**1. Introduction**

Cancer stands in the frontline among leading killers worldwide and the annual mortality rate is expected to reach 16.4 million by 2040 [1,2]. The marine environment has the potential to produce candidate compounds (structures) as leads to drugs, or actual drugs, as has been actively discussed for the last 50 or so years [3–5]. Nowadays, several compounds have led to drugs, especially in the area of cancer, such as trabectedin, and eribulin, which were discovered under the cytotoxic activity-guided approach [3–6]. Brefeldin A (BFA), a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, was first isolated from *Penicillium decumbens* in 1958 [7,8] and subsequently identified only from the marine-derived genus *Penicillium* [9]. Previous studies reported that BFA showed strong anticancer activity in a variety of cancers, including colorectal, prostate, lung, and breast cancers [10,11]. BFA is considered as a promising leading molecule for developing anticancer drugs.

**Citation:** Wang, C.-F.; Ma, J.; Jing, Q.-Q.; Cao, X.-Z.; Chen, L.; Chao, R.; Zheng, J.-Y.; Shao, C.-L.; He, X.-X.; Wei, M.-Y. Integrating Activity-Guided Strategy and Fingerprint Analysis to Target Potent Cytotoxic Brefeldin A from a Fungal Library of the Medicinal Mangrove *Acanthus ilicifolius*. *Mar. Drugs* **2022**, *20*, 432. https://doi.org/10.3390/ md20070432

Academic Editors: Wenhan Lin and Jing Xu

Received: 12 June 2022 Accepted: 27 June 2022 Published: 29 June 2022

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The mangrove forests are a complex ecosystem growing in tropical and subtropical intertidal estuarine zones and nourish a diverse group of microorganisms [12,13]. Microorganisms associated with mangrove environments are a major source of antimicrobial agents and also produce a wide range of important medicinal compounds, including enzymes, antitumor agents, insecticides, vitamins, immunosuppressants, and immune modulators [13–17]. Among the mangrove microbial community, mangrove associated fungi were the second-largest ecological group of the marine fungi [13,14]. Up to December 2020, at least 1387 new structures have been isolated and identified from a diverse range of mangrove-derived fungi (325 strains), which belong to about 69 genera. Furthermore, about 40.7% (530) of the 1300 new compounds displayed a wide range of pharmacological activities, and the antitumor (mainly cytotoxicity) function is noteworthy and visible, accounting for 34% (196 compounds) of the active compounds. Therefore, mangrove associated fungi are a rich source of structurally unique and diverse bioactive secondary metabolites [13].

*Acanthus ilicifolius* is widely distributed from India to southern China, tropical Australia and the Western Pacific islands, throughout Southeast Asia [18]. Various classes of bioactive compounds including alkaloids, benzoxazinoids, lignans, flavanoids, triterpenoids and steroids have been obtained from *A. ilicifolius* [18–20]. In addition, up to December 2020, a total of 22 strains belonging to 9 genera have been reported, which produced 95 new secondary metabolites. The endophytic fungi derived from *A. ilicifolius* are one of the most favored to be studied [13], ye<sup>t</sup> little attention has been paid to the fungal communities associated with *A. ilicifolius*.

Investigating new bioactive natural products from marine fungi is a major and constant research focus in our laboratory [21–24]. Natural product researchers also face the challenge of targeting the discovery of bioactive compounds from a microbial resource library. The present work aims to integrate activity-guided strategy and fingerprint analysis to target the potent cytotoxic compounds from a fungal library of the medicinal mangrove *A. ilicifolius* (Figure 1). The cultivable fungi associated with the medicinal mangrove *A. ilicifolius* from the South China Sea were firstly systematic evaluated for their diversity. Furthermore, integrating the cytotoxic activity-guided strategy, the target active strains were quickly identified. Combined with fingerprint analysis, a potent cytotoxic activity compound, brefeldin A, was isolated from the target active strains. The combination of activityguided strategy and fingerprint analysis could improve the efficiency of discovering active compounds in crude extracts from a complex and diverse fungal library.

**Figure 1.** The detailed flowchart of this study.
