*Article* **Transcriptomics and Proteomics Characterizing the Anticancer Mechanisms of Natural Rebeccamycin Analog Loonamycin in Breast Cancer Cells**

**Xiao Sun 1,†, Zhanying Lu 1,†, Zhenzhen Liang 2, Bowen Deng 2, Yuping Zhu 1, Jing Shi <sup>3</sup> and Xiaoling Lu 2,\***


**Abstract:** The present study is to explore the anticancer effect of loonamycin (LM) in vitro and in vivo, and investigate the underlying mechanism with combined multi-omics. LM exhibited anticancer activity in human triple negative breast cancer cells by promoting cell apoptosis. LM administration inhibited the growth of MDA-MB-468 tumors in a murine xenograft model of breast cancer. Mechanistic studies suggested that LM could inhibit the topoisomerase I in a dose-dependent manner in vitro experiments. Combined with the transcriptomics and proteomic analysis, LM has a significant effect on O-glycan, p53-related signal pathway and EGFR/PI3K/AKT/mTOR signal pathway in enrichment of the KEGG pathway. The GSEA data also suggests that the TNBC cells treated with LM may be regulated by p53, O-glycan and EGFR/PI3K/AKT/mTOR signaling pathway. Taken together, our findings predicted that LM may target p53 and EGFR/PI3K/AKT/mTOR signaling pathway, inhibiting topoisomerase to exhibit its anticancer effect.

**Keywords:** loonamycin; triple negative breast cancer; p53; PI3K/AKT/mTOR; O-glycan

#### **1. Introduction**

Triple negative breast cancer (TNBC) is a special subtype of breast cancer, accounting for about 12.7% of breast cancer [1], which is characterized by negative estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) with high recurrence rate, strong invasiveness, and poor prognosis characteristics. Due to the lack of corresponding targets and high heterogeneity, the treatment of TNBC is mainly limited to chemotherapy [2]. It is valuable to develop novel chemotherapeutics that can broadly target TNBCs to render this highly deadly disease subtype curable.

Marine natural products provide an important source of lead compounds for new drug research and development because of their unique structure and diversity biological activity. At present, more than 35,000 marine natural compounds have been discovered in the world, most of which possess unique structures and present diverse biological activity. Loonamycin (LM) is an indole carbazole compound rebeccamycin analog produced from *Nocardiopsis flavescens* NA01583 isolated from marine sediment in Yongxing Island, South China Sea [3] (Figure 1A). To the best of our knowledge, rebeccamycin is a cytotoxicity compound that binds to topoisomerase I to inhibit the reconnection at the DNA strand incision [4], leading to the break of DNA single strand and double strand. This compound showed an impressive cytotoxicity in vitro but could not be further developed because of poor water solubility. Some rebeccamycin analogues (e.g., becatecarin [5] and edotecacin [6]) have entered clinical research. LM has a rare sugar group and hydroxyl group

**Citation:** Sun, X.; Lu, Z.; Liang, Z.; Deng, B.; Zhu, Y.; Shi, J.; Lu, X. Transcriptomics and Proteomics Characterizing the Anticancer Mechanisms of Natural Rebeccamycin Analog Loonamycin in Breast Cancer Cells. *Molecules* **2022**, *27*, 6958. https://doi.org/10.3390/ molecules27206958

Academic Editor: Giovanni Ribaudo

Received: 31 July 2022 Accepted: 12 October 2022 Published: 17 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

compared with rebecamycin [3], which could increase its water solubility, and improve its pharmaceutical potential.

**Figure 1.** LM targets TNBC in vitro and in vivo. (**A**) The structure of LM; (**B**) the cell viability experiment of LM on human TNBC cell line MDA-MB-468. The MDA-MB-468 cells were treated with 0, 0.0625, 0.125, 0.25, 0.5, 1, 2,4 μM LM for 48 and 72 h, and the cell viability was detected by the CCK8 assay; (**C**) effects of LM at 0.8 μM and 1.6 μM on MDA-MB-468 cell cycle arrest after 48 h of incubation. Cells were stained with Annexin V and analyzed by flow cytometry. The vertical bars represent the standard deviation of means (SD) (*n* = 3 experiments); \* *p* < 0.05 and \*\* *p* < 0.01, vs. negative control. The G1, G2 and S represent the phase of the cell cycle. (**D**) The inhibitory effect of LM on topoisomerase I activity. The plasmid was treated with 5, 10, 20 μM LM and the 20 μM camptothecin (CPT) was used as positive control. (**E**) The quantification of electrophoretic band. The Y axis is SC DNA/total DNA. (**F**) The images of tumors from each group. Tumor-bearing mice were administered the vehicle (negative control), 15 mg/kg Paclitaxel (positive control), LM (10 or 20 mg/kg per day). (**G**) The average tumor weight in each group. Data are presented as the mean ± S.D. *n* = 4, \* *p* < 0.05 and \*\* *p* < 0.01, vs. negative control. (**H**) The average tumor volume in each group recorded during the treatments. Data are presented as the mean ± S.D., *n* = 4, \* *p* < 0.05 and \*\* *p* < 0.01, vs. negative control. (**I**) The average body weight in each group. (**J**) The HE staining on stripped tumor tissue.

Our previous studies showed that LM had strong cytotoxic activities against various tumor cell lines, especially to the human TNBC cell line MDA-MB-468. In this article, we used RNA-seq and TMT quantitative proteomics technology to uncover the mechanism of LM in TNBC cells.

#### **2. Results**

#### *2.1. LM Targets TNBC In Vitro and In Vivo*

First, LM was investigated for its inhibitory effects on several breast cancer cell lines (Table 1). LM displayed a preferential anticancer activity against the cell lines with IC50 values of 0.517 μM, 0.197 μM and 0.372 μM in the MCF-7 cell line, MDA-MB-231 cell line and MDA-MB-468 cell line. The IC50 of LM for LO2 was 1.022 μM. The LO2 was used as a control cell line and the result suggested that LM was less toxic to LO2 than the cancer cell line. Then, we further investigated the inhibitory effects of LM on TNBC MDA-MB-468 cells. As exhibited in Figure 1B, LM displayed a good anticancer activity against MDA-MB-468 in a dose-dependent manner with IC50 values of 0.372 μM and 0.235 μM after 48 h and 72 h.


**Table 1.** IC50 value of LM on multiple tumor cell lines in 48 h.

Second, we examined the effect of LM on cell cycle in MDA-MB-468 cells. In MDA-MB-468 cells, after 48 h of incubation, LM induced cell cycle arrest in the G2 phase in a dose-dependent manner (Figure 1C). At 1.6 μM LM, the value of G1-phase declined to 24.46%, while G2-phase increased to 48.90%. The values of the S phase showed a slight change. Growing lines of evidence indicate that eukaryotic topoisomerase activity is monitored and regulated throughout the cell cycle [7]. As rebeccamycin is a cytotoxicity compound which is bound to the topoisomerase I, we detected the inhibitory effect of LM on topoisomerase I activity (Figure 1D,E). Topoisomerase I (Topo I) relaxes the super helix structure of DNA by cutting its single strands [8]. The normal plasmid DNA is a closed double stranded DNA. In the process of electrophoresis, the plasmid may have three configurations: the superhelical DNA (SC DNA), the open circular DNA (OC DNA) and the linear DNA (L DNA). The SC DNA is at the front of gel, OC DNA is at the back, and L DNA is between SC DNA and OC DNA. After the cleavage of topoisomerase I, the structure of SC DNA will be destroyed. The experiment showed that LM could inhibit topoisomerase I activity in a dose-dependent manner.

Next, the effects of LM on the growth and formation of subcutaneous xenograft nodes derived from the inoculated MDA-MB-468 cells in vivo in BALB/c nude mice were investigated. Both the volumes and weights of the formed MDA-MB-468 cells tumor nodes were reduced by LM administration every 2 days for a total of 17 days at a concentration of 10 mg/kg or 20 mg/kg LM by i.v., compared to 15 mg/kg for paclitaxel as a positive control [9] by i.p. and 2% DMSO as vehicle by i.v. The low-dose group was of no statistical significance compared with the negative control group. The volumes and weights of tumors in high-dose group reduced significantly (Figure 1F–I). The frozen section and HE staining on the stripped tumor tissue was performed, and obvious tumor-like tissues were observed under the microscope, such as large cell volume, big nucleus and deformity loose arrangement of tumor cells (Figure 1J). There were no detectable toxic or necrotic effects on the heart, liver, spleen, lung or kidney tissues after LM treatment and no significant weight loss. Taken together, these data suggest that LM exhibits good therapeutic activity.

#### *2.2. Functional Annotation Enrichment of LM-Regulated Genes*

To uncover the LM regulatory mechanism in TNBC MDA-MB-468 cells, we performed RNA-seq analysis to profile the transcriptomes of MDA-MB-468 cells when treated with 1.6 μM LM. Differentially abundant genes (DAGs) were those meeting the qualified data (fold change ≥ 1.2 and *p* < 0.05) under comparison of LM vs. the control group. A total of 1764 DAGs were shown in the volcano map, of which 737 genes were upregulated and 1027 genes were down-regulated (Figure 2A). GO (Gene ontology) is a comprehensive database that describes gene functions. GO enrichment analysis basing on the DAGs including up-regulated genes and down-regulated genes, were mapped the differential genes to the entries in the three aspects of cell components (CC), molecular functions (MF), and biological processes (BP) (Figure 2B). Through GO enrichment analysis, we can roughly understand which biological functions, signal pathways, or cell locations are enriched of the DAGs. In terms of the CC, the up-regulated genes were mainly enriched in the extracellular matrix, anchored component of membrane, collagen trimer and MHC protein complex. The down-regulated genes were mainly enriched in postsynapse, synaptic membrane, receptor complex, ion channel complex. In terms of the MF, the up-regulated genes were mainly enriched in the receptor ligand activity, receptor regulator activity, cytokine receptor binding, extracellular matrix structural constituent. The down-regulated genes were mainly enriched in actin binding, ion gated channel activity, Ras GTPase binding, passive transmembrane transporter activity. In terms of the BP, the up-regulated genes were mainly enriched in the regulation of signaling receptor activity, regulation of endothelial cell proliferation, endothelial cell proliferation, positive regulation of locomotion and wound healing. The down-regulated genes were mainly enriched in trans-synaptic signaling, chemical synaptic transmission, anterograde trans-synaptic signaling, regulation of membrane potential and neurotransmitter levels.

