Secondary Metabolites of the Genus Didemnum: A Comprehensive Review of Chemical Diversity and Pharmacological Properties

Tunicates (ascidians) are common marine invertebrates that are an exceptionally important source of natural products with biomedical and pharmaceutical applications, including compounds that are used clinically in cancers. Among tunicates, the genus Didemnum is important because it includes the most species, and it belongs to the most speciose family (Didemnidae). The genus Didemnum includes the species D. molle, D. chartaceum, D. albopunctatum, and D. obscurum, as well as others, which are well known for their chemically diverse secondary metabolites. To date, investigators have reported secondary metabolites, usually including bioactivity data, for at least 69 members of the genus Didemnum, leading to isolation of 212 compounds. Many of these compounds exhibit valuable biological activities in assays targeting cancers, bacteria, fungi, viruses, protozoans, and the central nervous system. This review highlights compounds isolated from genus Didemnum through December 2019. Chemical diversity, pharmacological activities, geographical locations, and applied chemical methods are described.


Introduction
The biodiversity of organisms in the marine ecosystem has motivated researchers to discover many marine natural products that might ultimately be developed into therapeutics [1]. Among marine organisms, invertebrates such as ascidians, sponges, molluscs, and bryozoans represent the major source of organic extracts with significant bioactivities [2]. Currently, there are eight marine-derived drugs approved by different agencies, including the U.S. FDA, European Medicines Agency (EMEA), Japanese Ministry of Health, and Australia's Therapeutic Goods Administration (Figure 1). An additional 22 drug leads are currently in different phases (I-III) of drug development [3]. Considering that ≈1000 new molecules have been isolated from marine organisms annually for the past few years, the biotechnological and pharmaceutical potential of the sea remains awe-inspiring [4][5][6][7][8][9]. Of the marine invertebrates commonly investigated for marine natural products, as members of Phylum Chordata tunicates (ascidians) are the most closely related to humans. Ascidians represent the most diverse and biggest class of the sub-phylum Tunicata, comprising about 3000 described species [10]. Didemnidae is the largest tunicate family [10], and it has been confirmed to be monophyletic using molecular methods [11]. Didemnidae features many genera that are prolific and famous producers of bioactive natural products, including Diplosoma, Lissoclinum, Polysyncraton, and Trididemnum ( Figure 2). Among tunicates from family Didemnidae, the genus Didemnum stands out, with more described species than any other tunicate [11]. In addition, the number of described species is certainly an underestimate of the true biodiversity of genus Didemnum. For example [12], the widespread tunicate Didemnum molle exists in a variety of overlapping color morphs. These morphs are genetically monophyletic and deeply divergent, indicating that they are likely to be different species [12]. Further complicating this variety, tunicates from family Didemnidae are colonial, with up to thousands of individual animals known as zooids sharing a single tunic. The individual colonies are often comprised of hybrids, or mixtures, of genetically different zooids that have mixed by colony fusion [13]. Finally, the genus Didemnum harbors many different symbiotic bacteria, which are sometimes responsible for producing the bioactive secondary metabolites isolated from the whole animals [14]. As a result of these factors, the genus Didemnum provides an amazing array of biological and chemical diversity. Of the marine invertebrates commonly investigated for marine natural products, as members of Phylum Chordata tunicates (ascidians) are the most closely related to humans. Ascidians represent the most diverse and biggest class of the sub-phylum Tunicata, comprising about 3000 described species [10]. Didemnidae is the largest tunicate family [10], and it has been confirmed to be monophyletic using molecular methods [11]. Didemnidae features many genera that are prolific and famous producers of bioactive natural products, including Diplosoma, Lissoclinum, Polysyncraton, and Trididemnum ( Figure 2). Among tunicates from family Didemnidae, the genus Didemnum stands out, with more described species than any other tunicate [11]. In addition, the number of described species is certainly an underestimate of the true biodiversity of genus Didemnum. For example [12], the widespread tunicate Didemnum molle exists in a variety of overlapping color morphs. These morphs are genetically monophyletic and deeply divergent, indicating that they are likely to be different species [12]. Further complicating this variety, tunicates from family Didemnidae are colonial, with up to thousands of individual animals known as zooids sharing a single tunic. The individual colonies are often comprised of hybrids, or mixtures, of genetically different zooids that have mixed by colony fusion [13]. Finally, the genus Didemnum harbors many different symbiotic bacteria, which are sometimes responsible for producing the bioactive secondary metabolites isolated from the whole animals [14]. As a result of these factors, the genus Didemnum provides an amazing array of biological and chemical diversity.
