Structural Diversity and Biological Activities of the Cyclodipeptides from Fungi

Cyclodipeptides, called 2,5-diketopiperazines (2,5-DKPs), are obtained by the condensation of two amino acids. Fungi have been considered to be a rich source of novel and bioactive cyclodipeptides. This review highlights the occurrence, structures and biological activities of the fungal cyclodipeptides with the literature covered up to July 2017. A total of 635 fungal cyclodipeptides belonging to the groups of tryptophan-proline, tryptophan-tryptophan, tryptophan–Xaa, proline–Xaa, non-tryptophan–non-proline, and thio-analogs have been discussed and reviewed. They were mainly isolated from the genera of Aspergillus and Penicillium. More and more cyclodipeptides have been isolated from marine-derived and plant endophytic fungi. Some of them were screened to have cytotoxic, phytotoxic, antimicrobial, insecticidal, vasodilator, radical scavenging, antioxidant, brine shrimp lethal, antiviral, nematicidal, antituberculosis, and enzyme-inhibitory activities to show their potential applications in agriculture, medicinal, and food industry.

Among the organisms, fungi have been considered as the most important sources of novel and bioactive cyclodipeptides. More and more cyclodipeptides with interesting biological activities have been isolated and characterized from fungi. However, no detailed and comprehensive summary of the fungal cyclodipeptides on their occurrence, structures and biological activities has been reported though chemistry and biology of the cyclodipeptides from either all organisms or a certain class of cyclodipeptides have been documented [3,5,6]. In this review, we aim to describe the diversity of chemical structures and biological activities of the fungal cyclodipeptides and their analogs. A total of 635 fungal cyclodipeptides have been discussed and reviewed with literature covered up to July 2017. According to their biosynthetic origins and structural characters, these cyclodipeptides are classified as tryptophan-proline, tryptophan-tryptophan, tryptophan-Xaa (Xaa is indicated as an Table 1. Fungal tryptophan-proline cyclodipeptide analogs and their biological activities.

Name
Fungus and Its Origin Biological Activity Ref.

Tryptophan-Tryptophan Cyclodipeptides
The ditryptophan cyclodipeptides, which have two tryptophan units, are widely distributed in filamentous fungi, especially in the genera Penicillium and Aspergillus. Their occurrence and biological activities are listed in Table 2, and the structures are provided in Figure 2.

Tryptophan-Tryptophan Cyclodipeptides
The ditryptophan cyclodipeptides, which have two tryptophan units, are widely distributed in filamentous fungi, especially in the genera Penicillium and Aspergillus. Their occurrence and biological activities are listed in Table 2, and the structures are provided in Figure 2.

Tryptophan-Xaa Cyclodipeptides
Apart from Trp-Pro and Trp-Trp cyclodipeptides, other tryptophan cyclodipeptides are also abundant in fungi and represent a structurally diverse group of natural products. Their occurrence and biological activities are shown in Table 3, and their structures are provided in Figure 3.
Aspertryptanthrins A-C (156-158) were obtained from a terrestrial-derived fungus Aspergillus sp. These compounds all contain an anthranilate unit and a tryptophan residue. In addition, aspertryptanthrin C (158) contains a rare 16-membered ring [74].
Brevicompanines D-H (164-168) were isolated from a deep ocean sediment derived fungus Penicillium sp. [77]. Both brevicompanines E (165) and H (168) inhibited lipopolysaccharide (LPS)induced nitric oxide production in BV2 microglial cells. Further studies showed that brevicompanine E (165) reduced lipopolysaccharide-induced production of pro-inflammatory cytokines and enzymes in microglia by inhibiting activation of activator protein-1 and nuclear factor κB and, hence, may be potentially useful for modulating neuroinflammation [78].
Cycloechinulin (190) was isolated from the sclerotia of Aspergillus ochraceus, and it showed moderate insecticidal activity against the lepidopteran crop pest Helicoverpa zea [57].
Echinulin (206) is one of the simplest classes of isoprenylated tryptophan cyclodipeptides. It was toxic to rabbits, producing a significant degree of damage to lung and liver [79].
Fructigenines A (211) and B (212) are annulated derivatives of cyclo(L-Trp-L-Phe) (187), and were isolated from Penicillium fructigenum. Only fructigenine A (211) inhibited growth of Avena coleoptiles and L-5178Y (mouse lymphoma cells), and was subsequently found to have more potent anti-inflammatory activity than indomethacin in the mouse ear edema model [80]. Fructigenine A (211) was also named rugulosuvine B (211) in Penicillium rugulosum, and showed potent antiinflammatory and antitumor activities in vitro [81].
Neoechinulin A (234) had scavenging, neurotrophic factor-like and antiapoptotic activities. The protective properties of neoechinulin A (234) against SIN-1-induced neuronal cell death suggested that neoechinulin A (234) could protect against neuronal cell death in neurodegenerative diseases [82].