KEGG (Kyoto Encyclopedia of Genes and Genomes) is a comprehensive database integrating genome, chemistry and system function information. It stores information on gene pathways of different species. KEGG pathway enrichment analysis was conducted to describe the significant changes in signal pathways of DAGs (Figure 2C). The results showed that the up-regulated differential genes were mainly centered on the pathways in cancer, hippo signaling pathway, p53 signaling pathway, etc. The down-regulated proteins were mainly concentrated on the glucagon signaling pathway, calcium signaling pathway, phosphatidylinositol signaling system, propanoate metabolism, inositol phosphate metabolism, pyruvate metabolism, other types of O-glycan biosynthesis and so on.

GSEA enrichment analysis was conducted to explore the changes of gene expression in the pathway and find the upstream factors leading to these changes(Figure 2D). In our studies, the curated gene sets as the exploration set showed that p53 and downstream signal pathway were up-regulated, and O-glycan and PI3K-AKT-mTOR signaling pathway were down-regulated in LM treatment group.

#### *2.3. Proteomic Expression Profiling of LM-Treated TNBC Cells*

To further elucidate cellular mechanism and molecular function, we performed TMT quantitative proteomics analysis to assess the protein expression profiles in MDA-MB-468 cells treated with 1.6 μM LM. Differentially abundant proteins (DAPs) were those meeting the qualified data (fold change ≥ 1.2 and *p* < 0.05) under comparison of LM vs. control group. A total of 1314 DAPs were shown in the volcano map, of which 778 proteins were upregulated and 536 proteins were down-regulated (Figure 3A). Wolf PSORT software was used for localization analysis of differential proteins, which show that the DAPs were mainly distributed in cytoplasm, nucleus, mitochondria, and plasma membrane (Figure 3B). In total, 119 GO terms were obtained based on the DAPs including up-regulated proteins and down-regulated proteins (Figure 3C). GO enrichment analysis showed that in terms of the cell components (CC), the up-regulated proteins were mainly enriched in the cytosol (GO:0005829), ficolin-1-rich granule lumen (GO:1904813) and nucleus (GO:0005634). The down-regulated proteins were mainly enriched in the mitochondrial matrix (GO:0005759), integral component of plasma membrane, (GO:0005887) and nBAF complex (GO:0071565). In terms of biological process (BP), the up-regulated proteins were mainly enriched in the processes related to gluconeogenesis (GO:0006094), negative regulation of ryanodine-sensitive calcium-release channel activity (GO:0060315) and proteasomal ubiquitin-independent protein catabolic process (GO:0010499). The downregulated proteins were mainly enriched in the processes related to isoleucine catabolic process (GO:0006550), O-glycan processing (GO:0016266) and leucine catabolic process (GO:0006552). In terms of molecular function (MF), the up-regulated proteins were mainly enriched in S100 protein binding (GO:0044548), RAGE receptor binding (GO:0050786) and threonine-type endopeptidase activity (GO:0004298). The down-regulated proteins were mainly enriched in polypeptide N-acetylgalactosaminyl transferase activity (GO:0004653), biotin binding (GO:0009374) and signaling receptor activity (GO:0038023).

KEGG pathway enrichment analysis was conducted to describe the significant changes in pathways of DAPs (Figure 3D). The results showed that the DAPs including up-regulated proteins and down-regulated proteins were classified into 34 terms. The up-regulated differential proteins were mainly centered on the changes of metabolic pathway, like amino acid biosynthesis, glucose metabolism, nucleotide metabolism, glutathione metabolism, and p53 pathway, etc. The down-regulated proteins were mainly concentrated on the pathway of amino acid degradation, mTOR signaling pathway, PI3K-Akt signaling pathway, O-glycan biosynthesis, etc. Among these DAPs, the expressions of classical tumor related signaling pathway p53 were up-regulated and the EGFR/mTOR pathway were down-regulated.

GSEA enrichment analysis was conducted to explore the changes of gene expression in the pathway and find the upstream factors leading to these changes (Figure 3E). In our studies, the curated gene sets as the exploration set showed that p53 and downstream signal pathway were up-regulated, and the EGFR and mTOR related pathway, and O-glycan were down-regulated in LM treatment group.

#### *2.4. Validation of Transcriptomic and Proteomic Results*

According to the combined analysis of RNA-seq and TMT-based quantitative proteomic, it is suggested that LM may have a significant effect on O-glycan, p53-related signal pathway and EGFR/PI3K/AKT/mTOR signal pathway in enrichment of the KEGG pathway. The GSEA data also suggests that the TNBC cells treated with LM may be regulated by p53 and EGFR/PI3K/AKT/mTOR signaling pathway.

The Cys124 located in the loop1/sheet3 (L1/S3) pocket of the p53 protein plays a key role in maintaining p53 stable conformation (Figure 4A). The covalently binding model between LM and p53 (L1/S3) was investigated by molecular docking. The binding energy was −26.8 kJ/mol. The main combination modes were the hydrogen bonds, as shown in Figure 4D. The ether bond of LM formed hydrogen bond with Thr102 of p53. The methoxy group on the rare sugar group of LM formed hydrogen bond with Phe113 of p53. The hydroxyl group of LM formed one hydrogen bond with Leu114, His115, Cys124 and His 233 separately. The hydroxyl and ether group of LM formed three hydrogen bonds with Thr123. The above results suggested that LM can target the pockets of wild-type p53 l1/s3, improving the stability of p53 and activating p53 related pathways.

To verify the combined analysis of transcriptomic and proteomic results, the western blot of key proteins was performed. It is determined that expression of p53 was increased after treated with LM for 48 h on MDA-MB-468 (Figure 4B,C), but there was no statistical significance. The expressions of EGFR, PI3K, mTOR and BCL-2 were significantly down-regulated (Figure 4B,D). The expression of p-p53 was significantly up-regulated (Figure 4B,C), and the expression of p-EGFR, p-PI3K and p-mTOR were significantly downregulated (Figure 4B,D). It was shown that LM could activate p53 and inhibit the expression of EGFR, PI3K, mTOR and BCL-2.

**Figure 2.** Functional analysis of differential genes. (**A**) Scatter plots of differentially expressed genes. There were 737 up-regulated genes and 1027 down-regulated genes in the LM group. Abscissa is the difference multiple (logarithmic transformation based on 2); (**B**) The gene ontology annotation analysis between DAGs and classification of BP, MF, and CC; (**C**) The KEGG pathway analysis of related DAGs; (**D**) The GSEA results of differentially abundant genes.

**Figure 3.** Functional analysis of differential proteins. (**A**) Scatter plots of differentially expressed proteins. There were 778 up-regulated proteins and 536 down-regulated proteins in the LM group. Abscissa is the difference multiple (logarithmic transformation based on 2); (**B**) localization analysis of differentially expressed proteins; (**C**) the gene ontology annotation analysis between DAPs and classification of BP, MF, and CC; (**D**) the KEGG pathway analysis of related DAPs; (**E**) the GSEA results of differentially abundant proteins.

**Figure 4.** Validation of key proteins based on combined transcriptomic and proteomic results. (**A**) The docking picture of LM and p53 (L1/S3). (**B**) Western blot of key differentially abundant proteins. The MDA-MB-468 cells were treated with LM by 0, 0.8 and 1.6 μM. (**C**,**D**) Fold change in protein levels (LM treatment group/control group) from Western blot analysis. Significance: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus the control, *p* value based on *t*-test.

#### **3. Discussion**

In this study, we evaluated the anticancer activity of LM, an indole carbazole rebeccamycin analog from *Nocardiopsis flavescens* NA01583. Our data showed that LM could inhibit tumor cell growth both in vitro and in vivo. The inhibitory effect of LM in cancer cells demonstrated that it could inhibit the topoisomerase I in a dose-dependent manner in vitro experiments like other rebeccamycin analogs [10]. Topo I cut one strand of DNA to form single-strand breaks, allowing supercoiled DNA to relax, otherwise it can hinder DNA replication and transcription, and thus block cell growth [11–13]. For rapid cell division, cancer cells need high Topo I activity to finish the DNA metabolic processes. Topoisomerase I inhibitors can block the reconnection of DNA strands, lead to the accumulation of Topo I-breaking complexes, inhibit replication and transcription, and cause DNA damage, thus activating DNA damage checkpoints and inhibiting the progress of cell cycle [14]. Topoisomerase were recognized as promising targets in cancer, and various DNA Topos inhibitors have been on the market [15]. Some rebeccamycin analogues like becatecarin and edotecacin have entered clinical research. Becatecarin intercalates into DNA and inhibits the catalytic activity of topoisomerases I/II [16]. Edotecarin is a potent inhibitor of topoisomerase I and also has an effect on protein kinase C [17].

