Anti-Trypanosomal Bufadienolides from the Oocytes of the Toad Rhinella alata (Anura, Bufonidae).

Amphibians are widely known as a prolific source of bioactive metabolites. In this work, we isolated and characterized compounds with antiparasitic activity from the oocytes of the toad Rhinella alata collected in Panama. Bio-guided isolation and structural elucidation were carried out using chromatographic and spectroscopic techniques, respectively. The organic extract was subjected to solid phase extraction followed by HPLC purification of the fraction with in vitro activity against Trypanosoma cruzi trypomastigotes. Seven steroids (1-7) of the bufadienolide family were isolated, and their structures were determined using NMR and MS analyses; of these 19-formyl-dyscinobufotalin, (3) is reported as a new natural product. Compounds 1 and 3-7 resulted in a good anti-trypanosomal activity profile. Among these, 16ß-hydroxyl-hellebrigenin (1) and bufalin (7) showed significant selectivity values of >5 and 2.69, respectively, while the positive control benznidazole showed a selectivity of 18.81. Furthermore, molecular docking analysis showed compounds 1, 3 and 7 interact through H-bonds with the amino acid residues GLN-19, ASP-158, HIS-159 and TRP-177 from cruzipain at the catalytic site. Given the lack of therapeutic options to treat American trypanosomiasis, this work can serve as the basis for further studies that aim for the development of bufadienolides or their derivatives as drugs against Chagas disease.

Bufadienolides from the Skin Secretions of the Neotropical Toad Rhinella alata (Anura: Bufonidae): Antiprotozoal Activity against Trypanosoma cruzi.

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16ß-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16ß-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16ß-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1-10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells; however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.

New Eunicellin-Type Diterpenes from the Panamanian Octocoral Briareum Asbestinum.

Gorgonian octocorals are considered a prolific source of secondary metabolites with a wide range of biological activities, including anti-inflammatory activity. In particular, the genus Briareum is known for producing a wealth of diterpenes with complex chemical structures. The chemical study of the methanolic extract of Briareum asbestinum collected in Bocas del Toro, on the Caribbean side of Panama, led to the isolation of three new eunicellin-type diterpenes: briarellin T (1), asbestinin 27 (2), asbestinin 28 (3) and the previously described asbestinin 17 (4). The structures of the new compounds were determined by extensive NMR analyses and HRMS. Anti-inflammatory activity assays showed a significant reduction of the pro-inflammatory cytokines TNF-α, IL-6, IL-1ß and IL-8 as well as a downregulation of COX-2 expression in LPS-stimulated THP-1 macrophages. These findings support the potential use of these marine compounds as therapeutic agents in the treatment of inflammatory diseases.

Genome Mining, Microbial Interactions, and Molecular Networking Reveals New Dibromoalterochromides from Strains of Pseudoalteromonas of Coiba National Park-Panama.

The marine bacterial genus Pseudoalteromonas is known for their ability to produce antimicrobial compounds. The metabolite-producing capacity of Pseudoalteromonas has been associated with strain pigmentation; however, the genomic basis of their antimicrobial capacity remains to be explained. In this study, we sequenced the whole genome of six Pseudoalteromonas strains (three pigmented and three non-pigmented), with the purpose of identifying biosynthetic gene clusters (BGCs) associated to compounds we detected via microbial interactions along through MS-based molecular networking. The genomes were assembled and annotated using the SPAdes and RAST pipelines and mined for the identification of gene clusters involved in secondary metabolism using the antiSMASH database. Nineteen BGCs were detected for each non-pigmented strain, while more than thirty BGCs were found for two of the pigmented strains. Among these, the groups of genes of nonribosomal peptide synthetases (NRPS) that code for bromoalterochromides stand out the most. Our results show that all strains possess BGCs for the production of secondary metabolites, and a considerable number of distinct polyketide synthases (PKS) and NRPS clusters are present in pigmented strains. Furthermore, the molecular networking analyses revealed two new molecules produced during microbial interactions: the dibromoalterochromides D/D' (11-12).

Antimicrobial-producing Pseudoalteromonas from the marine environment of Panama shows a high phylogenetic diversity and clonal structure.

