Metabolites from marine invertebrates and their symbiotic microorganisms: molecular diversity discovery, mining, and application
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Metabolites from marine organisms have proven to be a rich source for the discovery of multiple potent bioactive molecules with diverse structures. In recent years, we initiated a program to investigate the diversity of the secondary metabolites from marine invertebrates and their symbiotic microorganisms collected from the South China Sea. In this review, representative cases are summarized focusing on molecular diversity, mining, and application of natural products from these marine organisms. To provide a comprehensive introduction to the field of marine natural products, we highlight typical molecules including their structures, chemical synthesis, bioactivities and mechanisms, structure–activity relationships as well as biogenesis. The mining of marine-derived microorganisms to produce novel secondary metabolites is also discussed through the OSMAC strategy and via partial chemical epigenetic modification. A broad prospectus has revealed a plethora of bioactive natural products with novel structures from marine organisms, especially from soft corals, gorgonians, sponges, and their symbiotic fungi and bacteria.
KeywordsMarine invertebrates Symbiotic microorganisms Marine natural products Molecular diversity Bioactivities Marine drugs
The marine environment has been proven to be a rich source of new bioactive natural products for drug discovery. Coral reefs are among the most productive ecosystems and are a source of a large group of structurally unique biosynthetic products. To date, more than 40,000 marine natural products (MNPs) have been identified from various marine organisms, such as sponges, cnidarians, tunicates, molluscs, echinoderms, bryozoans, red algae, brown algae, green algae, and microorganisms (Carroll et al. 2019; Deshmukh et al. 2017; Jiménez 2018; Leal et al. 2016; Newman and Cragg 2016b). The upward trend in the discovery of new MNPs sourced from marine microorganisms continues unabated and now represents 57% of the total new MNPs reported in 2017 (Carroll et al. 2019). Based upon the putative biogenetic origins, these MNPs can be classified as polyketides, terpenoids, alkaloids, steroids, lactones, peptides, phenols, and lipids. Also, a large proportion of MNPs display interesting pharmaceutical activities, such as cytotoxic, antimicrobial, hypolipidemic, anti-inflammatory, antimalarial, analgesic, and antiasthmatic activities (According to the following websites: http://marinepharmacology.midwestern.edu; Jiménez 2018). Hence, MNPs are considered as an excellent and potentially valuable source for new chemical entities with novel structures and distinct mechanisms of action. To date, there have been 13 approved therapeutic agents that could be considered derivatives of MNPs (Altmann 2017; Jiménez 2018; Newman 2019; Pereira et al. 2019). Moreover, more than 30 MNP derivatives constitute the global marine pharmaceutical clinical pipeline in Phases III, II or I of drug development (According to the following websites: http://marinepharmacology.midwestern.edu; http://pharma.id.informa.com (accessed on August 6, 2019); Jiménez 2018; Newman 2019; Pereira et al. 2019). The significant potential for new drug development based on MNPs in all disease areas has been previously discussed (Newman and Cragg 2016a).
Marine invertebrates have proven to be a primary source of bioactive MNPs, as many serve as chemical defense tools against predators, competitors and other ecological pressures. It has been demonstrated that the true origin of most MNPs appears to be the microorganisms living in concert with invertebrates. Most invertebrates are sessile, soft-bodied and move slowly, and are thus subject to potential parasite predation and detrimental microbial colonization. Therefore, they require a complex arsenal of secondary metabolites produced by their symbiotic microorganisms to facilitate a form of chemical defense (Jiménez 2018; Wang et al. 2008). This is likely the reason why MNPs from marine invertebrates and their symbiotic microorganisms are a rich sources of diverse and bioactive secondary metabolites (Martins et al. 2014; Mayer et al. 2010; Newman and Cragg 2016b). The chemical ecology underlying invertebrate–microorganism interactions provides a great opportunity for natural product chemists to mine for novel drug discovery. Therefore, the invertebrates and the abundant microorganisms in their ecosystems have attracted widespread attention for producing novel structural metabolites with potential bioactive applications (Blunt et al. 2018).
