**Marine Bioactive Peptides II: Structure, Function, and Therapeutic Potential**

Editor

**Tatiana V. Ovchinnikova**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Tatiana V. Ovchinnikova Institute of Bioorganic Chemistry, Russian Academy of Sciences Russia

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Marine Drugs* (ISSN 1660-3397) (available at: http://www.mdpi.com).

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## **Contents**

Reprinted from: *Mar. Drugs* **2019**, *17*, 531, doi:10.3390/md17090531 ................. **165**

#### **Linda Kornstad Nyg˚ard, Ingunn Mundal, Lisbeth Dahl, J ¯urat˙e Saltyt˙ ˇ e Benth and Anne Marie Mork Rokstad**

Limited Benefit of Marine Protein Hydrolysate on Physical Function and Strength in Older Adults: A Randomized Controlled Trial Reprinted from: *Mar. Drugs* **2021**, *19*, 62, doi:10.3390/md19020062 .................. **183**

#### **Milica Pavlicevic, Elena Maestri and Marta Marmiroli**

### **About the Editor**

**Tatiana V. Ovchinnikova**, Head of the Science-Educational Centre, M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Full Professor, Department of Bioorganic Chemistry, M.V. Lomonosov Moscow State University. She received her degree in Chemistry with Honors at the University of Kyiv (Ukraine) and then completed her Ph.D. in Bioorganic Chemistry at M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry (Moscow, Russia). She obtained a Second Doctorate (Habilitation) degree in Bioorganic Chemistry and Biotechnology in 2011. Her research interests focus on discovery, isolation, structural elucidation, functional characterization, structure–function relationship analysis, and the evaluation of the therapeutic potential of novel antimicrobial and host defense peptides of animal and plant origin, investigation of their antimicrobial and anticancer activity, as well as mechanisms of their participation in innate immunity, screening of marine organisms for the discovery of novel peptides with high activities against microorganisms and transformed human cells, bioengineering of peptide molecules with improved therapeutic potency and efficacy for using in medicine and veterinary, design of innovative antibiotics and anticancer drugs. She has published over 450 scientific papers, reviews, book chapters, patents, and communications at national and international conferences, including over 150 peer-reviewed publications in international journals.

### *Editorial* **Marine Peptides: Structure, Bioactivities, and a New Hope for Therapeutic Application**

**Tatiana V. Ovchinnikova 1,2,3**

Over the last years, plethora of bioactive peptides have been isolated from organisms which live in sea water. Taking in account that more than two-thirds of the global surface area is covered with the oceans, marine species constitute above a half of the total biodiversity. Long-term evolution of marine organisms was advanced in the midst of pathogens, and efficient defense mechanisms were the necessary condition of their survival. Many marine peptides play a key role in host defense as evolutionary ancient components of innate immunity system [1]. They are involved in basic mechanisms of living organisms survival, including growth, defense, reproduction, and homeostasis. Protective peptides were isolated from many marine invertebrates and vertebrates. Their antibacterial, antifungal, antiviral, antitumor, antioxidative, antihypertensive, antiatherosclerotic, anticoagulant, antidiabetic, analgesic, immune-modulating, and neuroprotective properties attract increasing attention of pharmaceutical, cosmeceutical, and nutraceutical industries which focus on the design of innovative antibiotics, anticancer drugs, analgesics, medicines for neurological disorders, etc.

Marine peptides are multifarious in structure and biological properties. The Special Issue «Marine Bioactive Peptides II: Structure, Function, and Therapeutic Potential» was aimed to collect papers on present-day information regarding structural elucidation, functional characterization, and therapeutic potential evaluation of peptides from marine organisms. Getting started with this book, we hope to assemble an interesting edition that would highlight new developments and current trends in marine peptide research.

Cyanobacteria are Gram-negative organisms, also called blue-green algae. They appeared over 3.5 billion years ago and live all over the world including fresh and ocean water, deserts and ice shelves. This evolutionary success is related to their ability to produce a wide variety of secondary metabolites [2]. Lipopeptides laxaphycins have been isolated from several species of cyanobacteria including *Hormothamnion enteromorphoides, Anabaena torulosa, Lyngbya confervoides* and *Anabaena laxa* [3]. In particular, laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. In this Special Issue, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod (sea hare) Stylocheilus striatus, and structures of both compounds were elucidated [4]. Activities of all the six peptides were determined towards SH-SY5Y human neuroblastoma cells. In this Special Issue, pro-apoptotic properties of cyclic laxaphycins B were indicated. Acyclic laxaphycins affected on autophagy-related protein expression by increasing AMPK phosphorylation and inhibiting mTOR. Gastropod-derived acyclic compounds were shown to undergo a biotransformation (ring opening and amino acid residues deletion) that had not been previously described [4].

Marine fungi represent a valuable source of bioactive peptides. In this Special Issue, the review [5] summarized data on structures and biological activities of 131 peptides

**Citation:** Ovchinnikova, T.V. Marine Peptides: Structure, Bioactivities, and a New Hope for Therapeutic Application. *Mar. Drugs* **2021**, *19*, 407. https://doi.org/10.3390/md19080 407

Received: 14 July 2021 Accepted: 20 July 2021 Published: 23 July 2021

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

**Copyright:** © 2021 by the author. 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/).

isolated from 17 fungal genera including *Acremonium, Ascotricha, Aspergillus, Asteromyces, Ceratodictyon, Clonostachs, Emericella, Exserohilum, Microsporum, Metarrhizium, Penicillium, Scytalidium, Simplicillium, Stachylidium, Talaromyces, Trichoderma,* and *Zygosporium.* 35 marine peptides revealed cytotoxic activities against different cancer cells, 23 ones displayed pronounced antimicrobial and antiviral activities. For example, asperterrestide A exerted the antiviral activity against influenza H1N1 and H3N2 virus strains. Some of the *Aspergillus* peptides revealed anti-inflammatory properties via inhibiting IL-10 expression of the LPS-induced THP-1 cells. Antidiabetic and lipid-lowering activities were also demonstrated some fungal peptides, such as terrelumamides A and B, which improved insulin sensitivity as determined by the utilization of mesenchymal cells of a bone marrow origin obtained from human adopting an adipogenesis model. Psychrophilin G exhibited a pronounced lipid-reducing activity. Simplicilliumtide D displayed an antifouling effect against the larvae of *Bugula neritina*. However, 47% of the isolated peptides did not show the examined biological activities and thus required further in-depth study [5].

Jellyfish are venomous marine invertebrates of the phylum Cnidaria. All members of this ancient phylum are generally toxic (jellyfish, sea anemones, hydra, and corals) [6]. The toxins of these mostly marine animals are of ever-increasing biomedical interest. In this Special Issue, the putative toxins of two species of jellyfish (*Rhopilema esculentum* Kishinouye, 1891, also known as a flame jellyfish, and Amuska jellyfish *Sanderia malayensis* Goette, 1886) were identified in nematocysts [7]. Using nano-flow liquid chromatography tandem mass spectrometry (nLC–MS/MS), in total 3000 proteins were found in the nematocysts in each of the above two jellyfish species. 40 and 41 putative toxins were identified in *R. esculentum* and *S. malayensis*, respectively. All the toxins were further classified into 8 families in accordance with their predicted functions. The most dominant toxins (>60%) were identified as hemostasis-impairing ones and proteases. For the first time ever, the authors studied the proteomes of nematocysts from two jellyfish species [7]. Earlier, this group reported high-quality de novo reference genomes for the above jellyfish, as well as their transcriptomes [8]. Herein, the authors identifed the putative toxins in these two jellyfish species at the protein level.

Many bioactive peptides were identified in the immune, neuroendocrine, and gut systems of mollusks [9]. The marine peptide Dolastatin 10 (Dol-10) was isolated from the Indian Ocean mollusk *Dolabella auricularia* [10]. The peptide induces apoptosis of lung cancer cells and other tumor cells at nanomolar concentrations. Dol-10 and its derivatives are highly effective in vitro towards L1210 leukemia cells, small cell lung cancer NCI-H69 cells, human prostate cancer DU-145 cells, etc. [11]. Dol-10 has been developed into commercial drugs for treating some specific lymphomas. With the implementation of Dol-10 derivatives and antibody-drug conjugates (ADCs), anticancer activity and tumor targeting were essentially improved, and the systemic toxicity was reduced. Adcetris® has been approved for the treatment of anaplastic large T-cell systemic malignant lymphoma and Hodgkin lymphoma [11]. Thus, Dol-10 is one of the most medically valuable marine peptides discovered up to now. By modifying the chemical structure of Dol-10 and combining the peptide with the application of ADCs technology, new antitumor drug candidates can be developed. In this Special Issue, the authors summarized data on biological activities and chemical structures of Dol-10 derivatives [11]. In their comprehensive review, the authors analyzed synthetic work with Dol-10 in the last 35 years, which provides the input for the development of novel antitumor drugs on the basis of marine peptides.

Molecular mechanisms of anticancer action of marine peptides are still obscure. Nevertheless, several marine peptides have been applied in preclinical treatment. This Special Issue presents the comprehensive review [12] that highlights mechanisms of anticancer action of linear and cyclic peptides from marine organisms. More than 10,000 bioactive molecules have been isolated from marine organisms [13]. Over recent years, many natural and synthetic peptides were characterized, and special databases were established, including database of anticancer peptides and proteins The CancerPPD [14]. 49 marinederived bioactive compounds or their derivatives have been applied for clinical trials or

approved for the market [15]. 11 marine drugs were approved for the market by European and American drug authorities. Four of them are anticancer drugs: Cytosar-U, Yondelis, Halaven, and Adcetris [12]. The review focus on small anticancer peptides of marine origin and molecular mechanisms of their action with a view to the future development of novel marine antitumor agents.

The large majority of polychaeta species are marine animals that inhabit all oceans and seas from the Arctic to the Antarctic. Marine polychaeta is an underinvestigated class of invertebrates in the context of discovery of new host defense peptides. Papers included in this Special Issue deal with marine polychaeta, providing good examples of their biological potential. The peptides arenicins and capitellacin, were isolated from *Arenicola marina* [16] and *Capitella teleta* [17], respectively. Earlier, structure-function relationships of arenicins have been extensively investigated [18–25]. Arenicin was shown to modulate the human complement system [26]. Here, the authors reported the property change in arenicin-1 derivative Ar-1-(C/A) structure and its antimicrobial, hemolytic and complement-modulating activities in comparison with those of the natural peptide. Despite the absence of a disulfide bond, the peptide possessed all important functional features, but its hemolytic activity reduced [26]. The use of marine peptides as new complement modulators has several advantages. These molecules are relatively small, and the immune response to them is limited. They do not resemble human peptides, which allows to avoid cross-reactivity and side effects. Besides, this approach promotes antimicrobial effects, taking into account the inhibited complement system [26]. In this Special Issue, a novel BRICHOS-domain related AMP from the marine polychaeta *Capitella teleta*, named capitellacin, was reported [17]. The peptide exhibits high homology with β-hairpin marine peptides tachyplesins and polyphemusins from the horseshoe crabs. The β-hairpin structure of capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide adopts a monomeric right-handed twisted β-hairpin without significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment. Capitellacin displays a pronounced antimicrobial activity in vitro against a wide panel of bacteria including drug-resistant strains. In contrast to other known β-hairpin antimicrobial peptides, capitellacin acts via non-lytic mechanism at concentrations inhibiting bacterial growth. Molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation. A low cytotoxicity towards human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make capitellacin a promising candidate compound for design of a novel anti-infective drug [17].