Tryptophan-Xaa Cyclodipeptides
Apart from Trp-Pro and Trp-Trp cyclodipeptides, other tryptophan cyclodipeptides are also abundant in fungi and represent a structurally diverse group of natural products. Their occurrence and biological activities are shown in Table 3, and their structures are provided in Figure 3.
Aspertryptanthrins A-C (156-158) were obtained from a terrestrial-derived fungus Aspergillus sp. These compounds all contain an anthranilate unit and a tryptophan residue. In addition, aspertryptanthrin C (158) contains a rare 16-membered ring [74].
Brevicompanines D-H (164-168) were isolated from a deep ocean sediment derived fungus Penicillium sp. [77]. Both brevicompanines E (165) and H (168) inhibited lipopolysaccharide (LPS)-induced nitric oxide production in BV2 microglial cells. Further studies showed that brevicompanine E (165) reduced lipopolysaccharide-induced production of pro-inflammatory cytokines and enzymes in microglia by inhibiting activation of activator protein-1 and nuclear factor κB and, hence, may be potentially useful for modulating neuroinflammation [78].
Cycloechinulin (190) was isolated from the sclerotia of Aspergillus ochraceus, and it showed moderate insecticidal activity against the lepidopteran crop pest Helicoverpa zea [57].
Echinulin (206) is one of the simplest classes of isoprenylated tryptophan cyclodipeptides. It was toxic to rabbits, producing a significant degree of damage to lung and liver [79].
Fructigenines A (211) and B (212) are annulated derivatives of cyclo(L-Trp-L-Phe) (187), and were isolated from Penicillium fructigenum. Only fructigenine A (211) inhibited growth of Avena coleoptiles and L-5178Y (mouse lymphoma cells), and was subsequently found to have more potent anti-inflammatory activity than indomethacin in the mouse ear edema model [80]. Fructigenine A (211) was also named rugulosuvine B (211) in Penicillium rugulosum, and showed potent anti-inflammatory and antitumor activities in vitro [81].
Neoechinulin A (234) had scavenging, neurotrophic factor-like and antiapoptotic activities. The protective properties of neoechinulin A (234) against SIN-1-induced neuronal cell death suggested that neoechinulin A (234) could protect against neuronal cell death in neurodegenerative diseases [82].

Name Fungus and its Origin Biological Activity
Ref.

Proline-Xaa Cyclodipeptides
Except Trp-Pro cyclodipeptides, other proline containing cyclodipeptides (Pro-Xaa) are also abundantly distributed in fungi. Their occurrence and biological activities are shown in Table 4, and their structures are provided in Figure 4.
Cyclo(L-Pro-L-Ala) (318) was isolated from Alternaria alternata [149] and the phytopathogenic fungus Colletotrichum gloesporoides [150]. This compound inhibited aflatoxin production in aflatoxigenic fungi without affecting fungal growth. Further investigation on the mode of action suggested that this cyclodipeptide inhibited aflatoxin biosynthesis by affecting glutathione Stransferase (GST) function in Aspergillus flavus to show its potency as the biocontrol agent [151].

Name
Fungus and its Origin Biological Activity Ref.

