Fungal Depsides—Naturally Inspiring Molecules: Biosynthesis, Structural Characterization, and Biological Activities

Fungi represent a huge reservoir of structurally diverse bio-metabolites. Although there has been a marked increase in the number of isolated fungal metabolites over the past years, many hidden metabolites still need to be discovered. Depsides are a group of polyketides consisting of two or more ester-linked hydroxybenzoic acid moieties. They possess valuable bioactive properties, such as anticancer, antidiabetic, antibacterial, antiviral, anti-inflammatory, antifungal, antifouling, and antioxidant qualities, as well as various human enzyme-inhibitory activities. This review provides an overview of the reported data on fungal depsides, including their sources, biosynthesis, physical and spectral data, and bioactivities in the period from 1975 to 2020. Overall, 110 metabolites and more than 122 references are confirmed. This is the first review of these multi-faceted metabolites from fungi.


Introduction
Fungi are widespread cosmopolitan organisms that represent the second-largest class of organisms after insects [1]. For decades, they have been seen as harmful, causing health hazards and spoiling foods. Today, the view on fungi has altered to take into account their advantageous effects, which have become apparent in biotechnological and industrial fields, as well as the production of structurally unique and life-saving metabolites [2][3][4][5]. In the past decades, the number of bioactive metabolites isolated from fungi has been rapidly increased due to their wide diversity of ecological and environmental niches across the globe, including marine, terrestrial, and water environments where they function as pathogens, symbionts, and saprobes [6,7]. Fungi-derived metabolites have made remarkable contributions to the process of drug discovery [8][9][10][11][12][13][14][15][16]. They have been used as antibiotics, herbicides, pesticides, anti-infectives, immuno-suppressants, and anticancer agents [6,7,17]. Depsides are simple polyketides that are formed by the condensation of two or more hydroxybenzoic acid moieties via ester linkage; the COOH group of one molecule is esterified with a phenolic OH group of the second molecule. They could be β-orcinol (β-orsellinic acid) or orcinol (orsellinic acid) derivatives, relying on the existence of the C 3 methyl group on both rings ( Figure 1). The ring with an ester-carbonyl is referred to as ring A and the other as ring B. Their major structural variations are the attached alkyl chains' length, the degree of chain oxidation, and the degree of methylation of OH and COOH groups [18]. The OH groups usually exist at the aromatic carbons, (NR-PKSs). Depsides consist of two orsellinic acid molecules, connected by an ester linkage. Therefore, orsellinic acid can be considered the constructing unit of all depsides [28]. Biosynthetically, orsellinic acid is produced from a linear tetraketide chain. This chain is formed through an acetate-malonate pathway that is catalyzed by PKSs [29,30]. The tetraketide chain forming β-orsellinic acid (methyl-3-orsellinate) is produced by introducing a CH 3 group obtained from SAM (S-adenosylmethionine) by the methyl transferase (CMeT) domain of the corresponding PKS [31]. Then, the non-enzymatic 2,7-aldol condensation of these chains produces orsellinic and β-orsellinic acids. Furthermore, the molecular skeleton is probably designed by post-biosynthetic tailoring enzymes, such as cyclases and hydrolases [18]. p-Depsides are produced by the condensation of either orsellinic acid and orcinol derivatives or by two methyl-3-orsellinate or orsellinate moieties, through the formation of an ester [12]. The consequent condensation of an additional unit produces a tri-depside, and two moieties yield a tetra-depside [32]. Moreover, depsides containing alkyl side-chains can be produced by the reduction of the terminal ketone groups, resulting in the required saturated alkyl moieties. m-Depsides are formed through the hydroxylation of the para-depside B-ring, subsequently followed by rearrangement [33] (Figure 2).

Biological Activities
Despite the unique structures of depsides, they have not been well investigated in terms of their pharmacological activities. The literature survey revealed that depsides have various biological activities (Table 2). Thus, an overview of their reported pharmacological activities is summarized in Table 2 and is described in detail below.

