Cnidarians as a Source of New Marine Bioactive Compounds—An Overview of the Last Decade and Future Steps for Bioprospecting

Marine invertebrates are rich sources of bioactive compounds and their biotechnological potential attracts scientific and economic interest worldwide. Although sponges are the foremost providers of marine bioactive compounds, cnidarians are also being studied with promising results. This diverse group of marine invertebrates includes over 11,000 species, 7500 of them belonging to the class Anthozoa. We present an overview of some of the most promising marine bioactive compounds from a therapeutic point of view isolated from cnidarians in the first decade of the 21st century. Anthozoan orders Alcyonacea and Gorgonacea exhibit by far the highest number of species yielding promising compounds. Antitumor activity has been the major area of interest in the screening of cnidarian compounds, the most promising ones being terpenoids (monoterpenoids, diterpenoids, sesquiterpenoids). We also discuss the future of bioprospecting for new marine bioactive compounds produced by cnidarians.


Introduction
In terms of biodiversity, marine environments are among the richest and most complex ecosystems. Harsh chemical and physical conditions in the environment have been important drivers for the production of a variety of molecules with unique structural features. These marine molecules exhibit various types of biological activities [1], with compounds of high economic interest having potential applications in the pharmaceutical and medical sectors. Although nearly 20,000 compounds have been discovered since the field of marine bioactive compound biochemistry began in the mid-1960s, only a very limited number have seen industrial application. It has been clear since marine bioprospecting began that the world's oceans and their diverse biota represent a significant resource, perhaps the greatest resource on Earth, for the discovery of new bioactive compounds. Early National Cancer Institute (NCI) programs in the USA demonstrated that marine invertebrates were a superb source of potential lead molecules. The decisive boost to this new age of bioprospecting was provided by the NCI when it was found that bioassays with marine organism extracts were far more likely to yield anticancer drugs than terrestrial sources [2]. In this way, it is not surprising that over the past 40 years major advances in the discovery of marine drugs have been recorded in clinical trials for cancer [3]. Apart from anticancer activity, these compounds have proven to be an abundant source of pharmacologically active agents for the production of therapeutic entities [4] against AIDS, inflammatory conditions and microbial diseases.
Marine bioactive compounds display varied potential applications, namely as molecular tools, in cosmetics, as fine chemicals, as nutraceuticals and in agrochemical industries [5].
Although only a few marine-derived products are currently on the market (e.g., Prialt ® and Yondelis ® ), several new compounds are now in the clinical pipeline and several more are in clinical development. The few approvals so far for the commercialization of drugs from the sea have not been due to a lack of discovery of novel marine bioactive compounds, but because of the complexity of issues raised upon the development of these products [4]. Faulkner [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], Blunt et al. [21][22][23][24][25][26][27][28][29], and Mayer [30][31][32][33][34][35][36][37][38] have provided extensive reviews on the total number of marine natural products (MNPs) discovered over the last 25 years, the most promising ones being produced by marine invertebrates. Sponges (phylum Porifera) have long been recognized as the most interesting group of marine invertebrates for the discovery of new drugs [5,39,40]. However, with growing bioprospecting efforts and the screening of previously unexplored marine habitats, the biotechnological potential of other groups of marine invertebrates has also started to attract the attention of researchers. The ability of cnidarians (such as jellyfish, sea anemones and corals) to produce powerful toxins and venoms [41] has been well documented. However, further research has demonstrated that MNPs produced by cnidarians are more than toxins and venoms. The phylum Cnidaria is a large, diverse and ecologically important group of marine invertebrates that includes over 11,000 extant species [42]. Over 3000 MNPs have been described from this phylum alone, mostly in the last decade.
In this work, we present an overview of the most promising marine bioactive compounds isolated from cnidarians in the first decade of the 21st century, which may have applications in the therapy of human diseases. The present study also discusses future perspectives for the bioprospecting of new MNPs produced by this speciose group of marine invertebrates.

