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

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

Compounds PW66, PW69 and PW72 inhibit ricin cytotoxicity.

<p>(Panels A, C, E) Vero cells were treated with ricin (0.2 nM; dashed lines) or pretreated with (A) PW66, (C) PW69, or (E) PW72 at the indicated concentrations for 30 min before ricin was added. Cell viability was measured at 24 h, as described in the Experimental Procedures. (Panels B, D, F) Vero cells were treated with indicated concentrations of ricin in the absence (solid symbols) or presence (open symbols) of (B) PW66, (D) PW69, and (F) PW72. Compounds were present at 25 µM. Each panel shows results of a representative experiment from three independent experiments that were done in triplicate and showed < 10% correlation of variation (% CV) for individual experiment.</p

Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

<div><p>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.</p> </div

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 ∼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 ≥20-fold lower in B. cereus and B. megaterium spores. Athough 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

Compound PW66 interferes with activation of p38 MAPK in ricin-treated cells.

<p>(A) J774A.1 cells were treated with ricin (0.2 nM) with or without indicated compounds for 6 h before cells were lysed. Phospho-p38 MAPK was immunoprecipitated from cell lysates and used in an <i>in vitro</i> ATF-2 phosphorylation assay, as described in the Experimental Procedures. Shown are the results of a dot-blot analysis. (B) J774A.1 cells were treated with ricin (0.2 nM) with or without PW66 at indicated concentrations as shown in Panel A, except that ATF-2 was subjected to SDS-PAGE and Western blotting. (C) Western blot analysis of total p38 MAPK from ricin- or ricin + compound-treated cells, as indicated. Shown are results of representative experiments from three or more independent experiments.</p

Compounds described in this study.

a<p>Source of the compound; <i>^b</i>PubChem ID; <i>^c</i>Chemspider ID; <i>^d</i>Vendor ID.</p

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