Secondary Metabolites Diversity of Aspergillus unguis and Their Bioactivities: A Potential Target to Be Explored

Aspergillus unguis belongs to the Aspergillus section Nidulantes. This species is found in soils and organisms from marine environments, such as jellyfishes and sponges. The first chemical study reported in the literature dates from 1970, with depsidones nidulin (1), nornidulin (2), and unguinol (3) being the first isolated compounds. Fifty-two years since this first study, the isolation and characterization of ninety-seven (97) compounds have been reported. These compounds are from different classes, such as depsides, depsidones, phthalides, cyclopeptides, indanones, diarylethers, pyrones, benzoic acid derivatives, orcinol/orsenillate derivatives, and sesterpenoids. In terms of biological activities, the first studies on isolated compounds from A. unguis came only in the 1990s. Considering the tendency for antiparasitic and antibiotics to become ineffective against resistant microorganisms and larvae, A. unguis compounds have also been extensively investigated and some compounds are considered very promising. In addition to these larvicidal and antimicrobial activities, these compounds also show activity against cancer cell lines, animal growth promotion, antimalarial and antioxidant activities. Despite the diversity of these compounds and reported biological activities, A. unguis remains an interesting target for studies on metabolic induction to produce new compounds, the determination of new biological activities, medicinal chemistry, structural modification, biotechnological approaches, and molecular modeling, which have yet to be extensively explored.


Introduction
Many fungi are capable of producing heterogeneous low-molecular-mass compounds, also called secondary metabolites. These metabolites are not directly necessary for organism growth, unlike primary metabolites [1,2]. The versatile metabolism of fungi allows different types of interactions with other organisms, ranging from bacteria to metazoa, and substrates, which play essential roles in ecosystems. Over hundreds of millions of years, fungi use these metabolites as chemical signals for communication, defending their habitat, or inhibiting the growth of competitors, leading to the evolution of natural products and, consequently, acting in the ecological success of fungi in colonizing approximately all habitats on the planet [3][4][5][6].
Penicillin, considered the "wonder drug" of World War II and the first broad-spectrum antibiotic, was discovered in the fungus Penicillium notatum by Alexander Fleming in 1928. This metabolite significantly changed the search for new natural products from plants to microorganisms, inaugurating "the golden age of antibiotics [7][8][9]. Currently, 45 % of the known microbial metabolites are of fungal origin such as filamentous fungi, including Penicillium, Trichoderma, and Aspergillus, which represent almost 99 % of the total known fungal metabolites [10].
Aspergillus is an ascomycetous fungus with the greatest bioactive potential in nature. Species of this genus have been studied extensively for years, and they produce metabolites of considerable medical, industrial, agricultural, and economic importance. For example, the phytohormone gibberellin produced by A. fumigatus improves plant growth, and the anti-cholesterol drug lovastatin produced by A. terreus, with worldwide sales of USD 10 billion annually. The metabolic roles of these metabolites have been increasingly explored in biotechnological research that seeks new fungal natural products of commercial interest [11][12][13].
Fungal species such as A. niger, A. oryzae, and A. terreus are considered the work horses in biotechnology [14]. However, other species that are not in the spotlight and are less well known have great biotechnological value that has already been described, such as A. unguis. Research into this fungus include its application for the removal of heavy metals from industrial wastewater [15], expression systems for heterologous protein expression [16], production of enzymes of industrial interest [17,18] and, since the last century, the rich production of secondary metabolites. Therefore, this review highlights the secondary metabolites of A. unguis and their biological activities, with a brief description of the biology of A. unguis.

