A Comprehensive Review of Bioactive Peptides from Marine Fungi and Their Biological Significance

Fungal marine microorganisms are a valuable source of bioactive natural products. Fungal secondary metabolites mainly comprise alkaloids, terpenoids, peptides, polyketides, steroids, and lactones. Proteins and peptides from marine fungi show minimal human toxicity and less adverse effects comparable to synthetic drugs. This review summarizes the chemistry and the biological activities of peptides that were isolated and structurally elucidated from marine fungi. Relevant fungal genera including Acremonium, Ascotricha, Aspergillus, Asteromyces, Ceratodictyon, Clonostachys, Emericella, Exserohilum, Microsporum, Metarrhizium, Penicillium, Scytalidium, Simplicillium, Stachylidium, Talaromyces, Trichoderma, as well as Zygosporium were extensively reviewed. About 131 peptides were reported from these 17 genera and their structures were unambiguously determined using 1D and 2D NMR (one and two dimensional nuclear magnetic resonance) techniques in addition to HRMS (high resolution mass spectrometry). Marfey and Mosher reactions were used to confirm the identity of these compounds. About 53% of the isolated peptides exhibited cytotoxic, antimicrobial, and antiviral activity, meanwhile, few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity. However 47% of the isolated peptides showed no activity with respect to the examined biological activity and thus required further in depth biological assessment. In conclusion, when searching for bioactive natural products, it is worth exploring more peptides of fungal origin and assessing their biological activities.