Methodology
The most relevant peer reviewed literature published during the first decade of the 21st century covering MNPs was surveyed for the present work [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. During this period alone, over 2000 molecules from cnidarians were described. In order to focus our study and address only those compounds displaying a high potential for industrial applications, we have decided to use as guidelines the values of IC 50 (half maximal inhibitory concentration). IC 50 is a quantitative measure which indicates how much of a particular substance (inhibitor) is needed to inhibit a given biological process or component of a process by half. It is important to highlight that the NCI has renamed the IC 50 to GI 50 [43] in order to emphasize the correction for cell count at time zero in cancer cells; in this way, some results on this quantitative measure are now also presented under these directives. Additionally, the ED 50 (the median dose that produces the desired effect of a drug in half the test population) was also used to identify promising marine bioactive compounds produced by cnidarians. Only the compounds displaying an IC 50 ≤ 10.0 µg/mL or µM (except where stated otherwise) and ED 50 ≤ 4.0 µg/mL were considered for the present study, as these values are commonly used in the surveyed literature to ascertain relevant bioactivity (e.g., [44,45]). In the few cases were neither IC 50 nor ED 50 values were described for a MNP in a manuscript, that compound was selected to be part of the present survey only if either the authors of that manuscript, or those citing that manuscript, clearly stated that the results recorded were highly promising for industrial applications. All species producing the compounds selected for the present work were grouped into classes and orders of phylum Cnidaria (Table 1) (according to the classification proposed in the World Register of Marine Species (WoRMS)) [46]. This approach allowed us to identify which taxonomic groups of cnidarians screened so far display the highest potential to yield new drugs or pharmacological products derived from marine bioactive compounds. Nonetheless, it is important to highlight that cnidarian species identification is a challenging task and it is possible that some of the species (or even genera) referred to in the scientific literature may not be correct [47]. In this way, it is of paramount importance that in future works the authors addressing marine bioactive compounds produced by cnidarians provide a detailed description on how target species have been identified.

Class Anthozoa
Class Anthozoa currently includes 10 orders and over 7500 valid species (about 2/3 of all known cnidarian species) ( Table 1). Within the Anthozoa, the order Alcyonacea (soft corals) and Gorgonacea (sea fans) are the ones which have contributed with the highest number of promising bioactive marine compounds, although other orders, such as Actiniaria (sea anemones) and Scleractinia (hard corals), have also yielded relevant compounds [48][49][50][51].
Another example of a potential new therapeutic anticancer agent is a cembranolide diterpene from Lobophytum cristagalli, which has shown a potent inhibitory activity (IC 50 0.15 μM) [59] over farnesyl protein transferase (FPT, an important protein in signal transduction and regulation of cell differentiation and proliferation [99]). This type of FPT inhibition enhanced interest in this group of metabolites [86]. Other species of this genus also showed cembranolide diterpenes (lobophytene) with significant cytotoxic activity against human lung adenocarcinoma (A549) and human colon adenocarcinoma (HT-29) cell lines [56]. Lobophytum durum and Lobophytum crassum produce durumolides A-C [60], durumhemiketalolide A-C [61] and crassumolides A and C [58], with anti-inflammatory effects. They have been shown to inhibit up-regulation of the pro-inflammatory iNOS and COX-2 proteins in LPS-stimulated murine macrophage cells at IC 50 < 10 µM [58,60]. The diterpenoids, lobohedleolide, (7Z)-lobohedleolide, and 17-dimethylaminolobohedleolide, were isolated from the aqueous extract of Lobophytum species and exhibited moderate HIV-inhibitory activity (IC 50 approximately 7-10 μg/mL) in a cell-based in vitro anti-HIV assay [57].
Klyxum simplex produces diterpene compounds, such as simplexin E, which at a concentration of 10 μM was found to considerably reduce the levels of iNOS and COX-2 proteins to 4.8 ± 1.8% and 37.7 ± 4.7%, respectively. These results have shown that this compound significantly inhibits the accumulation of the pro-inflammatory iNOS and COX-2 proteins in LPS-stimulated RAW264.7 macrophage cells [54]. This species also produces two diterpene compounds, klysimplexins B and H, exhibiting moderate cytotoxicity towards human carcinoma cell lines. Klysimplexin [55].
