New Micropeptins with Anti-Neuroinflammatory Activity Isolated from a Cyanobacterial Bloom.

Metabolite mining of environmentally collected aquatic and marine microbiomes offers a platform for the discovery of new therapeutic lead molecules. Combining a prefractionated chromatography library with liquid chromatography tandem mass spectrometry (LC-MS/MS)-based molecular networking and biological assays, we isolated and characterized two new micropeptins (1 and 2) along with the previously characterized micropeptin 996. These metabolites showed potency in anti-neuroinflammatory assays using BV-2 mouse microglial cells, showing a 50% reduction in inflammation in a range from 1 to 10 µM. These results show promise for cyanobacterial peptides in the therapeutic realm apart from their impact on environmental health and provide another example of the utility of large prefractionated natural product libraries for therapeutic hit and lead identification.

Cyanobufalins: Cardioactive Toxins from Cyanobacterial Blooms.

Cyanobufalins A-C (1-3), a new series of cardiotoxic steroids, have been discovered from cyanobacterial blooms in Buckeye Lake and Grand Lake St. Marys in Ohio. Compounds 1-3 contain distinctive structural features, including geminal methyl groups at C-4, a 7,8 double bond, and a C-16 chlorine substituent that distinguish them from plant- or animal-derived congeners. Despite these structural differences, the compounds are qualitatively identical to bufalin in their cytotoxic profiles versus cell lines in tissue culture and cardiac activity, as demonstrated in an impedance-based cellular assay conducted with IPSC-derived cardiomyocytes. Cyanobufalins are nonselectively toxic to human cells in the single-digit nanomolar range and show stimulation of contractility in cardiomyocytes at sub-nanomolar concentrations. The estimated combined concentration of 1-3 in the environment is in the same nanomolar range, and consequently more precise quantitative analyses are recommended along with more detailed cardiotoxicity studies. This is the first time that cardioactive steroid toxins have been found associated with microorganisms in an aquatic environment. Several factors point to a microbial biosynthetic origin for the cyanobufalins.

Microcystins Containing Doubly Homologated Tyrosine Residues from a Microcystis aeruginosa Bloom: Structures and Cytotoxicity.

Four new microcystin congeners are described including the first three examples of microcystins containing the rare doubly homologated tyrosine residue 2-amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa) (1-4). Large-scale harvesting and biomass processing allowed the isolation of substantial quantities of these compounds, thus enabling complete structure determination by NMR as well as cytotoxicity evaluation against selected cancer cell lines. The new Ahppa-toxins all incorporate Ahppa residues at the 2-position, and one of these also has a second Ahppa at position 4. The two most lipophilic Ahppa-containing microcystins showed 10-fold greater cytotoxic potency against human tumor cell lines (A549 and HCT-116) compared to microcystin-LR (5). The presence of an Ahppa residue in microcystin congeners is difficult to ascertain by MS methods alone, due to the lack of characteristic fragment ions derived from the doubly homologated side chain. Owing to their unexpected cytotoxic potency, the potential impact of the compounds on human health should be further evaluated.

Trichophycin A, a Cytotoxic Linear Polyketide Isolated from a Trichodesmium thiebautii Bloom.

In an effort to isolate and characterize bioactive secondary metabolites from Trichodesmium thiebautii blooms, collected cyanobacteria biomass was subjected to bioassay-guided extraction and fractionation using the human colon cancer cell line HCT-116, resulting in the isolation and subsequent structure characterization of a linear polyketide trichophycin A (1). The planar structure of 1 was completed using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). Trichophycin A was moderately toxic against the murine neuroblastoma cell line Neuro-2A (EC50: 6.5 µM) and HCT-116 cells (EC50: 11.7 µM). Trichophycin A was significantly more cytotoxic than the previously isolated polyketides trichotoxin A and trichotoxin B. These cytotoxicity observations suggest that toxicity may be related to the polyol character of these polyketide compounds.

Discovery of anabaenopeptin 679 from freshwater algal bloom material: Insights into the structure-activity relationship of anabaenopeptin protease inhibitors.

Cyanobacteria possess a unique capacity for the production of structurally novel secondary metabolites compared to the biosynthetic abilities of other environmental prokaryotes such as bacteria of the genus Streptomyces. Two different strategies to explore cyanobacteria-derived natural products have been explored previously: (1) cultivation of single cyanobacterial strains, in bioreactors for example; (2) bulk collections from the environment of so called 'algal blooms' that are dominated by cyanobacteria. In this study a new environmentally friendly collection technique for obtaining large quantities of algal bloom biomass was utilized. Algal biomass derived from eight million liters of lake water was concentrated using a novel continuous countercurrent filtration system. Analysis of this freshwater algal bloom from Grand Lake-Saint Marys, Ohio led to the discovery of anabaenopeptin 679 (1), as well as the known anabaenopeptins B, F, H and 908. Anabaenopeptin 679 is unusual in that it possesses the classical anabaenopeptin-like cyclic pentapeptide core, but lacks the typical sidechain attached to the constitutive ureido group. Screening of all anabaenopeptin derivatives in an enzymatic assay for inhibitory activity toward carboxypeptidase A identified anabaenopeptin 679 as a strong inhibitor of carboxypeptidase A with an IC50 value of 4.6µg/mL. This result defines a new minimal core structure for carboxypeptidase activity among the anabaenopeptin class, and provides further insight into the structure-activity relationship of anabaenopeptin-like carboxypeptidase A inhibitors.

