A guide to the use of bioassays in exploration of natural resources.

Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.

Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges.

Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.

Marine Drug Discovery through Computer-Aided Approaches.

Besides the importance of our oceans as oxygen factories, food providers, shipping pathways, and tourism enablers, oceans hide an unprecedented wealth of opportunities [...].

Impact of per- and polyfluorinated alkyl substances (PFAS) on the marine environment: Raising awareness, challenges, legislation, and mitigation approaches under the One Health concept.

Per- and polyfluorinated alkyl substances (PFAS) have long been known for their detrimental effects on the ecosystems and living organisms; however the long-term impact on the marine environment is still insufficiently recognized. Based on PFAS persistence and bioaccumulation in the complex marine food network, adverse effects will be exacerbated by global processes such as climate change and synergies with other pollutants, like microplastics. The range of fluorochemicals currently included in the PFAS umbrella has significantly expanded due to the updated OECD definition, raising new concerns about their poorly understood dynamics and negative effects on the ocean wildlife and human health. Mitigation challenges and approaches, including biodegradation and currently studied materials for PFAS environmental removal are proposed here, highlighting the importance of ongoing monitoring and bridging research gaps. The PFAS EU regulations, good practices and legal frameworks are discussed, with emphasis on recommendations for improving marine ecosystem management.

Exopolysaccharide Production from Marine-Derived Brevundimonas huaxiensis Obtained from Estremadura Spur Pockmarks Sediments Revealing Potential for Circular Economy.

Marine environments represent an enormous biodiversity reservoir due to their numerous different habitats, being abundant in microorganisms capable of producing biomolecules, namely exopolysaccharides (EPS), with unique physical characteristics and applications in a broad range of industrial sectors. From a total of 67 marine-derived bacteria obtained from marine sediments collected at depths of 200 to 350 m from the Estremadura Spur pockmarks field, off the coast of Continental Portugal, the Brevundimonas huaxiensis strain SPUR-41 was selected to be cultivated in a bioreactor with saline culture media and glucose as a carbon source. The bacterium exhibited the capacity to produce 1.83 g/L of EPS under saline conditions. SPUR-41 EPS was a heteropolysaccharide composed of mannose (62.55% mol), glucose (9.19% mol), rhamnose (19.41% mol), glucuronic acid (4.43% mol), galactose (2.53% mol), and galacturonic acid (1.89% mol). Moreover, SPUR-41 EPS also revealed acyl groups in its composition, namely acetyl, succinyl, and pyruvyl. This study revealed the importance of research on marine environments for the discovery of bacteria that produce new value-added biopolymers for pharmaceutical and other biotechnological applications, enabling us to potentially address saline effluent pollution via a sustainable circular economy.

Hydrophobic DES Based on Menthol and Natural Organic Acids for Use in Antifouling Marine Coatings.

Marine biofouling negatively impacts industries with off-shore infrastructures, such as naval, oil, and aquaculture. To date, there are no ideal sustainable, economic, and environmentally benign solutions to deal with this phenomenon. The advances achieved in green solvents, as well as its application in different industries, such as pharmaceutical and biotechnology, have promoted the emergence of deep eutectic systems (DES). These eutectic systems have applications in various fields and can be revolutionary in the marine-based industrial sector. In this study, the main objective was to investigate the potential use of hydrophobic DES (HDES) based on menthol and natural organic acids for their use as marine antifouling coatings. Our strategy encompassed the physicochemical characterization of different formulations, which allowed us to identify the most appropriate molar ratio and intermolecular interactions for HDES formations. The miscibility of the resulting HDES with the marine coating has been evaluated and proven to be successful. The Men/OL (1:1) system proved to be the most promising in terms of cost-production and thus was the one used in subsequent antifouling tests. The cytotoxicity of this HDES was evaluated using an in vitro cell model (HaCat cells) showing no significant toxicity. Furthermore, the application of this system incorporated into coatings that are used in marine structures was also studied using marine species (Mytilus edulis mussels and Patella vulgata limpets) to evaluate both their antifouling and ecotoxicity effects. HDES Men/OL (1:1) incorporated in marine coatings was promising in reducing marine macrofouling and also proved to be effective at the level of microfouling without viability impairment of the tested marine species. It was revealed to be more efficient than using copper oxide, metallic copper, or ivermectin as antifouling agents. Biochemical assays performed on marine species showed that this HDES does not induce oxidative stress in the tested species. These results are a strong indication of the potential of this HDES to be sustainable and efficiently used in marine fouling control technologies.

Improving awareness, understanding, and enforcement of responsibilities and regulations in Blue Biotechnology.