Antifouling agents from natural sources are of increasing interest since the International Maritime Organization (IMO) banned the use of certain antifouling agents, such as tri-n-butyltin (TBT), due to the ecological impacts of these biocides in the marine environment. Several studies have demonstrated that soft corals can yield large quantities of promising antifouling metabolites [102,103]. In fact, 17.95% of potential antifouling natural compounds are from cnidarians (e.g., soft coral) [104]. One of the most promising natural antifouling agent identified so far is an isogosterone isolated from an unspecified Dendronephthya [77].
The genus Clavularia contains secondary metabolites with unique structures and remarkable biological activities. Some of the species in this genus produce prostanoids (icosanoids) [45,72,73,105,106], steroids [75] and diterpenoids [70,107]. The bioactive marine diterpene, stolonidiol, isolated from an unidentified Clavularia, showed potent choline acetyltransferase (ChAT) inducible activity in primary cultured basal forebrain cells and clonal septal SN49 cells, suggesting that it may act as a potent neurotrophic factor-like agent on the cholinergic nervous system [69]. Cholinergic neurons in the basal forebrain innervate the cortex and hippocampus, and their function may be closely related to cognitive function and memory. The degeneration of neuronal cells in this brain region is considered to be responsible for several types of dementia including Alzheimer's disease. One of the neurotransmitters, acetylcholine, is synthesized from acetyl coenzyme A and choline by the action of ChAT. Therefore, induction of ChAT activity in cholinergic neurons may improve the cognitive function in diseases exhibiting cholinergic deficits [108][109][110].
Telesto riisei produces punaglandins, highly functional cyclopentadienone and cyclopentenone prostaglandins. Cyclopentenone prostaglandins have unique antineoplastic activity and are potent growth inhibitors in a variety of cultured cells. These punaglandins have been shown to inhibit P53 accumulation (a tumor suppressor protein) and ubiquitin isopeptidase activity (IC 50 between 0.04 and 0.37 µM) (enzyme involved in protein degradation system) in vitro and in vivo [76]. Since these proteasome inhibitors exhibit higher antiproliferative effects than other prostaglandins [112], they may represent a new class of potent cancer therapeutics.
Fuscosides, originally isolated from Eunicea fusca [138], selectively and irreversibly inhibited leukotriene synthesis. Leukotrienes are molecules of the immune system that contribute to inflammation in asthma and allergic rhinitis and its production is usually related to histamine release [143]. Pharmacological studies indicated that fuscoside B inhibits the conversion of arachidonic acid (AA) to leukotriene B 4 and C 4 (LTB 4 and LTC 4 ) [138,144] by inhibiting the 5-Lipoxygenase (5-LO), in the case of LTB 4 with an IC 50 of 18 μM [144]. These selective inhibitors of lipoxygenase isoforms can be useful as pharmacological agents, as nutraceuticals or as molecular tools [99]. Sesquiterpenoids metabolites isolated from Eunicea sp. display antiplasmodial activity against the malaria parasite P. falciparum W2 (chloroquine-resistant) strain, with IC 50 values ranging from 10 to 18 µg/mL [137].
The gorgonian Junceella fragilis produces secondary metabolites, frajunolides B and C, with anti-inflammatory effects towards superoxide anion generation and elastase release by human neutrophils, with an IC 50 > 10 µg/mL [121]. When properly stimulated, activated neutrophils secrete a series of cytotoxins, such as the superoxide anion (O 2 •− ), a precursor of other reactive oxygen species (ROS), granule proteases, and bioactive lipids [145,146]. The production of the superoxide anion is linked to the killing of invading microorganisms, but it can also directly or indirectly damage surrounding tissues. On the other hand, neutrophil elastase is a major secreted product of stimulated neutrophils and a major contributor to the destruction of tissue in chronic inflammatory disease [147]. The anti-inflammatory butenolide lipide [148] from the gorgonian Euplexaura flava [139] can be currently synthesized, opening the possibility of advancing into a new level of anti-inflammatory pharmaceuticals.