Augmenting anti-cancer natural products with a small molecule adjuvant.

Aquatic microbes produce diverse secondary metabolites with interesting biological activities. Cytotoxic metabolites have the potential to become lead compounds or drugs for cancer treatment. Many cytotoxic compounds, however, show undesirable toxicity at higher concentrations. Such undesirable activity may be reduced or eliminated by using lower doses of the cytotoxic compound in combination with another compound that modulates its activity. Here, we have examined the cytotoxicity of four microbial metabolites [ethyl N-(2-phenethyl) carbamate (NP-1), Euglenophycin, Anabaenopeptin, and Glycolipid 652] using three in vitro cell lines [human breast cancer cells (MCF-7), mouse neuroblastoma cells (N2a), and rat pituitary epithelial cells (GH4C1)]. The compounds showed variable cytotoxicity, with Euglenophycin displaying specificity for N2a cells. We have also examined the modulatory power of NP-1 on the cytotoxicity of the other three compounds and found that at a permissible concentration (125 µg/mL), NP-1 sensitized N2a and MCF-7 cells to Euglenophycin and Glycolipid 652 induced cytotoxicity.

High-throughput, cell-based screens to identify small-molecule inhibitors of ricin toxin and related category b ribosome inactivating proteins (RIPs).

Ricin is a member of the ubiquitous ribosome-inactivating protein (RIP) family of toxins. The Centers for Disease Control and Prevention (CDC) classify ricin and related toxins as Category B biothreat agents. There are currently no antidotes or therapeutics to counteract RIPs in humans. The discovery of effective small-molecule inhibitors of RIPs is increasingly possible, however, due to the availability and accessibility of diverse small-molecule chemical libraries coupled with robust robotics and automated screening methodologies. In this article, we describe a cell-based, high-throughput screening strategy and secondary assays that we have successfully used to identify compounds that target ricin toxin's enzymatic activity and intracellular trafficking, as well as stress-activated signaling pathways associated with cell death. The methods described in the protocol are amenable to the other RIPs.

Identification of small molecules that suppress ricin-induced stress-activated signaling pathways.

Ricin is a member of the ribosome-inactivating protein (RIP) family of plant and bacterial toxins. In this study we used a high-throughput, cell-based assay to screen more than 118,000 compounds from diverse chemical libraries for molecules that reduced ricin-induced cell death. We describe three compounds, PW66, PW69, and PW72 that at micromolar concentrations significantly delayed ricin-induced cell death. None of the compounds had any demonstrable effect on ricin's ability to arrest protein synthesis in cells or on ricin's enzymatic activity as assessed in vitro. Instead, all three compounds appear to function by blocking downstream stress-induced signaling pathways associated with the toxin-mediated apoptosis. PW66 virtually eliminated ricin-induced TNF-α secretion by J774A.1 macrophages and concomitantly blocked activation of the p38 MAPK and JNK signaling pathways. PW72 suppressed ricin-induced TNF-α secretion, but not p38 MAPK and JNK signaling. PW69 suppressed activity of the executioner caspases 3/7 in ricin toxin- and Shiga toxin 2-treated cells. While the actual molecular targets of the three compounds have yet to be identified, these data nevertheless underscore the potential of small molecules to down-regulate inflammatory signaling pathways associated with exposure to the RIP family of toxins.

Small-molecule inhibitors of ricin and Shiga toxins.

This review summarizes the successes and continuing challenges associated with the identification of small-molecule inhibitors of ricin and Shiga toxins, members of the RNA N-glycosidase family of toxins that irreversibly inactivate eukaryotic ribosomes through the depurination of a conserved adenosine residue within the sarcin-ricin loop (SRL) of 28S rRNA. Virtual screening of chemical libraries has led to the identification of at least three broad classes of small molecules that bind in or near the toxin's active sites and thereby interfere with RNA N-glycosidase activity. Rational design is being used to improve the specific activity and solubility of a number of these compounds. High-throughput cell-based assays have also led to the identification of small molecules that partially, or in some cases, completely protect cells from ricin- and Shiga-toxin-induced death. A number of these recently identified compounds act on cellular proteins associated with intracellular trafficking or pro-inflammatory/cell death pathways, and one was reported to be sufficient to protect mice in a ricin challenge model.

Identification of new classes of ricin toxin inhibitors by virtual screening.