Introduction
Hundreds of secondary metabolites obtained from marine fungal strains revealed potent pharmacological and biological activity [1]. These mainly comprise alkaloids, terpenoids, and peptides, in addition to polyketides, steroids, and lactones. Relevant bioactivities include antibacterial, anticancer, anti-inflammatory, and antiviral activity [2]. The great diversity in the structure and function of the metabolites derived from marine organisms is mainly attributed to the extensive variation in the chemical and physical conditions of the environment in which the marine organisms survive [3]. As many marine organisms are sessile, they need chemical protection against predators and pathogens.
Marine microorganisms represented by fungi and bacteria, but also marine invertebrates, are regarded as a valuable source of bioactive compounds. Marine microorganisms have the advantage
One more new cyclic hexapeptide, similanamide (33), was isolated from a sponge derived fungus, A. similanensis, which displayed weak cytotoxic activity versus MCF-7, A373, and NCI-H460 cancer cells with GI 50 values equal to 0.2, 0.18, and 0.18 µM, respectively, with no antibacterial activity as the (MIC value was found to be greater 256 µg/mL (0.4 µM)) [19]. Moreover, two new lumazine peptides namely terrelumamides A (34) and B (35), in addition to two new isomeric modified tripeptides, aspergillamides C (36) and D (37), and a cyclic tetrapeptide asperterrestide A (38) were isolated and unambiguously elucidated from A. terreus, which is a marine fungus. Compounds (34) and (35) displayed a promising improvement in insulin sensitivity as determined by the utilization of mesenchymal cells of a bone marrow origin obtained from human (hBM-MSCs) adopting an adipogenesis model. They exerted their action via increasing the formation of adiponectin during the process of adipogenesis in hBM-MSCs with EC50 values equal to 37.1 and 91.9 µM, for terrelumamides A (34) and B (35), respectively. It is noteworthy to highlight that the maximum elevation in adiponectin levels via terrelumamide A (34) induction was 56.9% comparable to that generated by glibenclamide (the standard anti-hyperglycaemic agent). Meanwhile compound (38) showed a potent cytotoxic effect versus U937 and MOLT4 human cancer cell lines with IC50 equal to 6.4 and 6.2 µM, respectively, in addition to a pronounced inhibitory potential versus an influenza virus of H1N1, as well as H3N2 strains with IC50 equal to 15 and 8.1 µM, respectively, that relied upon the presence of a 3-OH-N-CH3-Phe moiety that is rare in nature [20][21][22]. Besides, other known peptides were isolated from the latter Aspergillus species, which are aspergillamide A (39) and B (40) and cyclo-(l-Pro-l-Phe) (41) [21]. A. unguis is another marine fungus from which unguisin A (42), which is a cyclic peptide, was isolated [23]. In addition, A. unilateralis revealed a complex array of secondary metabolites comprising the heterocyclic dipeptides, aspergillazines A-E (43)(44)(45)(46)(47), which are dipeptides, in addition to trichodermamide A and B (48)(49) [24] ( Figure 3B).
Furthermore, four new compounds, psychrophilins E-H (50-53) were isolated from A. versicolor, and they are cyclic peptides characterized by the presence of a rare linkage of amide type between anthranilic acid through its carboxylic acid and the indole ring via its nitrogen. Additionally, a new cyclic hexapeptide versicotide C (54) was isolated from the same fungus. All the isolated peptides showed no cytotoxicity, however compound (52) exhibited a pronounced lipid-reducing activity at approximately 10 µM as determined by oil red O staining assay [25]. Moreover, secondary metabolites isolated from coral derived A. versicolor revealed the presence of a new centrosymmetric cyclohexapeptide, namely aspersymmetide A (55), in addition to asperphenamate (56) which is a known peptide. Noteworthy to mention is that compound (55) was the first centrosymmetric cyclohexapeptide to be isolated from fungi, however it showed weak cytotoxicity versus NCI-H292, as well as A431 cell lines, at 10 µM [26]. Besides, two new cyclopentapeptides, namely cotteslosins A (57) and B (58), were isolated from the same fungus, A. versicolor. Cotteslosin A (57) displayed weak cytotoxicity versus human melanoma (MM418c5), prostate (DU145), and breast (T47D) cells with EC 50 values of 0.1, 0.14, and 0.15 µM, respectively [27]. Concerning A. violaceofuscus, a sponge associated fungus, three peptides, which are cyclic, were isolated from its ethyl acetate extract and they were first to be isolated in nature. These cyclic peptides are termed, aspochracin-type cyclic tripeptide sclerotiotide L (59), diketopiperazine dimer (60), in addition to a cyclic tetrapeptide (61) where compounds (60-61) showed potent anti-inflammatory activity against IL-10 expression of the (Lipopolysaccharide) LPS-induced THP-1 cells (acute monocytic leukemia cell) as evidenced by its inhibition rates which were estimated to be 84.3 and 78.1%, respectively at 10 µM [28] ( Figure 3C). Concerning A. violaceofuscus, a sponge associated fungus, three peptides, which are cyclic, were isolated from its ethyl acetate extract and they were first to be isolated in nature. These cyclic peptides are termed, aspochracin-type cyclic tripeptide sclerotiotide L (59), diketopiperazine dimer (60), in addition to a cyclic tetrapeptide (61) where compounds (60-61) showed potent anti-inflammatory activity against IL-10 expression of the (Lipopolysaccharide) LPS-induced THP-1 cells (acute monocytic leukemia cell) as evidenced by its inhibition rates which were estimated to be 84. 3  Various peptides were isolated from miscellaneous Aspergillus species exemplified by psychrophilin E (50), a cyclic tripeptide isolated from two algae associated Aspergillus sp whose selectivity inhibited the proliferation of HCT116 (colon) cell line with an IC 50 value of 28.5 µM comparable to the standard drug cisplatin that showed IC 50 of 33.4 µM [29]. Additionally, many Aspergillus sp. derived peptides displayed antiviral activity such as aspergillipeptides D-G (62-65), in which aspergillipeptides D (62) and E (63) displayed a potent antiviral effect versus herpes simplex virus type 1 (HSV-1) displaying IC 50 of 9.5 and 19.8 µM, respectively, showing no cytotoxic effect at these concentrations versus Vero cell line, meanwhile aspergillipeptide D (62) also exhibited activity versus acyclovir resistant clinical isolates of HSV-1 [30]. Regarding the inhibition of nitric oxide production, it was found that the novel cyclic dipeptide isolated from an Aspergillus sp., 14-hydroxy-cyclopeptine (66), effectively inhibits nitric oxide production displaying IC 50 of 0.14 µM in recombinant mouse interferon-γ-activated macrophage-like cell line as well as in a lipopolysaccharide without cytotoxic effect at 0.34 µM [31]. Many other peptides were also isolated from Aspergillus sp. derived from Bruguiera sexangula var. rhynchopetala as aspergilumamide A (67) and penilumamide (68) [32] ( Figure 3D).