In the recent decade, the China Sea, especially the South China Sea, has become a hot spot in searching for novel bioactive MNPs. The invertebrates including sponges, soft corals, gorgonians and tunicates are prolific in the coral reefs in the South China Sea, and the microorganisms associated with these invertebrates have been demonstrated as a distinctive source for new bioactive MNPs.
Macrocyclic lactones derived from marine organisms have attracted much attention on account of their multiple potent biological activities including antitumor (Hirata and Uemura 1986; Isaka et al. 2002, 2009; Qi and Ma 2011; Suo et al. 2018), antifungal (Shier et al. 2001), and antimalarial activities (Isaka et al. 2009). For instance, caniferolides A and B, two glycosylated 36-membered polyol macrolides from marine-derived Streptomyces sp., showed pronounced antifungal activity (Perez-Victoria et al. 2019). Peloruside E, a macrolide from the New Zealand marine sponge Mycale hentscheli, displayed potent antiproliferative activity (Hong et al. 2018). As part of our current research, 88 macrolides with unique structures and significant biological properties have been obtained from marine invertebrates and their symbiotic microorganisms (Liu et al. 2014; Shao et al. 2011, 2015a, 2018). To obtain more valuable undiscovered natural compounds, multiple strategies and methods, including OSMAC and chemical epigenetic manipulation, were applied to microorganisms. Structural modification and chemical synthesis were performed to provide more derivatives, and the structure–activity relationships were discussed (Zhang et al. 2017). The molecular mechanisms of compounds with strong activities were also investigated (Wang et al. 2016).
The molecular mechanism underlying antifouling activity of 1 against the cyprids of barnacle A. amphitrite has been investigated using the isobaric tags for the relative or absolute quantification (iTRAQ) labeling proteomic method (Wang et al. 2016). Differentially expressed proteins were examined by analyzing the changes in the proteome of A. amphitrite cyprids in response to 1 treatment. The results suggested that compound 1 affected the cytochrome P450, glutathione S-transferase (GST) and NO/cGMP pathways. The results of real-time PCR further demonstrated that the NO/cGMP pathway was activated in response to 1. Larval settlement assays suggested that S-methylisothiourea sulfate (SMIS) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) rescued cyprids from 1 (cochliomycin A)-induced inhibition of larval settlement. These findings supported the hypothesis that 1 inhibited barnacle larval settlement through stimulating the NO/cGMP pathway (Wang et al. 2016).
However, further research on compound 1 and its analogues was hindered by strain-specific variation of the strain C. lunatus (M351), especially altering metabolite profiles when re-cultured. Fortunately, another fungal strain C. lunatus (TA26-46) derived from the sea anemone Palythoa haddoni was also found to produce 14-membered RALs. Three new 14-membered RALs, 4–6 (cochliomycins D–F) together with eight known analogues 1 (cochliomycin A), 7 ((7′E)-6′-oxozeaenol), 8 (deoxy-aigialomycin C), 9 (LL-Z1640-2), 10 (zeaenol), 11 (LL-Z1640-1), 12 (paecilomycin F) and 13 (aigialomycin B) (Fig. 3) were obtained (Liu et al. 2014). Compounds 4, 6–9, and the acetonide derivative 9a displayed strong anti-larval settlement activity against B. amphitrite (EC50 = 1.82 to 22.5 μg/mL; LC50/EC50 > 50), suggesting that they might be promising environmentally benign antifouling agents (Liu et al. 2014).
Noticeably, besides significant antifouling activity, compound 9 (LL-Z1640-2) produced by C. lunatus (TA26-46) exhibited promising inhibitory activity against phytopathogenic fungal strain Pestalotia calabae, with a minimum inhibitory concentration (MIC) value of 0.39 μmol/L, 25-fold stronger than that of the positive control, ketoconazole (MIC = 10 μmol/L) (Liu et al. 2014). A fungicide whole plant assay suggested that 9 exhibited pronounced activity on the phytopathogenic fungus Plasmopara viticola preventative test at 6 × 10−6 and concentration-dependent activity on the phytopathogenic fungus Phytophthora infestans preventative application at 200, 60, and 20 × 10−6. Compound 9 could be considered as a promising new natural fungicide lead (Liu et al. 2014).