Proline-Xaa Cyclodipeptides
Except Trp-Pro cyclodipeptides, other proline containing cyclodipeptides (Pro-Xaa) are also abundantly distributed in fungi. Their occurrence and biological activities are shown in Table 4, and their structures are provided in Figure 4.
Cyclo(L-Pro-L-Ala) (318) was isolated from Alternaria alternata [149] and the phytopathogenic fungus Colletotrichum gloesporoides [150]. This compound inhibited aflatoxin production in aflatoxigenic fungi without affecting fungal growth. Further investigation on the mode of action suggested that this cyclodipeptide inhibited aflatoxin biosynthesis by affecting glutathione S-transferase (GST) function in Aspergillus flavus to show its potency as the biocontrol agent [151].

Name
Fungus and its Origin Biological Activity Ref.

Non-Tryptophan-Non-Proline Cyclodipeptides
Non-tryptophan-non-proline cyclodipeptides mean neither tryptophan nor proline is incorporated into this group of cyclodipeptides in the fungi. Their occurrence and biological activities are shown in Table 5, and the structures are provided in Figure 5.
Cyclic phenylalanyl serine cyclo(Phe-Ser) (358) was isolated from the insect pathogenic fungus Verticillium hemipterigenum. It exhibited concentration-dependent atypical intestinal absorption in the small intestine of rats, which consisted of passive transport, carrier-mediated absorptive transport by PEPT1, and carrier-mediated excretive transport. It also exhibited weak inhibition of several cancer cell lines and selected microorganisms [173,174].
Diatretol (365) from the fungus Clitocybe diatreta exhibited a weak antibacterial activity. A single-crystal X-ray analysis showed that diatretol (365) has a nearly planar boat conformation in the solid state [175].
Dimerumic acid (368) has been isolated from the fungus Monascus anka, traditionally used for fermentation of food, and shown to be an antioxidant with hepatoprotective actions against chemically induced liver injuries [176], as well as protecting against oxidative stress-induced cytotoxicity in the isolated rat hepatocytes [177].
Phenylahistin (392) from the culture broth of Aspergillus ustus NSC-F038 exhibited a strong growth inhibition on various tumor cell lines for its microtubule binding function to show its potency as the tubulin depolymerizing agent [182]. Table 5. Non-tryptophan-non-proline cyclodipeptide analogs and their biological activities.

Name
Fungus and its Origin Biological Activity Ref.

1,4-Bridged Epiplythiodioxopiperazine Analogs
The sulfur-bridged cyclodipeptides are a class of metabolites mainly dominated by the epipolythiodioxopiperazines (ETPs) [1]. The toxicity of ETPs is due to the presence of a sulfide bridge, which can inactivate proteins via reaction with thiol groups and by generation of reactive oxygen species by redox cycling [5]. The ETPs are known for their cytotoxic effect on cancer cell lines, and it