The Spanish mackerel *Scomberomorous niphonius* belongs to the Scombridae family and is distributed in the Western North Pacific, including the East China Sea, the Yellow Sea, and the Bohai Sea of China. Recently, some bioactive ingredients have been prepared from the skins and bones of the Spanish mackerel [27]. Among them, antioxidant peptides (APs) are of particular interest. Eight APs including GPY, GPTGE, PFGPD, GPTGAKG, PYGAKG, GATGPQG, GPFGPM, and YGPM have been earlier isolated from the skin of the Spanish mackerel [28]. In this Special Issue, the authors reported isolation and characterization of APs from the protein hydrolysate of the Spanish mackerel muscle obtained by in vitro gastrointestinal (GI) digestion and evaluated biological properties of the isolated peptides [29]. The proteins of the Spanish mackerel muscle were hydrolyzed with different enzymes and by in vitro GI digestion. Four novel APs, designated as SMP-3, SMP-7, SMP-10, and SMP-11, were isolated and identified as PELDW, WPDHW, FGYDWW, and YLHFW, respectively. All these peptides displayed high radical scavenging activity, lipid peroxidation inhibition ability, and protective effects on plasmid DNA (pBR322DNA) against oxidative damage induced by H2O2 [29].

Marine peptides are approved as a food ingredient in Norway. The peptide compounds are manufactured by hydrolysis of the Atlantic cod Gadus morhua fillet [30]. The tablets are produced by Flexipharma AS and based on the marine peptide compound 565952 P from Firmenich Bjørge Biomarin AS. The double-blinded, randomized, controlled trial of the marine protein hydrolysate (MPH) was used to evaluate its effect o on measures

of physical function and strength in the elderly. This is one of the first long-term studies of MPH and age-related changes in muscle health. Despite a limited benefit of marine protein hydrolysate on physical function and strength in older adults was delivered, the authors came to the conclusion that a daily intake of 3 g MPH for 6 to 12 months would prevent loss of physical performance, compared with a placebo [30].

In this Special Issue, the review [31] analyzes structural features and biological activities of 253 peptides, mainly from marine food sources. The authors aimed to present the current state-of-art in marine peptides structures, biological activities, and applications, and also to compare them with those isolated from other animal food sources. In summary, the authors concluded that marine organisms have proven to be invaluable sources of peptides with unique structures and diverse bioactivities [31].

The papers included in this Special Issue deal with various marine-derived peptides, providing an informative view of their biomedical potential. The initial call resulted in 32 submissions, and 19 of them were accepted and included in the Special Issue I [32]. Following the success of the first Special Issue, we invited researchers in the field to contribute to the second edition entitled "Marine bioactive peptides II: structure, function, and therapeutic potential". A range of new marine peptides were isolated and characterized. Most of them displayed broad-spectrum biological activities and therapeutic potential for clinical trials in humans. All the papers presented in this Special Issue II underlined the central role of bioactive peptides in innate immunity of marine organisms as well as their potential for human health care.

In conclusion, the Guest Editor thanks all the Authors who contributed to this Special Issue, all the Reviewers for evaluating the submitted manuscripts, and the Editorial board of *Marine Drugs,* especially Prof. Dr. Orazio Taglialatela-Scafati, Editor-in-Chief of this journal for his continuous help in turning this Special Issue into reality.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

### *Article* **Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells**

#### **Rebeca Alvariño 1, Eva Alonso 1,2,\*, Louis Bornancin 3, Isabelle Bonnard 3,4, Nicolas Inguimbert 3,4, Bernard Banaigs 3,4 and Luis M. Botana 1**

Received: 8 June 2020; Accepted: 13 July 2020; Published: 15 July 2020

**Abstract:** Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria *Anabaena torulosa*. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod *Stylocheilus striatus*, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 μM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.

**Keywords:** biotransformation; laxaphycin; autophagy; apoptosis; cyanobacteria

#### **1. Introduction**

The phylum Cyanobacteria includes photosynthetic prokaryotes from terrestrial, freshwater and marine ecosystems. These Gram-negative organisms, also called blue-green algae, appeared on Earth over 3.5 billion years ago and can live as colonial or unicellular forms in almost all habitats (deserts, ice shelves, as endosymbionts, etc.). This high degree of adaptation is related to their ability to produce a wide range of secondary metabolites [1,2]. A significant part of these compounds are complex cyclic peptides, depsipeptides or lipopeptides that contain unusual amino acids and multiple N-methylations [3]. Many of these molecules are produced through non-ribosomal peptide synthase (NRPS) and/or polyketide synthase (PKS) enzymatic systems. The enzymes are organized in modules, each one carrying out the addition of a subpart of the final molecule and generating a great diversity of compounds [4,5]. As a result, non-ribosomal peptides exhibit a broad spectrum of biological activities, including, anticancer, antifungal and antimicrobial properties [6]. Furthermore, cyclic peptides are less flexible than their linear counterparts, and contain non-proteinogenic amino acids, two characteristics that give them greater selectivity and better resistance to hydrolysis by exo- and endopeptidases.

Laxaphycins are a large family of lipopeptides synthetized through a hybrid PKS/NRPS pathway by different species of cyanobacteria [7]. These compounds have been obtained from specimens of *Hormothamnion enteromorphoides*, *Anabaena torulosa*, *Lyngbya confervoides* and *Anabaena laxa* collected worldwide. The fact that different species could produce similar metabolites suggests horizontal gene transfer or an ancient common parent between them. Therefore, these peptides have been selected by evolution in both freshwater and oceanic cyanobacteria and may confer to them an ecological advantage. Laxaphycins are divided in two sub-families: laxaphycin A-type, undecapeptides with a segregation of hydrophobic and hydrophilic residues; and laxaphycin B-type, dodecapeptides in which hydrophobic and hydrophilic residues are alternated [8]. Laxaphycin B-type members such as laxaphycins B, B2 and B3 have presented antifungal, antimicrobial and cytotoxic activities [9–12]. Laxaphycin A-type compounds have shown weak cytotoxicity, with the exception of the compound hormothamnin A [13]. Moreover, we have recently reported evidence that points to an activation of the autophagic flux by laxaphycin A peptides [14].

Autophagy is a regulated process that leads to the clearance of misfolded or damaged proteins and dysfunctional organelles. This cellular machinery is activated by a variety of signals such as nutrient starvation, oxidative stress and energy depletion. Cells can degrade damaged components and restore substrates for energy metabolism through this pathway [15]. At basal levels, autophagy maintains cellular homeostasis and is an important mechanism in cell growth and development. This process plays an important role in many pathologies such as cancer, diabetes and neurodegenerative diseases [16]. Therefore, much effort has been made in the search for new compounds capable of targeting the autophagic flux [17,18].

When the autophagic flux is initiated, the mammalian target of rapamycin (mTOR), considered the master cell growth regulator, is inhibited. This inhibition leads to the activation of the Unc-51-like kinase 1 (ULK1) complex, which in turn stimulates the Beclin1-VPS34 complex. The components of this complex are phosphorylated and trigger the elongation of the phagophore. Two systems control this process: the ATG5-ATG12 and the microtubule-associated light chain 3 (LC3). During this step, LC3I is converted to LC3II, the lipidated form, considered the signature of the autophagic membranes [19]. Finally, the autophagosome undergoes maturation and fuses with the lysosome, leading to the formation of the autolysosome, with an internal acidic and hydrolytic environment that degrades the damaged cellular components [20,21].

Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor in cells and plays a key role in the activation of autophagy due to the significance of this process in the generation of metabolic intermediates to maintain ATP levels. AMPK is activated when energy levels decrease and can trigger autophagic flux either by phosphorylating the ULK1 complex or through the regulation of mTOR activity [22].

The human neuroblastoma cell line SH-SY5Y is a useful model for assessing neurotoxic and neuroprotective compounds, since these cells express intact genes implied in reactive oxygen species (ROS) metabolism, or calcium and mitochondrial signaling [23].

In this study, the biological activities of six B-type laxaphycins were analyzed in SH-SY5Y cells (Figure 1). The already known laxaphycins B (**1**) and B3 (**2**), and acyclolaxaphycins B (**3**) and B3 (**4**) were obtained from a marine specimen of *A. torulosa*, whereas two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycin B (**5**) and [des-(Ala4-Hle5)] acyclolaxaphycin B3 (**6**), were isolated from the herbivorous gastropod *Stylocheilus striatus*. The absolute configurations of **3** and **4**, as well as the complete structural elucidation of the new diet-derived peptides **5** and **6** are also provided.

**Figure 1.** Scheme of laxaphycin B sources and compound analysis

#### **2. Results**

#### *2.1. Structure Elucidation of Peptides* **3***,* **4***,* **5** *and* **6**

*S. striatus* was collected on *A*. cf *torulosa* in the lagoon of Moorea, French Polynesia, sealed underwater in a bag, freeze-dried and extracted. The crude extract was fractionated using flash chromatography and the resulting fraction containing new peptides was subjected to HPLC purification to yield compounds **5** (2.5 mg) and **6** (5 mg) as a white, amorphous powder. Compounds **5** and **6** responded positively to a ninhydrin test, suggesting a non-blocked N-terminus. The already described laxaphycins B (**1**) and B3 (**2**) [9], and acyclolaxaphycins B (**3**) and B3 (**4**) were repurified from *A. torulosa* as described in [7] (Figure 2).

**Figure 2.** Chemical structures of laxaphycin B-type compounds. As for (**1**) and (**2**), R=H for (**3**) and (**5**), and R=OH for (**4**) and (**6**).

Using the positive high-resolution electrospray ionization mass spectrometry (HRESIMS) spectra, the molecular formula was determined to be C56H100N12O17 (*m*/*z* 1213.7509) [M + H]+) for compound **5** and C56H100N12O18 (*m*/*z* 1229.7972) [M + H]+) for compound **6**.

All the NMR experiments were conducted in DMSO-*d6*. The signal distribution pattern observed in the 1H-NMR spectrum of **5** and **6** was characteristic of lipopeptides, displaying amide NH signals (δH7.40–7.90), CαH signals (δH3.5–4.7), aliphatic CH2 (δH1.1–1.3) and CH3 signals (δH0.7–0.9).

### • [des-(Ala4-Hle5)] acyclolaxaphycin B (**5**)

In the NH proton region, seven doublets and two singlets were observed. The values of chemical shifts (Table 1) were reported using 2D-NMR spectra including correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY),rotating frame nuclear magnetic resonance spectroscopy (ROESY), heteronuclear single quantum correlation (HSQC), HSQC-TOCSY, and heteronuclear multiple bond correlation (HMBC) (Figures S1–S8). Analysis of TOCSY correlations (Figure S4) revealed the presence of 10 amino acid residues: N-methylisoleucine (N-MeIle), 3-hydroxyasparagine (Has), two threonines (Thr), proline (Pro), leucine (Leu), β-aminodecanoic acid (β-Ade), valine (Val) and 3-hydroxyleucine (Hle). In comparison with laxaphycin B, analyses of TOCSY and HSQC (Figure S6) spectra revealed the absence of typical correlations within alanine and within one of the two hydroxyleucines, suggesting the lack of these two residues in **5**.

**Table 1.** NMR spectroscopic data for [des-(Ala4-Hle5)] acyclolaxaphycin B (**5**) and [des-(Ala4-Hle5)] acyclolaxaphycin B3 (**6**) (303 K) in DMSO-d6.