Antitumor Activity
Cancer is considered the second cause of death after cardiovascular diseases [34]. In 2020, around 10 million deaths were estimated to have been due to cancer, 70% of which were in middle-and low-income countries [35]. Most of the anticancer agents cannot distinguish between abnormal and normal cells; thus, researchers have been directed to develop selective and safe anticancer drugs that target the abnormal cancerous cells and have minimal effects on normal cells. Fungi represent an important source of anticancer agents, with significant benefits against various tumors [36]. It is noteworthy to mention that most of the reported depsides showed activity on cancer cells with no or little effect on normal cells.
CRM646-A (36) and CRM646-B (37) were discovered from Acremonium sp. that showed a potent anti-metastatic capacity toward B16-F10 melanoma cells, with an IC 50 value of 15 µM for 36 and IC 50 30 µM for 37 [43]. They also caused the dose-dependent inhibition of heparinase, with IC 50 values of 3 and 10 µM, respectively, in comparison to suramin (IC 50 value of 5 µM) [43,44]. Asami et al. established that CRM646-A (36) induced the inhibition of cells' invasion, migration, and growth in tumor cells, due to its induction of nucleus condensation, plasma membrane disruption, and morphological changes in result to the increase in Ca 2+ levels; thus, it could potentially be used as an effective anti-metastatic agent [45]. Compounds 45 and 46 in the MTT assay showed an antitumor effect against A549 and MAD-MB-435, with IC 50 values of 16.82 and 37.01 µM, and 20.75 and 37.73 µM, respectively, compared with epirubicin (IC 50 0.26 and 5.60 µM, respectively); however, 11 did not exhibit obvious activity [46]. Togashi et al. reported that 36 and 49 prohibited telomerase activity at doses of 3.2 and 32 µM, respectively. In addition, they inhibited viral reverse transcriptase activity at almost the same dose levels; therefore, they may inhibit universal RNA-dependent DNA polymerases [47]. Compound 50, a tridepside, was obtained from MSX 55526 fungus and showed moderate activity against the MCF-7, H460, and SF268 cell lines in the SRB assay, with IC 50 values of 7.3, 6.6, and 8.1 µM, respectively, compared to camptothecin (IC 50 0.07, < 0.01, and 0.04 µM, respectively) [48].

Antimicrobial Activity
The wide use of antibiotics leads to the development of resistant microbes [85]. Moreover, the number of efficient drugs against life-threatening fungal and bacterial infections has decreased dramatically because of emerging pathogens that are multidrug-resistant (MDR), which is the biggest obstacle to success during the treatment of infectious diseases [86]. Therefore, there is a growing demand for new antimicrobial compounds. Fungi are considered an important source of novel antimicrobials because of their rich secondary metabolites and abundant species diversity. Bacterial enoyl-ACP (acyl carrier protein) reductase accelerates the last and rate-limiting step in type II FAS (bacterial fatty acid synthesis) [87,88]. Enoyl-ACP reductase includes three isoforms, FabK, FabI, and FabL. It is found in most bacteria: S. aureus (FabI), Streptococcus pneumonia (FabK), P. aeruginosa and Enterococcus faecalis (FabK and FabI), B. subtilis (FabI and FabL), and Mycobacterium tuberculosis (InhA, a FabI homolog) [61,89]. This enzyme has been established as a novel target for treatment against infections produced by MDR pathogens.  [38]. Setophoma sp. associ-ated with guava fruits produced compounds 6-8 and 66-69 [19]. They did not have growth inhibition activity toward E. coli. However, 66-69 demonstrated inhibition of S. aureus with MIC values of 100, 6.25, 50, and 25 µg/mL, respectively, in comparison to tetracycline (MIC 3.12 µg/mL) [19]. Compounds 6 and 7 were inactive. Moreover, all compounds did not exhibit quorum-sensing inhibitory activity. Studying the structural activity relationship revealed that the activity increased with the full methylation of the B-ring; however, the additional CH 3 group at ring A, especially at C-2, resulted in a decrease in activity [19]. The agonodepsides A (12) and B (13) were isolated from the filamentous fungus, F7524 [50]. In the fluorometric InhA assay, 12 inhibited M. tuberculosis InhA with an IC 50 value of 75 µM, while 13 was inactive at 100 µM, compared with triclosan (IC 50 3.0 µM) [50].

Anti-Diabetic Activity
Diabetes is among the most prevalent chronic diseases and is characterized by hyperglycemia, which leads to damage of the blood vessels. This may produce macro-and micro-vascular disorders, as well as other complications, such as sexual dysfunction, dementia, lower-limb amputations, and depression [91]. Diabetes prevalence is expected to be at approximately 366 million cases by the year 2030 [92]. The side effects of the available hypoglycemic agents necessitate the discovery of efficient, low-side-effect, and affordable agents for treating diabetes.
Rivera-Chávez et al. reported that the tridepside, 59 (dose 3.1-31.6 mg/kg), reduced glucose blood levels after 30 min of oral administration of the sucrose load in mice (3.0 g/kg); however, only the highest dose (31.6 mg/kg) caused a marked reduction in blood glucose levels in NA-STZ (nicotinamide-streptozotocin) diabetic mice, indicating that 59 (doses of 3.1 and 10 mg/kg) reduced the blood glucose levels in both diabetic and normal mice [67].

