Secondary Metabolites from Fungi Microsphaeropsis spp.: Chemistry and Bioactivities

Microsphaeropsis, taxonomically classified within the kingdom fungi, phylum Ascomycota, subphylum Deuteromycotina, class Coelomycetes, order Sphaeropsidales, and family Sphaeropsidaceae, exhibit a ubiquitous distribution across various geographical regions. These fungi are known for their production of secondary metabolites, characterized by both structural novelty and potent biological activity. Consequently, they represent a significant reservoir for the advancement of novel pharmaceuticals. In this paper, a systematic review was present, marking the analysis of secondary metabolites synthesized by Microsphaeropsis reported between 1980 and 2023. A total of 112 compounds, comprising polyketones, macrolides, terpenoids, and nitrogen-containing compounds, were reported from Microsphaeropsis. Remarkably, among these compounds, 49 are novel discoveries, marking a significant contribution to the field. A concise summary of their diverse biological activities was provided, including antibacterial, antitumor, and antiviral properties and other bioactivities. This analysis stands as a valuable reference, poised to guide further investigations into the active natural products derived from Microsphaeropsis and their potential contributions to the development of medicinal resources.


Introduction
Microsphaeropsis, taxonomically classified within the phylum Ascomycetes, subphylum Deuteromycotina, class Coelenterata, order Sphaeropsidales, and family Sphaeropsidaceae in the fungal taxonomy [1], are common plant pathogens widely distributed in nature [2].For a long time, the genus Microsphaeropsis was not accurately recognized, leading to the misplacement of many fungi within the genus Coniothyrium.In 1980, Sutton meticulously elucidated the characteristics of the Microsphaeropsis genus.He subsequently reclassified species previously assigned to the genus Coniothyrium under Microsphaeropsis and renamed the genus [3,4].Subsequently, additional members of the genus Microsphaeropsis have been unearthed and taxonomically elucidated.As of now, the repository of the Species Fungorum database (https://speciesfungorum.org/Names/Names.asp(accessed on 2 November 2023)) registers a total of 54 species within the genus Microsphaeropsis.It is evident that fungi within the genus Microsphaeropsis exhibit remarkable biodiversity, with a high likelihood of containing structurally novel and biologically active secondary metabolites.
Fungi have been recognized for their remarkable capacity to synthesize secondary metabolites, and the extraction of natural products from fungal secondary metabolites offers significant advantages in terms of yield, economic viability, and environmental sustainability [5,6].Following the taxonomic redefinition of the Microsphaeropsis genus by Sutton in 1980, various structural categories of compounds, including polyketides, terpenoids, Note: "-"means the same as above.

Types of Chemical Structures 2.1. Polyketones
Polyketones represent a class of secondary metabolites characterized by their exceptionally diverse structures.These compounds are generated through a sequence of Claisen condensation reactions involving short-chain acyl-CoA molecules such as acetyl-CoA and malonyl-CoA.Their distinctive activities have positioned them as a prominent source for both the treatment of human diseases and the development of novel pharmaceuticals.Seventy-six polyketone compounds were found in Microsphaeropsis (Figure 1), encompassing pyranones, furanones, naphthoquinones, anthraquinones, phenylpropanoids, and coumarins.In terms of sheer chemical diversity, polyketones emerge as the most extensively documented natural products among the fungi within the Microsphaeropsis genus.

Polyketones
Polyketones represent a class of secondary metabolites characterized by their exceptionally diverse structures.These compounds are generated through a sequence of Claisen condensation reactions involving short-chain acyl-CoA molecules such as acetyl-CoA and malonyl-CoA.Their distinctive activities have positioned them as a prominent source for both the treatment of human diseases and the development of novel pharmaceuticals.Seventy-six polyketone compounds were found in Microsphaeropsis (Figure 1), encompassing pyranones, furanones, naphthoquinones, anthraquinones, phenylpropanoids, and coumarins.In terms of sheer chemical diversity, polyketones emerge as the most extensively documented natural products among the fungi within the Microsphaeropsis genus.

