Cyclotheonellazoles D-I, Potent Elastase Inhibitory Thiazole-Containing Cyclic Peptides from Theonella sp. (2131).

Six new thiazole-containing cyclic peptides, the cyclotheonellazoles D-I (1-6), were isolated from the Australian marine sponge Theonella sp. (2131) with their structures assigned by comprehensive 1D and 2D NMR spectroscopic and MS spectrometric analyses, Marfey's derivatization studies, and comparison with time-dependent density functional theory (TDDFT) calculated ECD data. The Type 2 azole-homologated peptides herein comprise up to five nonproteinogenic amino acids, including the protease transition state mimic α-keto-ß-amino acid residue 3-amino-4-methyl-2-oxohexanoic acid (Amoha), while 1-3 also contain a terminal hydantoin residue not previously found in cyclotheonellazoles. The keramamides A (7) and L (8) were reisolated affording expanded exploration of their biological activities. The peptides were examined for protease inhibitory activities against two mammalian serine proteases (elastase and chymotrypsin) and SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), a validated antiviral therapeutic target for COVID-19. Peptides 1-6 and keramamide A (7) displayed potent nanomolar inhibition of elastase (IC50 16.0 to 61.8 nM), while 7 also contained modest inhibition of chymotrypsin and SARS-CoV-2 3CLpro (IC50 0.73 and 1.1 µM, respectively). The cyclotheonellazoles D-E (1-3) do not affect the viability of human breast, ovarian, and colon cancer cells (>100 µM), with the cytotoxicity previously reported for keramamide L (8) not replicated (inactive >20 µM).

Amphiphilic Polyamine α-Synuclein Aggregation Inhibitors from the Sponge Aaptos lobata.

Bioassay-guided investigation of the sponge Aaptos lobata resulted in the isolation and identification of two new amphiphilic polyamines, aaptolobamines A (1) and B (2). Their structures were determined through analysis of NMR and MS data. MS analysis also indicated that A. lobata contained a complex mixture of aaptolobamine homologues. Both aaptolobamines A (1) and B (2) show broad bioactivity, including cytotoxicity against cancer cell lines, moderate antimicrobial activity against a methicillin-resistant strain of Staphylococcus aureus, and weak activity against a Pseudomonas aeruginosa strain. The mixtures of aaptolobamine homologues were shown to contain compounds that bind to the Parkinson's disease associated amyloid protein α-synuclein and inhibit its aggregation.

Marine indole alkaloid diversity and bioactivity. What do we know and what are we missing?

Covering: marine indole alkaloids (n = 2048) and their reported bioactivities up to the end of 2021Despite increasing numbers of marine natural products (MNPs) reported each year, most have only been examined for cytotoxic, antibacterial, and/or antifungal biological activities with the majority found to be inactive in these assays. In this context, why are natural products continuing to be examined in assays they are unlikely to show significant activity in, and what targets might be more useful for expanding knowledge of their biologically relevant chemical space? We have undertaken a meta-analysis of the biological activities for 2048 marine indole alkaloids (MIAs), a diverse sub-class of MNPs reported up to the end of 2021, and this has highlighted that the bioactivity potentials for up to 86% of published MIAs remains underexplored and/or undefined. Although most published MIAs are not cytotoxic or antimicrobial, there is a continued focus on using these assays to evaluate new structurally related analogues. Using cheminformatics analyses, the chemical diversity of the 2048 MIAs were clustered using fragment based fingerprints and their reported bioactivity potency towards specific disease targets was assessed for structure activity trends. These analyses showed that there are groups of MIAs that possess potent and diverse activities and that many analogues, previously tested only in cellular toxicity assays, could be better exploited to generate structure activity relationships associated with leads to treat emerging diseases. A collection of indole drug and drug-lead structures from non-natural sources were also incorporated into the dataset providing complementary bioactivity profiles that were further used to predict underexplored areas of potential new activity and to better direct future testing of MIAs. Our findings clearly suggest the biological evaluation of MIAs continues to be conducted on a narrow range of bioassays and disease targets, and that shifting the focus to non-toxic disease targets should provide expanded knowledge of biologically relevant chemical space aimed at maximising the potential of MIAs for drug discovery.