Blue Biotechnology is developing rapidly worldwide. However, the Nagoya Protocol (NP), Responsible Research and Innovation (RRI) and other regulatory requirements in this field are falling behind. This article identifies the main RRI, NP, and regulatory gaps and provides key recommendations to mitigate these challenges.

Advanced Methods for Natural Products Discovery: Bioactivity Screening, Dereplication, Metabolomics Profiling, Genomic Sequencing, Databases and Informatic Tools, and Structure Elucidation.

Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation.

Marine-Derived Actinomycetes: Biodegradation of Plastics and Formation of PHA Bioplastics-A Circular Bioeconomy Approach.

Plastics are present in the majority of daily-use products worldwide. Due to society's production and consumption patterns, plastics are accumulating in the environment, causing global pollution issues and intergenerational impacts. Our work aims to contribute to the development of solutions and sustainable methods to mitigate this pressing problem, focusing on the ability of marine-derived actinomycetes to accelerate plastics biodegradation and produce polyhydroxyalkanoates (PHAs), which are biodegradable bioplastics. The thin plastic films' biodegradation was monitored by weight loss, changes in the surface chemical structure (Infra-Red spectroscopy FTIR-ATR), and by mechanical properties (tensile strength tests). Thirty-six marine-derived actinomycete strains were screened for their plastic biodegradability potential. Among these, Streptomyces gougerotti, Micromonospora matsumotoense, and Nocardiopsis prasina revealed ability to degrade plastic films-low-density polyethylene (LDPE), polystyrene (PS) and polylactic acid (PLA) in varying conditions, namely upon the addition of yeast extract to the culture media and the use of UV pre-treated thin plastic films. Enhanced biodegradation by these bacteria was observed in both cases. S. gougerotti degraded 0.56% of LDPE films treated with UV radiation and 0.67% of PS films when inoculated with yeast extract. Additionally, N. prasina degraded 1.27% of PLA films when these were treated with UV radiation, and yeast extract was added to the culture medium. The main and most frequent differences observed in FTIR-ATR spectra during biodegradation occurred at 1740 cm-1, indicating the formation of carbonyl groups and an increase in the intensity of the bands, which indicates oxidation. Young Modulus decreased by 30% on average. In addition, S. gougerotti and M. matsumotoense, besides biodegrading conventional plastics (LDPE and PS), were also able to use these as a carbon source to produce degradable PHA bioplastics in a circular economy concept.

Responsible Research and Innovation Framework, the Nagoya Protocol and Other European Blue Biotechnology Strategies and Regulations: Gaps Analysis and Recommendations for Increased Knowledge in the Marine Biotechnology Community.

As the quest for marine-derived compounds with pharmacological and biotechnological potential upsurges, the importance of following regulations and applying Responsible Research and Innovation (RRI) also increases. This article aims at: (1) presenting an overview of regulations and policies at the international and EU level, while demonstrating a variability in their implementation; (2) highlighting the importance of RRI in biodiscovery; and (3) identifying gaps and providing recommendations on how to improve the market acceptability and compliance of novel Blue Biotechnology compounds. This article is the result of the work of the Working Group 4 "Legal aspects, IPR and Ethics" of the COST Action CA18238 Ocean4Biotech, a network of more than 130 Marine Biotechnology scientists and practitioners from 37 countries. Three qualitative surveys ("Understanding of the Responsible Research and Innovation concept", "Application of the Nagoya Protocol in Your Research", and "Brief Survey about the experiences regarding the Nagoya Protocol") indicate awareness and application gaps of RRI, the Nagoya Protocol, and the current status of EU policies relating to Blue Biotechnology. The article categorises the identified gaps into five main categories (awareness, understanding, education, implementation, and enforcement of the Nagoya Protocol) and provides recommendations for mitigating them at the European, national, and organisational level.

Predicting Antifouling Activity and Acetylcholinesterase Inhibition of Marine-Derived Compounds Using a Computer-Aided Drug Design Approach.

Biofouling is the undesirable growth of micro- and macro-organisms on artificial water-immersed surfaces, which results in high costs for the prevention and maintenance of this process (billion €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructure. To date, there are still no sustainable, economical and environmentally safe solutions to overcome this challenging phenomenon. A computer-aided drug design (CADD) approach comprising ligand- and structure-based methods was explored for predicting the antifouling activities of marine natural products (MNPs). In the CADD ligand-based method, 141 organic molecules extracted from the ChEMBL database and literature with antifouling screening data were used to build the quantitative structure-activity relationship (QSAR) classification model. An overall predictive accuracy score of up to 71% was achieved with the best QSAR model for external and internal validation using test and training sets. A virtual screening campaign of 14,492 MNPs from Encinar's website and 14 MNPs that are currently in the clinical pipeline was also carried out using the best QSAR model developed. In the CADD structure-based approach, the 125 MNPs that were selected by the QSAR approach were used in molecular docking experiments against the acetylcholinesterase enzyme. Overall, 16 MNPs were proposed as the most promising marine drug-like leads as antifouling agents, e.g., macrocyclic lactam, macrocyclic alkaloids, indole and pyridine derivatives.