Tunicates are a vital source of bioactive compounds with promising potential for biomedical applications, including several approved drugs. The production of active compounds in tunicates is thought to result from competition in the marine environment, especially to protect the sedentary animals from predation [15]. As the most speciose genus, Didemnum is also very rich in bioactive secondary metabolites [10]. While numerous chemical and biological studies investigate the genus Didemnum, most of these studies do not identify the animals to species. These studies show that the genus Didemnum is abundant in many classes of natural products, including peptides, alkaloids, indole/alkaloids, β-carboline alkaloids, spiroketals, polyketides, halogenated compounds, steroids, and many others ( Figure 3). Biological investigations of these entities have shown that some of these compounds possess anticancer, antimicrobial, and antimalarial activity [15].
Using an ecologically relevant assay as a guide for isolation of the feeding deterrent compounds from the active dichloromethane-methanol extract, four novel indole-maleimide-imidazole alkaloids, didemnimides A-D (97-100) ( Figure 12), were isolated from D. conchyliatum. Didemnimide (100) was found to deter feeding of the carnivorous wrasse Thalassoma bifasciatum at natural concentrations in aquarium assays [39]. Additionally, using ELISA-based high-throughput bioassay for targeting G2 cell cycle checkpoint inhibitors, the active extract of the Brazilian D. granulatum was selected. A bioassay-guided fractionation of the extract resulted in the isolation of the alkaloids granulatimide (101) and isogranulatimide (102) together with didemnimides A (97), D (99), E (103), and 6-bromogranulatimide (104) ( Figure 12). Compounds 101 and 102 displayed inhibitory activity for G2 cell cycle checkpoint and combined with a DNA damaging agent selectively kill p53-cancer cells [40,41]   exhibited antimicrobial effects towards Bacillus subtilis, Staphylococcus aureus, E. coli, and two strains of Saccharomyces cerevisiae (RS188N and RS322Y) with inhibition zones ranging from 7 to 23 mm at 100 µg/6 mm disc [42]. A β-carboline dimer (112) ( Figure 12) has been also isolated from an Australian Didemnum sp. [43]. From the cytotoxic (HCT 116) CHCl 3 fraction of the methanolic extract of the Fijian Didemnum sp., a β-carboline derivative, bengacarboline (113) (Figure 12), was isolated from the along with fascaplysin (114) ( Figure 12). Compound 113 displayed cytotoxic activity toward a 26 cell line human tumor panel with a mean IC 50   Using an ecologically relevant assay as a guide for isolation of the feeding deterrent compounds from the active dichloromethane-methanol extract, four novel indole-maleimide-imidazole alkaloids, didemnimides A-D (97-100) ( Figure 12), were isolated from D. conchyliatum. Didemnimide (100) was found to deter feeding of the carnivorous wrasse Thalassoma bifasciatum at natural concentrations in aquarium assays [39]. Additionally, using ELISA-based high-throughput bioassay for targeting G2 cell cycle checkpoint inhibitors, the active extract of the Brazilian D. granulatum was selected. A bioassay-guided fractionation of the extract resulted in the isolation of the alkaloids granulatimide (101) and isogranulatimide (102) together with didemnimides A (97), D (99), E (103), and 6bromogranulatimide (104) ( Figure 12). Compounds 101 and 102 displayed inhibitory activity for G2 cell cycle checkpoint and combined with a DNA damaging agent selectively kill p53-cancer cells [40,41].