Next, the RNA-Seq and TMT were conducted to investigate the changes in transcriptomic and proteomic profile of MDA-MB-468 cells treated with LM (LM). The combined transcriptome and proteome analyses revealed that LM may activate p53 and inhibit the O-glycans, inhibiting EGFR/PI3K/mTOR signaling pathway to exhibit its cytotoxicity activity. The tumor suppressor p53 functions mainly as a transcription factor. A mutation of the TP53 gene that encodes p53 protein is the main way of inactivating p53. p53 is a key tumor suppressor in the process of preventing tumorigenesis. The dysfunction of p53 often leads to cancer. When cells suffer from DNA damage, excessive proliferation, hypoxia, lack of nutrition, telomere loss, or in the environment of oxidative stress, lack of nucleotides or replication pressure, p53 will be activated to induce cell cycle arrest, apoptosis, aging or autophagy, preventing cells from growing and dividing and killing cells before they become cancerous [18]. p53 removes cells with high mutation risk in this way, inhibiting tumor formation. In normal cells, p53 remains at a low level and dormant state under non-stress conditions to prevent its adverse effects on cell growth. Its low expression is mainly by its interaction with ubiquitin E3 ligase MDM2 [19]. A stress response can prevent MDM2 mediated p53 degradation by various mechanisms, promoting the stability and activation of p53. About 50% of cancers still express wild-type p53, but these p53 proteins usually lose its function, because of the over activation of MDM2 and MDMX. The stability and transcriptional activity of p53 depend on its phosphorylation [20]. According to research, the phosphorylation of p53 protein Ser15, Ser20, Ser33 and Ser37 could enhance its binding to P300, thereby activating the transcriptional activity of p53.The phosphorylation of Thr18 can not only enhance p53-p300 binding, but also interfere with p53-MDM2 binding [21]. Reactivating p53 and restoring its function is a feasible and promising tumor treatment strategy. Moreover, p53 is the most common mutant gene in human cancer, of which p53 mutations are found in more than 50% of tumors. For example, approximately 80% of TNBCs express an inactive, mutant form of the p53 tumor suppressor protein (mtp53), resulting in rapid tumor growth and metastasis [22]. Many mutations occur in the DNA binding domain of p53 gene and the altered mutant p53 protein (mtp53) is subsequently not degraded, in which high levels of mtp53 protein accumulate within the cell, leading to the development of tumors. Therefore, converting the mtp53 protein back into its functional wild-type conformation is also a promising means to prevent or reverse tumor development. Restoring the function of wild-type p53 and developing drug candidates for mutant p53 to restore the normal function of p53 can activate p53 to inhibit tumor. At present, many anti-cancer drugs targeting p53 have been developed. For example, the arsenic trioxide, which is used for acute primary myeloid leukemia, can restore the transcriptional activity of p53 mutants by arsenic ions binding to three cysteine residues in the DNA-binding domain [23]. HDAC6 (Histone deacetylase 6) can promote the combination of HSP90 (heat shock protein 90) and mutant p53 protein by catalyzing HSP90 deacetylation and thus make mutant p53 molecule more stable. So, some HDAC6 inhibitors (e.g., statin) or HSP90 inhibitors (e.g., Ganetespib) have been found to induce mutant p53 degradation [24,25]. Small molecule compounds like RG7112 and some nucleic acid drugs can activate p53 by promoting the expression of TP53 gene or inhibiting the expression of MDM2 [26–28].

In 2002, PRIMA-1 was discovered as a mutant p53 reactivator based on the tumor cells screening of the mutated p53 [29]. The specific mechanism was not studied that it directly bound to the Cys124 of mutant p53 protein until 2009 [30]. Cys124 locating in loop1/sheet3 (L1/S3) pocket of p53 protein, plays a key role in maintaining stable conformation. Loop1 can directly interact with helix2 in the p53 DNA binding domain, suggesting that the stability of p53 can be improved by small molecules compounds to stabilize loop1. The compounds targeting wild-type p53 L1/S3 pockets could improve the stability of p53. L1/S3 pocket was a target for pharmaceutical reactivation of p53 mutants [31]. We constructed the computer virtual screening system with wild-type p53 as the target. In the computer docking experiment, LM could bind with L1/S3 pockets of p53 protein well, forming one hydrogen bond with Cys124. Subsequent WB experiments showed that LM could activate p53 signal pathway. Although, the molecular docking

did not provide direct proof that LM interacts with p53, but explored a potential route to influence the p53 pathways.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that belongs to the ErbB family and is involved in angiogenesis, cell proliferation, metastases as well as inhibition of apoptosis. It was demonstrated that EGFR is overexpressed in TNBC cells [32]. Its expression was an independent poor prognostic factor associated with worse DFS and OS [33,34]. In light of the high expression level of EGFR and its strong effect on cell proliferation and motility, EGFR has been considered as an attractive therapeutic target for TNBC [35]. EGFR is on the upstream of PI3K and activates PI3K/AKT/mTOR pathway. The PI3K/AKT/mTOR pathway is associated with cell metabolism, proliferation, differentiation, and survival. PI3Ks are heterodimers composed of regulatory (p85) and catalytic (p110) subunits and exist in four isoforms (α, β, δ, and γ) [36]. The signaling pathway is activated by stimulation of receptor tyrosine kinases, which in turn trigger PI3K activation, followed by phosphorylation of AKT and mTOR complex 1 (mTORC1). It is speculated that on one hand, LM could restore the function of wild-type p53 to activate p53, on the other hand, LM may inhibit the classical EGFR/PI3K/AKT/mTOR pathway to inhibit the growth of the cells; the specific mechanism needs to be further explored.

Glycosylation is one of the most important post-translational modifications of the protein, including N-glycosylation and O-glycosylation. O-linked glycosylation is considered more complicated than N-linked for its unknown initiation [37]. O-glycosylation added single monosaccharides one by one through enzymatic reaction. The linking monosaccharide GalNAc is added directly to Ser/Thr/Tyr residues in glycoproteins within the Golgi apparatus from the nucleotide sugar donor uridine diphospho-GalNAc (UDP-GalNAc). This linking sugar is commonly modified in all cells by the addition of galactose (Gal) from the donor UDP-Gal to create the disaccharide Galβ1-3GalNAcα1-O-Ser/Thr, known as core 1. Such O-glycans can be further modified and extended within the Golgi apparatus to generate an incredible diversity of many tens of thousands of different glycan structures. Meanwhile, the changes in the core structure of several types of O-glycans are related to multiple cancers, which abnormal O-linked glycosylation has been widely proved to act biological functions to directly result in cancer growth and progression. The T antigen and sialyl-Tn antigen (STn), tumor-associated carbohydrate antigens (TACAs), are truncated O-glycans commonly expressed by carcinomas on multiple glycoproteins which serve as potential biomarkers for tumor presence and stage both in immunohistochemistry and in serum diagnostics [38]. CA199 and CA125 are used as circulating tumor biomarkers. In 90% of breast cancers, altered O-glycosylation has been observed to have a correlation with cancer progression, worse prognosis, and metastatic potential; like the number of O-GalNAc glycans in glycoproteins changes, the core structure of O-GalNAc glycosylation changes, and breast cancer cells with shorter O-glycans (abnormal glycosyltransferase activity, premature sialylation of polylactosamine chain blocking the addition of more glycans or truncation of O-glycans at core 1 level). These abnormalities lead to the expression of TACA, such as Tn antigen, St antigen and STn antigen [39]. The polypeptide-N-acetylgalactosaminyl transferase (GT) is a key enzyme of O-linked glycosylation. In our validation experiment, LM down-regulated the GalNAc-T2 with no statistical difference. It is speculated that some other members of GTs should be validated in the future.

In general, the study showed that LM was a potential antitumor compound in vitro and in vivo. LM may target p53 and EGFR/PI3K/AKT/mTOR signaling pathway, inhibiting topoisomerase to exhibit its anticancer activity according to the combined transcriptome and proteome analyses. Our study provided important information for the specific cytotoxicity mechanism of LM and explanation of the anticancer activity of rebeccamycin analog.

#### **4. Materials and Methods**

#### *4.1. Reagents*

LM was isolated from Nocardiopsis flavescens NA01583 from marine sediment and provided by research group of Prof. Ge Huiming, Nanjing University. It was dissolved in dimethyl sulphoxide and stored at −20 ◦C until use. The Cell Cycle Analysis Kit, penicillin/streptomycin were acquired from Beyotime. The fetal bovine serum (FBS), phosphate-buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM) and Leibovitz's L-15 medium (L-15) were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The cell counting kit-8 (CCK8) was purchased from Dojindo Molecular Technology. Specific primary antibodies against β-actin, mTOR, P-mTOR, PI3K, P-PI3K, EGFR, p-EGFR, AKT, P-AKT, BCL-2, p53, p-p53 were acquired from CST (Cell Signaling Technology, Danvers, MA, USA); 0.25% trypsin and 0.2% EDTA were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA).

#### *4.2. Cell Culture*

TNBC cell line MDA-MB-468 (Cat.TCHu136) were cultured in 90% L-15 medium supplemented with 10% fetal bovine serum and 100 mg/L streptomycin–100 U/mL penicillin mixture. Cells were cultured at 37 ◦C in an incubator with controlled humidified atmosphere. The cell dispersed liquid was prepared by 0.25% trypsin plus 0.2% EDTA for subculturing and then spun down by centrifugation at 800 rpm for 5 min, after which the supernatant was removed and precipitated cells was resuspended in culture medium.

#### *4.3. Growth Curve Measured by CCK-8 Method*

The MDA-MB-468 cells were seeded in 96-well E-plates with the density of 5000 cells/well. After 24 h, the cells were treated with LM or 0.06% DMSO as control for 48 h or 72 h. LM treatment concentrations were 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM and 4 μM, and each group was performed in triplicate; 10 μL CCK-8 reagent was added for 1 h after treatment and the optical density value (OD450) was measured at the wavelength of 450 nm. The IC50 of LM was calculated by the cell growth curves drawn with Prism-GraphPad.

#### *4.4. Topoisomerase I-Mediated DNA Relaxation and Cleavage Assays*

The different concentrations of LM (5 μM, 10 μM, 20 μM) and camptothecin (CPT, 20 M) were incubated with supercoiled pUC19 plasmid DNA in relaxation buffer (50 mM Tris-HCl (pH7.5), 100 mM KCl, 0.5 mM EDTA and 30 pg/mL BSA) for 15 min at 37 ◦C to ensure binding equilibrium. Then, the recombinant topoisomerase I enzyme (from calf thymus, Beytime, China) was added for a further 30 min of incubation at 37 ◦C. The mixture of sodium dodecyl sulfate (SDS) and protease K (the final concentration was 0.25% and 250 g/mL, respectively) was added for a 30 min incubation at 50 ◦C to terminate the reaction. To obtain single stranded DNA, samples were loaded onto a 1% agarose gel lacking ethidium bromide at room temperature for 2 h at 120 V in TBE buffer. Gels were stained after migration using Gelred and then washed and finally photographed under UV light.