1,4-Bridged Epiplythiodioxopiperazine Analogs
The sulfur-bridged cyclodipeptides are a class of metabolites mainly dominated by the epipolythiodioxopiperazines (ETPs) [1]. The toxicity of ETPs is due to the presence of a sulfide bridge, which can inactivate proteins via reaction with thiol groups and by generation of reactive oxygen species by redox cycling [5]. The ETPs are known for their cytotoxic effect on cancer cell lines, and it has been shown mechanistically that ETPs were transcriptional antagonists that block the interaction of the p300/CBP coactivator with the hypoxia-inducible transcription factor HIF-1α by a zinc ejection mechanism, which resulted in rapid down regulation of hypoxia-inducible genes critical for cancer progression [208]. The occurrence and biological activities of 1,4-bridged epiplythiodioxopiperazine analogs from fungi are shown in Table 6, and their structures are provided in Figure 6.
The dimeric ETP chaetocin (420) was isolated from the fungus Chaetomium minutum, and, in addition to its antibacterial and cytostatic activity, was reported to have inhibitory activity against lysine-specific histone methyltransferases (HMTs), which are key enzymes in the epigenetic control of gene expression [209]. Chaetocins B (421) and C (422) from a Chaetomium sp. fermentation broth were potent inhibitors of Staphylococcus aureus, and exhibited potent cytotoxic activity against HeLa cells with IC 50 values of 0.03 and 0.02 µg/mL, respectively [210].
Three ETPs deoxyapoaranotin (432), acetylaranotin (408) and acetylapoaranotin (407) were isolated from Aspergillus sp. KMD 901 found in the marine sediment obtained from the East Sea of Korea. They had directly cytotoxic and apoptosis inducing effects toward HCT116 colon cancer cell lines [212].
The best known ETP was the small lipid-soluble gliotoxin (461), which exerted toxic effects on phagocytic cells and T-lymphocytes at low concentrations in vitro. This compound was the first ETP to be obtained from fungi. It has been isolated from a variety of fungi including species in the genera of Penicillium, Aspergillus, Gliocladium, Thermoascus, and Candida [2]. High levels of gliotoxin (461) were produced by Aspergillus fumigatus in vivo, and it appeared to be a virulence factor associated with invasive aspergillosis of immunocompromised patients [216]. Gliotoxin (461) was also a dual inhibitor of farnesyltransferase and geranylgeranyltransferase I with antitumor activity against breast cancer in vivo [217].
The antifungal macrolide MPC1001 (438) possessing an ETP ring was isolated from the fungus Cladorrhinum sp. KY4922, which was found in a soil sample collected in Indonesia. It possessed antiproliferative activity against the human prostate cancer cell line (DU145) with an IC 50 of 9.3 nM, and had antibacterial activity against Gram-positive bacteria [220].
Secoemestrin D (500) was obtained from the endophytic fungus Emericella sp. AST0036 isolated from a healthy leaf tissue of Astragalus lentiginosus. This compound showd cytotoxic activity with IC 50 values ranging from 0.06 to 0.24 µM on tumor cell lines [221].

Analogs with Sulfur-Bridge outside 2,5-DKP Ring
There are a group of sulfur-bridged cyclodipeptides where the sulfur linkage is outside the 2,5diketoperazine ring. The occurrence and biological activities of fungal analogs with sulfur-bridges outside 2,5-DKP ring are shown in Table 7, and their structures are provided in Figure 7.
Both aspirochlorine (524) and tetrathioaspirochlorine (551) were isolated from Aspergillus flavus and were potent antifungals that inhibited azole-resistant Candida albicans [107]. Aspirochlorine (524) was a rather potent and selective inhibitor of fungal protein synthesis that did not inhibit bacterial or mammalian protein synthesis [267].

Analogs with Sulfur-Bridge outside 2,5-DKP Ring
There are a group of sulfur-bridged cyclodipeptides where the sulfur linkage is outside the 2,5-diketoperazine ring. The occurrence and biological activities of fungal analogs with sulfur-bridges outside 2,5-DKP ring are shown in Table 7, and their structures are provided in Figure 7.
Both aspirochlorine (524) and tetrathioaspirochlorine (551) were isolated from Aspergillus flavus and were potent antifungals that inhibited azole-resistant Candida albicans [107]. Aspirochlorine (524) was a rather potent and selective inhibitor of fungal protein synthesis that did not inhibit bacterial or mammalian protein synthesis [267].

Nonbridged Methylthio-Containing Cyclodipeptide Analogs
Nonbridged methylthio-containing analogs were often isolated from fungi as co-metabolites with their sulfur-bridge 2,5-DKP parents. They had a related biosynthetic pathway [275]. The occurrence and biological activities of this group of fungal metabolites are shown in Table 8, and their corresponding structures are provided in Figure 8.
Alternarosin A (555) from Alternaria raphanin, a halotolerant marine fungus obtained from the sediment of the Hongdao sea salt field, showed very weak antimicrobial activity against Escherichia coli, Bacillus subtilis, and Candida albicans with MIC values ranging from 200 to 400 μM [276].
Plectosphaeroic acids A (620) and B (621) were obtained from marine-derived fungus Plectosphaerella cucumerina. They were inhibitors of indoeamine 2,3-dioxygenase (IDO), which existed in primary tumor cells. IDO has been considered as an important molecular target for cancer therapy [254].