We used two virtual screening programs, ICM and GOLD, to dock nearly 50,000 compounds into each of two conformations of the target protein ricin A chain (RTA). A limited control set suggests that candidates scored highly by two programs may have a higher probability of being ligands than those in a list from a single program. Based on the virtual screens, we purchased 306 compounds that were subjected to a kinetic assay. Six compounds were found to give modest, but significant, inhibition of RTA. They also tended to inhibit Shiga toxin A chain, with roughly the same IC(50). The compounds generally represent novel chemical platforms that do not resemble RTA substrates, as currently known inhibitors do. These six were also tested in a cell-based assay for their ability to protect cells from intact ricin. Two compounds were effective in this regard, showing modest to strong ricin inhibition, but also showing some cytotoxicity. RTA, with its large, polar active site is a difficult drug design target which is expected to bind small molecules only weakly. The ability of the method to find these novel platforms is encouraging and suggests virtual screening can contribute to the search for ricin and Shiga toxin inhibitors.

Identification of small-molecule inhibitors of ricin and shiga toxin using a cell-based high-throughput screen.

The Category B agents, ricin and shiga toxin (Stx), are RNA N-glycosidases that target a highly conserved adenine residue within the sarcin-ricin loop of eukaryotic 28S ribosomal RNA. In an effort to identify small-molecule inhibitors of these toxins that could serve as lead compounds for potential therapeutics, we have developed a simple Vero cell-based high-throughput cytotoxicity assay and have used it to screen approximately 81,300 compounds in 17 commercially available chemical libraries. This initial screen identified approximately 300 compounds with weak (>or=30 to <50%), moderate (>or=50 to <80%), or strong (>or=80%) ricin inhibitory activity. Secondary analysis of 244 of these original "hits" was performed, and 20 compounds that were capable of reducing ricin cytotoxicity by >50% were chosen for further study. Four compounds demonstrated significant dose-dependent ricin inhibitory activity in the Vero cell-based assay, with 50% effective inhibitory concentration (EC(50)) values ranging from 25 to 60microM. The same 20 compounds were tested in parallel for the ability to inhibit ricin's and Stx1's enzymatic activities in an in vitro translation reaction. Three of the 20 compounds, including the most effective compound in the cell-based assay, had discernible anti-toxin activity. One compound in particular, 4-fluorophenyl methyl 2-(furan-2-yl)quinoline-4-carboxylate ("compound 8"), had 50% inhibitory concentration (IC(50)) of 30microM, a value indicating >10-fold higher potency than is the case for previously described ricin-Stx1 inhibitors. Computer modeling predicted that compound 8 is capable of docking within the ricin active site. In conclusion, we have used a simple high-throughput cell-based method to identify several new small-molecule inhibitors of ricin and Stx.

Levels and localization of mechanosensitive channel proteins in Bacillus subtilis.

Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock > or =0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells' osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.

Characterization of spores of Bacillus subtilis that lack most coat layers.

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis.

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.

Release of small molecules during germination of spores of Bacillus Species.

Free amino acids, dipicolinic acid, and unidentified small molecules were released early in Bacillus spore germination before hydrolysis of the peptidoglycan cortex, but adenine nucleotides and 3-phosphoglycerate were not. These results indicate that early in germination there is a major selective change in the permeability of the spore's inner membrane.

Growth, osmotic downshock resistance and differentiation of Bacillus subtilis strains lacking mechanosensitive channels.

Previous work has shown that the mechanosensitive (MS) channel of large conductance (MscL) is essential for preventing lysis of Bacillus subtilis log phase cells upon a rapid, severe osmotic downshock. Growing cells of B. subtilis strains lacking MscL and one or more putative MS channel proteins of small conductance (YhdY, YkuT and YfkC) showed even higher sensitivity to an osmotic downshock. The effect was greatest for a strain lacking MscL and YkuT, and a strain lacking all four MS channel proteins had a similar phenotype. These defects were complemented by expression of either MscL or YkuT in trans. All MS channel mutant strains ultimately became resistant to osmotic downshock in stationary phase but at varying times, with mscL ykuT strains taking the longest time to become resistant. Expression of beta-galactosidase from gene fusions to lacZ showed modest expression of ykuT and lower levels of expression of yhdY and yfkC when strains were grown in medium containing high salt. Sporulation of all MS channel mutant strains was normal, and the mutant spores germinated normally with L-alanine or dodecylamine.

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis.

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL-lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL-lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of beta-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The DeltamscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase DeltamscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, DeltamscL cells had become resistant to a 0.9 M osmotic downshift.

Levels of glycine betaine in growing cells and spores of Bacillus species and lack of effect of glycine betaine on dormant spore resistance.

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

Terpenoids from Laurencia luzonensis.

Luzodiol (4), a diterpene possessing a new carbon skeleton, and five new sesquiterpenes (5-9) of the snyderane class have been isolated from the red alga Laurencia luzonensis and their structures determined by spectroscopic analysis. The relative stereochemistry of the known luzonensol (3) was assigned by its conversion to palisadin B (10).