Facing the challenges of discovery repetition and silent biosynthetic pathways, only limited mining approaches were applied in our work to date. As well known, microorganisms are easily manipulated on the genetic level. With gene sequencing technology blooming, genetic techniques and bioinformatics algorithms will continue to provide more opportunities for disclosing the novel pathways within marine microorganisms to then enable exploration of the untapped valuable bioactive molecules encoded within.
The diverse activities of anthraquinones isolated from marine-derived fungi with cytotoxic (Chen et al. 2013a; Xie et al. 2010; Zhu et al. 2012), antiviral (Huang et al. 2017), antioxidant (Li et al. 2016) and other activities have attracted many interests for drug discovery. For example, SZ-685C, a hydroanthraquinone isolated from the marine-derived Halorosellinia fungus, exhibited potent cytotoxic activity (Chen et al. 2013a; Xie et al. 2010; Zhu et al. 2012). In our investigation of bioactive anthraquinone derivatives from marine-derived fungi (Yang et al. 2012; Zheng et al. 2012), a total of 54 anthraquinone monomers and dimers have been isolated from Nigrospora sp., Alternaria sp. and other fungal genera. Analysis of anthraquinones with various chemical structures possessing interesting biological activities revealed the key structural features required for their activities.
The azaphilone molecules are a structurally variable family of fungal polyketide metabolites with a highly oxygenated pyranoquinone bicyclic core and exhibiting multiple bioactivities such as cytotoxic (Yamada et al. 2008), antimicrobial (Che et al. 2002), antiviral (Wang et al. 2011a), and anti-inflammatory (Yasukawa et al. 2008) activities. For instance, sclerketide C, an azaphilone analogous isolated from gorgonian-derived fungus Penicillium sclerotiorin CHNSCLM-0013, exhibited significant anti-inflammatory activity (Liu et al. 2019). Pleosporalone B from the culture of marine-derived fungus Pleosporales sp. CF09-1 and penicilazaphilone C from Penicillium sclerotiorum M-22 displayed potent antimicrobial activities (Cao et al. 2019; Zhou et al. 2016). Azaphilones have attracted much attention due to their fascinating structural features and distinguished bioactivities (Wei et al. 2017). Our previous work on metabolites produced by symbiotic microorganisms of marine invertebrates found 48 azaphilones, including 37 new compounds, many of which were reported to exhibit antifouling and antiviral activities (Wang et al. 2018; Wei et al. 2017; Zhao et al. 2015a). Desiring promising antifouling molecules, a one-step semisynthetic method was applied to discover more new azaphilonoids derivatives.
Marine-derived alkaloids represent a class of compounds with particularly privileged structures, which have been frequently encountered in a vast number of pharmaceuticals as well as agrochemicals (Bandini and Eichholzer 2009; Hibino and Choshi 2001; Humphrey and Kuethe 2006; Kochanowska-Karamyan and Hamann 2010; Lounasmaa and Tolvanen 2000). For example, raistrickindole A, an indole diketopiperazine alkaloid obtained from the marine-derived fungus Penicillium raistrickii, displayed antiviral activity against the hepatitis C virus (Li et al. 2019a). Agelastatin A, a bromopyrrole marine alkaloid isolated from the Mexican sponge, Agelas sp., exhibited strong antineoplastic activity (Pettit et al. 2005). Our group has isolated more than 100 alkaloids with promising biological activities from diverse fungal strains derived from marine invertebrates (Chen et al. 2014a; Jia et al. 2015; Shao et al. 2015b; Wang et al. 2015a). To obtain significant antibacterial compounds, efficient and environmental friendly methods for synthesis of unsymmetrical bisindolylmethanes or triarylmethane were developed (Wen et al. 2015).