#### *4.5. RNA-seq Analysis*

Total RNA was extracted from cells using TRIzol® reagent, and genomic DNA was removed using DNase I. RNA integrity was detected by Agilent 2100 BioAnalyzer. The library was built by the NEB method. AMPure XP Beads were used to screen cDNA, conduct PCR amplification, and purify PCR products. NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was used for Library construction. The library was initially quantified by Qubit2.0 Fluorometer and was diluted to 1.5 ng/uL. The insert size of the library was detected using Agilent 2100 BioAnalyzer. qRT-PCR was used to quantify the effective concentration of the library to ensure the quality of the library. Then, the Illumina sequencing was conducted. The basic principle is sequencing by synthesis.

The raw data was filtered by the removal of reads with adapter, the removal of reads with N (N indicates that the base information cannot be determined), and the removal of low-quality reads. At the same time, the Q20, Q30 and GC contents of clean data were calculated. All subsequent analyses were based on clean data. Hisat2v2.0.5 was used to compare clean reads of paired terminal with genomic species: human genes (GrCH38.p12). String Tie (1.3.3b) (Mihaela-Pertea et al., 2015) was used for new gene prediction. Feature Counts (1.5.0-P3) were used to calculate recounts mapped to each gene. The FPKM of each gene was calculated based on the length of the gene and the readout mapped to the gene was calculated to obtain the expression value matrix.

Gene expression analysis of the different groups was performed by The DESeq2 software (V1.16.1) (*n* = 3). The *p*-value was adjusted using Benjamini and Hochberg's method. Genes with an adjusted *p*-value (FDR) < 0.05 were defined as differentially expressed genes (DEGs). Gene Ontology analysis (GO) and KEGG pathway enrichment analysis of DEGs were implemented by cluster Profiler (3.4.4). GSEA enrichment analysis was performed using GSEA (V4.1.0).

#### *4.6. TMT Labeling and LC-MS/MS Analysis*

The MDA-MB-468 cells were treated with 1.6 μM LM or 0.06% DMSO control medium for 48 h. SDT buffer was added to the sample. The lysate was sonicated and then boiled for 15 min. After centrifuged at 14,000× *g* for 40 min, the supernatant was quantified with the BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). The protein was digested by Filter aided proteome preparation (FASP) method, and 100 μg peptide mixture of each sample was labeled using TMT reagent according to the manufacturer's instructions (Thermo Fisher Scientific, USA). TMT labeled peptides were fractionated by RP chromatography using the Agilent 1260 infinity II HPLC. The collected fractions were combined into 10 fractions and dried down via vacuum centrifugation at 45 ◦C.

Each fraction was injected for nano LC-MS/MS analysis. The peptide mixture was loaded onto the C18-reversed phase analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 μm × 15 cm, nano viper, P/N164943) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min. The linear gradient was as follows: 6% buffer B for 3 min, 6–28% buffer B for 42 min, 28–38% buffer B for 5 min, 38–100% buffer B for 5 min, hold in 100% buffer B for 5 min.

LC-MS/MS analysis was performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) that was coupled to Easy nLC (Thermo Fisher Scientific, Waltham, MA, USA) for 60 min. The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan (350–1800 *m*/*z*) for HCD fragmentation. Survey scans were acquired at a resolution of 60,000 at *m*/*z* 200 with an AGC target of 3 × <sup>10</sup><sup>6</sup> and a maxIT of 50 ms. MS2 scans were acquired at a resolution of 15,000 for HCD spectra at *<sup>m</sup>*/*<sup>z</sup>* 200 with an AGC target of 2 × <sup>10</sup><sup>5</sup> and a maxIT of 45 ms, and the isolation width was 2 *m*/*z*. Only ions with a charge state between 2 and 6 and a minimum intensity of 2 × <sup>10</sup><sup>3</sup> were selected for fragmentation. Dynamic exclusion for selected ions was 30 s. Normalized collision energy was 30 eV.

MS/MS raw files were processed using MASCOT engine (Matrix Science, London, UK; version 2.6) embedded into Proteome Discoverer 2.2. The protein database was Uniprot\_HomoSapiens\_20367\_20200226. The search parameters included trypsin as the enzyme used to generate peptides with a maximum of 2 missed cleavages permitted. A precursor mass tolerance of 10 ppm was specified and 0.05 Da tolerance for MS2 fragments. Except for TMT labels, carbamidomethyl (C) was set as a fixed modification. Variable modifications were Oxidation(M) and Acetyl (Protein N-term). A peptide and protein false discovery rate of 1% was enforced using a reverse database search strategy. Proteins with fold change > 1.2 and *p* value (Student's *t*-test) < 0.05 were considered to be differentially expressed proteins. Wolf PSORT software was used for localization analysis of differential proteins. Gene Ontology analysis (GO) and KEGG pathway enrichment analysis of DEGs were implemented by clusterProfiler (3.4.4). GSEA enrichment analysis was performed using GSEA (V4.1.0).

#### *4.7. Molecular Docking*

Molecular docking was performed using the Schrodinger software (Schrödinger, Inc., New York, NY, USA). The 3D structure of p53 were retrieved from the protein data bank (PDB ID: 1TSR). In LM-p53 covalent docking, the protein p53 was prepared by Protein Preparation Tool in Schrodinger including optimized hydrogen bond network at pH 7.0 with PROKA tool. The ligand LM was prepared by Avogadro Tool to obtain the structural optimization.

#### *4.8. Western Blot Analysis*

The MDA-MB-468 cells were treated with 1.6 μM LM or 0.06% DMSO control medium for 48 h. Cells were harvested with trypsin/EDTA and then total proteins were extracted by using RIPA lysis buffer (RIPA lysis buffer (Beyotime, Shanghai, China)), 100 μg/mL PMSF (Beyotime, Shanghai, China), and 1 × protease inhibitor (Sigma, St. Louis, MO, USA). The protein was quantified by BCA methods according to the instructions (Beyotime, Shanghai, China). The proteins extracted from cells were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. The membrane was incubated with primary antibodies and anti-rabbit IgG (HRP-linked). The bands were detected by ECL Western blot system (Kodak, Rochester, NY, USA). Western blotting bands from three independent measurements were quantified with ImageJ.

#### *4.9. Xenograft Model*

Twenty-five female BALB/c nude mice aged 6–8 weeks were kept at constant temperature and humidity. The body weight was 20–24 g. Animals were supplied by Laboratory Animal Business Department of Shanghai Family Planning (certificate of quality: 20210715Abzz0619000729). Each mouse was inoculated subcutaneously at the right flank with MDA-MB-468 tumor cells (1 × <sup>10</sup>7) in 0.2 mL of PBS with Matrigel (1:1) for tumor development. Treatments were started on day 27 after tumor inoculation when the average tumor size reached 160 mm3. The animals were assigned into groups randomly based upon tumor volumes. Each group consisted of 4 tumor-bearing mice. The vehicle was 2%DMSO + 15% Solutol + 83%Saline. The mice in control group were injected 2.5 mL/kg vehicle by i.v. The mice in the positive control group were injected 15 mL/kg Paclitaxel by i.p. The mice in the low-dose group were injected 10 mL/kg LM by i.v. The mice in the high dose group were injected 20 mL/kg LM by i.v. The animals were checked daily for any effects of tumor growth and treatments on normal behavior, and body weights were measured every 3 days. Tumor size was measured every 3 days by a caliper using the formula: V = 0.5a × b2 where a and b are the long and short diameters of the tumor, respectively.

#### *4.10. Statistical Analysis*

One-way ANOVA analysis was used for comparison between groups, and Student's t-test was used for pair-wise comparison within groups. All statistical analyses were processed with GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA). *p* < 0.05 was considered statistically significant.

**Author Contributions:** Conceptualization, X.L.; Cell culture and cell experiment, X.S. and Z.L. (Zhenzhen Liang); CCK-8 Method, X.S. and Z.L. (Zhenzhen Liang); Topoisomerase I-Mediated DNA Relaxation and Cleavage Assays, X.S.; RNA-seq Analysis, X.S.; TMT quantitative proteomics analysis, X.S.; Molecular Docking, B.D.; Western Blot Analysis, Z.L. (Zhanying Lu).; Xenograft Model, X.S. and Z.L. (Zhanying Lu); Statistical Analysis, X.S.; Data curation, Y.Z. and X.S.; writing—original draft preparation, X.S.; writing—review and editing, X.S and X.L., Resources, J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Project (2019YFC0312504).

**Institutional Review Board Statement:** The animal study was reviewed and approved by Naval Medical University Institutional Animal Care and Use Committee.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Research Advances of Bioactive Sesquiterpenoids Isolated from Marine-Derived** *Aspergillus* **sp.**

**Lixiang Sun 1,2,†, Huannan Wang 2,†, Maocai Yan 2, Chunmei Sai <sup>2</sup> and Zhen Zhang 2,\***

<sup>1</sup> School of Pharmacy, Binzhou Medical University, 346 Guanhai Road, Yantai 264003, China

<sup>2</sup> School of Pharmacy, Jining Medical University, 669 Xueyuan Road, Rizhao 276800, China

**\*** Correspondence: zhangz@mail.jnmc.edu.cn; Tel.: +86-633-2983683

† These authors contributed equally to this work.

**Abstract:** Marine fungi *Aspergillus* sp. is an important source of natural active lead compounds with biological and chemical diversity, of which sesquiterpenoids are an extremely important class of bioactive secondary metabolites. In this paper, we review the sources, chemical structures, bioactivity, biosynthesis, and druggability evaluation of sesquiterpenoids discovered from marine fungi *Aspergillus* sp. since 2008. The *Aspergillus* species involved include mainly *Aspergillus fumigatus*, *Aspergillus versicolor*, *Aspergillus flavus*, *Aspergillus ustus*, *Aspergillus sydowii*, and so on, which originate from sponges, marine sediments, algae, mangroves, and corals. In recent years, 268 sesquiterpenoids were isolated from secondary metabolites of marine *Aspergillus* sp., 131 of which displayed bioactivities such as antitumor, antimicrobial, anti-inflammatory, and enzyme inhibitory activity. Furthermore, the main types of active sesquiterpenoids are bisabolanes, followed by drimanes, nitrobenzoyl, etc. Therefore, these novel sesquiterpenoids will provide a large number of potential lead compounds for the development of marine drugs.

