Alban R. Pereira

Synergistic allelochemicals from a freshwater cyanobacterium

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.

The hoiamides, structurally intriguing neurotoxic lipopeptides from Papua New Guinea marine cyanobacteria.

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 alpha-methylated thiazolines and one thiazole, and a highly oxygenated and methylated C-15 polyketide unit. In neocortical neurons, the cyclic depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca(2+) oscillations with EC(50) values of 3.9 microM and 79.8 nM, respectively, while 3 had no significant effects in these assays.

Hoiamide A, a Sodium Channel Activator of Unusual Architecture from a Consortium of Two Papua New Guinea Cyanobacteria

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 alpha-methylated thiazolines and one thiazole, and a highly oxygenated and methylated C15-polyketide substructure. Pure hoiamide A potently inhibited [(3)H]batrachotoxin binding to voltage-gated sodium channels (IC(50) = 92.8 nM), activated sodium influx (EC(50) = 2.31 microM) in mouse neocortical neurons, and 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.

Palmyrolide A, an Unusually Stabilized Neuroactive Macrolide from Palmyra Atoll Cyanobacteria

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 a relatively potent sodium channel blocking activity in neuro-2a cells (IC(50) = 5.2 μM), without appreciable cytotoxicity.

A Concise Enantioselective Synthesis of the Chlorosulfolipid Malhamensilipin A

The first enantioselective synthesis of a member of the chlorosulfolipid family of natural products is reported. All of the polar substituents of malhamensilipin A are introduced with high stereoselectivity, and the unique (E)-chlorovinyl sulfate is created by a chemo-, regio-, and stereoselective E2 elimination of HCl in a reaction that likely has a counterpart in the biosynthesis of this fascinating natural product.

Cyanolide A, a glycosidic macrolide with potent Molluscicidal activity from the Papua New Guinea cyanobacterium Lyngbya bouillonii.

Over the last 50 years, molluscicides have played a critical role in the control of schistosomiasis transmission. Cyanolide A (2), isolated from extracts of a Papua New Guinea collection of Lyngbya bouillonii, is a new and highly potent molluscicidal agent against the snail vector Biomphalaria glabrata (LC(50) = 1.2 microM). The structure of cyanolide A (2) was elucidated through extensive NMR spectroscopic analyses, yielding a symmetrical dimer that represents the newest addition to the family of glycosidic macrolides from cyanobacteria.

The carmaphycins: new proteasome inhibitors exhibiting an α,β-epoxyketone warhead from a marine cyanobacterium.

Two new peptidic proteasome inhibitors were isolated as trace components from a Curaçao collection of the marine cyanobacterium Symploca sp. 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 and 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.

Exploiting adaptive laboratory evolution of Streptomyces clavuligerus for antibiotic discovery and overproduction.

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 unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in 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.

Heterologous production of 4-O-demethylbarbamide, a marine cyanobacterial natural product.

Heterologous expression of the barbamide biosynthetic gene cluster, obtained from the marine cyanobacterium Moorea producens, in the terrestrial actinobacterium Streptomyces venezuelae, resulted in the production of a new barbamide congener 4-O-demethylbarbamide, demonstrating the potential of this approach for investigating the assembly and tailoring of complex marine natural products.

Synthesis of Phosphatidylinositol 3-Kinase (PI3K) Inhibitory Analogues of the Sponge Meroterpenoid Liphagal

Analogues of the sponge meroterpenoid liphagal have been synthesized and evaluated for inhibition of PI3Kα and PI3Kγ as part of a program aimed at developing new isoform-selective PI3K inhibitors. One of the analogues, compound 24, with IC₅₀ values of 66 nM against PI3Kα and 1840 nM against PI3Kγ, representing a 27-fold preference for PI3Kα, exhibited enhanced chemical stability and modestly enhanced potency and selectivity compared with the natural product liphagal.