Pseudoalteromonas is a genus of marine bacteria often found in association with other organisms. Although several studies have examined Pseudoalteromonas diversity and their antimicrobial activity, its diversity in tropical environments is largely unexplored. We investigated the diversity of Pseudoalteromonas in marine environments of Panama using a multilocus phylogenetic approach. Furthermore we tested their antimicrobial capacity and evaluated the effect of recombination and mutation in shaping their phylogenetic relationships. The reconstruction of clonal relationships among 78 strains including 15 reference Pseudoalteromonas species revealed 43 clonal lineages, divided in pigmented and non-pigmented strains. In total, 39 strains displayed moderate to high activity against Gram-positive and Gram-negative bacteria and fungi. Linkage disequilibrium analyses showed that the Pseudoalteromonas strains of Panama have a highly clonal structure and that, although present, recombination is not frequent enough to break the association among alleles. This clonal structure is in contrast to the high rates of recombination generally reported for aquatic and marine bacteria. We propose that this structure is likely due to the symbiotic association with marine invertebrates of most strains analyzed. Our results also show that there are several putative new species of Pseudoalteromonas in Panama to be described.

Pumilacidins from the Octocoral-Associated Bacillus sp. DT001 Display Anti-Proliferative Effects in Plasmodium falciparum.

Chemical examination of the octocoral-associated Bacillus species (sp.) DT001 led to the isolation of pumilacidins A (1) and C (2). We investigated the effect of these compounds on the viability of Plasmodium falciparum and the mechanism of pumilacidin-induced death. The use of inhibitors of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) was able to prevent the effects of pumilacidins A and C. The results indicated also that pumilacidins inhibit parasite growth via mitochondrial dysfunction and decreased cytosolic Ca2+.

Dudawalamides A-D, Antiparasitic Cyclic Depsipeptides from the Marine Cyanobacterium Moorea producens.

A family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, named dudawalamides A-D (1-4), was isolated from a Papua New Guinean field collection of the cyanobacterium Moorea producens using bioassay-guided and spectroscopic approaches. The planar structures of dudawalamides A-D were determined by a combination of 1D and 2D NMR experiments and MS analysis, whereas the absolute configurations were determined by X-ray crystallography, modified Marfey's analysis, chiral-phase GCMS, and chiral-phase HPLC. Dudawalamides A-D possess a broad spectrum of antiparasitic activity with minimal mammalian cell cytotoxicity. Comparative analysis of the Dhoya-containing class of lipopeptides reveals intriguing structure-activity relationship features of these NRPS-PKS-derived metabolites and their derivatives.

MS/MS networking guided analysis of molecule and gene cluster families.

The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779(T). The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family-gene cluster families of hundreds or more diverse organisms in one single MS/MS network.

Uprolides N, O and P from the Panamanian Octocoral Eunicea succinea.

Three new diterpenes, uprolide N (1), uprolide O (2), uprolide P (3) and a known one, dolabellane (4), were isolated from the CH2Cl2-MeOH extract of the gorgonian octocoral Eunicea succinea, collected from Bocas del Toro, on the Caribbean coast of Panama. Their structures were determined using spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry (HRMS) together with molecular modeling studies. Compounds 1-3 displayed anti-inflammatory properties by inhibiting production of Tumor Necrosis Factor (TNF) and Interleukin (IL)-6 induced by lipopolysaccharide (LPS) in murine macrophages.

Antifungal Activity of Menisporopsin A against Relevant Plant Pathogens.

Current agrochemicals used in crop farming mainly consist of synthetic compounds with harmful effects on the environment and human health. Crop-associated fungal endophytes, which play many ecological roles including defense against pathogens, represent a promising source for bioactive and ecologically safer molecules in agrochemical discovery. The methanolic extract of the endophyte Menisporopsis sp. LCM 1078 was evaluated in vitro against the plant pathogens Boeremia exigua, Calonectria variabilis, Colletotrichum theobromicola, Colletotrichum tropicale, and Mycena cytricolor. Bioassay-guided isolation using chromatographic techniques followed by detailed chemical characterization by NMR and mass spectrometry led to the identification of menisporopsin A, which showed inhibitory activity in a dose-dependent manner against the five fungal pathogens including an endophytic strain (Colletotrichum tropicale), with MIC values in the range of 0.63-10.0 µg/mL showing a potency equivalent to the broadly employed agrochemical mancozeb.

