Helena Gaspar

Marketed Marine Natural Products in the Pharmaceutical and Cosmeceutical Industries: Tips for Success

The marine environment harbors a number of macro and micro organisms that have developed unique metabolic abilities to ensure their survival in diverse and hostile habitats, resulting in the biosynthesis of an array of secondary metabolites with specific activities. Several of these metabolites are high-value commercial products for the pharmaceutical and cosmeceutical industries. The aim of this review is to outline the paths of marine natural products discovery and development, with a special focus on the compounds that successfully reached the market and particularly looking at the approaches tackled by the pharmaceutical and cosmetic companies that succeeded in marketing those products. The main challenges faced during marine bioactives discovery and development programs were analyzed and grouped in three categories: biodiversity (accessibility to marine resources and efficient screening), supply and technical (sustainable production of the bioactives and knowledge of the mechanism of action) and market (processes, costs, partnerships and marketing). Tips to surpass these challenges are given in order to improve the market entry success rates of highly promising marine bioactives in the current pipelines, highlighting what can be learned from the successful and unsuccessful stories that can be applied to novel and/or ongoing marine natural products discovery and development programs.

Localization and ecological significance of oroidin and sceptrin in the Caribbean sponge Agelas conifera

The Caribbean sponge Agelas conifera was found to produce a mixture of previously described bromopyrrole alkaloids of which oroidin (1) and sceptrin (2) were predominant. This sponge harboured large populations of heterotrophic bacteria but no photosynthetic symbionts (cyanobacteria). However, 1 and 2 were not associated with the bacteria but with the sponge cells as shown by their distribution in enriched cell fractions obtained by differential centrifugation and Ficoll density gradients. Spherulous cells, found in great abundance in the sponge ectosome, were assumed to be involved in the production of 1 and 2. The target compounds were detected, although in small amounts, in short-term cultures of sponge cells, validating the possibility of a continuous cell culture source. Laboratory assays showed that organic sponge extracts affected the behaviour of the coral Madracis mirabilis in causing closure and retraction of the polyps at concentrations of the combined compounds 1 and 2 (1:3.3) as low as 0.7 mg/l (0.0125% of the concentration in whole sponges). At higher concentrations (1.4 mg/l) no recovery of the polyps occurred. The extracts, at almost natural concentrations of 1 and 2, deterred feeding by the predatory reef fish Stegastis partitus, supporting other reported research. In field experiments, wounding induced a sharp increase of 1 and 2 in the sponge tissues but prolonged predator exclusion by caging and forced confrontation with coral neighbours did not yield measurable changes in 1 and 2 concentrations. All sponges were found to release measurable amounts of bromopyrrole alkaloids in seawater conditioned for 30 min. Crude and fractionated sponge extracts and pure sceptrin (2) were active against bacteria, yeast and filamentous fungi. Taken together, these results support a role of oroidin (1) and sceptrin (2) in defence mechanisms against predators and possibly against space competitors and invading and fouling organisms.

Antimicrobial activity of heterotrophic bacterial communities from the marine sponge Erylus discophorus (Astrophorida, Geodiidae).

Heterotrophic bacteria associated with two specimens of the marine sponge Erylus discophorus were screened for their capacity to produce bioactive compounds against a panel of human pathogens (Staphylococcus aureus wild type and methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumanii, Candida albicans and Aspergillus fumigatus), fish pathogen (Aliivibrio fischeri) and environmentally relevant bacteria (Vibrio harveyi). The sponges were collected in Berlengas Islands, Portugal. Of the 212 isolated heterotrophic bacteria belonging to Alpha- and Gammaproteobacteria, Actinobacteria and Firmicutes, 31% produced antimicrobial metabolites. Bioactivity was found against both Gram positive and Gram negative and clinically and environmentally relevant target microorganisms. Bioactivity was found mainly against B. subtilis and some bioactivity against S. aureus MRSA, V. harveyi and A. fisheri. No antifungal activity was detected. The three most bioactive genera were Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%). Other less bioactive genera were Labrenzia, Acinetobacter, Microbulbifer, Pseudomonas, Gordonia, Microbacterium, Micrococcus and Mycobacterium, Paenibacillus and Staphylococcus. The search of polyketide I synthases (PKS-I) and nonribosomal peptide synthetases (NRPSs) genes in 59 of the bioactive bacteria suggested the presence of PKS-I in 12 strains, NRPS in 3 strains and both genes in 3 strains. Our results show the potential of the bacterial community associated with Erylus discophorus sponges as producers of bioactive compounds.

