**1. Introduction**

Cyanobacteria have been shown to be prolific producers of structurally diverse natural products with a wide range of ecological and pharmacological activities [1–3]. Many discovered marine natural products have gone through clinical trials and even been accepted by regulatory agencies as drugs, and these include several antibody-drug conjugates that use a dolastatin/symplostatin marine cyanobacterium natural product derivative as an

**Citation:** Ding, L.; Bar-Shalom, R.; Aharonovich, D.; Kurisawa, N.; Patial, G.; Li, S.; He, S.; Yan, X.; Iwasaki, A.; Suenaga, K.; et al. Metabolomic Characterization of a cf. *Neolyngbya* Cyanobacterium from the South China Sea Reveals Wenchangamide A, a Lipopeptide with In Vitro Apoptotic Potential in Colon Cancer Cells. *Mar. Drugs* **2021**, *19*, 397. https://doi.org/10.3390/md19070397

Academic Editor: Max Crüsemann

Received: 2 May 2021 Accepted: 13 July 2021 Published: 16 July 2021

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**Copyright:** © 2021 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/).

anti-cancer "warhead" [4]. Other cyanobacterial natural products have been advanced in anticancer drug discovery programs at the preclinical stage by means of total synthesis, medicinal chemistry analogue development, and pharmacological characterization of their mechanisms of action. Some notable lead molecules from cyanobacteria include the apratoxins, carmaphycins, coibamides, curacins, and largazoles, among others [1–3]. It is generally understood that the secondary metabolism of cyanobacteria, while energetically taxing, must serve some (often unknown) ecological function for the organisms. This has been demonstrated in a few specific cases, e.g., in the upregulation of microcystins production by some cyanobacteria in response to predation by grazers [5,6]. Filamentous cyanobacteria have also been reported to contain genetic information for biosynthesis of natural products comprising up to 20% of the genome, and even surpassing that in the example of some *Moorena* species, further supporting the importance to the organisms of this biosynthetic capacity on an evolutionary time scale [7]. However, it can be quite challenging to obtain or maintain filamentous cyanobacteria in axenic laboratory cultures, as well as perform molecular biology experiments with them [8]. A number of laboratory culture conditions is also understood to greatly impact not only the growth and survival of cyanobacteria, but also the associated natural product biosynthesis [9]. Accordingly, a majority of natural product chemicals reported historically from these organisms have come from larger environmental collections, or assemblages. A meta-analysis of all secondary metabolites reported from marine and microbial sources between 1941 and 2015 revealed that the chemistry of these samples is relatively source-specific, with the majority of cyanobacterial natural products being structurally dissimilar from those of all other producers examined [10].

The taxonomy of many documented filamentous cyanobacteria has come into question in the post-genomics era, and this is especially true for the *Lyngbya*-like and *Phormidium*like morphotype [11–13]. For example, *Phormidium* is formally accepted as a part of the family *Oscillatoriaceae*, but it still appears in some literature reports and databases under *Phormidiaceae* (the *Phormidium*-like family) following previous taxonomic assignment and reclassification [14,15]. The genus *Phormidium* once comprised some 200 species; however, about 90% of these organisms have been redistributed into other genera, such as *Lyngbya*, and even different families in the order Oscillatoriales, including both *Oscillatoriaceae* and *Phormidiaceae*, after molecular characterization studies in recent years [16–18]. The members of genus *Lyngbya* have also been re-evaluated and revised several times [14]. After molecular characterization, several newly formed genera have emerged for organisms previously described as members of *Lyngbya*, notably including *Leptolyngbya*, *Moorena*, and *Okeania* [12,13,19,20]. More recently, the new genus *Neolyngbya* has also been created for several newly described *Lyngbya*-like organisms [21]. Despite having a reported biotechnological potential for drug discovery and development, only one new natural product has yet been reported from assemblages with *Neolyngbya*, namely the neurotoxic sesquiterpenoid eudesmacarbonate [22,23]. *Neolyngbya* organisms have not been previously reported in the South China Sea. Meanwhile, the South China Sea is home to a vastly understudied biodiversity of marine filamentous cyanobacteria [24,25]. This biodiversity resource has been largely under-examined, especially when compared to the vast chemical study of other types of microorganisms in China (actinomycetes, fungi, etc.) [26].

Metabolomics is useful for the large-scale analysis of molecules within a biological sample [27]. In recent years, this field has taken a central role in many natural product research programs, especially for studying the chemical space and diversity using both untargeted and targeted metabolomics [28]. Untargeted metabolomics allows for the generation of a broad overview of the chemical diversity in even a complex extract. This can also be used for the comparative analysis of multiple samples, or various treatment conditions, to identify potential characteristic and chemical markers. In contrast, targeted metabolomics is useful when the focus can be specified to a single compound of interest or a set of pre-determined molecules for further qualitative and quantitative analysis. Mass spectrometry-based metabolomics in the past decade has shown immense utility in the field of natural product discovery, and has yielded major impacts, mainly because of the accuracy, sensitivity, speed, and robustness of these methods, along with newly developed cuttingedge downstream platforms for data analysis [29–36]. These platforms have been made to provide structural information based on the fragmentation patterns of each molecule, allowing for the comparison of each with other known and unknown compounds in spectrometric libraries, natural product databases, and public or private collections of raw data. Altogether, this has facilitated the characterization of putative structures based on a similarity between the fragmentation of different compounds, minimized the rediscovery of known structures by virtual dereplication, and allowed for a more efficient discovery of new natural products and new chemical scaffolds prior to the isolation and characterization effort [29–36].

In this study, a metabolomics-based approach was used to explore the chemistry of a cf. *Neolyngbya* sp. environmental collection and characterize novel natural product chemistry. Moreover, the concurrent bioactivity-guided fractionation of this extract was expected to yield pure compounds produced with potential anti-cancer effects, as evaluated in vitro using an immortalized colorectal cancer cell line. Reported herein is the chemical and biological exploration of an environmental collection from the South China Sea that is dominated by a marine filamentous cyanobacteria, cf. *Neolyngbya* sp. This report details the characterization of the microbiome, metabolome, and associated pharmacology that allowed for the directed isolation of a new bioactive natural product, wenchangamide A (**1**; Figure 1). The structure elucidation and investigation of this molecule as a potential anticancer drug lead is also described, along with the expansion of this class of compound to include a new proposed bioactive analogue based on available metabolomics and bioassay testing data.

**Figure 1.** Structure and numbering scheme of wenchangamide A (**1**).