Biology of A. unguis
The fungus A. unguis, described in 1935 by Emile-Weill and L. Gaudin, belongs to the Aspergillus section Nidulantes, forming the series Unguium with four other species: A. israelensis [19], A. croceus [20], A. longistipitatus [21], and A. croceiaffinis [21], all of which were described between 2016 and 2020 using polyphasic taxonomy; that is, with the application of phenotypic, genotypic (using multigenic DNA sequences), and chemotaxonomic techniques [19][20][21][22]. Aspergillus taxonomy is complex, and it changes over time as species identification technology reorganizes just as new species are described [23]. In recent years, many efforts have been made by the scientific community to revise the taxonomy and phylogeny of several species that are already known through polyphasic taxonomy, mainly using DNA sequence data, in order to reorganize these thousands of species in light of new knowledge and modern techniques [24].
Emericella unguis is the sexual state (teleomorph) of A. unguis (anamorph), and it became synonymous with A. unguis with the new nomenclatural rules based on a singlename system established by the International Commission of Penicillium and Aspergillus (ICPA) in 2012 [24]. A. unguis has other possible synonyms, such as Sterigmatocystis unguis and A. mellinus, and there may be others; however, the differentiation of A. unguis and non-cleistothecial A. nidulans isolates was problematic in the past [20].
The complete A. unguis genome was sequenced in 2016 as part of a project to sequence Aspergillus species (Joint Genome Institute, https://jgi.doe.gov, accessed on 24 April 2022) [24]. This information may soon contribute to the taxonomic reordering of the species mainly by using the supporting information on metabolite production.
A. unguis can be isolated from soils [25], lichens [26], and organisms in marine environments, such as jellyfishes [27], seaweeds [28], sponges [29], and others. This ubiquitous fungus has great potential for the "one strain many compounds" (OSMAC) strategy, as it is capable of growing in culture broth with different sources of carbon and nitrogen, such as potato dextrose, oatmeal, glycerol casein, yeast extract sucrose, Czapek-Dox, and malt extract [30,31]. Solid media, in addition to solid agar media, are also used as the main media [26]. On some occasions, the medium can be supplemented with specific salts to study the assimilation and modulation of the production of secondary metabolites or with sea water to mimic the environment of marine isolates [29,30]. The use of A. unguis biology and its different growth methods are excellent alternatives for the discovery of new metabolites of this fungus.

Secondary Metabolism
A review of the 52-year-long chemistry studies of A. unguis (1970-2022) was performed, from the first articles published by  to the last published by Cao in 2022 [32,33]. In this review, ninety-seven chemical structures of compounds isolated and identified from A. unguis are reported. In Table 1, these compounds are organized by code, presenting the following information: (I) the class of metabolites to which they belong, (II) the biological activities of each compound, and (III) the references that report the isolation and identification. The compounds found in A. unguis are classified into depsides, depsidones, phthalides, cyclopeptides, indanones, diarylethers, pyrones, benzoic acid derivatives, orcinol/orsenillate derivatives, and sesterpenoids. Most of the compounds are depsidones, representing approximately 30 % of the total. Among the ninety-seven compounds isolated from A unguis, only twelve were also isolated from other microorganisms: nidulin (1), nornidulin (2), unguinol (3), 2-chlorounguinol (8), (3S)-3-ethyl-5,7-dihydroxy-3,6-dimethylphthalide (9), folipastatin (13), unguisin E (20) aspergillusidone C (23), pilobolusate (37), (+)-montagnetol (38), averantin (75), and corynesidone D (84) (see Table 1). The chemical structure of each compound is also presented in Figure 1. Table 1. All the compounds identified in A. unguis were organized by code, showing for each one (I) the references that report the isolation and identification; (II) the chemical class to which they belong and (III) the biological activities of each compound.

Code
Compound Biological Activities Isolation and Identification

Depsidones
Depsidones consist of two aromatic rings (A and C rings) joined by a -CO-O-bridge (ester group) and an ether group, forming a third seven-membered ring (B ring). This class of compounds is biosynthesized through oxidative coupling of the depsides [42,70].
Three depsidones, nidulin (1), nornidulin (2), and yasimin (3), were isolated [33]. In the same year, the authors published a biosynthetic study of yasimin (3) by incorporating labeled acetate (1-14 C and 2-14 C) and malonate-1-14 C using a fungus culture growing in Czapek-Dox medium [54]. The labeled compound 3 was isolated, and the authors were able to make a series of considerations about the biosynthesis pathways of this compound, classifying them as polyketides [41,54]. Furthermore, in 1970, Kamal published another study describing the identification of four new depsidones from A. unguis: haiderin (4), rubinin (5), (-)-shirin (6), and nasrin (7). None of the four compounds described in this study were reisolated from A. unguis or any other species of fungus; therefore, this is the only report of these four compounds in literature.