Terpenoids constitute a class of broadly active natural products isolated from a diverse range of marine organisms. For example, 11R-methoxy-5,9,13-proharzitrien3-ol, obtained from an endophytic fungus Trichoderma harzianum X-5 derived from the marine brown alga Laminaria japonica, displayed growth inhibition of some marine phytoplankton species (Song et al. 2018). Nakijinol G, a meroterpenoid obtained from a sponge Hyrtios sp. collected from the South China Sea, showed protein tyrosine phosphatase (PTP1B) inhibitory activity (Wang et al. 2017). Trichodermanin C, a diterpenes obtained from a fungal strain Trichoderma harzianum OUPS-111D-4 derived from sponge Halichondria okadai, exhibited significant cytotoxic activity (Yamada et al. 2017). In our ongoing research on the marine invertebrates and their symboitic microorgnisms, 101 terpenoids including 43 new compounds with novel structures were isolated, which exhibited antibacterial, cytotoxic and antifouling activities (Cao et al. 2015, 2017; Li et al. 2012a).
Steroid derivatives from marine organisms are noted for diverse unusual structures with multiple potent biological properties. For instance, petasitosterone B, a steroid isolated from a Formosan marine soft coral Umbellulifera petasites exhibited promising anti-inflammatory activity (Huang et al. 2016). Two 9,11-secosteroidal glycosides, sinularosides A and B, isolated from the South China Sea soft coral Sinularia humilis, exhibited potent antimicrobial activity (Sun et al. 2012). In our previous reports, 86 steroidal compounds were obtained from marine invertebrates and their symbiotic fungi, which exhibited cytotoxic, antiviral and antibacterial activities (Cao et al. 2014; Sun et al. 2015; Zhao et al. 2013).
Phenylpropanoids contain many chemical structure types, such as chromone, coumarin, flavone and lignin, which possess various biological activities including antioxidant, cytotoxic, antimicrobial, and enzyme inhibitory activities. For example, arthone C, a chromone derivative isolated from a deep-sea-derived fungus Arthrinium sp., exhibited potent antioxidant activity (Bao et al. 2018). Two tetracyclic coumarin derivatives and two coumarin dimers isolated from the marine-derived fungus Eurotium rubrum showed significant tyrosinase inhibitory activity (Kamauchi et al. 2018). In our previous studies, 80 phenylpropanoid compounds with 54 new compounds were obtained, which showed antifouling, antibacterial and cytotoxic activities (Qi et al. 2013; Zhao et al. 2015b). These findings provide further insight into the chemical diversity and biological activities of this class of MNPs.
Peptides isolated from many marine species present various biological activities, such as antimicrobial, antiviral, antitumor, antioxidative and cardioprotective activities. For instance, mirabamide A, a cyclic depsipeptide obtained from the sponge Siliquariaspongia mirabilis, showed potent inhibitory activity on HIV-1 fusion (Plaza et al. 2007). Reniochalistatin E, an octapeptide isolated and characterized from the marine sponge Reniochalina stalagmitis, displayed significant cytotoxic activity (Zhan et al. 2014). In our studies, a total of 34 peptides were identified, of which more than half are cyclopeptides involved in 18 new peptides (Chen et al. 2014c; Hou et al. 2019b, 2019c). Particularly, LC–MS/MS-dependent molecular networking and 1H NMR techniques were applied in order to identify new peptides.
Until now, the molecular networking approach has only been applied to mine the undiscovered cycloheptapeptides from marine-derived fungus in our research on MNPs. Undoubtedly, the molecular networking strategy based on LC–MS/MS and relevant data libraries should be considered an effectively targeted isolation technique to speed the discovery of new cryptic secondary metabolites. It is worth applying the molecular networking approach to exploit novel molecules from the pools of MNPs in extracts from marine organisms.