**Table 1.** *Cont*.

The remaining non-identified spin system was identified as a glutamine residue with the presence of two carbonyl signals at 172.85 and 174.24 ppm, and by HMBC (Figure S8) correlation from Hα (δH4.50) to Cβ (δC29.95), from Hβ (δH1.88) to Cα (δC53.01) and Cγ (δC28.76) and from Hγ (δH2.20) to Cβ (δC29.95) and Cδ (δC174.24). However, the poor resolutions of glutamine correlation signals in 2D-NMR (HMBC, HSQC and ROESY) did not allow us to assign the two NH2 signals. This might be

explained by the presence of multiple conformations in solution due to an increase in the flexibility of the N-terminal residue. The HMBC spectrum provided information on sequence-specific assignments. Indeed, the cross-peaks between carbonyl carbons (residue i) and NH, NCH3 protons or Hα (residue i + 1) suggested the presence of two partial sequences including Gln-N-MeIle-Has-Thr (fragment 1) and Pro-Leu-Thr-β-Ade-Val-Hle (fragment 2). Analysis of the ROESY spectra (Figure S5) revealed a correlation between Hα (δH4.57) of Thr4 and Hδ (δH3.64/3.76) of Pro5, assembling fragments 1 and 2 and establishing the complete sequence as Gln-N-MeIle-Has-Thr-Pro-Leu-Thr-β-Ade-Val-Hle. Even if it does not constitute evidence, the lack of HMBC or ROESY correlations between Gln and Hle, observable in the case of laxaphycins B (**1)** and B3 (**2**), suggested that the peptide is linear. Positive arguments are provided by the molecular formula determined to be C56H100N12O17 by HRMS and by a positive response to a ninhydrin test suggesting a non-blocked N-terminus.

### • [des-(Ala4-Hle5)] acyclolaxaphycin B3 (**6**)

ESIMS-MS fragmentation of compound **6** led us to a y6-y8 ion series shifted to a higher mass by 16 amu in comparison to that of **5**, suggesting that the variable residue could be Pro. Compound **6** showed remarkable NMR spectral similarities (Figures S9–S16) to **5**, being the most significant differences being the presence of an additional hydroxyl group on proline (Hγ at 4.29 ppm vs. two Hγ at 1.81 and 1.89 ppm for [des-(Ala4-Hle5)] acyclolaxaphycin B (**5**); Cγ at 68.41 ppm vs. 24.07 ppm for **5**; Cβ and Cδ were also deblinded by the presence of the hydroxyl function (Δδ 8.8 and 8.4 ppm, respectively)). The NMR spectral analysis (Table 1) established the complete sequence as Gln-N-MeIle-Has-Thr-Hyp-Leu-Thr-β-Ade-Val-Hle.

ESIMS-MS fragmentations for **5** and **6** were consistent with the proposed amino acid sequences and the presence of y ions at *m*/*z* 416.3143 (y3), 517.3628 (y4), 727.5011 (y6), 828.5375 (y7) and 958.5770 (y8), and b ions at *m*/*z* 487.2525 (b4) and 256.1671 (b2) for compound **5**, as well as y ions at *m*/*z* 416.3174 (y3), 517.3633 (y4), 743.4884 (y6), 844.5478 (y7) and 974.5867 (y8), and b ions at *m*/*z* 814.4389 (b7), 487.2570 (b4) and 256.1678 (b2) for compound **6** (Figure 3).

**Figure 3.** Electrospray ionization mass spectrometry (ESIMS/MS) fragmentation of [des-(Ala4-Hle5)] acyclolaxaphycins B (**5**) and B3 (**6**)

#### *2.2. Absolute Configuration of Peptides* **3***,* **4***,* **5** *and* **6**

The absolute configuration of each amino acid residue in compounds **3**–**6** was established using the advanced Marfey's method after hydrolysis [24,25]. The LC-MS comparison between the Marfey's derivatives of the acid hydrolysate of laxaphycin B and those of acyclolaxaphycin B (**3**) established the 2*S* configuration of Val, Ala, Gln, Pro, N-MeIle, the 2*R* configuration of Leu, as well as the 3*R* configuration of Ade. The Marfey's analysis of the four stereoisomers of standard threonine revealed the (*2S,3R*) configuration of both threonines present in acyclolaxaphycin B (**3**). The Marfey's method also revealed the 2*R* configuration of the two 3-hydroxyleucines. The absolute configuration of Cβ of both 3-hydroxyleucines (2*R*,3*S*) was established through NOESY correlations between the Hγ and the NH observed.

As previously described [9], the elution order of the 3-hydroxyasparagine (HAsp), which results from the acid hydrolysis of Has, is another exception of Marfey's rule. Indeed, the D-FDLA-(2*R*)-HAsp derivative elutes after the L-FDLA-(2*R*)-HAsp derivative. Thus, we established that the Cα configuration of the Has residue was 2*R*. The configuration of the Cβ of Has was established to be 3*R* by a comparison with laxaphycin B Marfey's derivatives. Therefore, the complete structure of acyclolaxaphycin B (**3**) was established as (*2S)*- Ala-(2*R*,3*S*)- Hle-(*2S)*- Gln-(2*S*)-*N*-MeIle-(2*R*,3*R*)-Has-(*2S,3R)*-Thr-(*2S)*-Pro-(*2R)*-Leu-(*2S,3R*)-Thr-(3*R*)-Ade-(*2S)*-Val-(2*R*,3*S*)-Hle.

Regarding acyclolaxaphycin B3 (**4**), the configuration of Val, Ala, Gln, N-MeIle, Leu, Ade, Has, Thr (x2) and Hle (x2) were found to be the same as for acyclophycin B (**3**). The absolute configuration of the Cα of the Hyp residue appeared to be (2*S*) and a comparison with laxaphycin B3 derivative enabled the Cγ configuration to be assigned to *4R,* establishing the complete structure as (*2S)*-Ala-(2*R*,3*S*)-Hle-(*2S*)-Gln-(*2S*)-*N*-MeIle-(2*R*,3*R*)-Has-(*2S,3R)*-Thr-(*2S,4R)*-Hyp-(*2R)*-Leu-(*2S,3R)*- Thr-(3*R*)-Ade-(*2S*)-Val-(2*R*,3*S*)-Hle.

The chromatographic comparison between the Marfey's derivatives of the acid hydrolysate of [des-(Ala4-Hle5)] acyclolaxaphycin B (**5**) and those of acyclophycin B (**3**) established the 2*S* configuration of Val, Gln, Pro, N-MeIle, the 2*R* configuration of Leu, as well as the 3*R* configuration of Ade, and the (2*R*,3*R*) configuration of Has, *(2S,3R)* of Thr (x2), and (2*R*,3*S*) of Hle. The complete structure of **5** was defined as (2*S*)-Gln-(2*S*)-*N*-MeIle- (2*R*,3*R*)-Has-(*2S,3R*)-Thr-(*2S)*-Pro-(*2R)*-Leu-(*2S,3R*)-Thr-(3*R*)-Ade-(2*S*)-Val-(2*R*,3*S*)-Hle.

With regard to [des-(Ala4-Hle5)] acyclolaxaphycin B3 (**6**), the configuration of Val, Gln, N-MeIle, Leu, Ade, Has, Thr (x2) and Hle (x2) were found to be the same as for **5**. The absolute configuration of the Cα of the Hyp residue appeared to be (2*S*) and a comparison with laxaphycin B3 derivative enabled the Cγ configuration to be assigned to *4R,* establishing the complete structure as (*2S)*-Gln-(*2S)*-*N*-MeIle-(2*R*,3*R*)-Has-(*2S,3R)*-Thr-(*2S,4R)*-Hyp-(*2R)*-Leu-(*2S)*-Thr-(3*R*)-Ade-(*2S)*-Val- (*2R,3S*)-Hle.

#### *2.3. E*ff*ects of B-Type Laxaphycins on Cell Viability and Mitochondrial Function*

In order to make an initial evaluation of compounds activity, their effect on cell viability, metabolic activity and mitochondrial function were tested. Cell viability was assessed by monitoring lactate dehydrogenase (LDH) levels in cells supernatant [26], cell metabolic activity was analyzed with MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) [27], and tetramethylrhodamine methyl ester (TMRM) dye was used to determine the mitochondrial membrane potential (ΔΨm). The effect of laxaphycins over reactive oxygen species (ROS) and ATP levels was also monitored.

Cyclic compounds (**1** and **2**) turned out to be cytotoxic to neuroblastoma cells (Figure 4). Their half maximal inhibitory concentrations (IC50) were calculated for LDH and MTT assays. In LDH test, compound **2** presented an IC50 value of 0.8 μM, 95% confidence interval (CI): 0.24–3.0 μM, R2: 0.90, being was slightly more cytotoxic than compound **1** (IC50=1.8 μM, 95% CI: 0.65–5.1, R2: 0.93) (Figure 4a,f). The same was observed in MTT assay, compound **2** was the most toxic (IC50 =0.15 μM, 95 % CI: 0.06–0.37 μM, R2: 0.91), whereas compound **1** presented an IC50 of 0.3 μM (CI: 0.14–0.66, R2: 0.94) (Figure 4b,g). As expected, cyclic laxaphycins depolarized the mitochondria and reduced ROS and ATP levels at toxic concentrations (Figure 4c–e,h–j).

Acyclic laxaphycins obtained from *A*. *torulosa* (compounds **3** and **4**) did not display cytotoxicity at any of the concentrations tested (Figure 5a,f). Laxaphycin **3** significantly augmented cell metabolic activity at 0.1 and 1 μM and produced a decrease in ΔΨm (Figure 5b,c). This compound also reduced ROS and ATP levels at the highest concentrations (Figure 5d,e). Interestingly, the reduction in ATP was observed at 6 h, but it was recovered at 24 h, reaching levels of control cells. Compound **4** presented similar results, depolarizing the mitochondrial membrane at 10 μM, and diminishing ROS release and ATP levels at 6 h (Figure 5h–j).

**Figure 4.** Effect of cyclic laxaphycins B on cell viability and mitochondrial function. SH-SY5Y cells were treated with compounds **1** (**a**–**e**) and **2** (**f**–**j**) for 24 h. Then, cell viability was determined with LDH test, cell metabolic activity with MTT assay, ΔΨm with tetramethylrhodamine methyl ester (TMRM) dye, reactive oxygen species (ROS) levels with carboxy-H2DCFDA and ATP levels with a commercial kit. ATP levels were assessed both at 6 h (light bars) and 24 h (dark bars). Concentration expressed in μM. Mean ± SEM of three experiments performed by triplicate. Values are presented as percentage of control cells. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001

**Figure 5.** Effect of acyclic compounds isolated from *A. torulosa* on SH-SY5Y cells. Compounds **3** (**a**–**e**) and **4** (**f**–**j**) were added to neuroblastoma cells for 24 h and cellular viability, metabolic activity, ΔΨm, ROS release and ATP levels were determined. The levels of ATP were assessed at 6 and 24 h (light and dark bars, respectively). Concentration expressed in μM. Mean ± SEM of three experiments performed by triplicate. Values are presented as percentage of control cells. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001

The biological activities of compounds **5** and **6**, isolated from the gastropod *S. striatus*, were also determined (Figure 6). These laxaphycins did not show effects on cell survival (Figure 6a,f). Compound **5** increased cell metabolic activity, depolarized the mitochondria and reduced ROS levels at the highest concentrations (Figure 6b–d). With respect to ATP, its levels were reduced by **5** aftera6h incubation, and recovered at 24 h (Figure 6 e). Laxaphycin **6** only affected to ROS and ATP levels (Figure 6i–j), it decreased ROS release and reduced ATP content at 6 h, recovering it after 24 h, as happened with the other acyclic laxaphycins.