Understanding of black salve toxicity by multi-compound cytotoxicity assays.

BACKGROUND: Black salve is a controversial complementary and alternative medicine (CAM) associated with skin toxicity and skin cancer treatment failures. Black salve formulations vary between manufacturers and contain a number of botanical and synthetic constituents. The skin cancer cytotoxicity of a number of these constituents has not been assessed to date. The alkaloids from the rhizomes of Sanguinaria canadensis, a key black salve ingredient, have had their single compound cytotoxicity assessed; however, whether they possess synergistic cytotoxicity with other compounds has not been studied and is of direct clinical relevance. This research aimed to improve our understanding of the skin cancer cytotoxicity of black salve constituents. METHODS: The cytotoxicity of individual and combination black salve constituents were assessed against the A375 melanoma and A431 squamous cell carcinoma cell lines. Cytotoxicity was determined using the Resazurin assay with fluorescence measured using a Tecan Infinite 200 Pro Microplate reader, compound cytotoxicity being compared to that of the topical cancer therapeutic agent, 5- fluouracil. Docetaxal was used as a positive control. Dunnetts p value was used to determine whether significant synergistic cytotoxicity was present. RESULTS: Sanguinarine was the most cytotoxic compound tested with a 24-hour IC50 of 2.1 µM against the A375 Melanoma cell line and 3.14 µM against the A431 SCC cell line. All black salve constituents showed greater cytotoxicity against the two skin cancer cell lines tested than the skin cancer therapeutic 5-Fluouracil with 24 hours of compound exposure. Chelerythrine and minor Quaternary Benzophenanthridine Alkaloids (QBAs) present in black salve, at concentrations not having a cytotoxic effect by themselves, boosted the cytotoxic effects of sanguinarine. This could be a synergistic rather than additive cytotoxic effect although the synergistic effect was cell line and concentration dependent. CONCLUSIONS: Black salve contains several cytotoxic compounds, a number of which have been found to possess synergistic cytotoxicity for the first time against skin cancer cell lines. In addition, these compounds together increase the overall cytotoxic effect. Assessing multi-compound cytotoxicity in herbal medicine can provide additional information about both their therapeutic and toxicity potential. As black salve is currently being used by patients, further cytotoxicity work should be undertaken to assess whether synergistic cytotoxicity exists when tested in normal skin cells.

Echiumin E, an Aryl Dihydronaphthalene Lignan from the Australian Invasive Plant Paterson's Curse (Echium plantagineum).

A new aryl dihydronaphthalene lignan, echiumin E (1), and four known compounds, echiumin A, globoidnan A, (-)-rabdosiin, and rosmarinic acid (2-5), were isolated from the Australian invasive plant Echium plantagineum (Paterson's curse) for the first time. Echiumin E (1) was characterized by 1D/2D NMR spectroscopy and MS spectrometry, with its absolute configuration assigned through comparison of experimental and TDDFT-calculated ECD data. Echiumin E (1) along with compounds 3-5 were screened in vitro against three cancer cell lines (SH-SY5Y, HeLa, and PC-3) and a prostate stromal (normal) cell line (WPMY-1) using a resazurin reduction assay. Echiumin E (1) was found to be active toward HeLa cells (IC50 0.21 µM).

Tedaniophorbasins A and B-Novel Fluorescent Pteridine Alkaloids Incorporating a Thiomorpholine from the Sponge Tedaniophorbas ceratosis.

Two new fluorescent pteridine alkaloids, tedaniophorbasins A (1) and B (2), together with the known alkaloid N-methyltryptamine, were isolated, through application of mass directed purification, from the sponge Tedaniophorbas ceratosis collected from northern New South Wales, Australia. The structures of tedaniophorbasins A and B were deduced from the analysis of 1D/2D NMR and MS data and through application of 13C NMR DFT calculations. Tedaniophorbasin A possesses a novel 2-imino-1,3-dimethyl-2,3,7,8-tetrahydro-1H-[1,4]thiazino[3,2-g]pteridin-4(6H)-one skeleton, while tedaniophorbasin B is its 2-oxo derivative. The compounds show significant Stokes shifts (~14,000 cm-1) between excitation and emission wavelengths in their fluorescence spectra. The new compounds were tested for bioactivity against chloroquine-sensitive and chloroquine-resistant strains of the malaria parasite Plasmodium falciparum, breast and pancreatic cancer cell lines, and the protozoan parasite Trypanosoma brucei brucei but were inactive against all targets at 40 µM.