Asteromyces
A new pentapeptide compound, lajollamide A (69) was isolated from marine Asteromyces cruciatus. This newly isolated peptide showed a weak antimicrobial effect versus Gram-positive bacteria at 100 µM showing 61% and 30% inhibition in bacterial growth for Bacillus subtilis and Staphylococcus epidermidis, respectively, whereas it showed no inhibition of acetyl cholinesterase activity [33] (Figure 4).

Ceratodictyon
In depth chemical investigation of the red algae associated fungus, Ceratodictyon spongiosum, resulted in the isolation of two new peptides which are dictyonamides A and B (70-71). The former displayed a pronounced inhibition on cyclin-dependent kinase 4 with IC 50 value equals to 0.01 µM however the latter showed no inhibition [34] (Figure 4).

Clonostachys
Clonostachysins A and B (72-73) are two new cyclic peptides isolated from Clonostachys rogersoniana that revealed potent inhibitory activity on a dinoflagellate Prorocentrum micans at a concentration of 30 µM but revealed no activity to either bacteria or microalgae [35] (Figure 4).

Microsporum
The marine associated fungus, Microsporum cf. gypseum, yielded two new peptides microsporins, A and B (81-82), which effectively inhibit histone deacetylase with IC 50 values equal to 0.14 and 0.55 µM against (Histone Deacetylases) HDACs and HDAC8, respectively, in addition to displaying a considerable cytotoxic effect versus human colon adenocarcinoma (HCT-116) with IC 50 values of 1.17 and 16.5 nM, respectively. Additionally, microsporin A (81) displayed notable activity against the 60 cancer cell panel of the National Cancer Institute with mean IC 50 of 2.7 µM [39] (Figure 4).

Microsporum
The marine associated fungus, Microsporum cf. gypseum, yielded two new peptides microsporins , A and B (81-82), which effectively inhibit histone deacetylase with IC50 values equal to 0.14 and 0.55 μM against (Histone Deacetylases) HDACs and HDAC8, respectively, in addition to displaying a considerable cytotoxic effect versus human colon adenocarcinoma (HCT-116) with IC50 values of 1.17 and 16.5 nM, respectively. Additionally, microsporin A (81) displayed notable activity against the 60 cancer cell panel of the National Cancer Institute with mean IC50 of 2.7 μM [39] ( Figure  4).

Scytalidium
A marine fungus of the genus Scytalidium is highly popular for the production of linear, lipophilic peptides, termed the halovirs as halovirs A-E (102-106), which showed considerable in vitro antiviral activity against herpes simplex viruses of both type 1 and type 2. Further study on the structure activity relationship between the halovirs and their anti-viral activity showed that the presence of a N α -acyl chain, comprising of at least 14 carbons as well as the Aib-Pro dipeptide, are crucial to preserve the activity. Upon addition of Halovirs A (102), B (103), and C (104) to cells that are exposed to HSV-1 infection for 1 h, they displayed ED 50 values of 1.1, 3.5, and 2.2 µM, respectively. Meanwhile, halovirs D (105) and E (106) showed ED 50 values equal to 2.0 and 3.1 µM, respectively. Besides, halovir A (102) effectively prohibits HSV-1 replication showing ED50 value of 0.28 µM adopting the standard plaque reduction assay [44,45] (Figure 6).