**Keywords:** marine fungi; sesquiterpenoids; *Aspergillus*; bioactivity

#### **1. Introduction**

More than 70% area of the earth is covered by oceans, which is the largest known habitat for life. The marine environment is characterized by high salinity, high pressure, low oxygen, low temperature, darkness, scarce nutrients, etc. To adapt to the special environment and obtain advantages in the competition of limited resources, marine microorganisms could produce novel secondary metabolites with unique structures and potent biological activities during evolution [1,2]. Rich marine microorganisms, mainly derived from marine actinomycetes and marine fungi, are ubiquitous in the natural environment [3]. Diverse active natural products exist in endophytic fungi from the marine environment, which can be the resources for new lead compounds [4,5].

*Aspergillus* is a typical filamentous fungus, which is divided mainly into *Aspergillus fumigatus*, *Aspergillus versicolor*, *Aspergillus flavus*, *Aspergillus ustus*, *Aspergillus sydowii*, and so on [6]. Fumiquinazolines were isolated by Numata from marine *Aspergillus* sp. for the first time in 1992, which opened the door to the study of the metabolites of marine *Aspergillus* [7]. Recent studies have found that many organic compounds with unique structures, which showed a lot of physiological activities, were found in marine *Aspergillus* sp., including terpenoids, alkaloids, and polyketones [8]. Sesquiterpenoids, the most abundant among all the terpenoids skeletons, exhibit excellent biological activities, such as cytotoxicity, antibacterial, antifungal, antiviral, anti-inflammatory, and enzyme inhibitory activity, and have aroused widespread interest of many scholars [9,10]. This paper attempts to review the sources, bioactivities, biosynthesis, and other studies of sesquiterpenoids discovered from marine fungi *Aspergillus* sp. in the last 15 years.

**Citation:** Sun, L.; Wang, H.; Yan, M.; Sai, C.; Zhang, Z. Research Advances of Bioactive Sesquiterpenoids Isolated from Marine-Derived *Aspergillus* sp. *Molecules* **2022**, *27*, 7376. https://doi.org/10.3390/ molecules27217376

Academic Editor: Giovanni Ribaudo

Received: 8 October 2022 Accepted: 28 October 2022 Published: 30 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Characteristics of Sesquiterpenoids from Marine** *Aspergillus* **sp.**

Secondary metabolites of marine fungi have become one of the most active subfields of natural pharmaceutical discovery [11]. Sesquiterpenoids are an extremely important class of secondary metabolites and have been associated with a wide variety biological activities [12]. Approximately 268 sesquiterpenoids isolated from 56 strains of marine fungi are reviewed in this work. Furthermore, research has found that 37.5% of the sesquiterpenoid compounds came from marine animals (sponges, 21.4% and corals, 8.9%), 28.6% from marine plants (algae, 16.1% and mangroves, 12.5%), and the remaining compounds from the marine environment (21.4% from marine sediments and 1.8% from seawater), and 8.9% from unknown sources (see Figure 1).

Marine fungus *Aspergillus* is a huge community that occupies a great proportion in the fungus family, which is widely distributed in marine plants, marine organisms, marine sediments, and other environments. According to incomplete statistics, there were more than 180 species of fungus *Aspergillus*, such as *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus terreus*, and *Aspergillus versicolor* [13]. The proportions of the 56 species (Table 1) reviewed in this paper are as follows: *Aspergillus versicolor* (14.3%), *Aspergillus sydowii* (12.5%), *Aspergillus ustus* (10.7%), *Aspergillus fumigatus* (5.4%), *Aspergillus insulicola* (3.6%), *Aspergillus ochraceus* (3.6%), *Aspergillus carneus* (3.6%), *Aspergillus terreus* (3.6%), *Aspergillus flavus* (3.6%), *Aspergillus flavipes* (3.6%), and *Aspergillus* unknown (26.8%) (see Figure 2).






**Figure 2.** The proportions of marine fungi reviewed in this paper.

In recent years, more and more sesquiterpenoids were found in marine fungi *Aspergillus*, which consisted of the molecular skeleton structure with three isoprene units and contains 15 carbon atoms [56]. In addition, the number and skeleton types of sesquiterpenoids are the most abundant among all the terpenoids. According to the number of carbon rings, sesquiterpenoids can be divided into acyclic sesquiterpenes, monocyclic sesquiterpenoids, bicyclic sesquiterpenoids, tricyclic sesquiterpenoids, tetracyclic sesquiterpenoids, etc., [57]. Acyclic sesquiterpenes are also known as chain sesquiterpenes but rarely reported in fungi. The monocyclic sesquiterpenes referred mainly to bisabolanes, humaranes, and cybrodins, while the bicyclic sesquiterpenes consist mainly of drimanes, lacticinanes, and eudesmanes. This paper finds that the main types of sesquiterpenoids isolated from marine fungi *Aspergillus* were bisabolanes (46.6%), drimanes (27.2%), nitrobenzenes (6.3%), and unknown structure (9%) (see Figure 3).

**Figure 3.** The main types of sesquiterpenoids isolated from *Aspergillus* sp.

Recent studies have indicated that the metabolic pathway of marine fungi—that results in the production of a number of secondary metabolites with various chemical structures and specific physiological activities—is very different from that of terrestrial fungi [37]. This article concludes that 131 of the 268 sesquiterpenoids isolated from marine fungi *Aspergillus* have significant biological activities. Moreover, the structure types of inactive sesquiterpenoids are mostly bisabolanes and drimanes [58–62]. The relatively large number of sesquiterpenoids shows a variety of biological activities such as antitumor, antibacterial, anti-inflammatory, enzyme inhibitory, antioxidant, antiviral, and other activities. Overall, 30.5% of sesquiterpenoids exhibited antibacterial activity, followed by antitumor activity (29%), anti-inflammatory activity (22.9%), enzyme inhibitory activity (8.4%), and other activities (10.7%) (see Figure 4).

**Figure 4.** The bioactivity of sesquiterpenoids from *Aspergillus* sp.

#### **3. Bioactivity of Sesquiterpenoids from** *Aspergillus* **sp.**

#### *3.1. Antibacterial Activity*

In recent years, inappropriate and irrational use of antibiotics provides favorable conditions for resistant microorganisms to emerge and spread, which has become a global public health problem [63]. Therefore, it is urgent to develop new antibiotics with new structures and significant biological activities. To that end, the secondary metabolites of microorganisms in the marine environment are a great source for new antibacterial agents screening and much attention has been attracted to the relevant studies. This section covers 40 bioactive sesquiterpenoids (Figure 5) with antibacterial activity described to date from marine-derived *Aspergillus* sp.

Li et al. [14] isolated four new and one known bisabolane-type sesquiterpenoid from secondary metabolites of *Aspergillus* sp. from sponge. Compounds **1**–**5** showed different antibacterial activity against six pathogenic bacteria and two marine bacteria, and compounds **2** and **4** showed selective antibacterial activity. Compound **2** had strong inhibitory effects on *Staphylococcus albus* and *Micrococcus tetragenus*, with minimum inhibiting concentrations (MIC) values of 5.00 and 1.25 μM, respectively. The MIC values of compound **4** with *S. albus* and *Bacillus subtilis* were 5.00 μM and 2.50 μM, respectively. Notably, compound **1** represents the rare example of a bisabolane-type sesquiterpenoid with a 1, 4-disubstituted benzene ring isolated from marine organisms. Compounds **2** and **3** were the enantiomers of (+)-sydonol and (+)-sydonic acid, respectively. This fact suggests that fungi isolated from different marine organisms may produce different stereochemisty compounds. Furthermore, there were three sesquiterpenoids, **6**–**8**, from the sponge-associated fungus *Aspergillus sydowii* ZSDS1-F6, which has certain antibacterial activities; among them, compound **6** and **7** displayed antibacterial activities against *Klebsiella pneumonia*, with MIC values of 21.4 and 10.7μM, respectively [15]. In addition, compound 6 showed moderate antibacterial activity against *Aeromonas hydrophila* (MIC, 4.3 μM), while compound **8** showed moderate antibacterial activity against *Enterococcus faecalis* (MIC, 18.8 μM). Chen et al. [16] isolated two phenolic bisabolane sesquiterpenoids (PBS) compounds (**9**–**10**) from *Aspergillus flavipes* 297, including a pair of new enantiomers (±)-flavilane A (**9**). However, compounds **9** and

**10** represent the rare PBS-containing methylsulfinyl group and showed selective antibacterial activities against several pathogenic bacteria; their MIC values were 2–64 μg/mL. Furthermore, compound **10** exhibited mild antifungal activity against plant pathogenic fungus *Valsa mari*.

**Figure 5.** Chemical structures of antimicrobial compounds (**1**–**40**).

Aromatic bisabolene-type sesquiterpenoids **11**–**13** were isolated from the marine fungus *Aspergillus versicolor* SD-330 in the deep-sea sediments [17]. Compounds **11** and **12** had significant inhibitory activities against *A. hydrophilia*, *Escherichia coli*, *Edwardsiella tarda*, and *Vibrio harveyi*, with MIC values ranging from 2.0 to 8.0 μg/mL. Moreover, compound **13** had significant inhibitory activity against *E. coli* (MIC value was 1.0 μg/mL), which was better than the positive control chloramphenicol (MIC value was 2.0 μg/mL). A new aromatic bisabolene-type sesquiterpenoid (**14**) was discovered in *Aspergillus sydowii* SW9, whose absolute configuration is (*S*). Compound **14** had significant inhibitory effect on *E. coli*, and its MIC value was 2.0 μg/mL, which was similar to that of positive control chloramphenicol (MIC 2.0 μg/mL). Compound **14** also exhibited potent activity against *S. pneumonise*, with an MIC value of 4.0 μg/mL [18]. Wang et al. [19] obtained four sesquiterpenoids **15**–**18** with antibacterial activity from marine *Aspergillus versicolor* SD-330. Compounds **15** and **16** showed significant antibacterial activity against *E. coli*, *E. trada*, *V. harveyi*, and *Vibrio parahaemolyticus*, and the MIC values were less than or equal to 8.0 μg/mL. However, compound **17** exhibited significant antibacterial effect on *E. coli* with MIC value of 1.0 μg/mL, which was more potent than that of positive control chloramphenicol (MIC 2.0 μg/mL). Moreover, compound **17** showed strong inhibitory activity against *A. hydrophilia*, *E. tarda*, *Vibrio anguillarum*, and *V. harveyi*, each with MIC value of 4.0 μg/mL. Compound **17** showed a stronger antibacterial activity than compounds **15** and 16, suggesting that C-15 carboxyl group methyl ester or the methylated C-7 hydroxyl group could reduce their antibacterial activity.