Depsidones
Depsidones consist of two aromatic rings (A and C rings) joined by a -CO-O-brid (ester group) and an ether group, forming a third seven-membered ring (B ring). This cl of compounds is biosynthesized through oxidative coupling of the depsides [42,70].
Three depsidones, nidulin (1), nornidulin (2), and yasimin (3), were isolated [33]. the same year, the authors published a biosynthetic study of yasimin (3) by incorporati labeled acetate (1-14 C and 2-14 C) and malonate-1-14 C using a fungus culture growing Czapek-Dox medium [54]. The labeled compound 3 was isolated, and the authors w able to make a series of considerations about the biosynthesis pathways of this compoun classifying them as polyketides [41,54]. Furthermore, in 1970, Kamal published anoth study describing the identification of four new depsidones from A. unguis: haiderin ( rubinin (5), (-)-shirin (6), and nasrin (7). None of the four compounds described in t study were reisolated from A. unguis or any other species of fungus; therefore, this is t only report of these four compounds in literature.
Stodola et al. reisolated compound 3 in 1972 but did not publish the results; howev it was renamed unguinol (3) [56]. Since then, most articles have referred to this compou as unguinol, not yasimin, despite having exactly the same chemical structure. In 19 Turner and Aldridge identified nidulin (1), nornidulin (2), and unguinol (3) in E. nidula (anamorph A. nidulans) and A. mellinus; however, these two organisms have been reiden fied as A. unguis [27]. In 1988, Kawahara   Stodola et al. reisolated compound 3 in 1972 but did not publish the results; however, it was renamed unguinol (3) [56]. Since then, most articles have referred to this compound as unguinol, not yasimin, despite having exactly the same chemical structure. In 1983, Turner and Aldridge identified nidulin (1), nornidulin (2), and unguinol (3) in E. nidulans (anamorph A. nidulans) and A. mellinus; however, these two organisms have been reidentified as A. unguis [27]. In 1988, Kawahara et al. published two studies describing a series of new depsidones, including emeguisin A (10), emeguisin B (11), and emeguisin C (12) [38,39]. These compounds are the first examples of depsidones bearing two 1-methylprop-1-enyl groups in one molecule. Uchida et al. described the structure of a supposedly unprecedented compound, which they named 7-chlorofolipastatin (10). However, this compound was previously identified as emeguisin A (10) [47]. Sureram et al. (2013) subjected A. unguis to growth in medium containing different halogen salts (potassium bromide [KBr], potassium iodide [KI], and potassium fluoride). The fungus grown in medium containing KBr produced three new brominated depsidones, namely aspergillusidones D-F (26)(27)(28). Meanwhile, when the fungus was subjected to medium containing KI, the fungus did not incorporate iodine atoms into the compounds, but a new depsidone, 2,4-dichlorounguinol (32) was isolated and identified. Morshed et al. (2018) manipulated the concentration of halides in the culture medium, inducing A. unguis to produce a series of new compounds, by OSMAC strategy. When the fungus was grown in yeast extract with supplements (YES) medium without saline supplementation, several compounds were produced in addition to an unprecedented substance, 7-carboxyfolipastatin (45). When the YES medium was supple-mented with 0.5 % sodium chloride, it was possible to decrease the production of unguinol (3), in addition to producing new compounds such as 45 and 4,7-dichlorounguinol (46). In an experiment with 0.5 % KBr supplementation, the fungus produced new substances, such as 7-bromounguinol (48), 2-chloro-7-bromounguinol (49), and 7-bromofolipastatin (50). Sureram (2013) and Morshed (2018) explored the concept of precursor-directed biosynthesis, which is an attempt to exploit the metabolic potential of fungi in the face of a precursor. Modifying the concentration of the halides is a powerful tool for modulating secondary metabolite production and triggering quiescent pathways in the fungus. The fungus must be able to recognize, metabolize and generate new unnatural structures [71]. Sureram (2013) and Morshed (2018) showed that this approach was efficient in generating new structures of secondary metabolites. The structural diversity of depsidones has increased due to the recent discoveries of Saetang et al. (2021). They described two new depsidones (69 and 70), both harboring an interesting structure different from all other depsidones: the substitution of the 2-butenyl unit (C ring) for the hydroxy-3-butenyl moiety group. These recent discoveries show that the metabolic potential of A. unguis remains to be elucidated and may elicit surprise.