The Diversity, Metabolomics Profiling, and the Pharmacological Potential of Actinomycetes Isolated from the Estremadura Spur Pockmarks (Portugal).

The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat.

A Computer-Aided Drug Design Approach to Predict Marine Drug-Like Leads for SARS-CoV-2 Main Protease Inhibition.

The investigation of marine natural products (MNPs) as key resources for the discovery of drugs to mitigate the COVID-19 pandemic is a developing field. In this work, computer-aided drug design (CADD) approaches comprising ligand- and structure-based methods were explored for predicting SARS-CoV-2 main protease (Mpro) inhibitors. The CADD ligand-based method used a quantitative structure-activity relationship (QSAR) classification model that was built using 5276 organic molecules extracted from the ChEMBL database with SARS-CoV-2 screening data. The best model achieved an overall predictive accuracy of up to 67% for an external and internal validation using test and training sets. Moreover, based on the best QSAR model, a virtual screening campaign was carried out using 11,162 MNPs retrieved from the Reaxys® database, 7 in-house MNPs obtained from marine-derived actinomycetes by the team, and 14 MNPs that are currently in the clinical pipeline. All the MNPs from the virtual screening libraries that were predicted as belonging to class A were selected for the CADD structure-based method. In the CADD structure-based approach, the 494 MNPs selected by the QSAR approach were screened by molecular docking against Mpro enzyme. A list of virtual screening hits comprising fifteen MNPs was assented by establishing several limits in this CADD approach, and five MNPs were proposed as the most promising marine drug-like leads as SARS-CoV-2 Mpro inhibitors, a benzo[f]pyrano[4,3-b]chromene, notoamide I, emindole SB beta-mannoside, and two bromoindole derivatives.

Marine Anticancer Agents: An Overview with a Particular Focus on Their Chemical Classes.

The marine environment is a rich source of biologically active molecules for the treatment of human diseases, especially cancer. The adaptation to unique environmental conditions led marine organisms to evolve different pathways than their terrestrial counterparts, thus producing unique chemicals with a broad diversity and complexity. So far, more than 36,000 compounds have been isolated from marine micro- and macro-organisms including but not limited to fungi, bacteria, microalgae, macroalgae, sponges, corals, mollusks and tunicates, with hundreds of new marine natural products (MNPs) being discovered every year. Marine-based pharmaceuticals have started to impact modern pharmacology and different anti-cancer drugs derived from marine compounds have been approved for clinical use, such as: cytarabine, vidarabine, nelarabine (prodrug of ara-G), fludarabine phosphate (pro-drug of ara-A), trabectedin, eribulin mesylate, brentuximab vedotin, polatuzumab vedotin, enfortumab vedotin, belantamab mafodotin, plitidepsin, and lurbinectedin. This review focuses on the bioactive molecules derived from the marine environment with anticancer activity, discussing their families, origin, structural features and therapeutic use.

Antifouling Napyradiomycins from Marine-Derived Actinomycetes Streptomyces aculeolatus.

The undesired attachment of micro and macroorganisms on water-immersed surfaces, known as marine biofouling, results in severe prevention and maintenance costs (billions €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructures. To date, there are no sustainable, cost-effective and environmentally safe solutions to address this challenging phenomenon. Therefore, we investigated the antifouling activity of napyradiomycin derivatives that were isolated from actinomycetes from ocean sediments collected off the Madeira Archipelago. Our results revealed that napyradiomycins inhibited ≥80% of the marine biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae (EC50 < 5 µg/ml and LC50/EC50 >15), without viability impairment. In silico prediction of toxicity end points are of the same order of magnitude of standard approved drugs and biocides. Altogether, napyradiomycins disclosed bioactivity against marine micro and macrofouling organisms, and non-toxic effects towards the studied species, displaying potential to be used in the development of antifouling products.

Enriching cancer pharmacology with drugs of marine origin.