Wei et al. isolated three phenolic bisabolane-type sesquiterpenoids compounds **19**–**21** from *Aspergillus* sp., which is the first report of natural metabolites from marine fungus *Aspergillus* from gorgonian *Dichotella gemmacea* [20]. All of them exhibited weak antibacterial activity against *Staphylococcus aureus,* with the diameters of inhibition zones of 11, 7, and 5 mm at 100 μg/mL, respectively. Seven phenolic bisabolane sesquiterpenoids **22**–**28** were obtained from the endophytic fungus *Aspergillus* sp. xy02 from a Thai mangrove *Xylocarpus moluccensis* [21] and displayed moderate inhibitory activities against *S. aureus*, with IC50 values ranging from 31.5 to 41.9 μM. Two new phenolic bisabolane sequiterpenes, asperchondols A (**29**) and asperchondols B (**30**), were obtained from the sponge-derived fungus *Aspergillus* sp. and showed antibacterial activity against *S. aureus*, with the MICs of 50 and 25 μM, respectively [22]. Furthermore, structure–activity relationship found that the coexistence of phenolic bisabolane sesquiterpene and diphenyl ether moieties seems to be very important since the hybrid **30** was more active than phenolic bisabolane sesquiterpenoid **29** and phenyl esters.

A series of phenolic bisabolane-type sesquiterpenoids have been discovered from different marine invertebrates such as sponges [64] and gorgonians [65] in the last century. In addition, such compounds were also found in bacterium CNH-741 and fungus CNC-979 isolated from marine sediments [66]. These results indicate that the real producers of these compounds from marine invertebrates, sponges, and corals may be constituents of microorganisms. Albican-11,14-diol (**31**) is a sesquiterpene compound isolated from the cultures of the endophytic fungus *Aspergillus versicolor*, which is isolated from marine green alga Codium fragile [23]. The diameters of inhibition zones of compound **31** against *E. coli* and *S. aureus* were 7 and 10.3 mm, respectively, at the concentration of 30 μg/disk. Fang et al. isolated a drimane-type sesquiterpenoid (**32**) and three unknown-type sesquiterpenoids (**33**–**35**) from the algicolos fungus *Aspergillus* sp. RR-YLW-12, which exhibited little inhibitory activity against four marine-derived pathogenic bacteria, *V. anguillarum*, *V. harveyi*, *V. parahaemolytics*, and *Vibrio splendidus* [24]. Zheng et al. isolated and purified three bisabolane sesquiterpenes **36**–**38** from the fermentation products of *Aspergillus versicolor* ZJ-2008015, which were obtained from a soft coral *Sarcophyton* sp. [25]. The results showed that compounds **36**–**38** exhibited potent antibacterial activity with MICs of 5.3, 6.4, and 5.4 μM against *S. albus* and 2.6, 6.4, and 5.4 μM against *S. aureus*, respectively. Cohen et al. [26] isolated two drimane sesquiterpenes (**39**–**40**) from the sponge-derived fungus *Aspergillus insuetus* (OY-207), which exhibited anti-fungal activity against *Neurospora crassa*, with the MICs of 140 and 242 μM, respectively.

#### *3.2. Antitumor Activity*

The marine environment represents a unique resource that encloses a massive chemical and biological diversity, which leads to an important source of potential antitumor drugs [67]. Among antitumor compounds, sesquiterpenes (including bisabolane, drimane, illudalane, etc.) are obtained mainly from marine fungi, including *Aspergillus* sp. [68,69]. Therefore, more and more researchers pay close attention to looking for effective antitumor drugs from marine *Aspergillus*. In recent years, there were about 38 bioactive sesquiterpenoids (Figure 6) with antitumor activity isolated from marine-derived *Aspergillus* sp.

**Figure 6.** Chemical structures of antitumor compounds (**41**–**78**).

Orfali et al. [27] first discovered two illudalane sesquiterpenes, asperorlactone (**41**) and echinolactone D (**42**), from marine sediment ascomycete *Aspergillus oryzae*, in which compound **41** has an absolute configuration of (5R). Compounds **41** and **42** showed antiproliferative activity against human lung cancer (A549), liver cancer (HepG2), and breast cancer (MCF-7) cell lines, with half maximal inhibitory concentration (IC50) values of asperorlactone (**41**) <100 μM. Furthermore, compounds **9** and **10** isolated from *Aspergillus flavipes* 297 exhibited promising cytotoxic effects on MKN-45 and HepG2 cells, respectively, indicating that the methylsulfinyl substituent enhanced the cytotoxicity, to a certain degree [16]. Gao et al. [28] isolated four drimane sesquiterpene esters asperienes A-D (**43**–**46**) from marine-derived fungal *Aspergillus flavus* CF13-11, which was the first successful isolation of two pairs of C-6 /C-7 isoforms. Moreover, compounds **43**–**46** showed significant activity against four tumor cell lines (HeLa, MCF-7, MGC-803, and A549), with IC50 values of 1.4–8.3 μM. Notably, compounds **43** and **46** showed lower toxicity to normal GES-1 cells

than did **44** and **45**, suggesting their great potential for the development of an antitumor agent. Yurchenko et al. [29] isolated two drimane sesquiterpenes (**47**–**48**) from marinesediment-derived fungus *Aspergillus flocculosus*, which exhibited potent cytotoxic effect toward mouse neuroblastoma neuro-2A and human prostate cancer 22Rv1 cells, with the IC50 values were 24.1, 4.9 μM and 31.5, 3.0 μM, respectively. It is well known that human prostate cancer 22Rv1 cells are resistant to hormone therapy because of the expression of the androgen receptor splice variants AR-V7 [70]. Therefore, the results indicated that compounds **47** and **48** could be used in the treatment of human drug-resistant prostate cancer. Fang et al. [30] isolated two nitrobenzoyl sesquiterpenoids (**49**–**50**) from the marine-derived fungus *Aspergillus ochraceus* Jcma1F17, which was the first time nitrobenzoyl sesquiterpenoids obtained from this fungal were reported. Both compounds displayed significant cytotoxic effects on 10 human cancer cell lines (H1975, U937, K562, BGC-823, MOLT-4, McF-7, A549, Hela, HL60, and Huh-7), with IC50 values ranging from 1.95 to 6.35 μM.

Insulicolide A (Nitrobenzoyl substituted sesquiterpene, **51**) was isolated from the marine-sponge-associated endozoic fungus *Aspergillus insulicola* MD10-2 [31]. Compound **51** showed cytotoxic effects against human lung cancer cell line H-460, with an IC50 value of 6.9 μM. However, the cytotoxic activity of the acetylated derivatives of compound **51** decreased markedly, indicating that the double at C-7 might be involved in the cytotoxic activity. Tan et al. isolated three nitrobenzoyl sesquiterpenoids (**52**–**54**) from the marine fungus *Aspergillus ochraceus* Jcma 1F17 [32]. Compound **54** displayed potent cytotoxicities against three renal carcinoma ACHN, OS-RC-2, and 786-O cells lines (IC50 of 0.89–1.5 μM). The cytotoxic effects of compounds **52** and **53** on 786-O cells (IC50 of 2.3 and 4.3 μM, respectively) were exhibited more strongly than those of OS-RC-2 (IC50 5.3 and 8.2 μM) and ACHN (IC50 of 4.1 and 11 μM, respectively), suggesting that the C-9 hydroxy group may contribute more to the cytotoxic activities against renal carcinoma cells. Additionally, compound **52** showed stronger inhibitory activity at low concentration levels, compared with the positive control sorafenib, a drug approved for the treatment of primary kidney cancer (advanced renal cell carcinoma). Further investigation revealed that the cell cycle was arrested at G0/G1 phase after being treated with compound **52** at 1 μM, whereas after being treated at 2 μM for 72 h, the late apoptosis of 786-O cells were induced. Four nitrobenzoyl sesquiterpenoids (**55**–**58**) were isolated from an Antarctica-sponge-derived *Aspergillus insulicola* by Sun et al. [33], in which compounds **57** and **58** showed selective inhibitory activity against human pancreatic ductal adenocarcinoma (PDAC) cell lines, whereas compounds **55** and **56** were inactive, indicating that hydroxyl groups at C-9 is essential for cytotoxicity. Furthermore, the IC50 values of compounds **57** and **58** against PDAC cell lines AsPC-1 and PANC-1 were 2.7, 4.6 μM and 2.3, 4.2 μM, respectively. Numerous studies have shown that most of nitrobenzoyl sesquiterpenes were obtained from the marine-derived fungus *Aspergillus ochraceus*, suggesting that *Aspergillus ochraceus* may be a good resource for the production of these compounds.

Liu et al. [34] found three drimane sesquiterpenoids (**59**–**61**) from marine spongederived fungus *Aspergillus ustus*, which showed cytotoxic activities against mouse lymphoma cell line L5178Y, with half maximal effective concentration (EC50) values between 0.6 and 5.3 μM. In addition, the EC50 value of compound **60** against PC12 and HeLa cells were 7.2 μM and 5.9 μM, respectively. Zhou et al. [35] isolated drimane sesquiterpenoid (**62**) from mangrove-derived fungus *Aspergillus ustus* and exhibited moderate cytotoxic effects against the mice lymphocytic leukemia P388 cell line with IC50 value of 8.7 μM. Sun et al. [36] isolated three bisabolane sesquiterpenoid dimers (**63**–**65**) from the sponge-derived fungus *Aspergillus* sp., and the cytotoxic activity against HePG-2 human hepatoma cell line and Caski human cervical cell line were determined in vitro. Significantly, compounds **63** and **65** with (7S) and (7 S) configuration displayed better potent cytotoxicity toward the tumor cell lines than did compound **64**. The IC50 values of compound **63** and **65** were 9.31, 12.40 μM and 2.91, 10.20 μM, respectively. These results suggest that the cytotoxic activity of the compound may be weakened due to the mesomeric effect since the activity of the compounds is stereoselective. β-D-glucopyranosyl aspergillusene A (**66**) from the

sponge-derived fungus *Aspergillus sydowii* J05B-7F-4 exhibited mild cytotoxicity against KB (human nasopharyngeal carcinoma cells), HepG2 (human liver cancer cells), and HCT 116 (human colon cancer cells), with IC50 values between 50 and 70 μM [37].