Chronic pesticide exposure elicits a subtle carry-over effect on the metabolome of Aurelia coerulea ephyrae.

Chemical pollutants, such as pesticides, often leach into aquatic environments and impact non-target organisms. Marine invertebrates have complex life cycles with multiple life-history stages. Exposure to pesticides during one life-history stage potentially influences subsequent stages; a process known as a carry-over effect. Here, we investigated carry-over effects on the jellyfish Aurelia coerulea. We exposed polyps to individual and combined concentrations of atrazine (2.5 µg/L) and chlorpyrifos (0.04 µg/L) for four weeks, after which they were induced to strobilate. The resultant ephyrae were then redistributed and exposed to either the same conditions as their parent-polyps or to filtered seawater to track potential carry-over effects. The percentage of deformities, ephyrae size, pulsation and respiration rates, as well as the metabolic profile of the ephyrae, were measured. We detected a subtle carry-over effect in two metabolites, acetoacetate and glycerophosphocholine, which are precursors of the neurotransmitter acetylcholine, important for energy metabolism and osmoregulation of the ephyrae. Although these carry-over effects were not reflected in the other response variables in the short-term, a persistent reduction of these two metabolites could have negative physiological consequences on A. coerulea jellyfish in the long-term. Our results highlight the importance of considering more than one life-history stage in ecotoxicology, and measuring a range of variables with different sensitivities to detect sub-lethal effects caused by anthropogenic stressors. Furthermore, since we identified few effects when using pesticides concentrations corresponding to Australian water quality guidelines, we suggest that future studies consider concentrations detected in the environment, which are higher than the water quality guidelines, to obtain a more realistic scenario by possible risk from pesticide exposure.

Identification of compounds from terrestrial dissolved organic matter toxic to cyanobacteria.

There is emerging evidence for the phytotoxicity of terrestrial dissolved organic matter (DOM), however its sources, transformations and ecological effects in aquatic ecosystems are poorly understood. DOM characterization by Nuclear Magnetic Resonance (NMR) spectroscopy has typically involved solid-state techniques, but poor resolution has often precluded identification of individual components. This study is the first to directly identify individual phytotoxic components using a novel combined approach of preparative HPLC fractionation of DOM (obtained from leaves of two common riparian trees, Casuarina cunninghamiana and Eucalyptus tereticornis). This was followed by chemical characterization of fractions, using one-dimensional (1D) and two-dimensional (2D) solution-state 1H NMR analyses. Additionally, the phytotoxic effect of the fractions was determined using cultures of the cyanobacteria Raphidiopsis (Cylindrospermopsis) raciborskii. The amino acid, proline, from Casuarina leachate was identified as phytotoxic, while for Eucalyptus leachate, it was gallic acid and polyphenols. These phytotoxicants remained in the leachates when they were incubated in sunlight or the dark conditions over 5 days. Our study identifies phytotoxic compounds with the potential to affect algal species composition, and potentially control nuisance R. raciborskii blooms.

Polyps of the Jellyfish Aurelia aurita Are Unaffected by Chronic Exposure to a Combination of Pesticides.

Pesticides are a major contaminant in coastal waters and can cause adverse effects in marine invertebrates such as jellyfish. Most studies have investigated short-term responses of organisms to unrealistically high concentrations of pesticides; however, chronic exposure to persistent low concentrations, which are more likely to occur in the environment, are rarely analyzed. We tested the response of polyps of the moon jellyfish Aurelia aurita to environmental concentrations of the herbicide atrazine and the insecticide chlorpyrifos, individually and in combination, over 9 wk. We hypothesized that exposure to individual pesticides would reduce rates of asexual reproduction and alter polyps' metabolite profiles, and that the results would be more severe when polyps were exposed to the combined pesticides. Polyps survived and reproduced (through budding) in all treatments, and no differences among treatments were observed. Proton nuclear magnetic resonance spectroscopy revealed no difference in profiles of polar metabolites of polyps exposed to the individual or combined pesticides. Our results suggest that A. aurita polyps are unaffected by chronic exposure to atrazine and chlorpyrifos at concentrations recommended as being protective by current Australian water quality guidelines. Environ Toxicol Chem 2020;39:1685-1692. © 2020 SETAC.