A cyclic heptapeptide, mollamide (122) (Figure 13), was isolated from D. molle and displayed a cytotoxic effect with an IC 50 of 1.2 µM toward P388 (murine leukemia) and 3.0 µM toward A549 (human lung carcinoma). Mollamide also inhibited the RNA synthesis with an IC 50 of approximately at 1.23 µM [46]. Moreover, cyclic hexapeptides mollamides B and C (123 and 124) and keenamide A (125) ( Figure 13) were reported from the Indonesian D. molle. Mollamide B (123) displayed significant growth inhibition of 29%, 44%, and 42% when tested at 100 µM against the non-small cell lung cancer cell line (H460), the breast cancer cell line (MCF7), and the CNS cancer cell line (SF-268). However, when evaluated by the National Cancer Institute (NCI) in the 60-cell-line panel, none of the tested cell lines displayed any sensitivity to mollamide B that exceeded the mean [47]. By contrast, when tested in an in vitro disk diffusion assay that aims to identify differential cell killing among nine cell lines (two leukemias, five solid tumors, a murine, and a human normal cell line), mollamide C (124) showed against L1210, human colon HCT-116, and human lung H125 a unit zone differential value of 100 and against murine colon 38 a value of 250. Therefore, 124 was not considered as solid-tumor selective [47].  [48]. It was postulated that shishijimicins A-C (126-128) cleave DNA as in the case of other enediyne antibiotics including namenamicin (129) [48]. A pyrroloacridine alkaloid, plakinidine D (130) (Figure 14), was isolated from D. rubeum, along with 3,5-diiodo-4-methoxyphenethylamine (27) and ascididemin (131) (Figure 14). Compound 130 showed cytotoxicity to the human colon tumor cell line HCT-116 at 25 µM [49,50]. A unique pentacyclic aromatic alkaloid, ascididemin (131), isolated from Okinawan Didemnum sp., had strong antineoplastic activity toward L1210 murine leukemia cells with IC 50 value of 1.37 µM.

Compounds with Antiviral Activities
The chemical investigation of the D. guttatum resulted in the isolation of cyclodidemniserinol trisulfate (182) (Figure 20). This compound is closely related to didemniserinolipid A (183) (Figure 21) which is obtained from an Indonesian Didemnum sp. [71]. There are some notable variations between the structures of 182 and 183 with the presence of an additional ring containing a glycine unit and the presence of sulfate groups in 182 [72]. Furthermore, didemniserinolipids B and C (184 and 185) (Figure 21) are reported from the same tunicate species [71]. Tracing the active fraction in an HIV integrase assay through a bioassay-guided purification of the methanolic extract of Didemnum sp. led to the isolation of didemniserinolipid A (183). It displayed inhibitory effects against HIV-1 protease and MCV topoisomerase with an IC 50 of 100.5 and 120.6 µM, respectively [72]. A bioassay-guided fractionation of the extract of D. molle resulted in the isolation of two thiazoline peptides, mollamides E and F (186 and 187) (Figure 21), and the tris-phenethyl urea, molleurea A (5) (Figure 4) [72]. Compound 187 displayed a modest anti-HIV activity in both HIV integrase inhibition assay and a cytoprotective cell-based assay with IC 50 values of 39 and 78 µM, respectively, while compound 5 was active only in the cytoprotective cell-based assay with IC 50 of 60 µM [73]. A unique sulfated mannose homopolysaccharide, kakelokelose (188) (Figure 21), was reported during an investigation of mucous secretion of the Pacific D. molle. Compound 188 displayed a remarkable anti-HIV action determined 100% potential to inhibit infection with CEM cells by HIV strain RF at 0.20 µM, while no cytotoxicity against CEM cells at a concentration of 10.25 µM was observed [74].