Depsides
Depsides are substances that are biosynthesized from the union of two orsellinic acid derivative units or the union of an orsellinic acid derivative and an orcinol derivative [42]. Depsides are related to depsidones and are generally accepted as biosynthetic precursors of this depsidones [43]. Nielsen et al. (1999)

Cyclopeptides
Cyclic heptapeptides unguisin A (16) and unguisin B (17) isolated from a marinederived strain of E. unguis are considered to contain gamma-aminobutyric acid (GABA) in the ring. The only difference between the two peptides is that unguisin A (16) contains L-phenylalanine, while unguisin B (17) has L-leucine [55]. The cyclopeptide unguisin C (18) was also isolated as a minor component in 2002 [63]. This peptide is structurally similar to unguisin A (16), except for the substitution of D-alanine to D-serine, yielding a more hydrophilic peptide. Unguisin D (19) was produced when L-leucine was added to the fermentation medium and was detected only by liquid chromatography mass spectrophotometry analysis (LC-MS), with the structure cyclo-(tryptophyl-GABA-alanyl-valyl-leucylleucyl-alanyl). This compound is similar to unguisin B (17) with the replacement of valine-2 to leucine [63]. In 2011, Liu and Chen (2011) reported the isolation of a new cyclopeptide, unguisin E (20), from Aspergillus sp. AF119 [64]. The only difference was that the amino acid phenylalanine in unguisin A (16) was replaced by β-methylphenylalanine in 20.

Pyrone
Pyrones are a class of heterocyclic chemical compounds that contain an unsaturated six-membered ring containing one cyclic ester functional group. Despite its presence in fungi, the metabolism of pyrones is still not well known in A. unguis; only one compound of this class has been isolated and identified. The 3-methyl-4-hydroxy-6-(1-trans-methyl-1-propenyl)-2-pyrone (24) was isolated as a natural source for the first time. This pyrone was a des-O-methyl derivative of nectriapyrone, a metabolite previously isolated from the fungus Gyrostroma missouriense [29].

Indanones
Phainuphong described the isolation and structural characterization of a class of compounds that had not yet been identified in A. unguis: indanones. Two new indanones, asperunguisone A (39) and asperunguisone B (40), were described. Indanones structurally exhibit a junction between an aromatic ring and a cyclopropane ring with a ketone function [31].

Anthraquinones
Anthraquinones (polyketides compounds) are an important chemical group. The skeleton is basically two aromatic rings separated by two carbonyl groups. Although these compounds have already been isolated from other species of the genus Aspergillus, it was only in 2021 that the anthraquinones averantin (75), 7-chloroaverantin (76) and 1'-O-methylaverantion (77) were isolated from strains 158SC-067 of A. unguis [68].

Terpenoids and Sterols
Terpenoids belong to a class of compounds formed by the coupling of isoprene units (dimethylallyl pyrophosphate and isopentenyl pyrophosphate) and are classified into subclasses according to the multiplicity of five carbons. In fungi, these isoprene units originate from the mevalonate pathway. Li et al. (2019) isolated and identified six new asperane-type sesterterpenoids (terpenes with 25 carbon atoms), asperunguisins A-F (59)(60)(61)(62)(63)(64), together with a known analogue, aspergilloxide (65). These are rare asperane-type of sesterterpenoids, characterized by a unique hydroxylated 7/6/6/5 tetracyclic system. Compound 65 was discovered as the first asperane-type sesterterpenoid from the marine-derived fungus Aspergillus sp. in 2002 [73]. Rare ergostane-type sterols with an unusual unsaturated side chain (94-97) were reported by Cao and collaborators [32]. It was the first time that this type of compound had been isolated from A. unguis.