**Figure 6.** Effect of acyclic compounds obtained from *S. striatus* on cell viability and mitochondrial function in human neuroblastoma cells. SH-SY5Y cells were treated with compounds **5** (**a**–**e**) and **6** (**f**–**j**) for 24 h and their effects on cell viability, metabolic activity, ΔΨm, ROS and ATP levels were assessed with LDH assay, MTT test, TMRM dye, carboxy-H2DCFDA and a commercial kit, respectively. ATP assay was performed at 6 h (light bars) and 24 h (dark bars). Concentration expressed in μM. Mean ± SEM of three experiments performed by triplicate. Values are presented as percentage of control cells. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.01

#### *2.4. Cyclic Laxaphycin-B Peptides Induce Apoptosis in SH-SY5Y Cells*

In view of the cytotoxicity displayed by cyclic laxaphycins **1** and **2** in LDH and MTT assays, the type of cell death produced by compounds was determined. SH-SY5Y cells were treated with compounds for 24 h. Laxaphycins **1** and **2** were used at IC50 values obtained from MTT assay (0.3 and 0.15 μM, respectively). Otherwise, acyclic laxaphycins **3**, **4**, **5** and **6** were tested at 10 μM. Cells were co-stained with Annexin V-Fluorescein (FITC) and propidium iodide (PI) and the fluorescence was analyzed by flow cytometry (Figure 7a). The percentages of apoptotic cells, including early apoptotic cells (Annexin V positive and PI negative) and late apoptotic cells (Annexin V positive and PI positive), and necrotic cells (Annexin V negative and PI positive) were calculated. Cells treated with compounds **1** and **2** produced a significant decrease in cell survival (around 60% of control cells). The values of Annexin-V-positive cells in these treatments confirmed that cyclic B-type laxaphycins triggered an apoptotic process. The addition of **1** and **2** produced a 50.4% ± 4.8% (*p* < 0.001) and 46.4% ± 4.8% (*p* < 0.001) of apoptotic cells, respectively. As expected, treatment with staurosporine (STS) also generated apoptosis in neuroblastoma cells (63.3% ± 3.2%, *p* < 0.001). On the other hand, acyclic laxaphycins did not show any significant effect on cell viability, agreeing with our previous results.

To confirm the results obtained with flow cytometry, the activity of caspase 3 was analyzed. This enzyme is an executioner caspase, involved both in intrinsic and extrinsic apoptosis, which targets several apoptotic substrates and initiates a cascade of events that results in cell death [28]. SH-SY5Y cells were treated with laxaphycins for 24 h at the same concentrations used in flow cytometry assays and caspase 3 activity was evaluated in cell lysates. As Figure 7b shows, compounds **1** (0.3 μM) and **2** (0.15 μM) produced a significant increase in the activity of the executioner caspase (about 20% of control cells). Once again, acyclic laxaphycins did not affect to caspase 3 activity, which is in agreement with the results of flow cytometry. STS addition also augmented the enzymatic activity of the caspase (157.8% ± 6%, *p* < 0.001).

**Figure 7.** Cyclic laxaphycin B peptides induce apoptotic cell death in neuroblastoma cells. (**a**) Cells were treated for 24 h with compounds and stained with Annexin V-FITC and propidium iodide, and the fluorescence was analyzed by flow cytometry. Staurosporine (STS) was used as positive control. Apoptosis include early and late apoptotic cells. Data are expressed as percentage of total cells. (**b**) The activity of caspase 3 after treatment with laxaphycins during 24 h was assessed with a commercial kit. Results are expressed as percentage of control cells. Mean ± SEM of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001

#### *2.5. Acyclic B-Type Laxaphycins A*ff*ect to Autophagy in Human Neuroblastoma Cells*

Considering the results obtained with acyclic B-type laxaphycins in the mitochondrial function and our previous work with A-type laxaphycins [14], we decided to evaluate the effect of these compounds on the autophagic flux. With this purpose, SH-SY5Y cells were treated with laxaphycins B for 24 h, and the expression of proteins involved in autophagy was determined by Western blot. Bafilomycin A1 (Baf A1), an inhibitor of the fusion of autophagosomes and lysosomes, rapamycin (Rap), a mTOR inhibitor, and compound C (Comp C), an AMPK inhibitor, were used as positive controls in these assays [29]. Firstly, the effect of compounds on AMPK activation was determined, since this kinase is known to activate autophagy under circumstances of energy depletion [30]. Acyclic peptides produced a significant increase in the activation of AMPK, with compound **6** being the most active (Figure 8a). As expected, Comp C at 1 μM inhibited kinase activity. Then, we analyzed the expression of mTOR, considered the master regulator of the autophagic process. Cyclic laxaphycins **1** and **2** did not affect to the protein expression, whereas the acyclic peptides produced a significant reduction in its activation (Figure 8b). Treatment with 0.1 μM Rap also reduced mTOR activation.

**Figure 8.** Analysis of autophagy initiation-related proteins in SH-SY5Y cells treated with B-type laxaphycins. Compounds were added for 24 h and the protein expression of (**a**) AMPK and (**b**) mTOR was determined by Western blot. Left panels show the results of compounds and right panels present the results of the positive controls bafilomycin A1 (Baf A1), rapamycin (Rap) and compound C (Comp C). The activation of AMPK and mTOR was analyzed as the ratio between phosphorylated/total protein levels. Protein expression levels are normalized by β-actin. Values are mean ± SEM of three replicates performed in duplicate, expressed as percentage of control cells. \* *p* < 0.05, \*\* *p* < 0.01

The activation of p70 S6 kinase, an mTOR downstream target, was also determined (Figure 9a). The cyclic peptide **2** and the acyclic laxaphycins **4** and **6** diminished p70 S6 phosphorylation, reaching percentages among 53.5%–78.6% of untreated cells. Next, we analyzed the expression of beclin 1, a component of the complex that starts autophagosome formation [31]. As can be seen in Figure 9b, acyclic compounds **3**, **4**, **5** and **6** generated an increase in beclin1 expression. Treatment with the autophagy activator Rap also augmented its expression.

**Figure 9.** Analysis of p70 S6 kinase and Beclin 1 expression in neuroblastoma cells after treatment with B-type laxaphycins. The protein expression of (**a**) p70 S6 and (**b**) beclin 1 was determined by Western blot. Left panels show the results of compounds and right panels present the results of the positive controls bafilomycin A1 (Baf A1), rapamycin (Rap) and compound C (Comp C). The activation of the kinase was analyzed as the ratio between phosphorylated/total protein levels. Protein expression levels are normalized by β-actin. Values are mean ± SEM of three replicates performed in duplicate, expressed as percentage of control cells. \* *p* < 0.05, \*\* *p* < 0.01

Beclin 1 is implicated in other cellular mechanisms, such as apoptosis, so its quantification must be complemented with other proteins involved in autophagy [29]. In our case, the expression of LC3 and p62 was analyzed to further confirm the effects of laxaphycins over autophagy (Figure 10). As Figure 10a shows, LC3II/I ratio was increased when acyclic laxaphycins were added to neuroblastoma cells, confirming our previous results. Treatment with Baf A1 and Rap also increased LC3II/I ratio. With regard to p62, whose degradation is related to autophagy, treatment with acyclic laxaphycins produced a significant decrease in its expression, with levels between 37.7–48.6% of untreated cells, a greater degradation than that produced by Rap (Figure 10b). In summary, these results suggest that acyclic laxaphycins B affect to the autophagic flux in human neuroblastoma cells, which is probably mediated by their effects on mitochondria.

**Figure 10.** Determination of the effect of laxaphycins B on LC3 and p62 expression. SH-SY5Y cells were treated with compounds for 24 h and protein expression was analysed by Western blot. (**a**) LC3 expression, calculated as the ratio between LC3II (the lipidated form) and LC3I (the soluble form). (**b**) p62 expression. Left panels show the results of compounds and right panels present the results of the positive controls bafilomycin A1 (Baf A1), rapamycin (Rap) and compound C (Comp C). Protein expression levels are normalized by β-actin. Values are mean ± SEM of three replicates performed in duplicate, expressed as percentage of control cells. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001

#### **3. Discussion**

Laxaphycins are a large family of lipopeptides synthetized through a hybrid PKS/NRPS pathway by different cyanobacteria species. These peptides, selected by evolution in both freshwater and oceanic cyanobacteria, may confer to these primary producers an ecological advantage.

Laxaphycins are divided into two sub-families, laxaphycin A-type and laxaphycin B-type peptides. Laxaphycin B-type members such as laxaphycins B, B2, and B3 have presented antifungal, antimicrobial and cytotoxic activities [9–12], while laxaphycin A-type compounds have shown only weak cytotoxicity [10,12,14].

Here we describe the complete structural elucidation of four acyclic B-type laxaphycins, two of them (**3** and **4** with ring opening) corresponding to the acyclic analogues of laxaphycins B and B3, isolated from the cyanobacteria *A. torulosa*, and the other two (**5** and **6** with ring opening and deletion of two amino-acids) are diet-derived compounds isolated from the herbivorous gastropod *S. striatus* that feeds on the cyanobacteria. It is not unlikely that these acyclolaxaphycins B ensued from an adaptative biotransformation mechanism from a specific herbivorous species [32]. Cyclic lipopeptides are relatively widespread in cyanobacteria, but such biotransformation, ring opening and amino-acids deletion, had never been described.

Moreover, this study is the first description of the effects on mitochondria and autophagy produced by acyclic B-type laxaphycins, and also confirms that the toxic effects of cyclic laxaphycins B are mediated by an apoptotic process. The cytotoxicity of cyclic laxaphycins had been tested in previous works in which laxaphycin B showed cytotoxic effects against a panel of cancer cell lines (IC50 < 2 μM), and both compounds (laxaphycins B and B3) had displayed toxicity towards drug-sensitive and multidrug-resistant tumor cell lines (IC50 ≈ 1 μM) [9]. Our results in neuroblastoma cells are in agreement with these previous assays, with IC50 values of 1.8 and 0.8 μM for laxaphycins B and B3, respectively. A previous work had hypothesized that laxaphycin B toxicity could be mediated by the inhibition of topoisomerase II [10]. The inhibition of this enzyme causes DNA disorders that enhance apoptotic cell death [33]. In the current work, we provide new data that support the triggering of an apoptotic process by laxaphycins B and B3, as indicated by Annexin V staining and caspase 3 activation. The results obtained in p70 S6 kinase, whose inhibition is involved both in autophagy and apoptosis activation [34,35], suggest a different mechanism of action of these cyclic peptides, since only laxaphycin B3 reduced the phosphorylation of this enzyme. On the other hand, the biological activities of acyclic B-type laxaphycins had not been tested so far. Our results suggest that these compounds affect the mitochondrial function, producing a decrease in ATP levels, which may lead to the activation of AMPK. Moreover, acyclic laxaphycins have an impact on the autophagic flux, as evidenced by their effect on the expression of proteins related to this cellular event. Further experiments will help us to clarify if the effect on autophagy is being produced by the activation of AMPK.

AMPK is activated when ATP levels decrease, and maintains energy homeostasis through the inhibition of energy-consuming processes, such as protein and lipid biosynthesis, and the activation of ATP-producing pathways, such as glucose metabolism and mitochondrial biogenesis. Moreover, when an energy depletion occurs, AMPK triggers the autophagic flux, since this catabolic process provides energy and substrates for the synthesis of new biomolecules [22]. Thus, increasing AMPK activity will be a good strategy to increase cellular energy and avoid energetic failure in vulnerable cells. There are several examples of indirect AMPK activators that act through the decrease of ATP levels. Natural compounds such as quercetin, resveratrol, genistein and curcumin activate the kinase by targeting components of the oxidative phosphorylation and increasing the AMP/ATP ratio [36]. However, the most studied AMPK activator is the biguanide metformin, in clinical use for the treatment of Type 2 diabetes mellitus. Metformin inhibits the complex I of mitochondrial respiratory chain, reducing the proton gradient, the production of ROS and ATP levels. This leads to the activation of AMPK and the subsequent increase in glucose uptake [37]. Along with their use as antidiabetic agents, the therapeutic application of AMPK activators has been expanded to the treatment of cancer, inflammation and neurodegeneration [38].