Coloration and Defense in the Nudibranch Gastropod Hypselodoris fontandraui

The striking color patterns of chromodorid (and other) nudibranchs appear to be indicative of aposematism. In Müllerian mimicry, all the mimic species have a defense mechanism. It has been proposed that a group of blue, white, and yellow Mediterranean and northeastern Atlantic species of the genus Hypselodoris form a Müllerian mimetic circle. One of these, H. fontandraui, lacks the mantle dermal formations (repugnatorial glands) that are typically found in other members of this circle and are reservoirs of feeding deterrent compounds. It therefore seemed possible that this animal lacks chemical defense and acts like a Batesian mimic. Within this study, we found that this nudibranch contains the furanosesquiterpenoid tavacpallescensin, most probably derived from sponges of the genus Dysidea, upon which it probably feeds. The metabolite concentrations were measured from samples of the mantle rim, other external parts, and internal organs. Concentrations were about 4 times higher in the mantle rim than in the other external parts, and more than 20 times higher in the mantle rim than in the internal organs, considerably exceeding the threshold value of concentration showing the maximum dose effect as feeding deterrent against the crustacean Palaemon elegans. In conclusion, the reported data clearly demonstrate that H. fontandraui is chemically defended in much the same way as its aposematic, co-occurring, and blue-colored congeners within the Müllerian mimetic circle and is not a Batesian mimic.

Isomeric Furanosesquiterpenes from the Portuguese Marine Sponge Fasciospongia sp.

This paper reports the chemical study of a sample of Fasciospongia sp. collected along the Atlantic Portuguese coast. Three new isomeric furanosesquiterpenes, isomicrocionin-3 ( 1), (-)-microcionin-1 ( 2), and (-)-isomicrocionin-1 ( 3), were isolated along with the known (-)- ent-pallescensin A ( 4) and (-)-pallescensin-1 ( 5) from the ethyl acetate-soluble portion of the methanolic extract. The structures of the new compounds were elucidated by extensive spectroscopic studies. (-)-Microcionin-1 ( 2) tested positive against several Gram-negative bacteria.

The antimicrobial activity of heterotrophic bacteria isolated from the marine sponge Erylus deficiens (Astrophorida, Geodiidae)

Interest in the study of marine sponges and their associated microbiome has increased both for ecological reasons and for their great biotechnological potential. In this work, heterotrophic bacteria associated with three specimens of the marine sponge Erylus deficiens, were isolated in pure culture, phylogenetically identified and screened for antimicrobial activity. The isolation of bacteria after an enrichment treatment in heterotrophic medium revealed diversity in bacterial composition with only Pseudoalteromonas being shared by two specimens. Of the 83 selected isolates, 58% belong to Proteobacteria, 23% to Actinobacteria and 19% to Firmicutes. Diffusion agar assays for bioactivity screening against four bacterial strains and one yeast, revealed that a high number of the isolated bacteria (68.7%) were active, particularly against Candida albicans and Vibrio anguillarum. Pseudoalteromonas, Microbacterium, and Proteus were the most bioactive genera. After this preliminary screening, the bioactive strains were further evaluated in liquid assays against C. albicans, Bacillus subtilis and Escherichia coli. Filtered culture medium and acetone extracts from three and 5 days-old cultures were assayed. High antifungal activity against C. albicans in both aqueous and acetone extracts as well as absence of activity against B. subtilis were confirmed. Higher levels of activity were obtained with the aqueous extracts when compared to the acetone extracts and differences were also observed between the 3 and 5 day-old extracts. Furthermore, a low number of active strains was observed against E. coli. Potential presence of type-I polyketide synthases (PKS-I) and non-ribosomal peptide synthetases (NRPSs) genes were detected in 17 and 30 isolates, respectively. The high levels of bioactivity and the likely presence of associated genes suggest that Erylus deficiens bacteria are potential sources of novel marine bioactive compounds.