Mar. Drugs 2020, 18, x 25 of 37 The chemical investigation of the D. guttatum resulted in the isolation of cyclodidemniserinol trisulfate (182) (Figure 20). This compound is closely related to didemniserinolipid A (183) ( Figure  21) which is obtained from an Indonesian Didemnum sp. [71]. There are some notable variations between the structures of 182 and 183 with the presence of an additional ring containing a glycine unit and the presence of sulfate groups in 182 [72]. Furthermore, didemniserinolipids B and C (184 and 185) (Figure 21) are reported from the same tunicate species [71]. Tracing the active fraction in an HIV integrase assay through a bioassay-guided purification of the methanolic extract of Didemnum sp. led to the isolation of didemniserinolipid A (183). It displayed inhibitory effects against HIV-1 protease and MCV topoisomerase with an IC50 of 100.5 and 120.6 μM, respectively [72]. A bioassayguided fractionation of the extract of D. molle resulted in the isolation of two thiazoline peptides, mollamides E and F (186 and 187) (Figure 21), and the tris-phenethyl urea, molleurea A (5) (Figure 4) [72]. Compound 187 displayed a modest anti-HIV activity in both HIV integrase inhibition assay and a cytoprotective cell-based assay with IC50 values of 39 and 78 μM, respectively, while compound 5 was active only in the cytoprotective cell-based assay with IC50 of 60 μM [73]. A unique sulfated mannose homopolysaccharide, kakelokelose (188) (Figure 21), was reported during an investigation of mucous secretion of the Pacific D. molle. Compound 188 displayed a remarkable anti-HIV action determined 100% potential to inhibit infection with CEM cells by HIV strain RF at 0.20 μM, while no cytotoxicity against CEM cells at a concentration of 10.25 μM was observed [74].  Using a bioassay-guided fractionation of the anti-HIV extract of D. molle collected in the Eastern Fields of Papua New Guinea, two anti-HIV compounds, divamides A (189 and 190) ( Figure 22) were isolated [75]. Insufficient material was obtained for full structure elucidation, so metagenome sequencing revealed a biosynthetic pathway encoded in symbiotic Prochloron bacteria. The pathway was expressed in E. coli, leading to material for full structure elucidation and pharmacological testing. isolated [75]. Insufficient material was obtained for full structure elucidation, so metagenome sequencing revealed a biosynthetic pathway encoded in symbiotic Prochloron bacteria. The pathway was expressed in E. coli, leading to material for full structure elucidation and pharmacological testing.   (Table S2) During the chemical investigation of methanolic extract of Australian Didemnum sp., (R)-(E)-1-aminotridec-5-en-2-ol (191) was isolated, displaying modest activity toward Candida albicans (9-mm zone of inhibition at 50 µg/disk) [76] In addition, two minor compounds were characterized as their N-Boc derivatives, l-(N-Boc-amino)tridec-4-en-2-ol (192) and l-(N-Boc-amino)tridec-5-en-2-ol (193) [76]. (Table S3) Three decahydroquinolines metabolites were reported from tropical marine Didemnum sp., To understand the molecular interactions important for kinase inhibition by ningalins and lamellarins, docking studies were performed using the X-ray crystallographic structure of CDK5 D144N /p25 in complex with aloisine. It was predicted that the ningalins preferentially bind in the ATP binding site, which is consistent with their broad inhibitory effects across the three kinases. Lamellarins, on the other hand, are predicted to prefer to bind in the ATP binding pocket making the lamellarins' activity due to a nonspecific interaction with the kinases [83].

Discussion: The Chemistry and Chemical Potential of Didemnum
Since the first report of Didemnum secondary metabolites by Ireland, Durso, and Scheuer in 1981 [16], the genus has contributed at least 212 compounds. The field was most active during the period of 1993-2009, when 143 new compounds were reported (68%) from at least 45 species (65%) ( Figure  26). However, new discoveries continue apace even through 2019, with 54 compounds (25%) from at least 18 species (26%). These species have been collected from locations around the world, focused on tropical regions ( Figure 26). There is a notable scarcity of reports from the western coasts of all continents. These data indicate that genus Didemnum continues to be a rich source of secondary metabolites that are new to science and suggest potential locations for further discovery. To understand the molecular interactions important for kinase inhibition by ningalins and lamellarins, docking studies were performed using the X-ray crystallographic structure of CDK5 D144N /p25 in complex with aloisine. It was predicted that the ningalins preferentially bind in the ATP binding site, which is consistent with their broad inhibitory effects across the three kinases.