The phosphorylation of mTOR, considered the main regulator of autophagy, was also inhibited by treatment with acyclic laxaphycins B. mTOR plays a central role in integrating growth signals and controlling their physiological effects at a cellular level. Its activation upregulates anabolic processes such as synthesis of proteins, lipids and nucleotides, and downregulates catabolic mechanisms such as autophagy. However, mTOR hyperactivation is linked to several diseases such as cancer, since the kinase promotes tumor growth and proliferation, as well as diabetes, in which it contributes to insulin resistance [39]. Currently, mTOR inhibitors are in clinical use as immunosuppressants and anti-cancer drugs, and due to crucial role of the kinase in many illnesses, it is expected that mTOR inhibitors may have a broader application for other diseases [40,41].

Along with these illnesses, there is substantial evidence of autophagy dysregulation in neurodegenerative diseases, such as alterations in mTOR and beclin 1 expression, and accumulation of defective autophagosomes [42]. In this context, the activation of the autophagic flux has emerged as a therapeutic approach for these pathologies, because it represents a major pathway for clearance of aggregated proteins and damaged organelles. Several compounds such as metformin or Rap have shown promising effects in vivo [42], and the activation of mitophagy has been recently related to a reduction in cognitive deficits in Alzheimer's disease models [43]. Therefore, the stimulation of autophagy maybe a good strategy to face neurodegeneration. However, the use of activators must be handled carefully, as an excessive autophagy can lead to the destruction of essential cellular machinery [44]. In addition, intervention in early phases of the diseases would be more favorable than in an advanced state [21]. The results obtained with acyclic B-type laxaphycins in the human neuroblastoma cell line SH-SY5Y, which maintains certain characteristics of neuronal cells [23], could be used as a starting point to analyse their neuroprotective effects. In this sense it would be interesting to study the ability of laxaphycins to cross the blood brain barrier (BBB). There are several examples of non-ribosomal peptides capable of penetrating the BBB, such as polymyxins [45] and some cyanotoxins [46], which can reach the brain through specific peptide transporters. Moreover, the lipidic nature of laxaphycins could allow the compounds to cross the BBB, as lipid solubility is a crucial factor that facilitates transport across the barrier [47].

On the other hand, the data obtained in this work show a clear correlation among the chemical structure and the biological activities of laxaphycins B. Cyclic laxaphycins produce apoptotic cell death and exhibit a greater cytotoxicity than their acyclic analogues. Also, the results obtained in p70 S6 kinase with the cyclic laxaphycin B3 and their acyclic analogs suggest that the presence of an OH group in R is a key structural feature to the inhibition of this enzyme. Due to the supply problem associated with marine compounds, the chemical synthesis of B-type laxaphycins would be a good strategy for their pharmacological use. Laxaphycin B total synthesis has been previously published [48], as well as the synthesis of two simplified analogues of the parent compound [7], which opens a door for future synthesis of acyclic laxaphycins, which will help to better understand the structure-activity relationship of B-type laxaphycins.

In summary, this work provides new data that confirm the pro-apoptotic effects produced by cyclic laxaphycins B, and describes for first time the complete structural elucidation of four B-type acyclic laxaphycins and their biological activities. The acyclic peptides affect mitochondrial function and have an effect on the expression of key proteins involved in autophagic flux, suggesting an involvement of the compounds in the activation of this mechanism. Further experiments will help to clarify the mechanism of action of the B-type laxaphycins and the structural requirements for these activities.

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

#### *4.1. Chemicals and Solutions*

EnzCheck® Caspase-3 Assay Kit, TMRM, 5-(and-6)-carboxy-2 , 7 -dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), Pierce TM Protease Inhibitor Mini Tablets and PierceTM Phosphatase Inhibitor Mini Tablets were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC Apoptosis Detection Kit was obtained from Immunostep (Salamanca, Spain). Rap, Comp C and Baf A1 were obtained from Abcam (Cambridge, UK). Other chemicals were reagent grade and were purchased from Sigma-Aldrich (Madrid, Spain).

#### *4.2. Organism Collection*

The cyanobacterium *Anabaena torulosa*, as well as the herbivorous gastropod *Stylocheilus striatus,* were collected by SCUBA diving at a depth of 1–5 m in Moorea Atoll, French Polynesia (S 17◦29 22", W 149◦54 17") in Pacific Ocean. The cyanobacterium and the gastropod samples were sealed independently underwater in a bag with seawater and then frozen and freeze-dried.

#### *4.3. HPLC and LC-MS Analyses*

HPLC-PDA-ELSD analyses were performed with a Waters Alliance HPLC system (W 2695) coupled to a photodiodes array detector (PDA Waters 2998) and an evaporative light scattering detector (Waters ELSD 2424). The analyses were performed on a reversed-phase column (Thermo Hypersil Gold C-18, 150 × 2.1 mm, 3 μm) employing a gradient of 10% to 100% CH3CN over 40 min followed by 25 min at 100% CH3CN (all solvents buffered with 0.1% formic acid) with a flow rate of 0.3 mL/min. Semi-preparative HPLC purifications were performed on a binary HPLC pump system Waters 1525 with a dual λ absorbance detector Waters 2487, equipped with a reverse phase column (Interchim UP5ODB.25M, 250 × 10 mm, 5 μm) using isocratic elution (H2O-CH3CN) at a flow rate of 3 mL/min.

#### *4.4. Compound Isolation and Purification*

Six hundred grams of freeze-dried *A*. cf *torulosa* and 4.24 g of *S. striatus* (6 specimens) were extracted, separately but in the same manner, with a mixture of CH3OH-CH2Cl2 (1:1) and sonicated during 10 minutes to yield two organic extracts after evaporation under reduced pressure. Then the two crude extracts (38 g and 4 g, respectively) were subjected to flash RP18 silica gel column eluted with H20 (A), H2O-CH3CN (20:80) (B) and CH3OH-CH2Cl2 (80:20) (C) to afford 3 fractions (A, B and C). Fractions B, from *A. torulosa* and *S. Striatus*, were fractioned, in turn, with flash chromatography and RP18 silica gel column with a gradient of H2O-CH3CN to give 7 sub-fractions. Fraction B4 from the organic extract of *S. striatus* subjected to HPLC purification (Phenomenex Gemini C6-phenyl, 110Å, 250 × 10 mm, 5 μm) and eluted with 28% CH3CN in H2O with 0.1% formic acid at a flow rate of 4 mL/min, gave compounds **5** (2.5 mg) and **6** (5 mg). Laxaphycin A was found in sub-fraction B5, but none of the already described laxaphycins B or B3 could be detected. Fraction B4 from the organic extract of *A. torulosa* was eluted with 38% CH3CN and gave compounds **3** (3 mg) and **4** (4 mg). Laxaphycins A, B (**1**) and B3 (**2**) were found respectively in sub-fraction B6 and B5.

#### *4.5. Mass Spectrometry and NMR Spectroscopy*

LC-MS analyses were carried out using a Thermo Fisher Scientific LC-MS device, Accela HPLC coupled to an LCQ Fleet equipped with an electrospray ionization source and a 3D ion-trap analyzer. High-resolution ESI mass spectra were obtained on a Bruker Thermo Scientific Q-Tof Maxis mass spectrometer using electrospray ionization in positive mode. Compounds were solubilized in MeOH at 1 μg/mL and infused in mass spectrometer (collision energy: 50 eV).

1D-NMR and 2D-NMR experiments were acquired on a Brucker Avance 800 spectrometer equipped with a cryogenic probe (5 mm), all compounds solubilized in DMSO-*d*6 (500 μL) at 303 K. All chemical shifts were calibrated on the residual solvent peak (DMSO-*d*6, 2.50 ppm (1H) and 39.5 ppm ( 13C). The chemical shifts (δ), reported in parts per million (ppm) are referenced relatively to TMS.

#### *4.6. Advanced Marfey's Analyses*

The Marfey's analyses were carried out on compounds **3**, **4**, **5** and **6**. Approximately 0.3 mg of each compound were hydrolyzed with 1 mL of 6 N HCl for 20 h at 110 ◦C in sealed glass vials. The cooled hydrolysate mixtures were evaporated to dryness and traces of HCl were removed from the reaction mixtures by repeated evaporation. Each hydrolysate mixture was dissolved in H2O (100 μL). 110 μL of acetone, 20 μL of 1 N NaHCO3 and 20 μL of 1% L or D/L FDLA (1-fluoro-2,4-dinitrophenyl-5-L-leucinamide) in acetone were added to each 50 μL aliquot. The mixtures were then heated to 40 ◦C for 1 h. The cooled solutions were neutralized with 1 N HCl (20 μL), and then dried in vacuo. The residues were dissolved in 1:1 CH3CN-H2O and then analyzed by LC-MS. LC-MS analyses were performed on a reversed-phase column (Thermo Hypersil Gold C-18, 150 × 2.1 mm, 3 μm) with two linear gradients: (1) from 20% CH3CN-80% 0.01 M formic acid to 60% CH3CN-40% 0.01 M formic acid at 0.3 mL/min over 70 min and (2) from 10% CH3CN-90% 0.01 M formic acid to 50% CH3CN-50% 0.01 M formic acid at 0.3 mL/min over 70 min, then to 80% CH3CN-20% over 10 min. The configuration of the α carbon for each residue can be assigned in accordance with the elution order of the D- and L-FDLA derivatives [24,25]: amino acids for which the D-FDLA analogue elutes first have a D configuration, whereas those for which the L-FDLA analogue elutes first have an L configuration. Furthermore, the hydrolysates were compared to that of laxaphycin B.

#### *4.7. Cell Culture*

SH-SY5Y human neuroblastoma cell line was obtained from American Type Culture Collection (ATCC), number CRL2266. Cells were cultured in Dulbecco´s modified Eagle´s medium: Nutrient Mix F-12 (DMEM/F-12) with 10% fetal bovine serum (FBS), 1% glutamax, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2 and 95% air and dissociated weekly using 0.05% trypsin/EDTA. All the reagents were obtained from Thermo Fischer Scientific.

#### *4.8. Cytotoxicity Assay*

Cell viability was assessed with the LDH test [14]. Cells were seeded in 96-well plates and exposed to different compound concentrations (0.001–10 μM). Cells were incubated with compounds at 37 ◦C in humidified 5% CO2/95% air atmosphere for 24 h. Quillaja bark saponin was used as cellular death control. Then, cell medium was collected and LDH release was evaluated. PierceTM LDH-Cytotoxicity Assay Kit (Thermo Fisher Scientific) was used for LDH determination. Absorbance was measured at 490 nm to determine LDH release to the medium. Experiments were carried out in triplicate at least three independent times.

#### *4.9. Metabolic Activity Evaluation*

The effect of laxaphycins on cell metabolic activity was evaluated by MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay [27,49]. SH-SY5Y cells were cultured in 96-well plates and treated with compounds at concentrations ranging from 0.001 to 10 μM for 24 h. Next, cells were rinsed three times and incubated for 1 h with MTT (500 μg/mL) dissolved in saline buffer. MTT excess was washed and cells were disaggregated with 5% sodium dodecyl sulfate. Absorbance of the colored formazan salt was measured at 595 nm in a spectrophotometer plate reader. Cell death control signal was subtracted from the other data.