Terpenoids
Terpenoids represent a class of natural compounds characterized by their foundational isoprene or isopentane unit structures.They exhibit unparalleled structural diversity and comprise the most extensive group of natural compounds, offering a wide array of activities, including anti-inflammatory, antibacterial, and antitumor properties [32,33].Seven sesquiterpenes were discovered within Microsphaeropsis (Figure 2), and this collection includes eucalyptus sesquiterpenes, airimo phenolic sesquiterpenes, and sesterterpene.

Terpenoids
Terpenoids represent a class of natural compounds characterized by their foundational isoprene or isopentane unit structures.They exhibit unparalleled structural diversity and comprise the most extensive group of natural compounds, offering a wide array of activities, including anti-inflammatory, antibacterial, and antitumor properties [32,33].Seven sesquiterpenes were discovered within Microsphaeropsis (Figure 2), and this collection includes eucalyptus sesquiterpenes, airimo phenolic sesquiterpenes, and sesterterpene.

Macrolide
Macrolide compounds represent a class of multi-carbon compounds distinguished by the presence of a lactone ring within their molecular structure.They are notable for their pronounced anti-inflammatory, anti-tumor, and antibacterial activities [34].A comprehensive review of 10 macrolides (Figure 3), comprising two distinct structural types, 10-member macrolides and 16-member macrolides, is provided.These are the first natural-product-bearing three lactone groups in the molecule among the 16-membered macrocyclic antibiotics, showcasing remarkable anti-leukocyte activity.

Macrolide
Macrolide compounds represent a class of multi-carbon compounds distinguished by the presence of a lactone ring within their molecular structure.They are notable for their pronounced anti-inflammatory, anti-tumor, and antibacterial activities [34].A comprehensive review of 10 macrolides (Figure 3), comprising two distinct structural types, 10-member macrolides and 16-member macrolides, is provided.These are the first naturalproduct-bearing three lactone groups in the molecule among the 16-membered macrocyclic antibiotics, showcasing remarkable anti-leukocyte activity.

Terpenoids
Terpenoids represent a class of natural compounds characterized by their foundational isoprene or isopentane unit structures.They exhibit unparalleled structural diversity and comprise the most extensive group of natural compounds, offering a wide array of activities, including anti-inflammatory, antibacterial, and antitumor properties [32,33].Seven sesquiterpenes were discovered within Microsphaeropsis (Figure 2), and this collection includes eucalyptus sesquiterpenes, airimo phenolic sesquiterpenes, and sesterterpene.

Macrolide
Macrolide compounds represent a class of multi-carbon compounds distinguished by the presence of a lactone ring within their molecular structure.They are notable for their pronounced anti-inflammatory, anti-tumor, and antibacterial activities [34].A comprehensive review of 10 macrolides (Figure 3), comprising two distinct structural types, 10-member macrolides and 16-member macrolides, is provided.These are the first natural-product-bearing three lactone groups in the molecule among the 16-membered macrocyclic antibiotics, showcasing remarkable anti-leukocyte activity.

Nitrogen Compounds
Nitrogen-containing compounds represent another prevalent and exceptionally diverse class of secondary metabolites.They are distinguished by their intricate cyclic structures and demonstrate substantial biological activities against bacteria, fungi, and tumor cells [35].This study offers an extensive review of 14 nitrogen-containing compounds, encompassing a range of subtypes (Figure 4), including organic amines alkaloids, pyrrole alkaloids, pyrazine alkaloids, imidazolone alkaloids, indole alkaloids.

Nitrogen Compounds
Nitrogen-containing compounds represent another prevalent and exceptionally diverse class of secondary metabolites.They are distinguished by their intricate cyclic structures and demonstrate substantial biological activities against bacteria, fungi, and tumor cells [35].This study offers an extensive review of 14 nitrogen-containing compounds, encompassing a range of subtypes (Figure 4), including organic amines alkaloids, pyrrole alkaloids, pyrazine alkaloids, imidazolone alkaloids, indole alkaloids.

Other Classes
In addition to their primary secondary metabolites, Microsphaeropsis fungi also generate a limited quantity of fatty acids and chlorine-containing compounds (Figure 5).Among these, compound 112 has demonstrated noteworthy antibacterial activity.