Some of the most interesting compounds identified so far in the on-going search for new anti-fouling agents have been recorded in the order Gorgonacea. Good examples of such compounds are juncin ZII from Junceella juncea [122], homarine from Leptogorgia virgulata and Leptogorgia setacea [123], pukalide and epoxypukalide recorded so far only from L. virgulata [124].
Species of genus Briareum (family Briareidae) ( Figure 1D) (which commonly exhibit an incrusting appearance rather than the fan-like shape of many gorgonians) are widely abundant in Indo-Pacific and Caribean coral reefs. These organisms have been recognized as a valuable source of bioactive compounds with novel structural features. Briarane-related natural products are a good example of such promising compounds due to their structural complexity and biological activity [149,150]. Briaexcavatin E, from Briareum excavata (Nutting 1911), also occasionally referred to as Briarium excavatum, inhibited human neutrophil elastase (HNE) release with an IC 50 between 5 and 10 µM [118]. Briaexcavatolides L and P, diterpenoids from the same species exhibited significant cytotoxicity against mouse lymphocytic leukemia (P-388) tumor cells with ED 50 of 0.5 [119] and 0.9 μg/mL [151], respectively. Diterpenoids produced from Briareum polyanthes (presently accepted as Briareum asbestinum), namely Briarellin D, K and L, exhibited antimalarial activity against P. falciparum with an IC 50 between 9 and 15 µg/mL [120].

Other Orders
Sea anemones (order Actiniaria) are a rich source of biologically-active proteins and polypeptides. Several cytolytic toxins, neuropeptides and protease inhibitors have been identified from them [48]. In addition to several equinatoxins, potent cytolytic proteins and an inhibitor of papain-like cysteine proteinases (equistatin), were isolated from the sea anemone Actinia equina [152]. Equistatin has been shown to be a very potent inhibitor of papain and a specific inhibitor of the aspartic proteinase cathepsin D [153]. While papain-like cysteine proteases have been implicated in various diseases of the central nervous system, such as brain tumors, Alzheimer's disease, stroke, cerebral lesions, neurological autoimmune diseases and certain forms of epilepsy [154], aspartic proteinase cathepsin D is involved in the pathogenesis of breast cancer [155] and possibly Alzheimer's disease [156].
Cycloaplysinopsin C, a bis(indole) alkaloid isolated from Tubastrea sp. (order Scleractinia), was found to inhibit growth of two strains of P. falciparum, one chloroquine-sensitive (F32/Tanzania) and other chloroquine-resistant (FcB1/Colombia) with IC 50 1.48 and 1.2 μg/mL, respectively [51]. Cladocorans A and B, isolated from Cladocora caespitosa (order Scleractinia) [49], are marine sesterterpenoids which possess a γ-hydroxybutenolide moiety, which is thought to be responsible for the biological activity of these compounds. The potent anti-inflammatory activity of these natural metabolites was attributed to the inhibition of secretory phospholipase A 2 (sPLA 2 , IC 50 0.8-1.9 μM). Given the general role of inflammation in diseases that include bronchial asthma and rheumatoid arthritis, identifying and developing potent inhibitors of sPLA2 continues to be of great importance for the pharmaceutical industry, with this type of metabolite being of paramount importance for future research [50].

