Psammaplin A and Its Analogs Attenuate Oxidative Stress in Neuronal Cells through Peroxisome Proliferator-Activated Receptor γ Activation.

Psammaplins are sulfur containing bromotyrosine alkaloids that have shown antitumor activity through the inhibition of class I histone deacetylases (HDACs). The cytotoxic properties of psammaplin A (1), the parent compound, are related to peroxisome proliferator-activated receptor γ (PPARγ) activation, but the mechanism of action of its analogs psammaplin K (2) and bisaprasin (3) has not been elucidated. In this study, the protective effects against oxidative stress of compounds 1-3, isolated from the sponge Aplysinella rhax, were evaluated in SH-SY5Y cells. The compounds improved cell survival, recovered glutathione (GSH) content, and reduced reactive oxygen species (ROS) release at nanomolar concentrations. Psammaplins restored mitochondrial membrane potential by blocking mitochondrial permeability transition pore opening and reducing cyclophilin D expression. This effect was mediated by the capacity of 1-3 to activate PPARγ, enhancing gene expression of the antioxidant enzymes catalase, nuclear factor E2-related factor 2 (Nrf2), and glutathione peroxidase. Finally, HDAC3 activity was reduced by 1-3 under oxidative stress conditions. This work is the first description of the neuroprotective activity of 1 at low concentrations and the mechanism of action of 2 and 3. Moreover, it links for the first time the previously described effects of 1 in HDAC3 and PPARγ signaling, opening a new research field for the therapeutic potential of this compound family.

Accraspiroketides A-B, Phenylnaphthacenoid-Derived Polyketides with Unprecedented [6 + 6+6 + 6] + [5 + 5] Spiro-Architecture.

Two novel polyketides, accraspiroketides A (1) and B (2), which feature unprecedented [6 + 6+6 + 6] + [5 + 5] spiro chemical architectures, were isolated from Streptomyces sp. MA37 ΔaccJ mutant strain. Compounds 1-2 exhibit excellent activity against Gram-positive bacteria (MIC = 1.5-6.3 µg/mL). Notably, 1 and 2 have superior activity against clinically isolated Enterococcus faecium K60-39 (MIC = 4.0 µg/mL and 4.7 µg/mL, respectively) than ampicillin (MIC = 25 µg/mL).

A ribosomally synthesised and post-translationally modified peptide containing a ß-enamino acid and a macrocyclic motif.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally complex natural products with diverse bioactivities. Here we report discovery of a RiPP, kintamdin, for which the structure is determined through spectroscopy, spectrometry and genomic analysis to feature a bis-thioether macrocyclic ring and a ß-enamino acid residue. Biosynthetic investigation demonstrated that its pathway relies on four dedicated proteins: phosphotransferase KinD, Lyase KinC, kinase homolog KinH and flavoprotein KinI, which share low homologues to enzymes known in other RiPP biosynthesis. During the posttranslational modifications, KinCD is responsible for the formation of the characteristic dehydroamino acid residues including the ß-enamino acid residue, followed by oxidative decarboxylation on the C-terminal Cys and subsequent cyclization to provide the bis-thioether ring moiety mediated by coordinated action of KinH and KinI. Finally, conserved genomic investigation allows further identification of two kintamdin-like peptides among the kin-like BGCs, suggesting the occurrence of RiPPs from actinobacteria.

Inhibition of the Quorum Sensing System, Elastase Production and Biofilm Formation in Pseudomonas aeruginosa by Psammaplin A and Bisaprasin.

Natural products derived from marine sponges have exhibited bioactivity and, in some cases, serve as potent quorum sensing inhibitory agents that prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. In this study, the inhibitory activity of the psammaplin-type compounds, psammaplin A (1) and bisaprasin (2), isolated from the marine sponge, Aplysinellarhax, are evaluated in quorum sensing inhibitory assays based on the Pseudomonas aeruginosa PAO1 lasB-gfp(ASV) and rhlA-gfp(ASV) biosensor strains. The results indicate that psammaplin A (1) showed moderate inhibition on lasB-gfp expression, but significantly inhibited the QS-gene promoter, rhlA-gfp, with IC50 values at 14.02 µM and 4.99 µM, respectively. In contrast, bisaprasin (2) displayed significant florescence inhibition in both biosensors, PAO1 lasB-gfp and rhlA-gfp, with IC50 values at 3.53 µM and 2.41 µM, respectively. Preliminary analysis suggested the importance of the bromotyrosine and oxime functionalities for QSI activity in these molecules. In addition, psammaplin A and bisaprasin downregulated elastase expression as determined by the standard enzymatic elastase assay, although greater reduction in elastase production was observed with 1 at 50 µM and 100 µM. Furthermore, the study revealed that bisaprasin (2) reduced biofilm formation in P. aeruginosa.