Phenyl ether derivatives
An increasing number of marine-derived phenyl ether derivatives with novel structures have been reported with diverse biological activities, including antimicrobial, antitumor, cardiotonic, antidiabetic, antiinflammatory and antiviral activities. For instance, 4-carbglyceryl-3,30-dihydroxy-5,50-dimethyldiphenyl ether, a diphenyl ether isolated from the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502 showed potent antifouling activity (Huang et al. 2017). A series of antibacterial polybrominated diphenyl ethers were isolated from the Indonesian sponge Lamellodysidea herbacea (Hanif et al. 2007). In our studies, 35 phenyl ether derivatives were obtained in all, among them 9 compounds were first reported, showing antibacterial, antifouling and cytotoxic activities (Chen et al. 2013b; Shi et al. 2017). To clarify the structure–activity relationships, chemical synthesis was performed (Chen et al. 2013b).
The South China Sea possesses rich and unique species resources, with more than 95% of the invertebrates mainly existing in the coral reefs in this sea area in China (Zhang et al. 2006). Not surprisingly, many investigations on marine invertebrates and their symbiotic microorganisms target this “biodiversity hotspot” as the center of sample collection. It should be mentioned that the diverse abundance of MNPs derived from marine invertebrates and their symbiotic microorganisms from the South China Sea were discovered and studied. In recent decades, research on the diversity of MNPs discovered from the South China Sea by other Chinese researchers has been well underway. These groundbreaking research efforts contributed to the discovery of a great number of novel and promising bioactive molecules usually from such sources as sponges (Gui et al. 2019; Jiao et al. 2019; Wang et al. 2015b), corals (Li et al. 2019b; Wu et al. 2019), bryozoans (Yu et al. 2015), and associated microorganisms (Cheng et al. 2016; Nong et al. 2016). Regarding the abundance of MNPs with diverse structures and a wealth of biological activities, we believe that only the proverbial “tip of the iceberg” has been explored from the South China Sea, and the resulting novel active metabolites with potential pharmacology applications are worthy of further exploration.
In this review, we exemplified nine types of structurally unique MNPs including macrocyclic lactones, anthraquinones, azaphilones, alkaloids, terpenoids, steroids, phenylpropanoids, peptides, and phenyl ether derivatives obtained from marine invertebrates and their symbiotic microorganisms. Our research provides several typical representative MNPs from marine invertebrates, especially sponges, soft corals, gorgonian corals, and their symbiotic microorganisms (mainly fungi). These MNPs display various potent bioactivities involved in not only chemoecological effects such as antifouling, ichthyootoxic, and brine shrimp lethal activities but also pharmaceutical activities including antibacterial, antiviral, fungicidal, cytotoxic, and antimalarial activities. Our studies demonstrate that MNPs derived from marine invertebrates and their symbiotic microorganisms in the South China Sea are a prolific resource for the discovery of bioactive MNPs. It could be expected that the symbiotic microorganisms associated with marine invertebrates have great potential as a significant source of structurally interesting molecules. It should be noted that the application of multiple discovery strategies and methods could effectively promote the exploitation of novel MNPs with diverse structures. In our study, different methodological approaches, including single culture, OSMAC, chemical epigenetic manipulation, co-culture, structural modification and chemical synthesis, have been applied in the search for new MNPs. Among them, co-culture, structural modification and chemical synthesis were found to be effective approaches to obtain potential bioactive MNPs. Particularly, LC–MS/MS-dependent molecular networking has been applied as a promising approach to dereplicate complex natural product mixtures, contributing to the targeted isolation of a series of new compounds. It is anticipated that future genetic techniques and bioinformatics tools, especially metagenomic approaches, genome mining, and heterologue biosynthesis, could accelerate the exploration and accessibility of remaining undiscovered MNPs with novel structures and promising bioactivities from marine microorganisms.
This work is supported by the National Natural Science Foundation of China (nos. 41830535; 81673350; U1706210), the Fundamental Research Funds for the Central Universities of China (no. 201962002), and the Taishan Scholars Program, China.
LL and YYZ collected the data and wrote the paper; CLS and CYW designed, directed and revised the manuscript.
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Conflict of interest
The authors declare that they have no conflict of interest.
Animal and human rights statement
This article does not contain any studies with human participants or animals performed by any of the authors.
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