Class Hydrozoa
Class Hydrozoa includes seven orders and nearly 3500 valid species (Table 1) Immune escape plays an important role in cancer progression and, although not completely understood, it has been proposed that indoleamine 2,3-dioxygenase (IDO) plays a central role in evasion of T-cell-mediated immune rejection [157]. IDO catalyzes the oxidative cleavage of the 2,3 bond of tryptophan, which is the first and rate-limiting step in the kynurenine pathway of tryptophan catabolism in mammalian cells [158]. The polyketides annulins A, B, and C, purified from the marine hydroid Garveia annulata (order Anthoathecata), potently inhibited IDO in vitro (K i 0.12-0.69 μM) [159]. These annulins are more powerful than most tryptophan analogues known to be IDO inhibitors. These compounds are active at concentrations higher than ~10 μM and therefore more effective than 1-methyltryptophan (K i 6.6 μM), one of the most potent IDO inhibitors currently available [160]. Solandelactones C, D, and G are cyclopropyl oxylipins isolated from the hydroid Solanderia secunda (order Anthoathecata) and exhibit moderate inhibitory activity against farnesyl protein transferase (FPT, 69, 89, and 61% inhibition, respectively) at a concentration of 100 μg/mL [161]. Note that FPT is associated with cell differentiation and proliferation and its inhibition may be a target for novel anticancer agents (as already referred above for the soft coral L. cristagalli).

Class Scyphozoa
Approximately 200 species are currently classified in three orders in class Scyphozoa (Table 1). However, in the last decade, only a single MNP purified from the mesoglea of the jellyfish Aurelia aurita (order Semaeostomeae) was considered to be promising enough to be included in the present work. This compound is a novel endogenous antibacterial peptide, aurelin, which exhibited activity against Gram-positive and Gram-negative bacteria. As an example, aurelin displayed an IC 50 of 7.7 µg/mL for Esherichia coli (Gram negative bacteria) [162].

Other Classes
The classes Staurozoa, Cubozoa and Polypodiozoa are the least speciose in the phylum Cnidaria (Table 1). This fact may explain the current lack of data on secondary metabolites produced by these organisms. It is possible that with growing bioprospecting new MNPs may be revealed once these cnidarian species are screened. Cubozoa (box jellies), for example, produce some of the most harmful cnidarian toxins for humans [163].

Exploring the Unexplored and Being Creative: Future Perspectives for the Bioprospecting of Cnidarians
For several years, the bioprospecting of cnidarians was commonly limited to habitats that could be readily sampled by researchers, such as shallow coral reefs and the intertidal region. However, with improvements in SCUBA gear, researchers are now able to dive deeper and longer, allowing them to collect a wider range of cnidarian species for the screening of MNPs. The growing efforts to explore Earth's last frontier, the deep sea, made it possible to start bioprospecting several unique marine ecosystems that had remained either previously unrecorded or inaccessible to researchers [164]. New cnidarian species (some of them belonging to new genera and probably even to new families) (e.g., [165,166]) are currently being sampled from the deep sea. These findings suggest that many new species are yet to be discovered along deep continental margins [167] and open good perspectives for the discovery of new MNPs with ongoing surveys of deep sea fauna. Cnidarians are known to colonize unique deep sea biotopes, namely chemosynthetic sites (such as hydrothermal vents, cold seeps and whale falls [168]), as well as seamounts [169]. Some of these organisms are endemic to these habitats and display remarkable adaptations to extreme environments (e.g., chemosynthetic sea anemones) [170]. These species are certainly interesting candidates for the discovery of new MNPs [171]. However, some of these remarkable biotopes, namely deep sea coral reefs, are already facing serious threats to their conservation [169] and thus, the bioprospecting of these and other endangered habitats must be carefully addressed [164,172].
Another interesting source of cnidarian species for bioprospecting is the marine aquarium industry. Over 200 species of hard and soft corals, along with several other anemone, zoanthid and corallimorph species, are harvested every year from coral reefs to supply the marine aquarium trade [173]. However, researchers using these organisms in the bioprospecting of new MNPs must be aware that it is not commonly possible to get reliable information on either the place of origin or the scientific name of most traded specimens. With the advent of high-throughput screening (HTS) [174], it will be possible to rapidly survey these organisms for interesting MNPs, although HTS of natural sources may present several challenges (see [175,176]). If necessary, additional biomass of target organisms producing interesting MNPs can be achieved using inexpensive techniques [177,178] and eliminate problems commonly faced by researchers screening marine organisms for MNPs-the loss of the source and reproducibility [176].
