Deep-Sea-Derived Fungi as Valuable Producers of Cytotoxic Secondary Metabolites and Their Leads Potential

Cancer is the leading lethal disease worldwide. Natural products have contributed significantly to the development of approved therapeutic agents. Therefore, research into new bioactive naturally sourced metabolites with lead potential is urgently needed. It is well-known that marine microorganisms are by far one of the most notable and prolific sources of bioactive natural products. Among them, deep-sea-derived fungi are extraordinarily adapted and metabolically active under extreme environmental conditions, which enable them to produce a large number of novel secondary metabolites. Chemical examination of deep-sea-derived fungi has yielded enormous amounts of cytotoxic natural products and potential drug leads. This review summarizes a total of 229 cytotoxic compounds isolated from deep-sea-derived fungi from 2010 to 2021. The emphasis is on the unique chemical diversity of these metabolic products, together with their relevant cytotoxic properties. Among the isolated metabolites, 82 compounds have been found to possess moderate to potent cytotoxic activities. Meanwhile, we also highlight some compounds with potent cytotoxicities (namely “star molecules”) considering their high drug lead potential. This review reveals deep-sea-derived fungi as considerable resources for the development of new drugs and the potential of the newly discovered secondary metabolites as valuable antitumor lead compounds.


INTRODUCTION
Covering approximately 72% of the Earth's surface, the oceans are considered to make significant contributions to the development of novel pharmaceutical resources. Of the total sea areas, 60% are deep seas that are covered by seawater at a depth of more than 2,000 m. The deep sea is quite a complicated and extreme ecosystem characterized by elevated hydrostatic pressure, low or high temperature (such as hydrothermal vents), absence of light, fickle salinity, oligotrophy, and low oxygen concentration (Zeng et al., 2010). It is the largest remaining unexplored aqueous habitat on Earth, and organisms in this realm are confronted by various fundamental challenges (Wu J. et al., 2013). To overcome these multiple extreme stresses, organisms that reside in deep sea ecosystems have evolved specific genetic capabilities to produce a large number of metabolic products, including small molecules such as secondary metabolites, proteins and enzymes, saccharides, and so on. These deep-sea-derived metabolic products have played important roles in adaptation to species communications and biotechnological and pharmaceutical applications.
Fungi are regarded as the richest and most varied eukaryotes on Earth, and their existence in every possible extreme environment makes them a valuable source for new drug discovery (Zain Ul Arifeen et al., 2019). Marine-derived fungi have proven to be untapped sources of novel marine natural products for exploitation in medicine. In addition to fungi living in terrestrial environments, marine-derived fungi suffer from the abovementioned extreme environmental stresses, and therefore, they have enjoyed specific metabolic pathways to synthesize structurally creative metabolites with remarkable biological activities . However, although massive metabolites have been reported from marine-derived fungi thus far Carroll et al., 2021), it is a matter of fact that the search for new marine natural products is gradually approaching saturation. As a result, the discovery of new marine natural products from unexplored environments has become an alternative pathway. Extremophiles isolated from the deep sea, hydrothermal vents, cold water, and polar regions, have attracted much attention (Soldatou and Baker, 2017). They are extraordinarily adapted and metabolically active under extreme environmental conditions, which affords a large number of marine natural products.
Cancer is the leading lethal disease worldwide. Although localized surgery and radiation approaches play an important role in the treatment of cancer, it is impossible to prevent the dissemination of tumor cells. Chemotherapy has become the most preferred treatment of choice for patients, which has aroused an urgent necessity and priority to discover new molecules . Natural products have benefited greatly from the growth of the pharmaceutical industry, especially pharmacologically attractive leads and potential clinical therapeutic drugs. It is estimated that among all 75 small-molecule approved antitumor drugs from 1946 to 1980, 53.3% are derived from unaltered natural products or their derivatives (Newman and Cragg, 2020). Among them, marine natural product-originated drugs have attracted more and more attention. As for antitumor drugs, Figure 1 listed representative marine natural products originated antitumor drugs, which have been approved and in phase III, II, and I clinical trials. For example, cytarabine obtained from a marine sponge is mainly used to treat acute and chronic lymphocyte in clinic (Deshmukh et al., 2018) (Figure 1). Eribulin (E7389), which was isolated from a marine sponge, was approved by FDA for metastatic breast cancer. In addition, plinabulin, which was previously isolated from a marine-derived fungus, is in a phase II clinical trial for the treatment of non-small-cell lung cancer (Zhou et al., 2016).
As previously mentioned, deep-sea-derived fungi (depth > 1000 m) have been recognized as valuable treasure houses for structurally novel and biologically active secondary metabolites. Many interesting reviews of deep-sea-derived secondary metabolites have been published in recent years. For example, Sun et al. summarized 442 new molecules obtained from deepsea-derived fungi, actinomycetes, bacteria, and archaea, with emphasis on structural characteristics, biological activities, and biogenetic origins (Sun et al., 2020). Wang et al. described 98 secondary metabolites with various bioactivities such as antitumor, antibacterial, antiviral, and anti-inflammatory isolated from deep-sea fungi and bacteria during 2018-2020 (Wang et al., 2020a). Wang et al. reported 180 metabolites with anticancer,antimicrobial,antifungal,antiprotozoal,and antiviral activities from deep-sea fungi . However, to the best of our knowledge, there are no reviews particularly focused on cytotoxic secondary metabolites isolated from deepsea fungi. Considering their interesting chemical structures and potent lead potential, in this review, we summarize a total of 229 cytotoxic compounds isolated from deep-sea fungi from 2010 to 2021. The emphasis is on their unique chemical diversity, their relevant cytotoxic properties, and their potential as drug leads. This review will reveal deep-sea-derived fungi as considerable resources for the development of new drugs and the potential of newly discovered secondary metabolites as valuable antitumor lead compounds.

