Palmyrolide A (1) is a new neuroactive macrolide isolated from a marine cyanobacterial assemblage composed of Leptolyngbya cf. and Oscillatoria spp. collected from Palmyra Atoll. It features a rare N-methyl enamide and an intriguing t-butyl branch; the latter renders the adjacent lactone ester bond resistant to hydrolysis. Consistent with its significant suppression of calcium influx in cerebrocortical neurons (IC 50 =3.70 µM), palmyrolide A (1) showed relatively potent sodium channel blocking activity in neuro-2a cells (IC 50 =5.2 µM), without appreciable cytotoxicity.Suppression and/or activation of spontaneous Ca 2+ oscillations of murine cerebrocortical neurons1 has proven to be an extremely sensitive screening method for the discovery of new neurotoxins, including the recently reported cyanobacterial metabolites hoiamide A,2a alotamide A,2b and palmyramide A.2c In the case of hoiamide A, further pharmacological characterization found it to be a partial agonist at neurotoxin site 2 of voltage-gated sodium channels (VGSCs).2aPrimary cultures of cerebrocortical neurons allow the detection of two distinct actions when cells are loaded with the Ca 2+ sensitive fluorescent dye, fluo-3.1 First, those metabolites that trigger Ca 2+ influx can be easily revealed by monitoring for this ion using a Fluorometric Imaging Plate Reader (FLIPR). Secondly, because these cultures display spontaneous Ca 2+ oscillations, they provide a robust screening system for the discovery of small molecule ion wgerwick@ucsd.edu. Supporting Information Available: Experimental, full NMR data of 1, 2 and 4 (all stereoisomers), bioassay data, and taxonomic characterization. This material is available free of charge via the Internet at http://pubs.acs.org. (Table 1 and SI) was an intense singlet at δ0.86 (nine protons), which could be attributed to three isochronous methyl groups comprising a t-butyl moiety.8 Also present were the methyl doublets at δ0.90 and δ1.21, as well as the N-methyl singlet at δ3.04. The 1 H NMR of 1 was completed by a deshielded methine proton at δ4.88 and a terminal 1,2-disubstituted vinylic system represented by protons at δ5.27 (dt) and δ6.47 (d). The presence of a t-butyl moiety was supported in the 13 C NMR spectrum of 1 by a very intense resonance at δ26.1, as well as a quaternary carbon at δ35.2. Additionally, the deshielded carbons at δ117.3 and δ130.6 were in agreement with a 1,2-disubstituted double bond, whereas the carbonyls at δ172.9 and δ175.3 indicated a total of two ester or/and amide functionalities. As detailed below, extensive analysis of these 1 H and 13 C NMR resonances using HSQC, HMBC, COSY and NOESY led us to deduce the planar structure of 1. NIH Public AccessThe intense singlet at δ0.86 (H9, H10, H11) showed HMBC correlations with the quaternary carbon C8 (δ35.2), oxymethine C7 (δ76.9), and methylene carbon C6 (δ35.6) ( Figure 1A). This last carbon was found by HSQC to bear the diastereotopic proton resonances H6a (δ1.38) and H6b (δ1.66), which according to the COSY spectrum, participa...

The ability of cyanobacteria to produce complex secondary metabolites with potent biological activities has gathered considerable attention due to their potential therapeutic and agrochemical applications. However, the precise physiological or ecological roles played by a majority of these metabolites have remained elusive. Several studies have shown that cyanobacteria are able to interfere with other organisms in their communities through the release of compounds into the surrounding medium, a phenomenon usually referred to as allelopathy. Exudates from the freshwater cyanobacterium Oscillatoria sp. had previously been shown to inhibit the green microalga Chlorella vulgaris. In this study, we observed that maximal allelopathic activity is highest in early growth stages of the cyanobacterium, and this provided sufficient material for isolation and chemical characterization of active compounds that inhibited the growth of C. vulgaris. Using a bioassay-guided approach, we isolated and structurally characterized these metabolites as cyclic peptides containing several unusually modified amino acids that are found both in the cells and in the spent media of Oscillatoria sp. cultures. Strikingly, only the mixture of the two most abundant metabolites in the cells was active toward C. vulgaris. Synergism was also observed in a lung cancer cell cytotoxicity assay. The binary mixture inhibited other phytoplanktonic organisms, supporting a natural function of this synergistic mixture of metabolites as allelochemicals.allelopathy | chemical ecology | Oscillatoria | cyclic peptides | synergism C yanobacteria are a prolific source of nearly 800 diverse bioactive secondary metabolites, originating mainly from nonribosomal peptide synthetase (NRPS) or mixed polyketide synthase (PKS)-NRPS biosynthesis (1, 2). Efforts to isolate and characterize cyanobacterial metabolites have usually been motivated from a desire to describe either their natural toxicity toward animals in natural settings (e.g., ref.3) or promising activities from in vitro biomedical screening programs (e.g., ref. 4, 5). However, in general the ecological role played by the majority of these metabolites is not well known (6, 7). Functions established to date for cyanobacterial secondary metabolites include nitrogen storage (8), UV protection (9), metal chelation (10), defense against predation (11), and quorum sensing (12).Allelopathy refers to the chemically mediated interaction between plants or microorganisms (13). These interactions are characterized by the release of allelopathic compounds (allelochemicals) into the surrounding medium, eliciting either a positive or deleterious response in a target organism (13). In aquatic ecosystems, allelopathy is regarded as an important process influencing the shaping of microbial communities (14-16). Toxic properties have been attributed to cyanobacteria over the last 130 yr (e.g., ref. 17), and the allelopathic potential of these organisms was described through field-derived observations in the 1970s (18,19). Since th...