Lamellarin Sulfates from the Pacific Tunicate Didemnum ternerratum.

Six new lamellarin sulfates (1-6) were isolated from the methanolic extract of the Pacific tunicate Didemnum ternerratum, collected from the Kingdom of Tonga. Mass spectrometric molecular networking through the GNPS platform was used to target the isolation of 1-6. Planar structures were elucidated through a combination of NMR and MS experiments. Through comparison of experimental and calculated ECD spectra, the absolute configurations of atropisomers 2-5 were determined, with their energetic barriers to racemization also determined computationally. The cytotoxicity of the compounds was tested against the human colon carcinoma cell line HCT-116, where lamellarin D-8-sulfate (5) exhibited moderate activity with an IC50 of 9.7 µM.

Structural and conformational studies of the heparan sulfate mimetic PI-88.

The heparan sulfate mimetic PI-88 is a complex mixture of sulfated oligosaccharides with anti-metastatic and anti-angiogenic activity due to its potent inhibition of heparanase and heparan sulfate-dependent angiogenic growth factors. It was recently in Phase III clinical trials for postresection hepatocellular carcinoma. The major oligosaccharide constituents of PI-88 were prepared for the first time by sulfonation of individually purified phosphorylated oligosaccharides isolated from the PI-88 precursor. PI-88 and its components were subjected to detailed 1D and 2D NMR spectroscopic analysis. The spectra of the individual components greatly assisted the assignment of minor resonances in the 1H NMR spectrum of PI-88. The data also showed that the majority of the oligosaccharides in PI-88 are fully sulfated and that undersulfated species present are largely due to anomeric desulfation. The solution conformation of the phosphomannopentaose sulfate (major component) of PI-88 was then determined by a combination of molecular dynamics simulations and NOE measurements which may provide insights into its binding interactions with target proteins.

HSQC-TOCSY Fingerprinting for Prioritization of Polyketide- and Peptide-Producing Microbial Isolates.

Microbial products are a promising source for drug leads as a result of their unique structural diversity. However, reisolation of already known natural products significantly hampers the discovery process, and it is therefore important to incorporate effective microbial isolate selection and dereplication protocols early in microbial natural product studies. We have developed a systematic approach for prioritization of microbial isolates for natural product discovery based on heteronuclear single-quantum correlation-total correlation spectroscopy (HSQC-TOCSY) nuclear magnetic resonance profiles in combination with antiplasmodial activity of extracts. The HSQC-TOCSY experiments allowed for unfractionated microbial extracts containing polyketide and peptidic natural products to be rapidly identified. Here, we highlight how this approach was used to prioritize extracts derived from a library of 119 ascidian-associated actinomycetes that possess a higher potential to produce bioactive polyketides and peptides.

New structural insights into the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y2448 phosphomannan.

The oligosaccharide phosphate fraction (OPF) obtained from mild acid hydrolysis of P. holstii NRRL Y-2448 phosphomannan is the starting material for the preparation of the Phase III anticancer drug candidate PI-88. The OPF was for the first time successfully separated by preparative ion exchange chromatography and the major oligosaccharides isolated and characterized by NMR spectroscopy. The components were also acetylated and subjected to LC-MS analysis. These studies revealed that the OPF also contained all-α(1 â†’ 3)-linked oligosaccharides in addition to the known α(1 â†’ 3)/(1 â†’ 2)-linked species, most likely formed by hydrolysis of the latter. Contrary to previous assumptions, the only phosphorylated disaccharide present is α(1 â†’ 3)-linked. In addition, it was determined that a glycosylamine derivative previously isolated is, in fact, a manufacturing byproduct formed from exposure to aqueous ammonium bicarbonate during chromatographic purification. Based on these findings a new generic structure for PI-88 is proposed which more accurately reflects its composition.