Lamellarins, on the other hand, are predicted to prefer to bind in the ATP binding pocket making the lamellarins' activity due to a nonspecific interaction with the kinases [83].

Discussion: The Chemistry and Chemical Potential of Didemnum
Since the first report of Didemnum secondary metabolites by Ireland, Durso, and Scheuer in 1981 [16], the genus has contributed at least 212 compounds. The field was most active during the period of 1993-2009, when 143 new compounds were reported (68%) from at least 45 species (65%) ( Figure 26). However, new discoveries continue apace even through 2019, with 54 compounds (25%) from at least 18 species (26%). These species have been collected from locations around the world, focused on tropical regions ( Figure 26). There is a notable scarcity of reports from the western coasts of all continents. These data indicate that genus Didemnum continues to be a rich source of secondary metabolites that are new to science and suggest potential locations for further discovery. Didemnum has been an exceptional source of compounds with important therapeutic promise, with most interest so far involving potential anticancer compounds. Among these, the lamellarins have been the most extensively studied for their anticancer potential [85]. These compounds were initially discovered in a mollusk, Lamellaria sp., which likely concentrates the compounds from a tunicate diet [86]. The enediynes namenamicin and shishijimicins are exceptionally potent (picomolar) [48]. Structurally related enediynes have been FDA approved as their antibody-drug conjugates [87]. The structurally unusual, cyclic peptide vitilevuamide exhibited powerful, subnanomolar potency against a variety of cancer cell lines [55]. Another cyclic peptide, divamide A, inhibited HIV replication at ≈200 nM in cell lines [75].
There are many different chemical families found in Didemnum spp., but as in other tunicates a large percentage appear to be amino acid-derived. Virtually nothing is known about the biosynthetic origin of most of these compounds, with the exception of two classes of ribosomally synthesized and posttranslationally modified peptides (RiPPs) [88], which in both cases are produced by obligately symbiotic bacteria, Prochloron spp. [89]. One group of these belongs to a large family of mostly N-C circular peptides, the cyanobactins [90]. Didemnum spp. cyanobactins include didmolamides, anatollamides, minimide, mollamides, keenamide, comoramides, mayotamides, and hexmollamide, or a total of 16 compounds unique to genus Didemnum spp. Another class of Didemnum spp. RiPPs is a group of lanthipeptides, the divamides [75]. In both the cyanobactin and the divamide cases, the biosynthetic pathways were identified by metagenome sequencing. Bioinformatics methods Didemnum has been an exceptional source of compounds with important therapeutic promise, with most interest so far involving potential anticancer compounds. Among these, the lamellarins have been the most extensively studied for their anticancer potential [85]. These compounds were initially discovered in a mollusk, Lamellaria sp., which likely concentrates the compounds from a tunicate diet [86]. The enediynes namenamicin and shishijimicins are exceptionally potent (picomolar) [48]. Structurally related enediynes have been FDA approved as their antibody-drug conjugates [87]. The structurally unusual, cyclic peptide vitilevuamide exhibited powerful, subnanomolar potency against a variety of cancer cell lines [55]. Another cyclic peptide, divamide A, inhibited HIV replication at ≈200 nM in cell lines [75].