Antifungal, Antibacterial, Antimalarial and Larvicidal Activities
Considering the tendency for antibiotics to become ineffective against resistant strains, A. unguis compounds have also been extensively investigated and some of them are very promising against larvae and pathogenic microorganisms. A good example is the study by Yang et al. (2022) which show the potent activity of depsidones nornidulin (2), emeguisin A (10) and folipastatin (13) against vancomycin-resistant bacteria Enterococcus faecium [44]. Synthetic derivatives of nidulin (1) were also investigated for their significant activities against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus bacterial properties. These derivatives, in particular 8-O-aryl ether derivatives, proved to be very promising [74].
Saetang et al. pointed out the activity of the depsides 10 and 13 that were strongly active against the pathogenic yeast Candida neoformans with respective MIC values of 1 and 0.5 µg/mL, an identical value to the positive control, the antifungal amphotericin B. Another despside, aspergillusidone C (23) showed the strongest antifungal activity against Microsporum gypseum with the MIC value of 2 µg/mL, similar to positive control clotrimazole. Yang et al. (2018) considers that the compounds nidulin (1), nornidulin (2) and aspergillusidone F (28) could be used for the development of pesticides for their larvicidal activity. These depsidones exhibited potent larvicidal activity against brine shrimp, with close or even lower LC 50 values compared with the positive control mercury (II) nitrate, Hg(NO 3 ) 2 [31]. Klaiklay and colleagues also demonstrated the potent antimicrobial activities of the depsidone emeguisin A (13). This compound 13 exhibited the most potent antibacterial (against S. aureus and methicillin-resistant S. aureus), and antifungal (against C. neoformans) activities with MIC values of 0.5 µg/mL. Emeguisin A (13) also showed antimalarial activity, with MIC values of 2.2 µM against P. falciparum, identical to the positive control dihydroartemisinin [40].

Anti-Osteoclastogenic Activity
With the aging of the population, some diseases such as osteoporosis have become a concern in recent times. Therefore, the search for new bioactive molecules that can be applied to treatments and therapies becomes extremely important and some research groups have moved towards this. Zhang et al. (2022) evaluated several pure substances and guisinol (15) proved to be a further dose-dependent suppressed RANKL-induced osteoclast differentiation without any evidence of cytotoxicity in bone marrow macrophage cells. This is the first article that reports the identification of an A. unguis compound that has this type of activity [46].

Cytotoxic Activity against Cancer Cell Lines
Cancer is one of the most feared diseases and its treatment can still leave the patient debilitated and sometimes discouraged. Compounds isolated from A. unguis have been extensively evaluated against several tumor cell lines. As an example, Cao et al. (2022) evaluated ergosterane-type sterol against ACHN (renal), HCT-15 (colon), NUGC-3 (stomach), PC-3 (prostate), NCI-H23 (lung), and MDA-MB-231 (breast) cancer cell lines. The new substance aspersterol A (94) showed cytotoxicity against all the tested cell lines [32]. The orsellinate pilobolusate (37) exhibited potent activity against the KB cell line with an IC 50 value of 4.5 µM which was much stronger than the standard drug, ellipticin [40]. The asperane sesterterpenoid asperunguisin C (61) showed cytotoxicity against the human cancer cell line A549 with an IC50 value of 6.2 µM, a value very close to the positive control adriamycin (2.9 µM) [26].

Anti-Inflammatory Activity
Cao and colleagues monitored the anti-inflammatory potential of ergostanes and concluded that the compound aspersterol C (96) showed the most potent anti-inflammatory activity. This compound inhibited the production of inflammatory mediators, including IL-6 and iNOS in LPS-induced macrophages (RAW 264.7 cells). The authors consider that this molecule could be used as a lead for additional studies for anti-inflammatory models [32]. In a similar study, this research group isolated A. unguis and the anti-inflammatory properties of compound variotin B (90), the first linear nitrogenous secondary metabolite isolated from A. unguis [62].