#### *4.10. Mitochondrial Membrane Potential Measurement*

ΔΨm was evaluated with TMRM assay as previously described [49]. SH-SY5Y cells were seeded in 96-well plates at 5 × 104 cells per well. After 24 h, cells were treated with compounds (0.001–10 μM) for 24 h. Next, cells were washed twice with saline solution and incubated with 1 μM TMRM for 30 min. Then human neuroblastoma cells were solubilized with 50% DMSO–50% water. Fluorescence values were measured at 535 nm excitation, 590 nm emission with a plate reader. At least three independent replicates were performed in triplicate.

#### *4.11. Evaluation of Reactive Oxygen Species and ATP Levels*

ROS production was assessed with the fluorescence dye carboxy-H2DCFDA (5-(and-6)-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate) [49]. SH-SY5Y cells were seeded in 96-well plates and allowed to grow for 24 h. Then, cells were treated with laxaphycins (0.001–10 μM) for 24 h and ROS levels were measured. SH-SY5Y cells were washed twice with serum-free medium and loaded with 20 μM carboxy-H2DCFDA. Next, the plate was incubated for 1 h at 37 ◦C. After this time, phosphate buffered saline (PBS) was added to each well during 30 min. The fluorescence was read at 495 nm excitation and 527 nm emission. Experiments were carried out at least three times.

ATP levels were determined with the Luminescent ATP Detection Kit (Abcam), following manufacturer´s instructions. Briefly, human neuroblastoma cells were cultured in 96-well plates at 5 × 104 cells per well. Cells were treated with compounds as described above during 6 and 24 h, Then, cells were lysed and 50 μL of substrate solution were added to each well. The plate was incubated for 5 min, and the luminescence was measured in a plate reader. Rot at 1 μM was used as positive control.

#### *4.12. Flow Cytometry Analysis*

The Annexin V-FITC Apoptosis Detection Kit was used to determine the cell death produced by compounds as previously described [50]. SH-SY5Y cells were seeded in 12-well plates at 1 × 106 per well and incubated for 24 h with laxaphycins. Then, cells were washed with PBS and resuspended in Annexin binding buffer containing Annexin V-FITC and PI. Cells were incubated for 15 min at room temperature, resuspended in PBS and filtered. The fluorescence was determined by flow cytometry using the ImageStreamMKII (Amnis Corporation, Luminex Corp, Austin, TX, USA). 10,000 events were analyzed with IDEAS Application 6.0 software (Amnis Corporation, Luminex Corp, Austin, TX, USA). STS (Sigma Aldrich) at 0.01 μM was used as control in this assay.

#### *4.13. Evaluation of Caspase 3 Activity*

The detection of caspase 3 activity was carried out with the EnzCheck® Caspase-3 Assay Kit. Neuroblastoma cells were cultured in 12-well plates at 1 × 106 cells per well and treated with compounds. After 24 h, cells were lysed and an aliquot was collected to quantify protein concentration by the Bradford method. Then, the assay was performed following manufacturer's instructions. Briefly, 50 μL of each sample were mixed with equal volume of Z-DEVD-AMC substrate and incubated for 30 min at room temperature. Then, fluorescence at 342 nm excitation and 441 nm emission was measured. Caspase 3 activity data were corrected by protein concentration values. STS (0.01 μM) was used as positive control.

#### *4.14. Western Blotting*

SH-SY5Y cells were seeded in 12-well plates and treated with laxaphycins for 24 h. Next, cells were washed with PBS and lysis buffer (50mM Tris HCl, 150 mM NaCl, 1mM EDTA and 1% Triton x-100, supplemented with a complete phosphatase/protease inhibitor cocktail) was added to each well. Cells were scrapped, sonicated and centrifuged at 13,000 rpm at 4 ◦C for 20 min.

For p62 quantification, cells were lysed as previously described, with modifications [14]. A hypotonic buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, containing a complete phosphatase/protease inhibitor cocktail) was added to each well. Then, cells were incubated for 15 min on ice, sonicated and centrifuged at 3000 rpm at 4 ◦C for 15 min.

In both cases, the supernatant was collected as the cytosolic fraction and quantified with the Bradford method. Samples containing 15 μg were used for electrophoresis, resolved in 4–20% sodium dodecyl sulphate polyacrylamide gel (Biorad, Madrid, Spain). Proteins were transferred to PVDF membranes (Merck Millipore) with a Trans-Blot® semi-dry transfer cell (Biorad). Membrane blocking and antibody incubation was performed in Snap i.d. system (Merck Millipore). Protein bands were detected with Supersignal West Pico Luminiscent Substrate and Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Chemiluminiscence was determined with Diversity GeneSnap system and software (Syngene) [51]. LC3 II/I was detected with the primary antibody anti-LC3 II/I (1:1000, Abcam), the primary antibody anti-pmTOR (Ser2448) (1:1000, Merck Millipore) was used to recognize phospho-mTOR and the total levels of the kinase were determined with anti-mTOR antibody (1:10000, Abcam), beclin-1 was detected with anti-beclin-1 primary antibody (1:1000, Merck Millipore), p62 was quantified with anti-p62 antibody (1:1000, Merck Millipore), anti-phospho-p70 S6 kinase (Thr389) (1:1000, Merck Millipore) was used to detect phospho- p70 and the total levels were quantified using anti-p70 S6 kinase antibody (1:1000, Merck Millipore), anti-phospho-AMPK (Thr172) (1:500, Merck Millipore) was used to recognize the phosphorylated state of the kinase, and the total expression of the enzyme was detected with anti-AMPK antibody (1:500, Merck Millipore). Protein levels were normalized using β-Actin (1:20,000, Millipore). Baf A1 (0.001 μM) [29], Rap (0.1 μM) [52] and Comp C (1 μM) [53] were used as controls for autophagic flux measurements. All the experiments were performed at least three times in duplicate.

#### *4.15. Statistical Analysis*

Data are presented as mean ± SEM. Statistical differences were evaluated by one-way ANOVA or Student´s *t*-tests with Graph Pad Prism 6 software. Statistical significance was considered at *p* < 0.05.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/7/364/s1, Figure S1: 1H-NMR spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S2: 13C-NMR spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S3: DEPT135-NMR spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S4: TOCSY spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S5: ROESY spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S6: HSQC spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S7: HSQC-TOCSY spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S8: HMBC spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B (5) in DMSO (303K), Figure S9: 1H-NMR spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), FigureS10: 13C-NMR spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), Figure S11: DEPT135-NMR spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), Figure S12: TOCSY spectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), Figure S13: ROESYspectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), Figure S14: HSQCspectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), Figure S15: HSQC-TOCSYspectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K), Figure S16: HMBCspectrum of [des-(Ala4-Hle5)] acyclolaxaphycin B3 (6) in DMSO (303K).

**Author Contributions:** Funding acquisition, L.M.B.; investigation, R.A., E.A., L.B., I.B., N.I. and B.B.; supervision, E.A. and L.M.B.; writing—original draft, R.A.; writing—review and editing, E.A., B.B. and L.M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support was provided to LB, IB, NI and BB by the Laboratoire d'Excellence (LabEx) "CORAIL" (Keymicals and Cyanodiv) and the GDR CNRS 3658 MediatEC (https://www.gdr-mediatec.cnrs.fr/english-version/). Chromatographic, spectroscopic, and structural analyses were performed using the facilities of the Biodiversité et Biotechnologies Marines platform at the University of Perpignan (Bio2Mar, http://bio2mar.obs-banyuls.fr/fr/index.html).

**Acknowledgments:** The research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. We thank Olivier Thomas and Stephane Greff for HRMS facilities and Christian Roumestand for NMR measurements.

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

### *Review* **A Comprehensive Review of Bioactive Peptides from Marine Fungi and Their Biological Significance**

#### **Fadia S. Youssef 1, Mohamed L. Ashour 1,2, Abdel Nasser B. Singab 1,\* and Michael Wink 3,\***

Received: 28 August 2019; Accepted: 28 September 2019; Published: 29 September 2019

**Abstract:** Fungal marine microorganisms are a valuable source of bioactive natural products. Fungal secondary metabolites mainly comprise alkaloids, terpenoids, peptides, polyketides, steroids, and lactones. Proteins and peptides from marine fungi show minimal human toxicity and less adverse effects comparable to synthetic drugs. This review summarizes the chemistry and the biological activities of peptides that were isolated and structurally elucidated from marine fungi. Relevant fungal genera including *Acremonium*, *Ascotricha*, *Aspergillus*, *Asteromyces*, *Ceratodictyon*, *Clonostachys*, *Emericella*, *Exserohilum*, *Microsporum*, *Metarrhizium*, *Penicillium*, *Scytalidium*, *Simplicillium*, *Stachylidium*, *Talaromyces*, *Trichoderma*, as well as *Zygosporium* were extensively reviewed. About 131 peptides were reported from these 17 genera and their structures were unambiguously determined using 1D and 2D NMR (one and two dimensional nuclear magnetic resonance) techniques in addition to HRMS (high resolution mass spectrometry). Marfey and Mosher reactions were used to confirm the identity of these compounds. About 53% of the isolated peptides exhibited cytotoxic, antimicrobial, and antiviral activity, meanwhile, few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity. However 47% of the isolated peptides showed no activity with respect to the examined biological activity and thus required further in depth biological assessment. In conclusion, when searching for bioactive natural products, it is worth exploring more peptides of fungal origin and assessing their biological activities.

**Keywords:** biological activity; chemistry; marine derived fungi; peptides

#### **1. Introduction**

Hundreds of secondary metabolites obtained from marine fungal strains revealed potent pharmacological and biological activity [1]. These mainly comprise alkaloids, terpenoids, and peptides, in addition to polyketides, steroids, and lactones. Relevant bioactivities include antibacterial, anticancer, anti-inflammatory, and antiviral activity [2]. The great diversity in the structure and function of the metabolites derived from marine organisms is mainly attributed to the extensive variation in the chemical and physical conditions of the environment in which the marine organisms survive [3]. As many marine organisms are sessile, they need chemical protection against predators and pathogens.

Marine microorganisms represented by fungi and bacteria, but also marine invertebrates, are regarded as a valuable source of bioactive compounds. Marine microorganisms have the advantage that they can be cultured and thus offer high reproducibility and an everlasting source of natural

products [4]. A considerable number of drugs already exist in the market that are of fungal or bacterial origin such as griseofulvin, fucidin, penicillins, and many ergot alkaloids containing products. However, the number of marine fungal metabolites is still quite small [5].

Besides, a number of marine fungal metabolites are characterized by appropriate oral-bioavailability and suitable physico-chemical properties that meet the criteria of formulating effective pharmaceuticals [1]. Furthermore, most fungal proteins and peptides show minimal human toxicity and less adverse effects compared to drugs of synthetic origin [6].

Regarding the history of bioactive peptide isolation from marine organisms, it is noteworthy to highlight that during the past half century hundreds of peptide antibiotics have been explored. They are classified as synthetic peptides (non-ribosomal) and natural (ribosomal) ones. The former are represented by glycopeptides, gramicidins, bacitracins, and polymyxins as well and they are extensively modified and produced mainly by bacterial strains, meanwhile, the latter are equally produced by all species comprising fungi and bacteria and considered as a primary line of defence elicited by these organisms [7].

However, naturally occurring peptides obtained from marine sources possess strongly modified structures either in its backbone or side chain compared to peptides of human origin that undoubtedly do due to the environmental conditions in which they live. This ultimately makes them good candidates for drug design offering great stability from enzymatic degradation as well as thermal conditions. Most of these peptides are taken from ascidians, sponges, as well as mollusks. Besides, wide array of bioactive peptides are produced as the result of the association that exists between microorganisms and the marine organisms. This symbiosis, in turn, generated biochemical pathways in both the marine organisms and its associated microorganisms with concomitant production of many beneficial pharmaceuticals that are of natural origin [8].