Symbiodinium mitigate the combined effects of hypoxia and acidification on a noncalcifying cnidarian.

Anthropogenic nutrient inputs enhance microbial respiration within many coastal ecosystems, driving concurrent hypoxia and acidification. During photosynthesis, Symbiodinium spp., the microalgal endosymbionts of cnidarians and other marine phyla, produce O2 and assimilate CO2 and thus potentially mitigate the exposure of the host to these stresses. However, such a role for Symbiodinium remains untested for noncalcifying cnidarians. We therefore contrasted the fitness of symbiotic and aposymbiotic polyps of a model host jellyfish (Cassiopea sp.) under reduced O2 (~2.09 mg/L) and pH (~ 7.63) scenarios in a full-factorial experiment. Host fitness was characterized as asexual reproduction and their ability to regulate internal pH and Symbiodinium performance characterized by maximum photochemical efficiency, chla content and cell density. Acidification alone resulted in 58% more asexual reproduction of symbiotic polyps than aposymbiotic polyps (and enhanced Symbiodinium cell density) suggesting Cassiopea sp. fitness was enhanced by CO2 -stimulated Symbiodinium photosynthetic activity. Indeed, greater CO2 drawdown (elevated pH) was observed within host tissues of symbiotic polyps under acidification regardless of O2 conditions. Hypoxia alone produced 22% fewer polyps than ambient conditions regardless of acidification and symbiont status, suggesting Symbiodinium photosynthetic activity did not mitigate its effects. Combined hypoxia and acidification, however, produced similar numbers of symbiotic polyps compared with aposymbiotic kept under ambient conditions, demonstrating that the presence of Symbiodinium was key for mitigating the combined effects of hypoxia and acidification on asexual reproduction. We hypothesize that this mitigation occurred because of reduced photorespiration under elevated CO2 conditions where increased net O2 production ameliorates oxygen debt. We show that Symbiodinium play an important role in facilitating enhanced fitness of Cassiopea sp. polyps, and perhaps also other noncalcifying cnidarian hosts, to the ubiquitous effects of ocean acidification. Importantly we highlight that symbiotic, noncalcifying cnidarians may be particularly advantaged in productive coastal waters that are subject to simultaneous hypoxia and acidification.

Surviving but not thriving: inconsistent responses of zooxanthellate jellyfish polyps to ocean warming and future UV-B scenarios.

Complex changes to UV radiation at the Earth's surface are occurring concurrently with ocean warming. Despite few empirical tests, jellyfish are hypothesised to be increasing in some parts of the world because they are robust to environmental stressors. Here we examine the effects of UV-B and ocean warming projections on zooxanthellate jellyfish polyps. We exposed Cassiopea sp. polyps to three levels of UV-B (future-low (1.43 Wm(2)), current (1.60 Wm(2)), future-high (1.77 Wm(2))) and two levels of temperature (current-day (25 °C) and future (28 °C)) over 6 weeks. The intensity of UV-B was varied throughout the day to mimic diel variation in UV-B irradiance. Polyp survival, asexual reproduction and YII were measured. In the current and future-high UV-B treatments, more polyps were produced in 25 °C than 28 °C. This pattern, however, was reversed under future-low UV-B conditions, where more polyps were produced at 28 °C. YII was highest under current summer conditions and future conditions of low UV-B and increased temperature. YII, however, was reduced under high UV-B conditions but was further reduced with warming. Our results suggest that although Cassiopea polyps may survive elevated UV-B and warming conditions, they are unlikely to thrive. If, however, UV-B radiation decreases then ocean warming may facilitate increases in Cassiopea populations.

Pteridine-, thymidine-, choline- and imidazole-derived alkaloids from the Australian ascidian, Leptoclinides durus.