Azaphilones
A total of 23 azaphilones with an oxabicyclic core were isolated from deep-sea fungi ( Figure 6).  . 103 shows the strongest activity toward HepG2 cells, with an IC 50 value of 3.9 mM, while 102 and 104 were found to be active against HeLa cells, with IC 50 values of 5−8 mM.

Structural Diversity of the Described Compounds Isolated From Deep-Sea Fungi
It is estimated that over 500 secondary metabolites have been isolated from deep-sea-derived fungi (> 1000 m). However, these microorganisms remain a relatively untapped source of bioactive molecules both structurally and biologically compared to the 24000 reported marine natural products (Carroll et al., 2021).  Figure 13A, among the 229 active compounds, approximately 56.33% are polyketides, which include 10.04% azaphilones, 8.30% tetramic acid and sorbicillinoid derivatives, 12.66% chromones, 3.93% benzophenones, and 21.40% other polyketides. These findings indicated that molecules grouped as polyketides are one of the most promising compounds as novel antitumor drug leads. Alkaloids are also the main structure t y p e f o r t h e s e c o m p o u n d s . T a k i n g i n t o a c c o u n t diketopiperazines (13.54%) and peptides (3.93%), alkaloids account for 26.64% of the isolated compounds. In addition, compounds isolated from deep-sea fungi often contain heteroatoms, such as sulfur and chlorine. For example, compounds 1−9, 17−20, and 27−31 are rare sulfur-containing diketopiperazines, which were isolated exclusively from extreme marine environments. As mentioned earlier, the extreme marine environment can produce more natural products with novel structures, which is a potential resource for new antitumor drugs.