Other Classes
In addition to their primary secondary metabolites, Microsphaeropsis fungi also generate a limited quantity of fatty acids and chlorine-containing compounds (Figure 5).Among these, compound 112 has demonstrated noteworthy antibacterial activity.

Nitrogen Compounds
Nitrogen-containing compounds represent another prevalent and exceptionally diverse class of secondary metabolites.They are distinguished by their intricate cyclic structures and demonstrate substantial biological activities against bacteria, fungi, and tumor cells [35].This study offers an extensive review of 14 nitrogen-containing compounds, encompassing a range of subtypes (Figure 4), including organic amines alkaloids, pyrrole alkaloids, pyrazine alkaloids, imidazolone alkaloids, indole alkaloids.

Other Classes
In addition to their primary secondary metabolites, Microsphaeropsis fungi also generate a limited quantity of fatty acids and chlorine-containing compounds (Figure 5).Among these, compound 112 has demonstrated noteworthy antibacterial activity.

Biological Activities
Numerous studies have revealed the isolation of a plethora of bioactive compounds from the secondary metabolites of Microsphaeropsis fungi [7,9].These compounds encompass a wide spectrum of activities, notably including antifungal, antibacterial, cytotoxic, cell adhesion inhibition, antiviral, antimalarial, and antioxidant activities [15,18,31].Furthermore, some of these compounds exhibit a substantial potential for their development into novel pharmaceuticals.
Two novel metabolites, microketide A (74) and microketide B (75), were successfully isolated from the fungus Microsphaeropsis sp.RA10-14, which inhabits the gorgonian Anthogorgia ochracea from the South China Sea.Both compounds exhibited significant antibacterial and antifungal activities, with a notable emphasis on their antibacterial effects.Specifically, compound 74 demonstrated significant antibacterial activity against B. subtilis, B. megaterium, E. aerogenes, K. rhizophila, and P. aeruginosa, all with equal MIC values of 0.19 µg/mL, mirroring the potency of ciprofloxacin [21].Ciprofloxacin is used for the treatment of a wide range of infections and has been shown to be active against various Gram-positive and Gram-negative bacteria [36][37][38].
Three novel metabolites, TAN-1496 A (99), C (101), and E (103), in conjunction with two known compounds, TAN-1496 B (100) and D (102), were successfully isolated from the fungus Microsphaeropsis sp.FL-16144.Extensive research has revealed that these compounds act as specific inhibitors of calf thymus Topo I [40].Notably, even at high concentrations, these compounds did not inhibit the function of Topo II.They exhibited a significant inhibition of the growth of various murine and human tumor cells, including P815 murine mastcytoma, EL4 murine lymphoma, Bl6 murine melanoma, WiDr human colon adenocarcinoma, and A549 human lung carcinoma [29].Additionally, these compounds displayed activity against Gram-positive bacteria [29].
It has been widely reported that cell adhesion molecules play pivotal roles in numerous physiological and biochemical processes, ranging from cancer [41][42][43] to inflammation [44].In a remarkable discovery, Hayashi et al. (1995) isolated two new 16-membered macrolides, namely macrosphelide A (87) and macrosphelide B (88), from the fungus Microsphaeropsis sp.FO-5050 [24].To explore the impact of macrosphelide A and B on cell adhesion, experiments were conducted using the dose-dependently inhibited adhesion of HL-60 cells to human umbilical vein endothelial