaplidiaquinones A and B and their respective non-natural dioxothiazine regioisomers have been shown to inhibit mammalian and protozoal farnesyltransferase (FTase) with the regioisomers exhibiting activity in the nanomolar range. In order to explore the structure-activity relationship (SAR) of this class of marine natural products, analogues of thiaplidiaquinones A and B and their regioisomers were synthesised, with variation in the number of isoprene units present in their side chains to afford prenyl and farnesyl analogues. The previously reported geranyl series of compounds were found to be the most potent FTase inhibitors closely followed by the novel farnesyl series. The prenyl series exhibited the most potent anti-plasmodial activity but the series was also the most cytotoxic. Overall, the farnesyl series exhibited moderate anti-plasmodial activity with one analogue, 14 also exhibiting low cytotoxicity, identifying it as a scaffold worthy of further exploration.

Thermoactinoamide A, an Antibiotic Lipophilic Cyclopeptide from the Icelandic Thermophilic Bacterium Thermoactinomyces vulgaris.

The thermophilic bacterium Thermoactinomyces vulgaris strain ISCAR 2354, isolated from a coastal hydrothermal vent in Iceland, was shown to contain thermoactinoamide A (1), a new cyclic hexapeptide composed of mixed d and l amino acids, along with five minor analogues (2-6). The structure of 1 was determined by one- and two-dimensional NMR spectroscopy, high-resolution tandem mass spectrometry, and advanced Marfey's analysis of 1 and of the products of its partial hydrolysis. Thermoactinoamide A inhibited the growth of Staphylococcus aureus ATCC 6538 with an MIC value of 35 µM. On the basis of literature data and this work, cyclic hexapeptides with mixed d/l configurations, one aromatic amino acid residue, and a prevalence of lipophilic residues can be seen as a starting point to define a new, easily accessible scaffold in the search for new antibiotic agents.

Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases.

A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.

Discovery and preliminary structure-activity relationship studies on tecomaquinone I and tectol as novel farnesyltransferase and plasmodial inhibitors.

Biological screening of a library of synthesized benzo[c]chromene-7,10-dione natural products against human farnesyltransferase (FTase) has identified tecomaquinone I (IC50 of 0.065±0.004µM) as being one of the more potent natural product inhibitors identified to date. Anti-plasmodial screening of the same library against a drug-resistant strain of Plasmodium falciparum identified the structurally-related dichromenol tectol as a moderately active growth inhibitor with an IC50 3.44±0.20µM. Two novel series of analogues, based on the benzo[c]chromene-7,10-dione scaffold, were subsequently synthesized, with one analogue exhibiting farnesyltransferase inhibitory activity in the low micromolar range. A preliminary structure-activity relationship (SAR) study has identified different structural requirements for anti-malarial activity in comparison to FTase activities for these classes of natural products. Our results identify tecomaquinone I as a novel scaffold from which more potent inhibitors of human and parasitic FTase could be developed.

Chloromethylhalicyclamine B, a Marine-Derived Protein Kinase CK1δ/ε Inhibitor.

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.