Dermacozine N, the First Natural Linear Pentacyclic Oxazinophenazine with UV-Vis Absorption Maxima in the Near Infrared Region, along with Dermacozines O and P Isolated from the Mariana Trench Sediment Strain Dermacoccus abyssi MT 1.1T.

Three dermacozines, dermacozines N-P (1-3), were isolated from the piezotolerant Actinomycete strain Dermacoccus abyssi MT 1.1T, which was isolated from a Mariana Trench sediment in 2006. Herein, we report the elucidation of their structures using a combination of 1D/2D NMR, LC-HRESI-MSn, UV-Visible, and IR spectroscopy. Further confirmation of the structures was achieved through the analysis of data from density functional theory (DFT)-UV-Visible spectral calculations and statistical analysis such as two tailed t-test, linear regression-, and multiple linear regression analysis applied to either solely experimental or to experimental and calculated 13C-NMR chemical shift data. Dermacozine N (1) bears a novel linear pentacyclic phenoxazine framework that has never been reported as a natural product. Dermacozine O (2) is a constitutional isomer of the known dermacozine F while dermacozine P (3) is 8-benzoyl-6-carbamoylphenazine-1-carboxylic acid. Dermacozine N (1) is unique among phenoxazines due to its near infrared (NIR) absorption maxima, which would make this compound an excellent candidate for research in biosensing chemistry, photodynamic therapy (PDT), opto-electronic applications, and metabolic mapping at the cellular level. Furthermore, dermacozine N (1) possesses weak cytotoxic activity against melanoma (A2058) and hepatocellular carcinoma cells (HepG2) with IC50 values of 51 and 38 µM, respectively.

The chemical profile of activated secondary metabolites by overexpressing LaeA in Aspergillus niger.

Although the mechanisms of regulating secondary metabolism by LaeA remains unclear, the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation. In our previous sutdy, RNA-seq data has showed that the transcriptional level of many SM backbone genes could be upregulated by overexpressing LaeA. Herein, we analyzed the chemical profile of activated secondary metabolites in the variant of A. niger FGSC A1279 by overexpressing LaeA (OElaeA). 14 compounds were activated in A. niger FGSC A1279 OElaeA variant in the WATM medium. Chemical workup of organic extracts of the culture broth from the A. niger OElaeA mutant identified three pure compounds, flaviolin, orlandin and kotanin. The structures of these compounds were confirmed by HR-ESIMS, 1D/2D NMR, and computer assisted structure elucidation (CASE). Based on homologous alignment and comparison of literatures, the biosynthetic gene cluster (fla) of flaviolin was identified. The in vivo function of the backbone gene, flaA, encoding a multidomain non-reducing polyketide synthase (SAT-KS-AT-PT-ACP), was verified via gene knockout and chemical analysis. Finally, a biosynthetic model for fungal flaviolin was proposed.

Tavarua Deoxyriboside A and Jasplakinolide as Potential Neuroprotective Agents: Effects on Cellular Models of Oxidative Stress and Neuroinflammation.

The oceans harbor a great reservoir of molecules with unknown bioactivities, which could be useful for the treatment of illnesses that nowadays have no cure, such as neurodegenerative diseases. In this work, we evaluated the neuroprotective potential of the marine Fijian compounds tavarua deoxyriboside A and jasplakinolide against oxidative stress and neuroinflammation, crucial mechanisms in neurodegeneration. Both metabolites protected SH-SY5Y human neuroblastoma cells from H2O2 damage, improving mitochondrial function and activating the antioxidant systems of cells. These effects were mediated by their ability of inducing Nrf2 translocation. In BV2 microglial cells activated with lipopolysaccharide, Fijian metabolites also displayed promising results, decreasing the release of proinflammatory mediators (ROS, NO, cytokines) through the reduction of gp91 and NFkB-p65 expression. Finally, we performed a coculture among both cell lines, in which treatment with compounds protected SH-SY5Y cells from activated microglia, corroborating their neuroprotective effects. These results suggest that tavarua deoxyriboside A and jasplakinolide could be used as candidate molecules for further studies against neurodegeneration.