Deng et al. [38] found four sesquiterpenoids containing 16 carbon atoms (**67**–**70**) from the mangrove endophytic fungus *Aspergillus terreus* GX3-3B, of which compound **67** showed inhibitory activity against human breast cancer cells (MCF-7) and human promyelocytic leukemia cells (HL-60), with the IC50 values were 4.49 and 3.43 μM, respectively. In addition, compound **68** exhibited promising inhibitory effect on MCF-7 cells, with an IC50 value of 2.79 μM, whereas compound **70** showed potent inhibitory effect on HL-60 cells, with an IC50 value of 0.6 μM. The structure–activity relationship indicated that the presence of C or D lactone ring may be helpful for the inhibitory against the human breast cancer cell line MCF-7. Compounds **67** and **70** showed stronger activities than did compounds 68 and 69, indicating that hydroxyl group at the C-7 position could improve the cytotoxicity toward HL-60 cell.

Aspergiketone (**71**) is the first sesquiterpenoid derivative isolated from *Aspergillus fumigatus*, which exhibited obvious cytotoxicity against HL-60 and A-549 cells, with IC50 values of 12.4 and 22.1 μM, respectively [39]. Oxalicine B (**72**), a unique pyridino-αpyrone sesquiterpenoid, was obtained from the sea-urchin-derived fungus *Aspergillus fumigatus* and exhibits moderate cytotoxicity to murine P388 leukemia cells, with IC50 of 55.9 μM [40]. Three drimane sesquiterpenes (**73**–**75**) were isolated from marine *Aspergillus ustus* 094102 [41], of which compounds **74** and **75** showed moderate cytotoxicity against A549 and HL-60 cells, with IC50 values of 10.5 and 9.0 μM, respectively. Moreover, compound **73** exhibited weak cytotoxic effect to A549 and HL-60 cells, with IC50 values of 20.6 and 30.0 μM, respectively. Proksch et al. found a drimane sesquiterpene (**76**) from marine-sponge-derived fungus *Aspergillus ustus*, which exhibited selective inhibition on lymphoma cell line L5178Y cells (median effective dose (ED50), 1.9 μM) [42]. Wang et al. found a β-bergamotane sesquiterpenoids (**77**) from marine-sediment-derived fungus *Aspergillus fumigatus* YK-7, which exhibited weak inhibitory activities against U937 cells, with an IC50 value of 84.9 μM [43]. Asperflavinoid A (**78**), a drimane-type sesquiterpenoids, was isolated from *Aspergillus flavipes* 297 and exerted toxic effect on HepG2 and MKN-45 cells, with the IC50 values of 38.5 and 26.8 μM, respectively [44].

#### *3.3. Anti-Inflammatory Activity*

Inflammation is a comprehensive array of physiological response to a foreign organism, which has been considered as a major factor for the progression of various chronic diseases/disorders [71]. Therefore, development of effective and economical anti-inflammatory drugs (NSAIDs) is an area of importance in drug discovery while natural anti-inflammatory supplements are becoming more popular and have been the focus of many scientific investigations. This section covers 30 sesquiterpenoids (Figure 7) with anti-inflammatory activity which isolated from marine-derived *Aspergillus* sp.

**Figure 7.** Chemical structures of anti-inflammatory compounds (**79**–**108**).

Cui et al. [45] isolated a sesquiterpene derivative (**79**) from the mangrove endophytic fungus *Aspergillus versicolor* SYSU-SKS025, which was found to inhibit nitric oxide (NO) production RAW 264.7 macrophages, with an IC50 value of 12.5 μM (positive control, indomethacin, IC50 = 37.5 μM). Wang et al. [46] found four triketide-sesquiterpenoids A−D (**80**–**83**) from the marine-algal-associated fungus *Aspergillus* sp. ZL0-1B14, which exhibited anti-inflammatory activity in LPS-stimulated RAW264.7 macrophages. In addition, compound **83** inhibited the production of IL-6 with an inhibition rate of 69% at 40 μM. Wu et al. [47] firstly discovered two brasilane sesquiterpenoids (**84**–**85**) with α and β unsaturated ketones from marine-derived fungus *Aspergillus terreus*, both of which showed moderate inhibitory effects; the inhibitory rates of nitric oxide were 47.7% and 37.3%, respectively, at 40 μM. Chung et al. [48] isolated five sesquiterpenoids (**86**–**90**) with anti-inflammatory activity from *Aspergillus sydowii* in marine sediments. Among them, compounds **88** and **90** displayed selective inhibition against fMLP/CB-induced superoxide anion generation by human neutrophils, with IC50 values of 5.23 and 6.11 μM, respectively. At the same time, they also exhibited the most potent inhibitory activity against the release of elastase induced by fMLP/CB, with the IC50 values of 16.39 and 8.80 μM, respectively. Interestingly, the anti-inflammatory activity of compound **88** was better than that of compound **86** indicating the important role of hydroxy group on C-7. Moreover, compounds containing

methylene alcohol on C-3 (**86**, **88**, and **90**) showed more potent anti-inflammatory activity compared with the derivatives with carboxylic acid functional groups (**87** and **89**). Four Eremophilane sesquiterpenoids (**91**–**94**) were isolated from deep-marine-sediment-derived fungus *Aspergillus sp.* SCSIOW2, and all showed inhibitory activity of NO production in a dose-dependent manner [49]. Additionally, five sesquiterpenoids (**95**–**99**) were isolated from the mangrove endophytic fungus *Aspergillus sp.* GXNU-MA1 by Zhou et al., which exhibited moderate inhibitory activities against NO production, with IC50 values ranging from 16.15 to 27.08 μM [50]. Niu et al. isolated six phenolic bisabolane (**100**–**105**) and two cuparene sesquiterpenoids (**106**–**107**) from *Aspergillus sydowii* MCCC3A00324 derived from deep sea sediments [51]. Compounds **100**, **101**, and **103**–**105** showed anti-inflammatory activity against NO secretion in LPS-activated BV-2 microglia cells, with the inhibition rates of more than 45% at 10 μM, while those of compounds **102**, **106**, and **107** were 32.8%, 32.6% and 45.4%, respectively. Furthermore, compound **101** exerted an anti-inflammatory effect by inhibiting NF-κB activation pathway in a dose-dependent manner. Tan et al. isolated a new nitrobenzoyl sesquiterpenoid (**108**) from *Aspergillus ochraceus*, which could suppress the RANKL-induced osteoclats formation and bone resorption by targeting NF-κB [52]. Additionally, compound **108** attenuated inflammatory bone loss in vivo.

#### *3.4. Enzymatic Inhibitory Activity*

Enzyme inhibitors are of value in treating many diseases in clinical use, and have become a very attractive target for drug development and discovery. In recent years, the prominence of various enzyme inhibitors has been discussed extensively by many researchers in comprehensive systematic reviews [72]. In this section, the inhibitory activities of sesquiterpenoids (Figure 8) from marine *Aspergillus* sp. against three enzymes (α-glucosidase, cholinesterase, and neuraminidase) are briefly reviewed.

**Figure 8.** Chemical structures of enzymatic inhibitory compounds (**109**–**118**).

α-Glucosidase is a membrane-bound enzyme present in the small intestinal epithelium [73], whose role is to promote the absorption of glucose in the small intestine by catalyzing the hydrolysis of oligosaccharides into absorbable glucose. α-Glucosidase inhibitors are the most widely used drugs in the clinical treatment of diabetes in China. By inhibiting the activity of α-glucosidase, the formation and absorption of glucose can be reduced to achieve the goal of lowering blood glucose. At the same time, it can also reduce the stimulation of blood glucose on the pancreas, effectively preventing and relieving diabetic complications [74]. 7-Deoxy-7,14-didehydrosydonol (**79**) was found from the mangrove endophytic fungus *Aspergillus versicolor* and possessed a significant inhibitory effect on α-glucosidase, with an IC50 value of 7.5 μM (acarbose as 350 μM), and the terminal ethylene group at C-7 may play a key role in α-glucosidase inhibition activity [45]. Wu et al. [53] isolated four phenolic bisabolane sesquiterpenoids (**109**–**112**) from the mangrove endophytic

fungus *Aspergillus flavus* QQSG-3. The inhibitory activity studies of α-glucosidase showed that the compounds (**109**–**112**) had strong inhibitory effects, with IC50 values of 4.5, 3.1, 1.5, and 2.3 μM, respectively (all lower than the positive control drug acarbose).

Alzheimer's Disease (AD) is a degenerative disease with unknown causes, mainly involving cerebral cortical neurons, which is the major cause of dementia [75]. The currently accepted pathogenesis is the cholinergic deficiency hypothesis [76]. Cholinesterase inhibitors (ChEI) are a class of drugs that can bind to cholinesterase (ChE) and inhibit ChE activity; they are also approved as first-line drugs for the treatment of mild-to-moderate AD [77]. Feng et al. firstly isolated the potential reversible cholinesterase inhibitor cyclopentapentalane sesquiterpenoid subergorgic (**113**) and its analogues 2-deoxy-2β-hydroxysubergorgic (114) from the soft-coral-derived fungus *Aspergillus* sp. EGF15-0-3 [54].

Neuraminidase (NA) is the most critical enzyme for influenza virus replication and diffusion in host cells and has become an important target for anti-influenza virus drug design [78]. Li et al. [55] isolated four drimane sesquiterpenoids (**115**–**118**) from the ascidian endophytic fungus *Aspergillus ustus* TK-5, which showed significant inhibitory activity against neuraminidase, with IC50 values of 31.8, 37.3, 28.4, and 36.8 μM, respectively. Further results showed that the degree of unsaturation of 11-OH and C-6 linked side chains, which can improve their neuraminidase inhibitory activity.