Imaging mass spectrometry of a coral microbe interaction with fungi.

Fungal infections are increasing worldwide, including in the aquatic environment. Microbiota that coexist with marine life can provide protection against fungal infections by secretion of metabolites with antifungal properties. Our laboratory has developed mass spectrometric methodologies with the goal of improving our functional understanding of microbial metabolites and guiding the discovery process of anti-infective agents from natural sources. GA40, a Bacillus amyloliquefaciens strain isolated from an octocoral in Panama, displayed antifungal activity against various terrestrial and marine fungal strains. Using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), the molecular species produced by this microbe were visualized in a side-by-side interaction with two representative fungal strains, Aspergillus fumigatus and Aspergillus niger. The visualization was performed directly on the agar without the need for extraction. By evaluating the spatial distributions, relative intensities and m/z values of GA40 secreted metabolites in the fungal interactions and singly grown control colonies, we obtained insight into the antifungal activity of secreted metabolites. Annotation of GA40 metabolites observed in MALDI-IMS was facilitated by MS/MS networking analysis, a mass spectrometric technique that clusters metabolites with similar MS/MS fragmentation patterns. This analysis established that the predominant GA40 metabolites belong to the iturin family. In a fungal inhibition assay of A. fumigatus, the GA40 iturin metabolites were found to be responsible for the antifungal properties of this Bacillus strain.

seco-Briarellinone and briarellin S, two new eunicellin-based diterpenoids from the Panamanian octocoral Briareum asbestinum.

Two new eunicellin-based diterpenes, seco-briarellinone (1) and briarellin S (2), and a known seco-asbestinin (3) have been isolated from the methanolic extract of the common octocoral Briareum asbestinum collected in Bocas del Toro, Caribbean of Panama. The structures and relative stereochemistry of the compounds were defined using extensive spectroscopic analysis including 1D, 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Compounds 1 and 2 displayed anti-inflammatory properties inhibiting nitric oxide (NO) production induced by lipopolisacharide (LPS) in macrophages with an Inhibitory concentration 50% (IC50) of 4.7 µM and 20.3 µM, respectively. This is the first report of briarellin diterpenes containing a ketone group at C-12.

Antitrypanosomal alkaloids from the marine bacterium Bacillus pumilus.

Fractionation of the ethyl acetate extract of the marine bacterium Bacillus pumilus isolated from the black coral Antipathes sp. led to the isolation of five compounds: cyclo-(L-Leu-L-Pro) (1), 3-hydroxyacetylindole (2), N-acetyl-ß-oxotryptamine (3), cyclo-(L-Phe-L-Pro) (4), and 3-formylindole (5). The structures of compounds 1-5 were established by spectroscopic analyses, including HRESITOF-MS and NMR (1H, 13C, HSQC, HMBC and COSY). Compounds 2, 3 and 5 caused the inhibition on the growth of Trypanosoma cruzi (T. cruzi), with IC50 values of 20.6, 19.4 and 26.9 µM, respectively, with moderate cytotoxicity against Vero cells. Compounds 1-5 were found to be inactive when tested against Plasmodium falciparum and Leishmania donovani, therefore showing selectivity against T. cruzi parasites.

Cannabinomimetic lipid from a marine cyanobacterium.

NMR-guided fractionation of two independent collections of the marine cyanobacteria Lyngbya majuscula obtained from Papua New Guinea and Oscillatoria sp. collected in Panama led to the isolation of the new lipids serinolamide A (3) and propenediester (4). Their structures were determined by NMR and MS data analysis. Serinolamide A (3) exhibited a moderate agonist effect and selectivity for the CB1 cannabinoid receptor (Ki=1.3 µM, >5-fold) and represents the newest addition to the known cannabinomimetic natural products of marine origin.

Malyngolide dimer, a bioactive symmetric cyclodepside from the panamanian marine cyanobacterium Lyngbya majuscula.