Enzyme Inhibitors
Biological tests involving natural products were greatly advanced in the 1990s. Among all the studies analyzed, the first one that shows the attribution of biological activity to a compound isolated directly from A. unguis was reported by Hamano and collaborators in 1992, wherein depsidone folipastatin (13) inhibited the enzyme phospholipase A 2 enzyme isolated from rabbit peritoneal exudate. These enzymes are involved in inflammatory processes [52]. Studies suggest that unguinol inhibits C4 plant enzyme pyruvate phosphate dikinase (PPDK) via a novel mechanism of action which also translates to a herbicidal effect on whole plants. This compound had deleterious effects on a model C4 plant but no effect on a model C3 plant [49].
The depsidone aspergillusidone C (23) showed the inhibitory activity of the aromatase enzyme with an IC 50 value of 0.74 µM, being more potent than the positive standard ketoconazole (IC 50 value 2.4 µM). It was concluded, from the structure activity relationship view, that the depsidone structure is important for this type of activity. The aromatase enzyme participates in aromatization reactions from androgens to the aromatic ring of estrogens and the inhibition of this enzyme reduces the incidence of breast cancer [29]. The same research group showed in another publication that unguinol (3) and aspergillusidone A (21) are also inhibitors of this enzyme [50]. The depsidones unguinol (3) and aspergillusidone D (26) were selected for further studies. Unguinol (3) induced apoptosis and cell cycle arrest in the breast cancer cell line-MB 231. Unguinol (3) and aspergillusidone D (26) also inhibit lifetime cell viability at low concentrations (µM) [51].
Depsidones were also evaluated for their potential to inhibit the enzyme acetylcholinesterase (AChE). Aspergillusidone A (21) showed AChE inhibition with IC50 value of 56.8 µM, with donepezil used as a positive control (IC50 = 0.3 µM). Docking studies were also performed, showing that this depsidone interacts with the enzyme in different ways [28].

Summary and Future Perspectives
In the past fifty-two years , since the first chemical study of A. unguis, ninety-seven compounds from different classes of microbial secondary metabolites have been isolated and identified by spectrometric and spectroscopic techniques. Many of the reported compounds are produced exclusively by A. unguis. The substances isolated from A. unguis have promising biological activity against pathogens such as larvae, bacteria and yeasts, with several examples where these substances are even more active in in vitro assays than the positive controls. Considering the tendencies of antiparasitic and antibiotics to become less effective, it is very important to continue these studies in order to investigate their action in vivo. In addition to antimicrobial and larvicidal activities, these substances also demonstrate promising activities against the cancer cell lineage, anti-inflammatory, and antimalarial activities. Several compounds of A. unguis have also been shown to be enzyme inhibitors, such as aromatase and AChE. The metabolic potential of A. unguis is still largely untapped; therefore, it has great potential for innovation. Variations in the concentration or depletion of salts in the culture media proved to be an extremely interesting approach to produce new structures. The OSMAC strategy, despite having been successfully applied by several authors in A. unguis, can still be widely explored, changing the parameters of microorganism cultures, in order to allow the fungus to produce new chemical structures [28,30,68]. Approaches using epigenetic regulation also have not been widely used in A. unguis. Epigenetic regulation is critical for fungal secondary metabolism biosynthesis and the activation of gene clusters.The modulation of epigenetic regulation is an interesting alternative for discovering new secondary metabolites and improving their production [75]. Another tool that could also accelerate the discovery of new minority metabolites as well as understand the metabolic capacity of A. unguis are metabolomicsbased dereplication methods. These approaches have not yet been applied to this fungus and deserve attention [76]. Medicinal chemists and molecular modeling specialists could also contribute significantly to the understanding of the interaction of secondary metabolite X enzyme inhibition. Despite the diversity of chemical structures, few studies have focused on the structural modification of these compounds which deserve attention in order to obtain semisynthetic derivatives even more potent than the natural substances.