Two new peptidic proteasome inhibitors were isolated as trace components from a Curaçao collection of Symploca sp. marine cyanobacteria. Carmaphycin A (1) and carmaphycin B (2) feature a leucine-derived α, β -epoxyketone warhead directly connected to either methionine sulfoxide or methionine sulfone. Their structures were elucidated on the basis of extensive NMR/MS analyses and confirmed by total synthesis, which in turn provided more material for further biological evaluations. Pure carmaphycins A and B were found to inhibit the β5 subunit (chymotrypsin-like activity) of the S. cerevisiae 20S proteasome in the low nanomolar range. Additionally, they exhibited strong cytotoxicity to lung and colon cancer cell lines, as well as exquisite antiproliferative effects in the NCI60 cell line panel. These assay results as well as initial structural biology studies suggest a distinctive binding mode for these new inhibitors.

Summary Hoiamide A, a novel bioactive cyclic depsipeptide, was isolated from an environmental assemblage of the marine cyanobacteria Lyngbya majuscula and Phormidium gracile collected in Papua New Guinea. This stereochemically complex metabolite possesses a highly unusual structure which likely derives from a mixed peptide-polyketide biogenetic origin, and includes a peptidic section featuring an acetate extended and S-adenosyl methionine modified isoleucine moiety, a triheterocyclic fragment bearing two a-methylated thiazolines and one thiazole, as well as a highly oxygenated and methylated C15-polyketide substructure. Pure hoiamide A potently inhibited [3H]batrachotoxin binding to voltage-gated sodium channels (IC50 = 92.8 nM) and activated sodium influx (EC50 = 1.73 μM) in mouse neocortical neurons, as well as exhibited modest cytotoxicity to cancer cells. Further investigation revealed that hoiamide A is a partial agonist of site 2 on the voltage gated sodium channel.

Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition‐based adaptive laboratory evolution scheme is presented whereby an antibiotic‐producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces clavuligerus replicates were adaptively evolved against methicillin‐resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the wild‐type strain. Moreover, genome re‐sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid. This and other strains also acquired numerous single nucleotide polymorphisms, some of which are located within genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition‐based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.

Crude extracts of the marine hydroid Garveia annulata show potent inhibition of indoleamine 2,3-dioxygenase (IDO). Fractionation of the extract led to the identification of the new polyketides annulin C (1), 2-hydroxygarveatin E (4), garveatin E (5), and garvin C (9). Annulins A (2), B (3), and C (1) were found to be submicromolar inhibitors of IDO.

Tauramamide (1), a new lipopeptide antibiotic, is produced by cultures of the marine bacterial isolate Brevibacillus laterosporus PNG276 obtained from Papua New Guinea. Tauramamide was isolated as its methyl and ethyl esters 2 and 3, whose structures were elucidated by analysis of NMR, MS, and chemical degradation data. A total synthesis of tauramamide (1) and tauramamide ethyl ester (3) confirmed the structure proposed from spectroscopic analysis and provided the natural product for antimicrobial testing. Tauramamide (1) and ethyl ester 3 show potent and relatively selective inhibition of pathogenic Enterococcus sp.

Two related peptide metabolites, one a cyclic depsipeptide, hoiamide B (2), and the other a linear lipopeptide, hoiamide C (3), were isolated from two different collections of marine cyanobacteria obtained in Papua New Guinea. Their structures were elucidated by combining various techniques in spectroscopy, chromatography and synthetic chemistry. Both metabolites belong to the unique hoiamide structural class, characterized by possessing an acetate extended and S-adenosyl methionine modified isoleucine unit, a central triheterocyclic system comprised of two α-methylated thiazolines and one thiazole, as well as a highly oxygenated and methylated C-15 polyketide unit. In neocortical neurons, the cyclic depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca2+ oscillations with EC50 values of 3.9 μM and 79.8 nM, respectively, while 3 had no significant effects in these assays.