Nonbridged Methylthio-Containing Cyclodipeptide Analogs
Nonbridged methylthio-containing analogs were often isolated from fungi as co-metabolites with their sulfur-bridge 2,5-DKP parents. They had a related biosynthetic pathway [275]. The occurrence and biological activities of this group of fungal metabolites are shown in Table 8, and their corresponding structures are provided in Figure 8.
Alternarosin A (555) from Alternaria raphanin, a halotolerant marine fungus obtained from the sediment of the Hongdao sea salt field, showed very weak antimicrobial activity against Escherichia coli, Bacillus subtilis, and Candida albicans with MIC values ranging from 200 to 400 µM [276].
Dehydroxybisdethiobis(methylthio)gliotoxin (577) has been isolated from the broth of a marine-derived fungus Pseudallescheria sp. and exhibited weak antibacterial activity against methicillin-resistant and multidrug-resistant Staphylococcus aureus with MIC values of 31.2 µg/mL [280].
Plectosphaeroic acids A (620) and B (621) were obtained from marine-derived fungus Plectosphaerella cucumerina. They were inhibitors of indoeamine 2,3-dioxygenase (IDO), which existed in primary tumor cells. IDO has been considered as an important molecular target for cancer therapy [254]. Table 8. Nonbridged methylthio-containing cyclodipeptide analogs from fungi and their biological activities.

Name
Fungus and its Origin Biological Activity Ref.

Conclusions and Future Perspectives
A large number of cyclodipeptides have been identified in fungi, and many have received attention not only as challenging synthetic targets but also because some of these compounds displayed diverse and interesting biological activities. Since then, interest has increased in the biosynthesis, genetics, total synthesis, biological activities, and medicinal properties of this class of natural products. Some cyclodipeptides such as tryprostatins A (103)

Conclusions and Future Perspectives
A large number of cyclodipeptides have been identified in fungi, and many have received attention not only as challenging synthetic targets but also because some of these compounds displayed diverse and interesting biological activities. Since then, interest has increased in the biosynthesis, genetics, total synthesis, biological activities, and medicinal properties of this class of natural products. Some cyclodipeptides such as tryprostatins A (103)

Conclusions and Future Perspectives
A large number of cyclodipeptides have been identified in fungi, and many have received attention not only as challenging synthetic targets but also because some of these compounds displayed diverse and interesting biological activities. Since then, interest has increased in the biosynthesis, genetics, total synthesis, biological activities, and medicinal properties of this class of natural products. Some cyclodipeptides such as tryprostatins A (103) and B (104), cyclo(L-Pro-L-Ala) (318), cyclo(L-Pro-L-Phe) (334), cyclo(L-Pro-L-Tyr) (337), phenylahistin (392), and FR106969 (590) have displayed their potential applications in agriculture and medicinal industry [16,17,149,151,182,281].
The fungal cyclodipeptides are mainly distributed in the genera of Aspergillus and Penicillium. However, the cyclodipeptides in the remaining genera seem to be less explored. Further identification and exploration of the cyclodipeptides from all of the fungal genera are needed. In recent years, more and more cyclodipeptides have been isolated from marine-derived and plant endophytic fungi [297][298][299][300]. These fungi inhabiting particular environments could be rich sources of biologically active cyclodipeptides that are indispensable for medicinal and agricultural applications.
The biological activities (shown in Tables 1-8) of the cyclodipeptides reported by each investigator were random and limited. Systematical screening of biological activities for each cyclodipeptide should be necessary. In most cases, the biological activities as well as the mode of action of fungal cyclodipeptides have been investigated based on in vitro studies or animal modes. Few studies have been performed at the level of clinical trials in patients. Effective research and development methods for these compounds should be explored to maximize their usefulness in the drug discovery and development processes [6]. For the diverse biological activities, the cyclodipeptides from fungi are expected to inspire medicinal chemists in their search for better agents such as antitumors, antifungals, and antibacterials than existing ones [297].
It is very important to understand biosynthetic mechanisms of the cyclodipeptides in fungi. These need to combine their biochemical and genetic approaches. More and more designed biologically active cyclodipeptides will be expected to be produced by genetic manipulation. With a good understanding of the biosynthetic pathways of bioactive cyclodipeptides, we can not only increase outputs of the beneficial cyclodipeptides but also block biosynthesis of some harmful cyclodipeptides by specific interferences [6].