Antiparasitic Activity of Bromotyrosine Alkaloids and New Analogues Isolated from the Fijian Marine Sponge Aplysinella rhax.

Ten bromotyrosine alkaloids were isolated and characterised from the marine sponge Aplysinella rhax (de Laubenfels 1954) collected from the Fiji Islands, which included one new bromotyrosine analogue, psammaplin P and two other analogues, psammaplin O and 3-bromo-2-hydroxy-5-(methoxycarbonyl)benzoic acid, which have not been previously reported from natural sources. HR-ESI-MS, 1D and 2D NMR spectroscopic methods were used in the elucidation of the compounds. Bisaprasin, a biphenylic dimer of psammaplin A, showed moderate activity with IC50 at 19±5 and 29±6 µM against Trypanzoma cruzi Tulahuen C4, and the lethal human malaria species Plasmodium falciparum clone 3D7, respectively, while psammaplins A and D exhibited low activity against both parasites. This is the first report of the antimalarial and antitrypanosomal activity of the psammaplin-type compounds. Additionally, the biosynthesis hypotheses of three natural products were proposed.

Whole Genome Sequence of Dermacoccus abyssi MT1.1 Isolated from the Challenger Deep of the Mariana Trench Reveals Phenazine Biosynthesis Locus and Environmental Adaptation Factors.

Dermacoccus abyssi strain MT1.1T is a piezotolerant actinobacterium that was isolated from Mariana Trench sediment collected at a depth of 10898 m. The organism was found to produce ten dermacozines (A‒J) that belonged to a new phenazine family and which displayed various biological activities such as radical scavenging and cytotoxicity. Here, we report on the isolation and identification of a new dermacozine compound, dermacozine M, the chemical structure of which was determined using 1D and 2D-NMR, and high resolution MS. A whole genome sequence of the strain contained six secondary metabolite-biosynthetic gene clusters (BGCs), including one responsible for the biosynthesis of a family of phenazine compounds. A pathway leading to the biosynthesis of dermacozines is proposed. Bioinformatic analyses of key stress-related genes provide an insight into how the organism adapted to the environmental conditions that prevail in the deep-sea.

Enzymatic Reconstitution and Biosynthetic Investigation of the Bacterial Carbazole Neocarazostatin A.

Tricyclic carbazole is an important scaffold in many naturally occurring metabolites, as well as valuable building blocks. Here we report the reconstitution of the ring A formation of the bacterial neocarazostatin A carbazole metabolite. We provide evidence of the involvement of two unusual aromatic polyketide proteins. This finding suggests how new enzymatic activities can be recruited to specific pathways to expand biosynthetic capacities. Finally, we leveraged our bioinformatics survey to identify the untapped capacity of carbazole biosynthesis.

LC-HRMS-Database Screening Metrics for Rapid Prioritization of Samples to Accelerate the Discovery of Structurally New Natural Products.

In order to accelerate the isolation and characterization of structurally new or novel secondary metabolites, it is crucial to develop efficient strategies that prioritize samples with greatest promise early in the workflow so that resources can be utilized in a more efficient and cost-effective manner. We have developed a metrics-based prioritization approach using exact LC-HRMS, which uses data for 24 618 marine natural products held in the PharmaSea database. Each sample was evaluated and allocated a metric score by a software algorithm based on the ratio of new masses over the total (sample novelty), ratio of known masses over the total (chemical novelty), number of peaks above a defined peak area threshold (sample complexity), and peak area (sample diversity). Samples were then ranked and prioritized based on these metric scores. To validate the approach, eight marine sponges and six tunicate samples collected from the Fiji Islands were analyzed, metric scores calculated, and samples targeted for isolation and characterization of new compounds. Structures of new compounds were elucidated by spectroscopic techniques, including 1D and 2D NMR, MS, and MS/MS. Structures were confirmed by computer-assisted structure elucidation methods (CASE) using the ACD/Structure Elucidator Suite.