Four new acylated pteridine alkaloids, duramidines A-D, two new acylated thymidine alkaloids, leptoclinidines A and B, two new 1-acylglyceryl-3-(O-carboxyhydroxymethylcholine) alkaloids, durabetaines A and B, three new 1,3-dimethyl-5-methylsulfanylimidazole alkaloids, leptoclinidamines D-F, and the known alkaloids leptoclinidamines B and C and 6-bromo-1H-indolo-3-yl-oxoacetic acid methyl ester were isolated from the Australian ascidian Leptoclinides durus. The duramidines are the first pteridine alkaloids, possessing a three carbon side chain esterified at C-1' with a 4-hydroxy-2'-methoxycinnamic acid, and are either hydroxylated or sulfated at C-2'. The leptoclinidines are the first 3'-indole-3-carboxylic acid ester derivatives of thymidine to be reported in the literature. The durabetaines are the first glyceryl-3-(O-carboxyhydroxymethylcholine) alkaloids to be reported from an animal source and are also the only known derivatives from this class to be acylated with aromatic carboxylic acids. MS and NMR data analysis established the structures of the new compounds. All compounds were shown to be inactive when tested for cytotoxic activity against prostate (LNCaP) and breast (MDA-MB-231) cancer cell lines and antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus.

Albopunctatone, an antiplasmodial anthrone-anthraquinone from the Australian ascidian Didemnum albopunctatum.

Chemical investigation of a MeOH extract of the Great Barrier Reef ascidian Didemnum albopunctatum has led to the isolation and identification of a new anthrone-anthraquinone, albopunctatone (1), together with the known 1,8-dihydroxy-9,10-anthraquinone (2). The structure of 1 was established from interpretation of 1D and 2D NMR spectroscopic and mass spectrometric data. The compounds were screened for antiplasmodial activity against chloroquine-resistant and -sensitive strains of the malaria parasite, Plasmodium falciparum. Albopunctatone (1) was moderately active against both strains (IC(50) 5.3 and 4.4 ± 0.5 µM, respectively), while 2 was inactive at doses up to 40 µM. Both compounds were also inactive up to 40 µM when tested against a variety of cancerous and normal human cell lines and the kinetoplastid Trypanosoma brucei brucei, indicating selectivity for the malaria parasite, P. falciparum.

Twilight zone sponges from Guam yield theonellin isocyanate and psammaplysins I and J.

From the organic extracts of two Guam sponges, Rhaphoxya sp. and Suberea sp., determined to have cytotoxic and chemopreventive activities, three new compounds, theonellin isocyanate (1) and psammaplysins I and J (5, 6), and six previously reported compounds (2-4, 7-9) were isolated and characterized spectroscopically ((1)H and (13)C NMR, MS, IR, UV, [α](D)). The two new metabolites (5 and 6) isolated from the Suberea sp. sponge are rare examples of compounds containing a bromotyramine moiety rather than the more usual dibromo analogue. For the compounds isolated from the Rhaphoxya sp., this is the first report of the known compounds 2-4 being found in a single sponge. For previously reported compounds 2-4 complete unambiguous (1)H and (13)C NMR data are provided.

Caelestines A-D, brominated quinolinecarboxylic acids from the Australian ascidian Aplidium caelestis.

Four new brominated natural products, caelestines A-D (1-4), have been isolated from the Australian ascidian Aplidium caelestis. The structures of 1-4 were determined by analysis of their NMR and MS data. This is the first report of brominated quinolinecarboxylic acids from nature. Compound 1 has been previously synthesized but not spectroscopically characterized. Compounds 1-4 were tested against three mammalian cell lines (MCF-7, NFF, and MM96L) and a panel of microbial strains and showed only minor cytotoxicity.

Pestalactams A-C: novel caprolactams from the endophytic fungus Pestalotiopsis sp.

Chemical investigations of a fermentation culture from the endophytic fungus Pestalotiopsis sp. yielded three novel caprolactams, pestalactams A-C (). The structures of were determined by analysis of 1D and 2D-NMR, UV, IR, and MS data. The structure of pestalactam A was confirmed following single crystal X-ray diffraction analysis. Pestalactams A-C are the first C-7 alkylated caprolactam natural products to be reported. Pestalactams A () and B () were tested against two different strains of the malaria parasite Plasmodium falciparum (3D7 and Dd2), and the mammalian cell lines, MCF-7 and NFF, and showed modest in vitro activity in all assays.