cells (HUVECs) stimulated with LPS.These compounds exhibited significant cytotoxicity, with IC 50 values of 3.5 µM and 36 µM, respectively.Notably, in vitro cell growth assays showed that these compounds had no discernible effect on the growth of cells, including P388 leukemia, human prostate tumor cells, and L929 fibroblast cells, even when administered at a high dose of 200 mg/kg for 5 days [45].Takamatsu's (1997) group isolated two more 16-membered macrolide metabolites, macrosphelide C (89) and macrosphelide D (90) from the fungus Microsphaeropsis sp.FO-5050 [25].Macrosphelide C (89) and macrosphelide D (90) exhibited moderate cytotoxic activity, with IC 50 values of 67.5 µM and 25 µM, respectively.Fukami et al. (1998) continued their exploration of the fungus Microsphaeropsis sp.FO-5050 and made further discoveries [10].They isolated three new metabolites, macrosphelide J (91), macrosphelide K (92), and 6-epi-5'hydroxymycosporulone (12).Although these compounds displayed cytotoxic activity, their IC 50 values exceeded 100 µg/mL.Collectively, these findings underscore the exceptional potential of the fungus Microsphaeropsis sp.FO-5050 as a source of novel macrolides with cytotoxic properties.These substances hold high promise for future development in the treatment of leukemia.Singh et al. (1994) previously reported that compounds 18, 19, and 20 possess the capability to inhibit Ras farnesyl-protein transferase [46].Seephonkai et al. (2002) corroborated this finding in their research [12].Furthermore, compounds 16-21 have demonstrated significant cytotoxicity against a spectrum of cell lines, including human epidermoid carcinoma (KB cells), human breast cancer (BC-1 cells), and African green monkey kidney fibroblast (vero cells).Compound 28, sourced from the endophyte fungus M. arundinis E12-2112, has exhibited moderate cytotoxicity against human bladder carcinoma cells (T24) and human lung carcinoma cells (A549), characterized by IC 50 values of 35.4 µg/mL and 81.6 µg/mL, respectively [14].
Bradykinin, an endogenous peptide, exerts its effects through specific cell receptors, giving rise to a plethora of physiological reactions, including pain, allergic responses, and muscle contractions.Bradykinin antagonists hold the potential to inhibit these physiological reactions, offering prospects for treating conditions such as inflammatory edema, rhinitis, and asthma.In a significant discovery, a novel non-peptide bradykinin-binding inhibitor, L-755,807 (95), was isolated from the endophyte fungus Microsphaeropsis sp.MF6057, found within the plant Prosopis glandulosa.Compound 95 demonstrated an IC 50 value of 71 µM in binding to a cloned human B2 receptor with 3 H-bradykinin, marking a noteworthy development in the pursuit of potential treatments [26].
Two novel γ-pyrone derivatives, microsphaerones A (96) and microsphaerones B (97), were successfully isolated from the fungus Microsphaeropsis sp.KMPB W-22.Interestingly, neither of these compounds exhibited significant cytotoxicity.Additionally, they displayed either no activity or only moderate activity against S. littoralis and A. salina [27].
From a global vantage point, malaria continues to rank among the foremost causes of human mortality.Consequently, the current paramount emphasis remains on research and the development of targeted pharmaceutical interventions.Seephonkai et al. (2002) first reported on the in vitro activity of preussomerins against Plasmodium falciparum [12].Compound 16-21 showed significant antiplasmodial activity, with an IC 50 range of 0.32-3.44µg/mL.