Deletion of the epigenetic regulator GcnE in Aspergillus niger FGSC A1279 activates the production of multiple polyketide metabolites.

Epigenetic modification is an important regulatory mechanism in the biosynthesis of secondary metabolites in Aspergillus species, which have been considered to be the treasure trove of new bioactive secondary metabolites. In this study, we reported that deletion of the epigenetic regulator gcnE, a histone acetyltransferase in the SAGA/ADA complex, resulted in the production of 12 polyketide secondary metabolites in A. niger FGSC A1279, which was previously not known to produce toxins or secondary metabolites. Chemical workup and structural elucidation by 1D/2D NMR and high resolution electrospray ionization mass (HR-ESIMS) yielded the novel compound nigerpyrone (1) and five known compounds: carbonarone A (2), pestalamide A (3), funalenone (4), aurasperone E (5), and aurasperone A (6). Based on chemical information and the literature, the biosynthetic gene clusters of funalenone (4), aurasperone E (5), and aurasperone A (6) were located on chromosomes of A. niger FGSC A1279. This study found that inactivation of GcnE activated the production of secondary metabolites in A. niger. The biosynthetic pathway for nigerpyrone and its derivatives was identified and characterized via gene knockout and complementation experiments. A biosynthetic model of this group of pyran-based fungal metabolites was proposed.

Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A2 and Azaspirofuran A.

In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A2, pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A2 and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A2 and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A2 and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A2 and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.

Targeted Dereplication of Microbial Natural Products by High-Resolution MS and Predicted LC Retention Time.

A new strategy for the identification of known compounds in Streptomyces extracts that can be applied in the discovery of natural products is presented. The strategy incorporates screening a database of 5555 natural products including 5098 structures from Streptomyces sp., using a high-throughput LCMS data processing algorithm that utilizes HRMS data and predicted LC retention times (tR) as filters for rapid identification of known compounds in the natural product extract. The database, named StrepDB, contains for each compound the structure, molecular formula, molecular mass, and predicted LC retention time. All identified compounds are annotated and color coded for easier visualization. It is an indirect approach to quickly assess masses (which are not annotated) that may potentially lead to the discovery of new or novel structures. In addition, a spectral database named MbcDB was generated using the ACD/Spectrus DB Platform. MbcDB contains 665 natural products, each with structure, experimental HRESIMS, MS/MS, UV, and NMR spectra. StrepDB was used to screen a mutant Streptomyces albus extract, which led to the identification and isolation of two new compounds, legonmaleimides A and B, the structures of which were elucidated with the aid of MbcDB and spectroscopic techniques. The structures were confirmed by computer-assisted structure elucidation (CASE) methods using ACD/Structure Elucidator Suite. The developed methodology suggests a pipeline approach to the dereplication of extracts and discovery of novel natural products.

Spongionella Secondary Metabolites, Promising Modulators of Immune Response through CD147 Receptor Modulation.

The modulation of the immune system can have multiple applications such as cancer treatment, and a wide type of processes involving inflammation where the potent chemotactic agent cyclophilin A (Cyp A) is implicated. The Porifera phylum, in which Spongionella is encompassed, is the main producer of marine bioactive compounds. Four secondary metabolites obtained from Spongionella (Gracilin H, A, L, and Tetrahydroaplysulphurin-1) were described to hit Cyp A and to block the release of inflammation mediators. Based on these results, some role of Spongionella compounds on other steps of the signaling pathway mediated by this chemotactic agent can be hypothesized. In the present paper, we studied the effect of these four compounds on the surface membrane CD147 receptor expression, on the extracellular levels of Cyp A and on the ability to migrate of concanavalin (Con A)-activated T lymphocytes. Similar to a well-known immunosuppressive agent cyclosporine A (CsA), Gracilin H, A, L, and tetrahydroaplysulphurin-1 were able to reduce the CD147 membrane expression and to block the release of Cyp A to the medium. Besides, by using Cyp A as chemotactic agent, T cell migration was inhibited when cells were previously incubated with Gracilin A and Gracilin L. These positive results lead us to test the in vivo effect of Gracilin H and L in a mouse ear delayed hypersensitive reaction. Thus, both compounds efficiently reduce the ear swelling as well as the inflammatory cell infiltration. These results provide more evidences for their potential therapeutic application in immune-related diseases of Spongionella compounds.