Simplicillium
A series of simplicilliumtide peptides were isolated from the marine fungus Simplicillium obclavatum namely; simplicilliumtides A-M (107-119) in addition to verlamelins A and B (120-121). They showed variable activities including antibacterial, antifungal, antiviral, antifouling, cytotoxic, and acetyl cholinesterase inhibitory activity. Simplicilliumtide D (110) displayed a potent antifouling effect versus the larvae of Bugula neritina with EC 50 equal to 0.02 µM and LC 50 /EC 50 equal to 100; however, simplicilliumtides A (107), E (111), G (113), and H (114) exhibited weak cytotoxic effect against human leukemia HL-60 and K562 cell lines with IC 50 above 100 µM. In addition, simplicilliumtide J (116) exhibited potent antifungal activity versus Curvularia australiensis as well as Aspergillus versicolor in addition to a potent anti -HSV-1 displaying IC 50 value of 14.0 µM that could be interpreted in virtue of the presence of lactone linkage and a fatty acid chain moiety [10,46] (Figure 6).

Stachylidium
Many N-methylated peptides have been isolated from Stachylidium sp., a fungus associated with marine sponge, which include endolides A-D (122-125) which showed a wide range of biological activities [47]. Endolide A (122) revealed potent binding activity to the vasopressin receptor 1A displaying a Ki of 7.04 µM, whereas endolide B (123) showed a pronounced binding to serotonin receptor 5HT2b evidenced by its Ki value, which is 0.77 µM [48] (Figure 6).

Talaromyces
Talarolide A (126), in addition to a series of extensively N-methylated linear peptides named talaropeptides A-D (127-130), were isolated from Talaromyces sp., a fungus derived from a marine tunicate. Biological evaluation of the previously mentioned compounds revealed that compounds (127-128) only displayed pronounced antibacterial activity against the growth of Bacillus subtilis, a Gram positive bacterium, displaying IC 50 values of 1.5 and 3.7 µM, respectively. Additionally compound talaropeptide A (127) showed a high stability to various rat proteases existing in plasma [49] (Figure 7).

Trichoderma
Trichoderma strains, derived from marine origin, afforded large amounts peptaibols, which are characterized by being small antimicrobial peptides (AMPs) that are expected to contribute to the antimicrobial defense of Trichoderma. Trichoderma strains produced 11-and 20-residue peptaibols; optimal yields with 1.4% and 2.3% of the fungal biomass for the 11-and 20-residue, respectively, were obtained on day 9 [50]. Generally, AMPs represent a large group of naturally occurring cationic and amphiphilic short peptides that act as first-line defense for many living organisms and are part of the innate immune system [51]. They act primarily via offering a protection to the host organisms versus the invasion of harmful organisms such as bacteria or fungi [52]. Their mode of action is completely different from traditional antibiotics as they modulate membrane stability and permeability. On the contrary, traditional antibiotics exert their effect mainly by interfering with bacterial metabolism, protein biosynthesis, or cell wall formation. Thus, low cross-resistance and an effective synergism could be achieved between AMPs and traditional antibiotics [53].

Zygosporium
Zygosporium masonii, a marine fungus that produced a cyclic depsipeptide termed zygosporamide (131) showed potent cytotoxic activity in the NCI's 60 cell line panel with median GI 50 equal to 9.1 µM. However, it revealed a notable selectivity versus the (Central Nervous System) CNS cancer cell SF-268 with GI 50 equal to 6.5 nM, as well as to the renal cancer cell line RXF 393 showing GI 50 less than 5.0 nM [54,55] (Figure 7).