D-Glucose-6-Phosphate Phosphohydrolase Inhibitory Activity
G6Pase (D-glucose-6-phosphate phosphohydrolase) is a hepatic metabolism-regulating enzyme, that catalyzes the last steps of glycogenolysis and gluconeogenesis pathways [93]. Its inhibition decreases the output of hepatic glucose from both pathways, leading to lowering the blood glucose levels in diabetes. The tetra-depside, 97 isolated from Chloridium sp. CL48903 prohibited G6Pase in rat liver microsomes (IC 50 1.6 µM) at a concentration of 133 µM, using a colorimetric assay and hepatocyte glucose output (81% inhibition), indicating the role of 97 as a G6Pase inhibitor [77].

α-Glucosidase Inhibitory (αGI) Activity
The α-glucosidase enzyme is an important therapeutic target for treating carbohydratemediated diseases. It catalyzes the breakdown of oligo-and disaccharides into monosaccharides in the final stage of carbohydrate digestion, leading to a rise in glucose levels [94][95][96][97]. Several studies revealed that α-glucosidase inhibitors (αGIs) slow down the digestion and absorption of carbohydrates, and thus reduce the postprandial blood glucose level [94][95][96][97]. The serious side effects of the current αGIs, such as liver injuries and gastrointestinal damage, have directed research efforts toward discovering and developing new and safer anti-diabetic agents.

Protein Tyrosine Phosphatase Inhibitory (PTP1BI) Activity
PTP1B (protein-tyrosine phosphatase 1B) is a negative regulator of the insulin signaling pathway. The inhibition of PTP1B activity has great promise for alleviating insulin and leptin resistance; hence, PTP-1BIs (PTP1B inhibitors) show potential for treating T2DM and other metabolic disorders [98].

Diacylglycerol Acyltransferase Inhibitory (DGATI) Activity
Postprandial hypertriglyceridemia is considered the main risk factor for cardiovascular functions. Thus, triglyceride synthesis inhibition has remarkable therapeutic potential in metabolic disorder treatment. The enzymes known as diacylglycerol acyltransferases (DGATs) catalyze the final and only committed step in the biosynthesis of triglycerides [99]. Therefore, these enzymes could be a potential therapeutic target to combat cardio-metabolic disorders [71,82,99]. Compound 9 also inhibited TG synthesis (IC 50 91 µM), as well as PC and PE syntheses, indicating that it had a non-specific DGATI effect [76]. The compounds 86 and 88-90 were purified from Humicola sp. by Tomoda et al. [74]. Compound 88 was the most potent DGATI, with an IC 50 of 10.

Activity of 11β-Hydroxysteroid Dehydrogenase Inhibitory (11β-HSDI) Enzyme
High levels of glucocorticoid produce insulin resistance and glucose intolerance, leading to metabolic syndrome (MS) [100]. The enzyme 11β-HSD (11β-hydroxysteroid dehydrogenase) is accountable for the production of glucocorticoids in tissues, thus it plays a remarkable role in T2DM and MS. The 11β-HSD1 inhibitors (11β-HSD1Is) could be considered promising therapeutics in treating MS. Compounds 38-41 exhibited powerful and selective inhibitory activities against 11β-HSD1 in the HTRF immunoassay. They inhibited human 11β-HSD1 activity in a dose-dependent manner with IC 50 values ranging from 240 to 6600 nM. Compounds 38 and 40 were the most active with IC 50 s 240 and 230 nM, respectively, while they did not prohibit 11β-HSD2 (IC 50 > 10,000 nM) [64].

Antimalarial Activity
Malaria is among many prevalent health concerns and is caused by the Plasmodium parasite in several of the world's tropical regions [105]. The emergence of malaria strains that are drug-resistant to the available therapeutics makes the discovery of new antimalarial agents a great scientific challenge [14,106].