Marine natural products have proven, over the last half-century, to be effective biological modulators. These molecules have revealed new targets for cancer therapy as well as dissimilar modes of action within typical classes of drugs. In this scenario, innovation from marine-based pharmaceuticals has helped advance cancer chemotherapy in many aspects, as most of these are designated as first-in-class drugs. Here, by examining the path from discovery to development of clinically approved drugs of marine origin for cancer treatment-cytarabine (Cytosar-U®), trabectedin (Yondelis®), eribulin (Halaven®), brentuximab vedotin (Adcetris®), and plitidepsin (Aplidin®)- together with those in late clinical trial phases-lurbinectedin, plinabulin, marizomib, and plocabulin-the present review offers a critical analysis of the contributions given by these new compounds to cancer pharmacotherapy.

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites.

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC-MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 (1) against S. aureus was confirmed.

In Silico HCT116 Human Colon Cancer Cell-Based Models En Route to the Discovery of Lead-Like Anticancer Drugs.

To discover new inhibitors against the human colon carcinoma HCT116 cell line, two quantitative structure⁻activity relationship (QSAR) studies using molecular and nuclear magnetic resonance (NMR) descriptors were developed through exploration of machine learning techniques and using the value of half maximal inhibitory concentration (IC50). In the first approach, A, regression models were developed using a total of 7339 molecules that were extracted from the ChEMBL and ZINC databases and recent literature. The performance of the regression models was successfully evaluated by internal and external validations, the best model achieved R² of 0.75 and 0.73 and root mean square error (RMSE) of 0.66 and 0.69 for the training and test sets, respectively. With the inherent time-consuming efforts of working with natural products (NPs), we conceived a new NP drug hit discovery strategy that consists in frontloading samples with 1D NMR descriptors to predict compounds with anticancer activity prior to bioactivity screening for NPs discovery, approach B. The NMR QSAR classification models were built using 1D NMR data (¹H and 13C) as descriptors, from 50 crude extracts, 55 fractions and five pure compounds obtained from actinobacteria isolated from marine sediments collected off the Madeira Archipelago. The overall predictability accuracies of the best model exceeded 63% for both training and test sets.

A Computer-Driven Approach to Discover Natural Product Leads for Methicillin-Resistant Staphylococcus aureus Infection Therapy.

The risk of methicillin-resistant Staphylococcus aureus (MRSA) infection is increasing in both the developed and developing countries. New approaches to overcome this problem are in need. A ligand-based strategy to discover new inhibiting agents against MRSA infection was built through exploration of machine learning techniques. This strategy is based in two quantitative structure⁻activity relationship (QSAR) studies, one using molecular descriptors (approach A) and the other using descriptors (approach B). In the approach A, regression models were developed using a total of 6645 molecules that were extracted from the ChEMBL, PubChem and ZINC databases, and recent literature. The performance of the regression models was successfully evaluated by internal and external validation, the best model achieved R² of 0.68 and RMSE of 0.59 for the test set. In general natural product (NP) drug discovery is a time-consuming process and several strategies for dereplication have been developed to overcome this inherent limitation. In the approach B, we developed a new NP drug discovery methodology that consists in frontloading samples with 1D NMR descriptors to predict compounds with antibacterial activity prior to bioactivity screening for NPs discovery. The NMR QSAR classification models were built using 1D NMR data (¹H and 13C) as descriptors, from crude extracts, fractions and pure compounds obtained from actinobacteria isolated from marine sediments collected off the Madeira Archipelago. The overall predictability accuracies of the best model exceeded 77% for both training and test sets.

Metabolomic Fingerprinting of Salinispora From Atlantic Oceanic Islands.

Salinispora (Micromonosporaceae) is an obligate marine bacterium genus consisting of three species that share over 99% 16S rRNA identity. The genome and biosynthetic pathways of the members of this genus have been widely investigated due to their production of species-specific metabolites. However, despite the species' high genetic similarity, site-specific secondary metabolic gene clusters have been found in Salinispora strains collected at different locations. Therefore, exploring the metabolic expression of Salinispora recovered from different sites may furnish insights into their environmental adaptation or their chemical communication and, further, may lead to the discovery of new natural products. We describe the first occurrence of Salinispora strains in sediments from the Saint Peter and Saint Paul Archipelago (a collection of islets in Brazil) in the Atlantic Ocean, and we investigate the metabolic profiles of these strains by employing mass-spectrometry-based metabolomic approaches, including molecular networking from the Global Natural Products Social Molecular Networking platform. Furthermore, we analyze data from Salinispora strains recovered from sediments from the Madeira Archipelago (Portugal, Macaronesia) in order to provide a wider metabolomic investigation of Salinispora strains from the Atlantic Oceanic islands. Overall, our study evidences a broader geographic influence on the secondary metabolism of Salinispora than was previously proposed. Still, some biosynthetic gene clusters, such as those corresponding to typical chemical signatures of S. arenicola, like saliniketals and rifamycins, are highly conserved among the assessed strains.