Trichoderma
Trichoderma strains, derived from marine origin, afforded large amounts peptaibols, which are characterized by being small antimicrobial peptides (AMPs) that are expected to contribute to the antimicrobial defense of Trichoderma. Trichoderma strains produced 11-and 20-residue peptaibols; optimal yields with 1.4% and 2.3% of the fungal biomass for the 11-and 20-residue, respectively, were obtained on day 9 [50]. Generally, AMPs represent a large group of naturally occurring cationic and amphiphilic short peptides that act as first-line defense for many living organisms and are part of the innate immune system [51]. They act primarily via offering a protection to the host organisms versus the invasion of harmful organisms such as bacteria or fungi [52]. Their mode of action is completely different from traditional antibiotics as they modulate membrane stability and permeability. On the contrary, traditional antibiotics exert their effect mainly by interfering with bacterial metabolism, protein biosynthesis, or cell wall formation. Thus, low cross-resistance and an effective synergism could be achieved between AMPs and traditional antibiotics [53].

Zygosporium
Zygosporium masonii, a marine fungus that produced a cyclic depsipeptide termed zygosporamide (131) showed potent cytotoxic activity in the NCI's 60 cell line panel with median GI50 equal to 9.1 μM. However, it revealed a notable selectivity versus the (Central Nervous System)

Discussion and General Perspectives
In our analysis we found that 131 peptides from marine sources were isolated from 17 fungal strains. These peptides are either cyclic, composed of two amino acids (dipeptide), as in case of Ascotricha up to nine amino acids (nonapeptides) in Emericella or linear pentadecapeptide as presented in Acremonium. Many depsipeptides (from Acremonium, Metarrhizium, and Zygosporium), peptaibols in Trichoderma and N-methylated peptides from Acremonium, Stachylidium, and Talaromyces are also characterized. It is clearly noticed that genus Aspergillus was the most extensively studied and it might represent a rich source of peptides with promising biological activity. However, there is no evidence about the chemotaxonomic relation between the production of a certain class of peptides in a specified genus. That could be explained based on the fact that the number of the isolated compounds is not enough to make an in-depth study about this relation. Besides, peptides from marine sources possess strongly modulated structures either in its backbone or side chain comparable to peptides of plant or human origin, which undoubtedly is due to the harsh requirements of the environment in which they live. It worthy to mention that although most of these peptides contain many functional groups, such as carbonyl and amide groups, beside esters as in the case of depsipeptides which gave them the ability to interact with many molecular targets in the cells, most of these peptides are either inactive or showed weak activity. That could be explained by the fact that most of these peptides are weakly soluble in physiological fluids that limit most of their in vivo activity [56].
It was found that about 53% of the isolated peptides exhibited various biological activities represented mainly by cytotoxic, antimicrobial, and antiviral activity, while few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity (Table 1). Table 1. Bioactive peptides isolated from marine associated fungi, their sources, and biological activities.