Peptides are generally isolated from the fermented fungal biomass culture media via extraction of the culture media using appropriate solvents, most commonly ethyl acetate, and then subjected to evaporation till dryness under vacuum at 40 ◦C to a semi solid residue. The obtained residue was subjected to a series of chromatographic fractionation using traditional stationary phases such as silica gel and sephadex as well, with concomitant purification using high performance liquid chromatography (HPLC) to fully purify and isolate individual peptides in their single forms [9].

Structural elucidation and characterization of the isolated peptides are unambiguously determined by spectroscopic analysis comprising 1D and 2D NMR techniques, in addition to mass spectrometry. Their absolute configurations were further ascertained by Marfey's methods, chemical structural modification, as well as Mosher's reaction [10]. In addition, ESIMS (Electrospray Ionization Mass Spectrometry) analyses of the free amino acids obtained by acid hydrolysis, as well as HPLC analysis of Marfey products prepared from the acid hydrolysate are also used for determination of peptides structures. However, the peptide amino acid sequence can be also unambiguously determined by MSn ion-trap ESI mass spectrometry [11].

Thus, in this review we comprehensively explore the chemistry and the biological activity of the peptides that were isolated and structurally elucidated from marine fungi of the genera *Acremonium*, *Ascotricha*, *Aspergillus*, *Asteromyces*, *Ceratodictyon*, *Clonostachys*, *Emericella*, *Exserohilum*, *Microsporum*, *Metarrhizium*, *Penicillium*, *Scytalidium*, *Simplicillium*, *Stachylidium*, *Talaromyces*, *Trichoderma*, and *Zygosporium.* Collection of data was done until July 2019 and classification was performed based upon the fungal genera, which are arranged in an alphabetical order. Additionally, a table summarizing the bioactive peptides isolated from marine associated fungi, their sources, and biological activities was added for better illustration of the collected data.

#### **2. Bioactive Peptides in Particular Fungal Genera**

#### *2.1. Acremonium*

From a fermenter-culture of *Acremonium persicinum*, three new compounds of cycloheptapeptide skeleton were isolated, namely cordyheptapeptides C–E (**1**–**3**). Only compounds (**1**) and (**3**) showed a substantial cytotoxicity versus MCF-7, SF-268, and NCI-H460 cancer cells displaying IC50 values between 2.5 and 12.1 μM [12]. Moreover, two new linear pentadecapeptides (efrapeptins Eα (**4**) and H (**5**)) in addition to known efrapeptin F–G (**6**–**7**) were isolated from an atypical *Acremonium* species. Additionally, RHM1 (**8**), RHM2 (**9**), RHM3 (**10**), and RHM4 (**11**), N-methylated linear octapeptides, were also isolated and structurally elucidated using different spectroscopic techniques from the same species (Figure 1A–C). Promising cytotoxic activity was exhibited by efrapeptins Eα (**4**), F (**6**), and G (**7**) versus H125 cells with an IC50 value about 1.3 nM; the latter also showed potent cytotoxic activity versus murine L1210 cells as well as versus HCT-116 with IC50 values equal to 0.002 nM. Pronounced antibacterial activity was observed for RHM1 (**8**) as well as for efrapeptin G (**7**) against *Staphylococcus epidermidis* with MIC (Minimum Inhibitory Concentration) values equal to 0.015 and 0.049 μM, respectively [13,14].

#### *2.2. Ascotricha*

In depth chemical investigation of the marine fungus *Ascotricha* using different chromatographic techniques resulted in the isolation of 10 compounds that are fully elucidated using various spectroscopic techniques in addition to their physicochemical characteristics. Among them are five peptides namely cyclo (Pro-Ala) (**12**), cyclo (Ile-Leu) (**13**), cyclo (Leu-Pro) (**14**), cyclo (Pro-Gly) (**15**), and cyclo (Pro-Val) (**16**) (Figure 2) that were first to be isolated from genus *Ascotricha* [15].

**&RUG\KHSWDSHSWLGH &** 5 + **&RUG\KHSWDSHSWLGH '** 5 2+

**&RUG\KHSWDSHSWLGH (**

(**A**)

**Figure 1.** *Cont*.

**Figure 1.** (**A**) Cordyheptapeptides isolated from *Acremonium* species. (**B**) Efrapeptins isolated from *Acremonium* species. (**C**) RHM family isolated from *Acremonium* species.

**Figure 2.** Peptides isolated from *Ascotricha* species.

#### *2.3. Aspergillus*

Five new depsipeptides, aspergillicins A–E (**17**–**21**), were isolated from *Aspergillus carneus* upon comprehensive chemical investigation of a fermenter culture that showed no anti-parasitic activity versus *Haemonchus contortus* in contrast to modest cytotoxic activity with LD99 values ranging between 0.03–0.07 μM [11]. Regarding *A. niger*, which was obtained from sediments from the northeast coast of Brazil, then cultured in various growth media, many cyclopeptides were obtained comprising cyclo (l-Pro-l-Phe) (**22**), cyclo (*trans*-4-hydroxy-l-Pro-l-Leu) (**23**), cyclo (l-Pro-l-Leu) (**24**), cyclo (*trans*-4-hydroxy-l-Pro-l-Phe) (**25**), cyclo (l-Pro-l-Val) (**26**), as well as cyclo (l-Pro-l-Tyr) (**27**). The isolated cyclopeptides exhibited no cytotoxic effect against HCT-116 cell line [16]. In addition, other three cyclopeptides namely cyclo-(l-Trp-l-Ile) (**28**), cyclo-(l-Trp-l-Phe) (**29**), and cyclo-(l-Trp-l-Tyr) (**30**) were

also reported. The first two cyclopeptides were considered to be plant growth regulators whereas the latter effectively stimulates the differentiation of HT-29 cancer cells. However, neither compounds (**28**–**30**) showed pronounced antimicrobial activity when tested against either *Staphylococcus aureus*, *Escherichia coli*, *A. niger*, or *Candida albicans* [17]. In addition, two novel hexapeptides were isolated and structurally elucidated from the fungus *A. sclerotiorum*. These hexapeptides were sclerotide A (**31**) and sclerotide B (**32**) which showed modest antifungal activity versus *C. albicans* showing MIC values of 7.0 and 3.5 μM, respectively. Additionally, sclerotide B (**32**) revealed selective activity versus *Pseudomonas aeruginosa* with MIC equals to 35.3 μM, meanwhile, it exerted weak cytotoxic activity versus HL-60 cell line with IC50 equal to 56.1 μM [18] (Figure 3A).

One more new cyclic hexapeptide, similanamide (**33**), was isolated from a sponge derived fungus, *A. similanensis*, which displayed weak cytotoxic activity versus MCF-7, A373, and NCI-H460 cancer cells with GI50 values equal to 0.2, 0.18, and 0.18 μM, respectively, with no antibacterial activity as the (MIC value was found to be greater 256 μg/mL (0.4 μM)) [19]. Moreover, two new lumazine peptides namely terrelumamides A (**34**) and B (**35**), in addition to two new isomeric modified tripeptides, aspergillamides C (**36**) and D (**37**), and a cyclic tetrapeptide asperterrestide A (**38**) were isolated and unambiguously elucidated from *A. terreus*, which is a marine fungus. Compounds (**34**) and (**35**) displayed a promising improvement in insulin sensitivity as determined by the utilization of mesenchymal cells of a bone marrow origin obtained from human (hBM-MSCs) adopting an adipogenesis model. They exerted their action via increasing the formation of adiponectin during the process of adipogenesis in hBM-MSCs with EC50 values equal to 37.1 and 91.9 μM, for terrelumamides A (**34**) and B (**35**), respectively. It is noteworthy to highlight that the maximum elevation in adiponectin levels via terrelumamide A (**34**) induction was 56.9% comparable to that generated by glibenclamide (the standard anti-hyperglycaemic agent). Meanwhile compound (**38)** showed a potent cytotoxic effect versus U937 and MOLT4 human cancer cell lines with IC50 equal to 6.4 and 6.2 μM, respectively, in addition to a pronounced inhibitory potential versus an influenza virus of H1N1, as well as H3N2 strains with IC50 equal to 15 and 8.1 μM, respectively, that relied upon the presence of a 3-OH-N-CH3-Phe moiety that is rare in nature [20–22]. Besides, other known peptides were isolated from the latter *Aspergillus* species, which are aspergillamide A **(39)** and B (**40**) and cyclo-(l-Pro-l-Phe) (**41**) [21]. *A. unguis* is another marine fungus from which unguisin A (**42**), which is a cyclic peptide, was isolated [23]. In addition, *A. unilateralis* revealed a complex array of secondary metabolites comprising the heterocyclic dipeptides, aspergillazines A–E (**43**–**47**), which are dipeptides, in addition to trichodermamide A and B (**48**–**49**) [24] (Figure 3B).

Furthermore, four new compounds, psychrophilins E–H (**50**–**53**) were isolated from *A. versicolor*, and they are cyclic peptides characterized by the presence of a rare linkage of amide type between anthranilic acid through its carboxylic acid and the indole ring via its nitrogen. Additionally, a new cyclic hexapeptide versicotide C (**54**) was isolated from the same fungus. All the isolated peptides showed no cytotoxicity, however compound (**52**) exhibited a pronounced lipid-reducing activity at approximately 10 μM as determined by oil red O staining assay [25]. Moreover, secondary metabolites isolated from coral derived *A. versicolor* revealed the presence of a new centrosymmetric cyclohexapeptide, namely aspersymmetide A (**55**), in addition to asperphenamate (**56**) which is a known peptide. Noteworthy to mention is that compound (**55**) was the first centrosymmetric cyclohexapeptide to be isolated from fungi, however it showed weak cytotoxicity versus NCI-H292, as well as A431 cell lines, at 10 μM [26]. Besides, two new cyclopentapeptides, namely cotteslosins A (**57)** and B (**58**), were isolated from the same fungus, *A. versicolor.* Cotteslosin A (**57**) displayed weak cytotoxicity versus human melanoma (MM418c5), prostate (DU145), and breast (T47D) cells with EC50 values of 0.1, 0.14, and 0.15 μM, respectively [27]. Concerning *A. violaceofuscus*, a sponge associated fungus, three peptides, which are cyclic, were isolated from its ethyl acetate extract and they were first to be isolated in nature. These cyclic peptides are termed, aspochracin-type cyclic tripeptide sclerotiotide L (**59**), diketopiperazine dimer (**60**), in addition to a cyclic tetrapeptide (**61**) where compounds (**60**–**61**) showed potent anti-inflammatory activity against IL-10 expression of the

(Lipopolysaccharide) LPS-induced THP-1 cells (acute monocytic leukemia cell) as evidenced by its inhibition rates which were estimated to be 84.3 and 78.1%, respectively at 10 μM [28] (Figure 3C).

**Figure 3.** *Cont*.

**Figure 3.** *Cont*.

**Figure 3.** (**A**) Peptides isolated from Aspergillus carneus, A. niger, A. sclerotiorum, and A. terreus. (**B**) Peptides isolated from A. terreus, A. unguis, and A. unilateralis. (**C**) Peptides isolated from A. versicolor and A. violaceofuscus. (**D**) Peptides isolated from miscellaneous *Aspergillus* species.