#### *3.5. Other Activities*

Hu et al. isolated an aromatic bisabolane sesquiterpenoid (7S,8S)-8-hydroxysydowic acid (**119,** Figure 9)) from the marine red algae endophytic fungus *Aspergillus sydowii* EN-434, which exhibited DPPH free radical scavenging activity, with an IC50 value of 113.5 μM [79]. An et al. found two sesquiterpenoids (**120**–**121,** Figure 9) with weak DPPH radical scavenging activity, with IC50 values of 1.8 mM and 0.6 mM, respectively (VC as 0.04 mM) [80]. Zhong et al. isolated three sesquiterpenoids (**122**–**124,** Figure 9) from the marine-offshore-mud-derived fungus *Aspergillus pseudoglaucus* [81]. Among them, compounds **122** and **123** showed strong DPPH radical scavenging activity, with IC50 values of 2.42 and 1.86 μg/mL (VC was 3.25 μg/mL), respectively, while compound **124** exhibited moderate antioxidant activity (IC50 was 10.89 μg/mL).

**Figure 9.** Chemical structures of other biological compounds (**119**–**127**).

Two bisabolane-type sesquiterpenoids (**4**–**5**) were derived from sponge-derived fungus *Aspergillus* sp., among which compound **4** completely inhibited larval settlement at 25.0 μg/mL, while compound **5** displayed an obvious toxic effect on larvae at the same concentration [14]. Compound **7** also showed weak anti-H3N2 activity, with IC50 values of 57.4 μM [15]. (−)-(7S)-10-hydroxysydonic acid (**28**) was found to have a mild DPPH radical scavenging activity, with an IC50 value of 72.1 μM [21]. Nitrobenzoyl sesquiterpenoids (**49**) also showed moderate antiviral activities against H3N2 and EV71, with IC50 values of

17.0 and 9.4 μM, respectively [30]. Liu et al. [82] isolated three drimane sesquiterpenoids (**125**–**127**, Figure 9) from the marine-green-alga-derived fungus *Aspergillus ustus*. In the brine shrimp (*Artemia salina*) toxicity assay, there was more than 75% lethality at the concentration of 100 μg/mL, and the LC50 values were 41.8, 62.2 and 48.9 μg/mL, respectively.

#### **4. Chemical Synthesis and Biosynthesis of Sesquiterpenoids from Marine** *Aspergillus* **sp.** *4.1. Chemically Induced Synthesis*

*Aspergillus* sp. is the important source for the discovery of natural active products with novel and diverse structures. However, in recent years, the continual study of secondary metabolites of marine fungi has led to a high frequency of repeated discovery of known compounds [83]. This encourages us to develop new strategies to obtain new natural products. Studies have found that a large number of secondary metabolite biosynthesis gene clusters exist in the genome of *Aspergillus* fungi. Furthermore, the genome can be segmented into active and silent clusters, while the silent clusters are inactive under normal environmental conditions [84–86]. In order to obtain more active metabolites, researchers have applied a variety of methods to activate silenced biological genetic gene clusters, such as transcription factor regulation, targeted genome mining, heterologous expression of gene clusters, and chemical epigenetic regulation [87–89]. Because of its simplicity and effectiveness, chemical epigenetic regulation has been widely used in marine fungi to activate silenced gene clusters, which could lead to the production of new secondary metabolites or known components with a higher concentration. Wang et al. [90] cultivated the gorgonian-derived fungus *Aspergillus* sp. SC-20090066 with a DNA methyltransferase inhibitor 5-azacytidae (5-AZA) in the culture medium and led to the isolation of six new bisabolane-type sesquiterpenoids (Figure 10). Among them, compounds (**128**–**130**) exhibited broad spectrum activities against *S. aureus*, *Bacillus cereus*, *Rhizophila*, *Pseudomonas putida*, and *Pseudomonas aeruginosa*, with MICs of less than 25 μM. In particular, compound **130** exhibited significant antibacterial activity against *S. aureus*, with MIC value of 3.13 μM, which was close to the positive control ciprofloxacin (MIC value was 2.5 μM). In order to trigger the chemical diversity of marine-derived fungus *Aspergillus versicolor* XS-2009006, epigenetic agents (histone deacetylase inhibitor SAHA and DNA methyltransferase inhibitor 5-AZA) were added to the culture medium by Wu et al. [91] Interestingly, the secondary metabolites was significantly increased and a new bisabolane sesquiterpene aspergillusene E (**131, Figure 10**) was isolated, which showed anti-larval attachment activity against bryozoan *B. neritina*, with the EC50 and (lethal concentration 50%) LC50 values of 6.25 μg/mL and 25 μg/mL, respectively. In addition, compound **131** showed certain antibacterial activities against *Staphylococcus epidermidis* and *S. aureus*, with MIC values ranging from 8 to 16 μM. By adding DNA methyltransferase inhibitors to the medium of *Aspergillus sydowii*, the composition of secondary metabolites was further changed and new bisabolane sesquiterpenoids (**86**–**87**) were isolated [48]. In addition, Wang et al. [49] applied chemical epigenetic manipulation to *Aspergillus* sp. SCSIOW2 and obtained four eremophilane sesquiterpenes with anti-inflammatory activity (**91**–**94**).

**Figure 10.** Structures of sesquiterpenoids obtained from chemical synthesis and biosynthesis from the *Aspergillus* sp. (**128**–**132**).

#### *4.2. Biosynthetic Pathways*

The skeleton structures of sesquiterpenoids were derived from farnesyl diphosphate (FPP) and underwent a series of reaction steps, including intramolecular rearrangement, cyclysis, and other biosynthetic transformations, leading to their structural diversity [92]. Ingavat et al. [93] studied the proposed biosynthesis of sesquiterpene compound **132** in *Aspergillus aculeatus*, which starts from a double-bond migration (C1/C2 to C2/C3) of silphineneene intermediate 2, and then the double bond of C2/C3 undergoes oxidative cleavage to generate intermediate 3, which, in turn, undergoes a series of oxidation and lactonizations to finally give **132** (Figure 10).

Wang et al. [46] proposed a biogenetic pathway for the synthesis of aspertetranones A-D (**80**–**83**). Common drimane-type merosesquiterpene were obtained by cyclization of farnesylated pyrone, followed by oxidation and retro-aldo/aldo rearrangement to produce the unique terpenoid part of aspertetranones. After nucleophilic attack and dehydration, the leaborate preaspertetranone was obtained. Illudalanes derive biosynthetically from a humulene precursor after cyclization, producing a protoilludanes, which is eventually rearranged to form the irudane derivative [94]. According to this report, Orfali et al. speculated a biosynthetic pathway of asperorlactone (**41**), in which illudol was a key intermediate. The iluane-type sesquiterpene asperorlactone can be synthesized by dehydration, oxidation, and four-membered ring opening [27].

#### **5. Potency of Sesquiterpenoids from Marine** *Aspergillus* **sp.**

Secondary metabolites of microorganisms in the marine environment, mainly derived from marine fungi, are a great source for new drug screening. Currently, the marine drug library includes 15 approved drugs (primarily for cancer treatment), 7 phase I compounds, 12 phase II compounds, and 5 compounds in phase III clinical trials, the latter including a recently recommended drug for symptomatic treatment of COVID-19 (Plitidepsin) [95,96]. Compound **13** displayed significant inhibitory activity against *E. coli* (MIC 1.0 μg/mL), and its antibacterial effect was more potent than that of the positive control chloramphenicol (MIC 2.0 μg/mL), which was expected to be a lead compound for antibiotics [17]. The sesquiterpene compound (**79**) isolated from *Aspergillus versicolor* exhibited better inhibitory effect on α-glucosidase than acarbose, while its anti-inflammatory effect was also stronger than that of indomethacin [45]. Compound **88** derived from marine sediments, showed a significant anti-inflammatory effect and hypoglycemic effect. In addition, compound **88** could also inhibit fat accumulation in adipocytes [48]. These results indicated compound **79** and **88** has the potential to be a lead compound targeting the vicious diabetes-inflammation cycle. Feng et al. found that sesquiterpene compound **113**, the reversible cholinesterase inhibitor, is a promising new drug candidate for the treatment of Alzheimer's Disease and a preclinical trial is already under way [54].

#### **6. Conclusions and Perspective**

In this paper, the biosources, bioactivities, structural types, biosynthetic, and pharmacogenic potential of sesquiterpenoids found from marine fungi *Aspergillus* sp. were reviewed. A total of 268 sesquiterpenes were isolated, including 131 bioactive sesquiterpenes, most of which were bisabolanes, followed by drimanes and nitrobenzoyl, etc. Most *Aspergillus* species derived from sponges, marine sediments, algae, mangroves, corals, etc. The main *Aspergillus* species involved are as follows: *Aspergillus fumigatus*, *Aspergillus versicolor*, *Aspergillus flavus*, *Aspergillus ustus*, *Aspergillus sydowii*, and so on. These sesquiterpenes exhibited excellent pharmacological activities such as antibacterial, antitumor, antiinflammatory, and enzyme inhibitory activities. Additionally, the biosynthesis and total synthesis of sesquiterpenes derived from marine *Aspergillus* sp. have also promoted the in-depth understanding of these sesquiterpenes. Because of the chemical and biological activity of these sesquiterpenoids, it is worthwhile to find promising lead compounds for the development of marine drugs in further studies from marine fungi.

**Author Contributions:** Conception and design of the manuscript: Z.Z.; conducting literature search and analysis of the information: L.S.; draft and revision of the manuscript: L.S. and H.W.; editing the manuscript: M.Y. and C.S.; finalization and approval of the revised manuscript for submission: Z.Z. and H.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Projects of Medical and Health Technology Development Program in Shandong Province (grant No 2019WS358, 202013050864), Research Fund for Lin He's Academician Workstation of New Medicine and Clinical Translation in Jining Medical University (grant No JYHL2021MS17).

**Conflicts of Interest:** The authors declare no potential conflict of interest.

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