Compound
Genus
A considerable number of fungal peptides showed powerful antiviral activity exemplified by asperterrestide A (38) which exerted potent potential antiviral activity versus an influenza virus of H1N1 as well as H3N2 strains that could be relied upon the presence of a 3-OH-N-CH3-Phe moiety which is rare in nature. Moreover, aspergillipeptides D-G (62-65), showed a pronounced antiviral effect versus herpes simplex virus type 1 (HSV-1) displaying IC 50 of 9.5 and 19.8 µM, respectively. Halovirs A-E (102-106) showed considerable in vitro antiviral activity against herpes simplex viruses of both type 1 and type 2 which is ultimately attributed to the presence of a N α -acyl chain, comprising of at least 14 carbons as well as the Aib-Pro dipeptide. However, simplicilliumtide J (116) exerted a potent anti -HSV-1 displaying IC 50 value of 14.0 µM which could be interpreted in virtue of the presence of lactone linkage and a fatty acid chain moiety.
The anti-inflammatory activity of some of the isolated peptides can be explained via exerting different mechanisms where compounds (60-61) obtained from Aspergillus at 10 µM effectively inhibited IL-10 expression of the LPS-induced THP-1 cells showing 84.3% and 78.1% inhibition, respectively. Besides, 14-hydroxy-cyclopeptine (66) effectively inhibits nitric oxide production displaying IC 50 of 0.14 µM in recombinant mouse interferon-γ -activated macrophage-like cell line.
Antidiabetic and lipid lowering activity was also observed for some of the isolated fungal peptides, such as terrelumamides A (34) and B (35), which displayed a promising improvement in insulin sensitivity as determined by the utilization of mesenchymal cells of a bone marrow origin obtained from human adopting an adipogenesis model. Additionally, psychrophilin G (52) exhibited pronounced lipid-reducing activity.
Additionally, miscellaneous activities were reported for the isolated bioactive peptides in which Simplicilliumtide D (110) displayed a potent antifouling effect versus the larvae of Bugula neritina. Moreover, endolide A (122) revealed potent binding activity to the vasopressin receptor 1A displaying a Ki of 7.04 µM whereas endolide B (123) showed a pronounced binding to serotonin receptor 5HT2b evidenced by its Ki value which is 0.77 µM. Talaropeptide A (126) showed a high stability to various rat proteases existing in plasma, however, microsporins A and B (81-82) effectively inhibit histone deacetylase.
Noteworthy to mention is that about 47% of the isolated peptides showed no activity with respect to the examined biological activity and thus required further in depth biological assessment. A pie chart summarizing the percentages of isolated peptides with respect to their biological activity is represented in Figure 8. The anti-inflammatory activity of some of the isolated peptides can be explained via exerting different mechanisms where compounds (60-61) obtained from Aspergillus at 10 μM effectively inhibited IL-10 expression of the LPS-induced THP-1 cells showing 84.3% and 78.1% inhibition, respectively. Besides, 14-hydroxy-cyclopeptine (66) effectively inhibits nitric oxide production displaying IC50 of 0.14 μM in recombinant mouse interferon-γ -activated macrophage-like cell line.
Antidiabetic and lipid lowering activity was also observed for some of the isolated fungal peptides, such as terrelumamides A (34) and B (35), which displayed a promising improvement in insulin sensitivity as determined by the utilization of mesenchymal cells of a bone marrow origin obtained from human adopting an adipogenesis model. Additionally, psychrophilin G (52) exhibited pronounced lipid-reducing activity.
Additionally, miscellaneous activities were reported for the isolated bioactive peptides in which Simplicilliumtide D (110) displayed a potent antifouling effect versus the larvae of Bugula neritina. Moreover, endolide A (122) revealed potent binding activity to the vasopressin receptor 1A displaying a Ki of 7.04 μM whereas endolide B (123) showed a pronounced binding to serotonin receptor 5HT2b evidenced by its Ki value which is 0.77 μM. Talaropeptide A (126) showed a high stability to various rat proteases existing in plasma, however, microsporins A and B (81-82) effectively inhibit histone deacetylase.
Noteworthy to mention is that about 47% of the isolated peptides showed no activity with respect to the examined biological activity and thus required further in depth biological assessment. A pie chart summarizing the percentages of isolated peptides with respect to their biological activity is represented in Figure 8.

Conclusion
Herein, we concluded that about 131 peptides were isolated from marine sources from seventeen fungal strains. Some of them exhibited much biological activity represented mainly by cytotoxic, antimicrobial, and antiviral activity, meanwhile, few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity; however, others should be deeply assessed for their biological potential. Thus, if active natural products are needed for drug development, further

Miscellenous activity 5%
Inactive and requires further studies 47% Figure 8. Percentages of isolated peptides with respect to their biological activity represented by a pie chart.

Conclusions
Herein, we concluded that about 131 peptides were isolated from marine sources from seventeen fungal strains. Some of them exhibited much biological activity represented mainly by cytotoxic, antimicrobial, and antiviral activity, meanwhile, few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity; however, others should be deeply assessed for their biological potential. Thus, if active natural products are needed for drug development, further investigations of marine fungi are recommended. In addition, it is a challenge to explore more peptides from fungal origin and so it is recommended to confirm their biological activities.