There are many different chemical families found in Didemnum spp., but as in other tunicates a large percentage appear to be amino acid-derived. Virtually nothing is known about the biosynthetic origin of most of these compounds, with the exception of two classes of ribosomally synthesized and posttranslationally modified peptides (RiPPs) [88], which in both cases are produced by obligately symbiotic bacteria, Prochloron spp. [89]. One group of these belongs to a large family of mostly N-C circular peptides, the cyanobactins [90]. Didemnum spp. cyanobactins include didmolamides, anatollamides, minimide, mollamides, keenamide, comoramides, mayotamides, and hexmollamide, or a total of 16 compounds unique to genus Didemnum spp. Another class of Didemnum spp. RiPPs is a group of lanthipeptides, the divamides [75]. In both the cyanobactin and the divamide cases, the biosynthetic pathways were identified by metagenome sequencing. Bioinformatics methods localized the biosynthetic pathways to symbiotic Prochloron bacteria, and not to the host itself. The pathways were reconstructed using chemical synthesis of DNA, and the resulting plasmids were expressed in Escherichia coli in the laboratory, leading to lab-based synthesis of the natural products. This represents strong evidence that the symbiotic bacteria, and not the host, are responsible for making bioactive compounds isolated from the whole animals.
Beyond the RiPPs made by symbiotic Prochloron bacteria, several other Didemnum metabolites likely have a symbiotic origin, but no biosynthetic studies have yet been reported [87]. For example, enterocins and enediynes are very similar to, or even identical to, products isolated from cultivated bacteria [91,92]. These, and also compounds such as didemnaketals, appear to be of polyketide origin, of a class normally associated with bacterial metabolism. Vitilevuamide has several features that are hallmarks of both RiPP and nonribosomal peptide biosynthesis in bacteria. For most other Didemnum spp. compounds, the biosynthetic origin is not clear. In many of those cases, the hosts themselves may likely synthesize many of the compounds, rather than symbiotic bacteria. Biosynthesis of secondary metabolites by animal biochemistry is a barely explored, blooming field [93]. In those cases, in contrast to finding pathways from symbiotic bacteria, the tool required is transcriptomics, so that genes expressed by the animal are analyzed and subsequently their products are biochemically characterized [94].
Overall, the chemistry of Didemnum spp. is distinct, with several classes of compounds that have not yet been found in other organisms. However, many compounds bear striking similarities to those from other didemnid ascidians. For example, Prochloron spp. symbionts are widespread in Didemnidae, where they produce many cyanobactins [90]. Interestingly, vitilevuamide and enediynes have been found in at least two different genera of colonial tunicates, implicating potentially as-yet unidentified symbiotic bacteria that might be widespread within family Didemnidae [55,87].
Given the interest in compounds from Didemnum, it could be asked why there are not even more studies reported for this widespread genus. A major challenge stems from the biology of the organisms. While some Didemnum spp. grow as massive sheets that can coat shallow substrates in the sea, most animals consist of very small colonies ( Figure 2). In the colonial didemnid ascidians, chemistry can vary between seemingly identical colonies collected in the same location [95]. Such variation can arise from the cryptic biodiversity prevalent in didemnid tunicates: most importantly, tunicates can be quite different species even though their appearances are identical. This has slowed the development of the field.
The methods of metagenome sequencing/transcriptomics and synthetic biology offer one potential avenue to access the diverse chemistry found in these abundant, yet tiny and variable, animal colonies. So far, in two cases involving RiPP biosynthesis within tunicates, painstaking efforts have led to the production of three Didemnum genus compounds-which are actually produced by symbiotic bacteria-in E. coli [23,75]. Developments in biotechnology may help to further access the potent, pharmaceutically promising agents from this ubiquitous genus. One key factor that has been found in studies of didemnid tunicates (although not focused on Didemnum spp.) was that the host taxonomy is most predictive of chemistry [14,96]. When chemical variation is observed in two identical looking didemnid tunicates, it is most likely that they are actually different species. This holds true even when the chemistry is made by bacteria, and not by the host animal. Thus, a better understanding of biology and ecology is a crucial ingredient in the discovery of new potential drugs from tunicates.
In summary, Didemnum spp. tunicates have been exceptional sources of biosynthetic and biochemical novelty applied to drug discovery. Even facing significant headwinds, new discoveries from Didemnum spp. and other tunicates from family Didemnidae continue apace.
Funding: Publication costs for this manuscript were provided by the William R. Droschkey Endowed Chair.