Ca 2+ /CaM Dependent Phosphodiesterase Inhibitory (CaM-PDEI) Activity
Calmodulin (CaM) is a prevalent Ca 2+ -binding protein that regulates several Ca 2+dependent cellular functions in physiological and pathophysiological processes [107]. It is implicated in the cytoskeleton function and architecture, cell motility, apoptosis, cell proliferation, autophagy, the dephosphorylation/phosphorylation of proteins, reproductive processes, ion channel function, the relaxation/contraction of smooth muscle, and gene expression [108]. CaM can regulate these processes via modulating various proteins, including enzymes: phosphodiesterase, kinases, NOS (nitric oxide synthases), phosphatases, and ion channels. CaM-PDE is a key enzyme that is embroiled in the complex interactions between the cyclic nucleotide and Ca 2+ -second messenger systems [108]. Moreover, CaM is linked with several pathological states, including smooth muscle malfunctions and unregulated cell growth. CaM-PDEIs may play an important role in treating various disorders, such as neurodegenerative diseases and cancer [109].
Nakanishi et al. reported that 34 and 35, purified from Sporothrix sp., inhibited heart and bovine brain PDEs (IC 50 4.3 and 1.8 µM and 5.9 and 15.0 µM, respectively) [63]. Moreover, they prohibited the CaM-dependent activities of CaM-PDEs but had a low effect against their CaM-independent effects, suggesting that these compounds interacted with CaM to inhibit Ca 2+ /CaM-dependent enzymes. On the other hand, they had no inhibitory activities on protein kinase C [63]. Moreover, PS-990 (47), isolated from Acremonium sp., inhibited brain CaM-PDE with an IC 50 value of 3 µg/mL and did not elevate the intracellular cyclic AMP level. It markedly induced the neurite extension of Neuro2A (mouse neuroblastoma) at concentrations ranging from 10 to 30 µg/mL, suggesting its neuritogenic effect. It inhibited both cell growth and thymidine incorporation into the cells at the same concentration range. Interestingly, 47 reversibly induced neurite formation, with cell growth arrest through a mechanism other than increasing the intracellular cyclic AMP concentration [66,110].
3.14. Antiviral Activity HCMV (human cytomegalovirus) is the most familiar viral cause of congenital infections, which can lead to severe birth defects. Its current treatments include viral DNA polymerase inhibitors, which block the late stages of HCMV replication; however, they do not prohibit the viral induction of multiple cell activation events [111]. Thus, it may be beneficial to discover new treatments for HCMV infections.

Human Leukocyte Elastase (HLE) Inhibitory Activity
HLE is one of the most destructive enzymes that can degrade tissue matrix proteins, such as collagen, elastin, fibronectin, proteoglycan, and laminin, by activating progelatinase, procollagenase, and prostromelysin [25]. It is released from PMNLs (polymorphonuclear leukocytes) as a result of inflammatory mediators and stimuli. HLE is considered an important therapeutic target for treating many inflammation-linked disorders [112].

Indoleamine 2,3-Dioxygenase Inhibitory (IDOI) Activity
IDO (indoleamine 2,3-dioxygenase) catalyzes the tryptophan catabolism initial step via the KP (kynurenine pathway) [68]. Dysregulation of the KP is accompanied by the IDO activity elevation and production of quinolinic acid (an excitotoxin), which has been engaged in the pathogenesis of neurodegenerative disorders, neuroinflammatory, HIV encephalitis, age-related cataract, and depression [68]. Therefore, IDO is a promising target of new therapeutics for treating neurological disorders and cancer, as well as other disorders characterized by a defect in tryptophan metabolism.

Adenosine Triphosphatase Inhibitory Activity
Na + /K + -ATPase (sodium/potassium adenosine triphosphatase) is an integral membrane protein that is accountable for maintaining Na + and K + gradients across the plasma membrane, an important process for mammalian cell survival. Currently, it is extensively studied as a potential target for cancer treatment, especially in glioblastoma and lung cancer [113]. The proton pump, H + /K + ATPase, plays an important role in the stomach acidification process. Its inhibition in gastric parietal cells decreases gastric acid overpro-duction [114]. H + /K + ATPase inhibitors can be utilized as a target for developing drugs against gastric acid production disturbances.

Conclusions
Recently, more focus has been given to fungi as they are excellent platforms for the biosynthesis of a huge number of structurally diverse metabolites. The knowledge of these metabolites offers a virtually untapped source of new bioactive metabolites with potential agrochemical and pharmaceutical uses. Fungi use these metabolites for defense, and many of these metabolites demonstrate a broad range of bioactivities. Among these metabolites are depsides that possess remarkable bioactivities. According to the listed results, there are 110 depsides that have been isolated from fungi. Most of them are reported from Thielavia (26.6%), Stereum (17.4%), Chaetomium (15%), and Humicola (12%) species (Figure 15).  Most of the reported depsides have been evaluated for their α-GI (α-glucosidase inhibitory), antimicrobial, antitumor, antifouling, PLA2 (phospholipase A2), and DGATI (diacylglycerol acyltransferase inhibitory) abilities ( Figure 17). Thus, these studies revealed that fungal depsides are a rich source for the discovery of effective and novel pharmaceutical leads and should be further exploited. They also demonstrate inhibitory activities against various enzymes that can be utilized as targets for the treatment of various diseases. These metabolites could have potential as lead compounds for treating metabolic syndrome, obesity, and diabetes via the inhibition of various enzymes, such as HSD, PTP1BI, α-GI, G6Pase, and DGAT. However, extensive explorations of their mechanism of action, as well as structure modification, chemical synthesis, and structure/activity relationship analysis are needed. Despite the extensive structural diversity of depsides, none of them has been approved by the FDA, and none of them has as yet progressed to clinical trials. Therefore, the impact of fungal depsides on human health concerns has to be considered in several ways.