Conclusions
Since Sutton reclassified the genus Microsphaeropsis in 1980, fifty-four different species have been included.Currently, this genus has yielded an impressive array of secondary metabolites, with 112 secondary metabolites reported, of which 49 (41.96%) were identified for the first time.These compounds are distributed across various structural classes, including 76 polyketones (67.86%), 14 nitrogenous compounds (12.5%), 10 macrolides (8.9%), 7 terpenoids (6.25%), and 5 other compounds (4.46%), indicating polyketones are the predominant structural type (Figure 6A).Moreover, a significant proportion (57.89%) of these compounds exhibit notable biological activities, of which 42 compounds (60.6%) demonstrate both antibacterial and antifungal properties, while 21 (37.8%)compounds exhibit antitumor activities.Furthermore, several compounds display distinct activities, including scavenging oxygen-free radicals, algae removal, resistance to aging, and acetylcholinesterase inhibition (Figure 6B).It is important to highlight that many compounds exhibit multiple activities.For instance, compound 44 demonstrates antibacterial, antitumor, and certain oxygen-free radical scavenging activities.

Conclusions
Since Sutton reclassified the genus Microsphaeropsis in 1980, fifty-four different species have been included.Currently, this genus has yielded an impressive array of secondary metabolites, with 112 secondary metabolites reported, of which 49 (41.96%) were identified for the first time.These compounds are distributed across various structural classes, including 76 polyketones (67.86%), 14 nitrogenous compounds (12.5%), 10 macrolides (8.9%), 7 terpenoids (6.25%), and 5 other compounds (4.46%), indicating polyketones are the predominant structural type (Figure 6A).Moreover, a significant proportion (57.89%) of these compounds exhibit notable biological activities, of which 42 compounds (60.6%) demonstrate both antibacterial and antifungal properties, while 21 (37.8%)compounds exhibit antitumor activities.Furthermore, several compounds display distinct activities, including scavenging oxygen-free radicals, algae removal, resistance to aging, and acetylcholinesterase inhibition (Figure 6B).It is important to highlight that many compounds exhibit multiple activities.For instance, compound 44 demonstrates antibacterial, antitumor, and certain oxygen-free radical scavenging activities.Fungi occupy an exceptionally pivotal role in drug discovery [47].Many renowned drug molecules, including penicillin and lovastatin, have originated from fungal sources [48].However, the challenge of unearthing novel compounds of exceptional activity and expanding the repertoire of known compound activities remains unresolved.Given the abundance and significant innovation rate (41.96%) of secondary metabolites, Microsphaeropsis can be used as a potential repository for exploiting secondary metabolites.Accordingly, the following recommendations were proposed: (i) Employ non-directional activation strategies to activate the biosynthesis of secondary metabolites, such as the A B Fungi occupy an exceptionally pivotal role in drug discovery [47].Many renowned drug molecules, including penicillin and lovastatin, have originated from fungal sources [48].However, the challenge of unearthing novel compounds of exceptional activity and expanding the repertoire of known compound activities remains unresolved.Given the abundance and significant innovation rate (41.96%) of secondary metabolites, Microsphaeropsis can be used as a potential repository for exploiting secondary metabolites.Accordingly, the following recommendations were proposed: (i) Employ non-directional activation strategies to activate the biosynthesis of secondary metabolites, such as the OSMAC (one strain many compounds) strategy [49], co-culture strategy [50], and epigenetic regulation strategy [51].(ii) Harness targeted activation strategies to activate numerous dormant gene clusters within fungal genomes, such as target sequence promoter replacements [52], transcription regulatory factor knockouts [53], the heterologous expression of biosynthetic gene clusters [54], DNA-assembly technology [55], and ribosome engineering [56].However, the genomic information of Microsphaeropsis has not been reported in the literature so far.Thus, whole gene sequencing and analysis will be the focus of further work.(iii) Expand the scope of activity screening of existing compounds and delve into their mechanism of action to verify their potential for pharmaceutical applications.For instance, microbetides A (74), a novel polyketone compound, demonstrates remarkable antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, with an IC 50 of 0.19 µg/mL, comparable to ciprofloxacin.Polyketone 10-norparvulone (76) exhibits the potential to effectively inhibit virus replications, positioning it as a promising candidate for a new anti-influenza drug.Macrolide compounds like macrosphelide A (87) and macrosphelide B (88), characterized by their unique chemical structures and cytotoxicity against leukemia, hold promise as novel anticancer drugs.

Figure 5 .
Figure 5.Chemical structures of compounds 108-112 of other classes.Figure 5. Chemical structures of compounds 108-112 of other classes.

Figure 5 .
Figure 5.Chemical structures of compounds 108-112 of other classes.Figure 5. Chemical structures of compounds 108-112 of other classes.

Figure 6 .
Figure 6.Structural classification (A) and activity classification (B) of secondary metabolites of Microsphaeropsis spp.

Figure 6 .
Figure 6.Structural classification (A) and activity classification (B) of secondary metabolites of Microsphaeropsis spp.

Table 1 .
Summary of secondary metabolites of Microsphaeropsis spp.
Antibacterial activity against Escherichia coli and B. megaterium, and antialgae activity against C. fusca [20] Microsphaeropsones B Antibacterial activity and antialgae activity against C. fusca Microsphaeropsones C Antibacterial activity against E. coli and B. megaterium, and antialgae activity against C. fusca Citreorosein Antibacterial activity against E. coli, B. megaterium, and M. violaceum, and antialgae activity against C. fusca Emodin Antibacterial activity and antialgae activity against C. fusca Fusidienol A