CARBON/NUTRIENT BALANCE IN RELATION TO BIOMASS PRODUCTION AND HALOGENATED COMPOUND CONTENT IN THE RED ALGA ASPARAGOPSIS TAXIFORMIS (BONNEMAISONIACEAE)1

We tested how the availability of carbon and nitrogen determines both the production of Asparagopsis taxiformis (Delile) V. Trevis. and content of the two major halocarbons, bromoform and dibromoacetic acid. The halogenated secondary metabolites of Asparagopsis species are particularly interesting from an applied perspective due to their remarkable antimicrobial activity. Terrestrial ecologists named the relationship between resources and secondary metabolites as the carbon (C)/nutrient balance (CNB) hypothesis. This relationship was tested both in the laboratory, with a factorial analysis using different concentrations of total ammonia (TAN) and dissolved inorganic carbon (DIC), and in an integrated aquaculture system where TAN and DIC fluxes of fish effluent were manipulated. The total C/N content of A. taxiformis biomass cultivated in laboratory was highly significantly linearly related to the content of both halocarbons, as predicted by the CNB hypothesis. A. taxiformis cultivated at low levels of carbon and high levels of nitrogen (N) (lowest C/N ratio) had the lowest content in both halogenated metabolites. Increased availability of CO2 in the medium resulted in a general higher halocarbon content in the biomass, even though the effect was only statistically significant for bromoform at high levels of N. The farm experiments supported the results of the laboratory experiments. DIC fluxes had the highest effect on the production of both bromoform and biomass, as shown by multiple regression analysis. In A. taxiformis integrated aquaculture, C, rather than N, is the most important factor affecting the production of biomass and of valuable halocarbon secondary metabolites.

Sterols from Teucrium abutiloides and T. betonicum

Abstract The sterol composition of two Teucrium species ( abutiloides and betonicum ) was studied. Both plants were shown to contain three 24-ethylcholestane derivatives, all of them having the 24β-configuration. These were identified as (24 S )-24-ethylcholesta-5,22( E ),25-trien-3β-ol, (24 S )-24-ethylcholesta-5,25-dien-3β-ol (clerosterol) and (24 R )-24-ethylcholesta-5,22( E )-dien-3β-ol (poriferasterol) by spectroscopic analysis of their acetates and by comparison of their physical data (mp, [ α ] D ) with those reported in the literature for these compounds. Some disagreements in the previously published data of these sterols are clarified and chemotaxonomic aspects are briefly discussed.

Biosynthetic evidence supporting the generation of terpene chemodiversity in marine mollusks of the genus Doriopsilla.

This paper reports the study of terpene biosynthesis in the marine nudibranch Doriopsilla pelseneeri. In vivo feeding experiments with 13C-glucose proved the de novo origin of the terpene metabolites via a mevalonate pathway. Characterization of the stereochemical relationship of the new acetyl pelseneeriols (7 and 8) suggests a two-step mechanism for the sesquiterpene cyclization, leading to a slight refinement of terpene tailoring in this family of marine invertebrates.

Turning the game around: toxicity in a nudibranch-sponge predator–prey association

Escalation theory proposes enemy-related selection as the most relevant factor of natural selection among individual organisms. When hazardous to predators, prey might be considered enemies that influence predator evolution. Opisthobranch molluscs that prey on chemically defended prey are an interesting study case on this subject. Predation on chemically defended species paved the way for opisthobranchs to enter in an arms race, developing means to detoxify and/or excrete harmful compounds, which led to the sequestration of those compounds and their self-defensive use, an escalation of defenses. Here we aim to understand whether the opisthobranch predator is better protected than its chemically defended prey, using as predator–prey model, a nudibranch (Hypselodoriscantabrica) and the sponge it preys upon (Dysidea fragilis), and from which it obtains deterrent chemical compounds. Specimens of both species were collected on the Portuguese coast, and their crude extracts were analyzed and used in palatability tests. Nudibranchs revealed a higher natural concentration of crude extract, probably due to a progressive accumulation of the compounds. Both predator and prey extracts revealed similar mixtures of deterrent metabolites (furanosesquiterpenes). Palatability tests revealed a more effective deterrence in the nudibranch extracts because significant rejection rates were observed at lower concentrations than those necessary for the sponge extracts to have the same effect. We concluded that the predator is chemically better protected than its prey, which suggests that its acquisition of chemical defenses reveals a defensive escalation.