Diverse Fungal Species as Producers of Isolated Compounds
As shown in Figure 13B, a total of 15 fungal species in this review, including Acrostalagmus, Alternaria, Aspergillus, Botryotinia, Chaetomium, Cladosporium, Diaporthe, Dichotomomyces, Engyodontium, Epicoccum, Penicillium, Phialocephala, Phomopsis, Trichobotrys, and Simplicillium, have been reported as producing strains for these cytotoxic compounds. Among them, Penicillium, Phomopsis and Aspergillus are the most prolific fungal strains, with 55 (accounting for 23.40%), 38 (accounting for 16.17%), and 34 (accounting for 14.47%) compounds produced, respectively. The genera Aspergillus and Penicillium are regarded as the most widely studied fungal groups in nature. Interestingly, the deepsea environment contains rare fungal species, such as Diaporthe, Dichotomomyces, and Engyodontium, which are rarely observed in terrestrial environments. Moreover, the distributions of the producing strains are shown in Figure 13C. The deep-sea is defined as that more than 1000 m below the water surface. Among these compounds, a total of 25, 60, 64, 36, and 25 compounds were isolated from deep-sea samples at depths of 1000−2000 m, 2001−3000 m, 3001−4000 m, 4001−5000 m, and > 5000 m, respectively. It is clear that more compounds can be obtained at depths of 2000−4000 m. Organisms living in this area are considered to be more adapted to the environment. Therefore, fungi have evolved diverse metabolic pathways to produce more novel bioactive metabolites. Conversely, deeper depths mean much more demanding environments, which is disadvantageous for marine-derived fungi, although they still can produce metabolites when subjected to extreme environmental stresses. Regarding the geographic origins of deep-sea natural products in Figure 13D, 31.65% of them were isolated from the Indian Ocean, followed by the South China Sea (25.32%) and Pacific Ocean (25.32%).

Some "Star Molecules" With High Drug Lead Potential
Among the 229 isolated metabolites, a total of 82 compounds were found to possess moderate to potent cytotoxic activities. Among them, as shown in Figure 14 and Supplementary  Table 1, we highlight some compounds with potent cytotoxicities and name them "star molecules" considering their high drug lead potential. These molecules include diketopiperazines with sulfur bridges (such as compounds 3, 4, 17, and 18), anthranilic acid derivatives (such as compounds 47, 51, and 52), nitrogenated azaphilones (such as compounds 84, 85, and 88), aphidicolin (compound 69), and meroterpenoids (compounds 79 and 80). Their structures are classified into polyketides, alkaloids, and terpenoids according to their putative biogenetic sources. A variety of chemical structures helps synthetists design better antitumor molecules. All of these compounds not only possess diverse chemical structures but also show significant activities, even higher than those of the positive controls (usually clinical drugs such as adriamycin, cisplatin, taxol, and doxorubicin). For example, compound 3 shows remarkable cytotoxicity at the nanomolar or low micromolar level (IC 50 values of 0.191, 0.015, and 0.008 mM against K562, A549, and MCF-7 cells), which are 10−100 times higher than that of the positive control. Compound 80, in particular, shows potent activities against SF-268, MCF-7, HepG-2, and A549 cell lines, with IC 50 values of 0.01−0.04 mM, approximately 100 times stronger than the positive control adriamycin. Table 1 lists the common target cell types and cytotoxic activity of the isolated compounds. The A549, HepG2, and MCF-7 cell lines are the main tested cell lines. Meanwhile, the structure-activity relationship and mechanism of action has been studied for some of the isolated compounds. For the diketopiperazines, the polysulfide bridge contributes significantly to their cytotoxicities . Aphidicolin A8 (69) is found to observably induce apoptosis in T24 and HL-60 cells by causing DNA damage (Niu et al., 2019a). Nitrogenated azaphilone 85 arrests the cell cycle in the G1 phase, while 84 and 85 induced apoptosis in a concentration-dependent manner (Wang et al., 2020b). These "star molecules", with potent activities and clear mechanisms of action, are considered to be potential alternatives to antitumor drugs.

CONCLUSIONS
In summary, deep-sea fungi are an untapped source of valuable marine natural products. Although a large number of metabolites have been isolated from deep-sea fungi, the further excavation of novel metabolites is expected. This review first summarizes 229 cytotoxic compounds isolated from deep-sea fungi. Among them, 82 members have been found to possess moderate to potent cytotoxic activities.
Most importantly, some of these compounds, namely, "star molecules" herein, show potent cytotoxic activities (higher than that of the positive controls). It is believed that in the near future, studies of cytotoxic compounds isolated from deep-sea fungi will become more prolific, which will certainly be beneficial for the discovery of new antitumor drugs.

AUTHOR CONTRIBUTIONS
GZ, WT, and JZ wrote this manuscript; PS, YL, JW, QS, HS, LJ, XY, HZ, and GC collected and reorganized the literature data; JZ, XZ, and HJ supervised the research work and revised the manuscript; all authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.