Evaluation of the Antioxidant Activity of the Marine Pyrroloiminoquinone Makaluvamines.

Makaluvamines are pyrroloiminoquinones isolated from Zyzzya sponges. Until now, they have been described as topoisomerase II inhibitors with cytotoxic effects in diverse tumor cell lines. In the present work, seven makaluvamines were tested in several antioxidant assays in primary cortical neurons and neuroblastoma cells. Among the alkaloids studied, makaluvamine J was the most active in all the assays. This compound was able to reduce the mitochondrial damage elicited by the well-known stressor H2O2. The antioxidant properties of makaluvamine J are related to an improvement of the endogenous antioxidant defenses of glutathione and catalase. SHSY5Y assays proved that this compound acts as a Nrf2 activator leading to an improvement of antioxidant defenses. A low concentration of 10 nM is able to reduce the reactive oxygen species release and maintain a correct mitochondrial function. Based on these results, non-substituted nitrogen in the pyrrole plus the presence of a p-hydroxystyryl without a double bond seems to be the most active structure with a complete antioxidant effect in neuronal cells.

Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica).

Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 µg/mL) and unreported antimicrobial activity against Bcc strains.

Accurate quantification of modified cyclic peptides without the need for authentic standards.

There is a growing interest in the use of cyclic peptides as therapeutics, but their efficient production is often the bottleneck in taking them forward in the development pipeline. We have recently developed a method to synthesise azole-containing cyclic peptides using enzymes derived from different cyanobactin biosynthetic pathways. Accurate quantification is crucial for calculation of the reaction yield and for the downstream biological testing of the products. In this study, we demonstrate the development and validation of two methods to accurately quantify these compounds in the reaction mixture and after purification. The first method involves the use of a HPLC coupled in parallel to an ESMS and an ICPMS, hence correlating the calculated sulfur content to the amount of cyclic peptide. The second method is an NMR ERETIC method for quantifying the solution concentration of cyclic peptides. These methods make the quantification of new compounds much easier as there is no need for the use of authentic standards when they are not available.

Biosynthesis of Neocarazostatin A Reveals the Sequential Carbazole Prenylation and Hydroxylation in the Tailoring Steps.

Neocarazostatin A (NZS) is a bacterial alkaloid with promising bioactivities against free radicals, featuring a tricyclic carbazole nucleus with a prenyl moiety at C-6 of the carbazole ring. Here, we report the discovery and characterization of the biosynthetic pathway of NZS through genome mining and gene inactivation. The in vitro assays characterized two enzymes: NzsA is a P450 hydroxylase and NzsG is a new phytoene-synthase-like prenyltransferase (PTase). This is the first reported native PTase that specifically acts on the carbazole nucleus. Finally, our in vitro reconstituted experiment demonstrated a coupled reaction catalyzed by NzsG and NzsA tailoring the NZS biosynthesis.

Legonaridin, a new member of linaridin RiPP from a Ghanaian Streptomyces isolate.

Linaridins are rare linear ribosomally-synthesized and post-translationally modified peptides (RiPPs) and only two, cypemycin and SGR-1832, in this family have been identified so far. Legonaridin 1 has been discovered as a new member of linaridins through chemical isolation, peptidogenomics, comprehensive 1- and 2-D NMR and advanced Marfey's analyses from the soil bacterium Streptomyces sp. CT34, an isolate collected from Legon, Ghana. Bioinformatics analysis of the gene cluster suggested that the biosynthesis of legonaridin 1 is different from those of cypemycin and SGR-1832. Consistent with bioinformatics and peptidogenomics analyses, 1 has a total of nine post-modifications, 8 dehydrobutyrine residues and a N,N-dimethylated N-terminus with a carboxylic acid at the C-terminus. Legonaridin 1 is structurally different from the two known linaridins comprising a new subfamily. This is the first time that NMR spectroscopy is used to establish the 2-D structure of a linaridin RiPP.