Fractionation of the extract of the marine cyanobacterium Lyngbya majuscula collected from Panama led to the isolation of malyngolide dimer (1). The planar structure of 1 was determined using 1D and 2D NMR spectroscopy and HRESI-TOFMS. The absolute configuration was established by chemical degradation followed by chiral GC-MS analyses and comparisons with an authentic sample of malyngolide seco-acid (4). Compound 1 showed moderate in vitro antimalarial activity against chloroquine-resistant Plasmodium falciparum (W2) (IC(50) = 19 microM) but roughly equivalent toxicity against H-460 human lung cell lines. Furthermore, because the closely related cyanobacterial natural product tanikolide dimer (5) was a potent SIRT2 inhibitor, compound 1 was evaluated in this assay but found to be essentially inactive.

Antimicrobial Secretions of Toads (Anura, Bufonidae): Bioactive Extracts and Isolated Compounds against Human Pathogens.

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo, Bufotes, Duttaphrynus, and Rhinella. Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells.

19-Hydroxy-bufalin, a major bufadienolide isolated from the parotoid gland secretions of the Panamanian endemic toad Rhinella centralis (Bufonidae), inhibits the growth of Trypanosoma cruzi.

American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.

Metabolites from Microbes Isolated from the Skin of the Panamanian Rocket Frog Colostethus panamansis (Anura: Dendrobatidae).

The Panamanian rocket frog Colostethus panamansis (family Dendrobatidae) has been affected by chytridiomycosis, a deadly disease caused by the fungus Batrachochytrium dendrobatidis (Bd). While there are still uninfected frogs, we set out to isolate microbes from anatomically distinct regions in an effort to create a cultivable resource within Panama for potential drug/agricultural/ecological applications that perhaps could also be used as part of a strategy to protect frogs from infections. To understand if there are specific anatomies that should be explored in future applications of this resource, we mapped skin-associated bacteria of C. panamansis and their metabolite production potential by mass spectrometry on a 3D model. Our results indicate that five bacterial families (Enterobacteriaceae, Comamonadaceae, Aeromonadaceae, Staphylococcaceae and Pseudomonadaceae) dominate the cultivable microbes from the skin of C. panamansis. The combination of microbial classification and molecular analysis in relation to the anti-Bd inhibitory databases reveals the resource has future potential for amphibian conservation.

Antibacterial Activity of Volatile Organic Compounds Produced by the Octocoral-Associated Bacteria Bacillus sp. BO53 and Pseudoalteromonas sp. GA327.

The present research aimed to evaluate the antibacterial activity of volatile organic compounds (VOCs) produced by octocoral-associated bacteria Bacillus sp. BO53 and Pseudoalteromonas sp. GA327. The volatilome bioactivity of both bacteria species was evaluated against human pathogenic antibiotic-resistant bacteria, methicillin-resistant Staphylococcus aureus, Acinetobacter baumanni, and Pseudomonas aeruginosa. In this regard, the in vitro tests showed that Bacillus sp. BO53 VOCs inhibited the growth of P. aeruginosa and reduced the growth of S. aureus and A. baumanni. Furthermore, Pseudoalteromonas sp. GA327 strongly inhibited the growth of A. baumanni, and P. aeruginosa. VOCs were analyzed by headspace solid-phase microextraction (HS-SPME) joined to gas chromatography-mass spectrometry (GC-MS) methodology. Nineteen VOCs were identified, where 5-acetyl-2-methylpyridine, 2-butanone, and 2-nonanone were the major compounds identified on Bacillus sp. BO53 VOCs; while 1-pentanol, 2-butanone, and butyl formate were the primary volatile compounds detected in Pseudoalteromonas sp. GA327. We proposed that the observed bioactivity is mainly due to the efficient inhibitory biochemical mechanisms of alcohols and ketones upon antibiotic-resistant bacteria. This is the first report which describes the antibacterial activity of VOCs emitted by octocoral-associated bacteria.

Reproducible molecular networking of untargeted mass spectrometry data using GNPS.

Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS2) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS2 data with sample information (metadata) and annotated MS2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90- to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.