The discovery of a new compound commonly requires only small amounts of biomass. However the production of these compounds at a scale large enough to fulfill commercial applications is still nearly impossible [179]. In theory, large-scale production of bioactive compounds can be achieved by chemical synthesis or through extraction from marine animals, either harvested from the sea or maricultured. The existence of ecophysiological diversity (e.g., differences between individuals often due to differences in environmental interactions) can interfere with the production of MNPs and must be carefully addressed in future efforts for large-scale production of these compounds. The harvest of target animals from the wild for the production of chemical compounds is commonly an unsustainable solution, while mariculture has proven to be more technically challenging and expensive than previously assumed [180]. In other considerations, chemical synthesis is not yet developed to synthesize complex molecules at the kilogram scale and, in cases where this may already be technically possible, most of the compounds cannot be synthesized at a price affordable for commercial applications [179]. Potential solutions for such bottlenecks may be the use of diverted total synthesis [181] and/or metabolic engineering [182].
There is growing evidence that microbes associated with marine invertebrates may be the true producers of some of the bioactive compounds isolated from these animals [179]. Whether this is the case of bioactive compounds currently assumed to be produced by cnidarians remains unanswered [183,184]. If so, we face another constraint for the commercial use of these compounds, as the culture of symbiotic microorganisms is generally not possible using classic/standardized methodologies.

Conclusions
The intense pressure to find and develop more profitable molecules for all sorts of industries continues to fuel the bioprospecting of marine invertebrates. Although the phylum Cnidaria is not the most significantly bioprospected at present, this review shows that some cnidarian species are promising sources of marine bioactive compounds of medical, economic and scientific interest. Green fluorescent protein (GFP), GPF-like proteins, red fluorescent and orange fluorescent protein (OPF) are good examples of biotechnological metabolites currently employed as molecular biomarkers. They were first purified from a fluorescent hydrozoan medusa [185] and since then have been recorded in other cnidarian species [186][187][188][189][190][191].
In the present survey, only about 0.31% of extant cnidarian species are represented, with class Anthozoa displaying by far the highest number of promising MNPs (Figure 2). This result is probably due to the fact that this class is the most speciose in the phylum (Table 1). Additionally, many anthozoans occupy marine habitats which can be readily accessed for the collection of biomass (e.g., coral reefs and intertidal regions), which facilitates bioprospecting. Of all the compounds presented in this review, 84% were detected in cnidarians collected from tropical waters (mostly from Southeast Asia and the Caribbean Sea) and the remaining 16% were recorded from species mostly occupying temperate waters (e.g., European countries and Japan).
Antitumor drugs are the main area of interest in the screening of MNPs from cnidarians (41%, Figure 3). This is not surprising, as the major financial effort for the screening of new marine compounds is made in cancer research [192]. Terpenoids (terpenoid, diterpenoid, sesquiterpenoid, sesterterpenoid, cembranoid) [193] (Figure 4) are the main chemistry group within the MNPs analyzed in this survey.     Even though most pharmaceutical industries abandoned their natural product-based discovery programs over a decade ago, the lack of new compounds in their pipelines in some strategic areas (e.g., antibiotics) suggests that renewed interest in this field is imminent. The establishment of small biotech companies can play a decisive role in the initial discovery of promising marine bioactive compounds, as these enterprises will work closely together with academics and governmental agencies performing the initial steps in the discovery of new MNPs. Collaboration between private companies and public institutions can be of paramount importance for financial support in the discovery process. On the other side, crude extracts and pure compounds produced by academic laboratories may be screened by diverse bioassays as a part of broader collaboration programs, nationally and internationally, with private biotech companies. One challenge for universities is to devise mechanisms that protect intellectual property and simultaneously encourage partnerships with the private sector, by recognizing that the chances of a major commercial pay-off are small if drug discovery is pursued by a single institution [3].
The commercial use of some promising marine bioactive compounds isolated from cnidarians may be several years away. New compounds other than toxins and venoms produced by members of this highly diverse group of marine invertebrates may be discovered in the quest for new marine products.