Various peptides were isolated from miscellaneous *Aspergillus* species exemplified by psychrophilin E (**50**), a cyclic tripeptide isolated from two algae associated *Aspergillus* sp whose selectivity inhibited the proliferation of HCT116 (colon) cell line with an IC50 value of 28.5 μM comparable to the standard drug cisplatin that showed IC50 of 33.4 μM [29]. Additionally, many *Aspergillus* sp. derived peptides displayed antiviral activity such as aspergillipeptides D–G (**62**–**65**), in which aspergillipeptides D (**62**) and E (**63**) displayed a potent antiviral effect versus herpes simplex virus type 1 (HSV-1) displaying IC50 of 9.5 and 19.8 μM, respectively, showing no cytotoxic effect at these concentrations versus Vero cell line, meanwhile aspergillipeptide D (**62**) also exhibited activity versus acyclovir resistant clinical isolates of HSV-1 [30]. Regarding the inhibition of nitric oxide production, it was found that the novel cyclic dipeptide isolated from an *Aspergillus* sp., 14-hydroxy-cyclopeptine (**66**), effectively inhibits nitric oxide production displaying IC50 of 0.14 μM in recombinant mouse interferon-γ-activated macrophage-like cell line as well as in a lipopolysaccharide without cytotoxic effect at 0.34 μM [31]. Many other peptides were also isolated from *Aspergillus* sp. derived from *Bruguiera sexangula* var. *rhynchopetala* as aspergilumamide A (**67**) and penilumamide (**68**) [32] (Figure 3D).

#### *2.4. Asteromyces*

A new pentapeptide compound, lajollamide A (**69**) was isolated from marine *Asteromyces cruciatus*. This newly isolated peptide showed a weak antimicrobial effect versus Gram-positive bacteria at 100 μM showing 61% and 30% inhibition in bacterial growth for *Bacillus subtilis* and *Staphylococcus epidermidis*, respectively, whereas it showed no inhibition of acetyl cholinesterase activity [33] (Figure 4).

#### *2.5. Ceratodictyon*

In depth chemical investigation of the red algae associated fungus, *Ceratodictyon spongiosum*, resulted in the isolation of two new peptides which are dictyonamides A and B (**70**–**71**). The former displayed a pronounced inhibition on cyclin-dependent kinase 4 with IC50 value equals to 0.01 μM however the latter showed no inhibition [34] (Figure 4).

#### *2.6. Clonostachys*

Clonostachysins A and B (**72**–**73**) are two new cyclic peptides isolated from *Clonostachys rogersoniana* that revealed potent inhibitory activity on a dinoflagellate *Prorocentrum micans* at a concentration of 30 μM but revealed no activity to either bacteria or microalgae [35] (Figure 4).

#### *2.7. Emericella*

Unguisins A (**42**) and B (**74**) are two cyclic heptapeptides isolated from the fungus *Emericella unguis* [36]. Besides, emericellamides A and B (**75**–**76**) were also isolated from certain *Emericella* sp. that is co-cultured from *Salinispora arenicola*. Both compounds (**42**) and (**76**) showed encouraging antimicrobial activity against MRSA (methicillin resistant *Staphylococcus aureus* strains) displaying MIC values equal to 3.8 and 6.0 μM, respectively [37] (Figure 4).

#### *2.8. Exserohilum*

Rostratins A–D (**77**–**80**) were first to be isolated from the broth of *Exserohilum rostratum*, a marine associated fungus that showed pronounced cytotoxic activity versus (HCT-116) the human colon carcinoma showing 20, 4.48, 1.6, and 34 nM as IC50 values, respectively [38] (Figure 4).

#### *2.9. Microsporum*

The marine associated fungus, *Microsporum* cf. *gypseum*, yielded two new peptides microsporins, A and B (**81–82**), which effectively inhibit histone deacetylase with IC50 values equal to 0.14 and 0.55 μM against (Histone Deacetylases) HDACs and HDAC8, respectively, in addition to displaying a considerable cytotoxic effect versus human colon adenocarcinoma (HCT-116) with IC50 values of 1.17

and 16.5 nM, respectively. Additionally, microsporin A (**81**) displayed notable activity against the 60 cancer cell panel of the National Cancer Institute with mean IC50 of 2.7 μM [39] (Figure 4).

**Figure 4.** Peptides isolated from Asteromyces, Ceratodictyon, Clonostachys, Emericella, Exserohilum, and Microsporum species.

#### *2.10. Metarrhizium*

Destruxin cyclic depsipeptides, including A (**83**) and B (**84**), were reported to be produced by the sponge associated fungus, *Metarrhizium* sp., in addition to efrapeptins Eα (**4**), F (**6**), and G (**7**) [14] (Figure 5).

**Figure 5.** Peptides isolated from *Metarrhizium* and *Penicillium* species.

#### *2.11. Penicillium*

Many peptides were isolated from various *Penicillium* species, such as *Penicillium citrinum*, through the stimulation of silent genes using scandium chloride that resulted in the isolation of three new peptide derivatives (**85**–**87**). Compound (**85**) showed potent antibacterial activity against *S. aureus* with MIC value equals to 0.04 μM. However, compounds (**86–87**) displayed potent cytotoxic activity against MCF-7 and HCT115 cell lines showing IC50 equal to 38 and 37 nM, respectively, while compound (**85**) showed no activity [39]. Furthermore, penicimutide (**88**), which is a novel dipeptide in a cyclic form in addition to other previously reported dipeptides, namely cyclo (l-Phe-l-Pro) (**89**), cyclo (l-Leu-l-Pro) (**90**), cyclo (l-Ile-l-Pro) (**91**), and cyclo (l-Val-l-Pro) (**92**) were isolated from *P. purpurogenum*. Penicimutide **(88)** effectively inhibited growth of HeLa cells showing 39.4% as an inhibition rate at 100 μg/mL (0.5 μM) approaching that of the 5-fluorouracil that showed 41.4% at 100 μg/mL (0.77 μM) [40] (Figure 5).

Besides, penilumamide (**93**), as well as penilumamides B–D (**94**–**96**), were isolated from miscellaneous *Penicillium sp.* [41] in addition to gliocladine C (**97)**, cyclo-(Trp-Ala) (**98**), cyclo-(Phe-Pro) (**99**), and cyclo-(Gly-Pro) (**100**). Compound**s** (**97**–**100**) were assessed for their ability to inhibit HepG2 cells proliferation with different degrees in which gliocladine C (**97**) effectively inhibited HepG2 cells growth displaying IC50 value of 19.6 μM, whereas compounds (**98**–**100**) showed no relevant cytotoxic activity [42]. Moreover, *cis*-cyclo (Leucyl-Tyrosyl) (**101**) isolated from a sponge derived *Penicillium* species showed a significant inhibition to the biofilm formation estimated by 85% which consequently prevents bacterial growth that was further ascertained by a scanning electron microscope [43] (Figure 5).

#### *2.12. Scytalidium*

A marine fungus of the genus *Scytalidium* is highly popular for the production of linear, lipophilic peptides, termed the halovirs as halovirs A–E (**102**–**106**), which showed considerable in vitro antiviral activity against herpes simplex viruses of both type 1 and type 2. Further study on the structure activity relationship between the halovirs and their anti-viral activity showed that the presence of a *N*α-acyl chain, comprising of at least 14 carbons as well as the Aib-Pro dipeptide, are crucial to preserve the activity. Upon addition of Halovirs A (**102**), B (**103**), and C (**104**) to cells that are exposed to HSV-1 infection for 1 h, they displayed ED50 values of 1.1, 3.5, and 2.2 μM, respectively. Meanwhile, halovirs D (**105**) and E (**106**) showed ED50 values equal to 2.0 and 3.1 μM, respectively. Besides, halovir A (**102**) effectively prohibits HSV-1 replication showing ED50 value of 0.28 μM adopting the standard plaque reduction assay [44,45] (Figure 6).

**Figure 6.** Peptides isolated from *Scytalidium*, *Simplicillium*, and *Stachylidium* species.

#### *2.13. Simplicillium*

A series of simplicilliumtide peptides were isolated from the marine fungus *Simplicillium obclavatum* namely; simplicilliumtides A–M (**107**–**119**) in addition to verlamelins A and B (**120**–**121**). They showed variable activities including antibacterial, antifungal, antiviral, antifouling, cytotoxic, and acetyl cholinesterase inhibitory activity. Simplicilliumtide D (**110**) displayed a potent antifouling effect versus the larvae of *Bugula neritina* with EC50 equal to 0.02 μM and LC50/EC50 equal to 100; however, simplicilliumtides A (**107**), E (**111**), G (**113**), and H (**114**) exhibited weak cytotoxic effect against human leukemia HL-60 and K562 cell lines with IC50 above 100 μM. In addition, simplicilliumtide J (**116**) exhibited potent antifungal activity versus *Curvularia australiensis* as well as *Aspergillus versicolor* in addition to a potent anti - HSV-1 displaying IC50 value of 14.0 μM that could be interpreted in virtue of the presence of lactone linkage and a fatty acid chain moiety [10,46] (Figure 6).

#### *2.14. Stachylidium*

Many N-methylated peptides have been isolated from *Stachylidium* sp., a fungus associated with marine sponge, which include endolides A–D (**122**–**125**) which showed a wide range of biological activities [47]. Endolide A (**122**) revealed potent binding activity to the vasopressin receptor 1A displaying a Ki of 7.04 μM, whereas endolide B (**123**) showed a pronounced binding to serotonin receptor 5HT2b evidenced by its Ki value, which is 0.77 μM [48] (Figure 6).

#### *2.15. Talaromyces*

Talarolide A (**126**), in addition to a series of extensively N-methylated linear peptides named talaropeptides A–D (**127**–**130**), were isolated from *Talaromyces* sp., a fungus derived from a marine tunicate. Biological evaluation of the previously mentioned compounds revealed that compounds (**127**–**128**) only displayed pronounced antibacterial activity against the growth of *Bacillus subtilis*, a Gram positive bacterium, displaying IC50 values of 1.5 and 3.7 μM, respectively. Additionally compound talaropeptide A (**127)** showed a high stability to various rat proteases existing in plasma [49] (Figure 7).

#### *2.16. Trichoderma*

*Trichoderma* strains, derived from marine origin, afforded large amounts peptaibols, which are characterized by being small antimicrobial peptides (AMPs) that are expected to contribute to the antimicrobial defense of *Trichoderma. Trichoderma* strains produced 11- and 20-residue peptaibols; optimal yields with 1.4% and 2.3% of the fungal biomass for the 11- and 20-residue, respectively, were obtained on day 9 [50]. Generally, AMPs represent a large group of naturally occurring cationic and amphiphilic short peptides that act as first-line defense for many living organisms and are part of the innate immune system [51]. They act primarily via offering a protection to the host organisms versus the invasion of harmful organisms such as bacteria or fungi [52]. Their mode of action is completely different from traditional antibiotics as they modulate membrane stability and permeability. On the contrary, traditional antibiotics exert their effect mainly by interfering with bacterial metabolism, protein biosynthesis, or cell wall formation. Thus, low cross-resistance and an effective synergism could be achieved between AMPs and traditional antibiotics [53].

#### *2.17. Zygosporium*

*Zygosporium masonii*, a marine fungus that produced a cyclic depsipeptide termed zygosporamide (**131**) showed potent cytotoxic activity in the NCI's 60 cell line panel with median GI50 equal to 9.1 μM. However, it revealed a notable selectivity versus the (Central Nervous System) CNS cancer cell SF-268 with GI50 equal to 6.5 nM, as well as to the renal cancer cell line RXF 393 showing GI50 less than 5.0 nM [54,55] (Figure 7).

**Figure 7.** Peptides isolated from *Talaromyces* and *Zygosporium* species.

#### **3. Discussion and General Perspectives**

In our analysis we found that 131 peptides from marine sources were isolated from 17 fungal strains. These peptides are either cyclic, composed of two amino acids (dipeptide), as in case of *Ascotricha* up to nine amino acids (nonapeptides) in *Emericella* or linear pentadecapeptide as presented in *Acremonium*. Many depsipeptides (from *Acremonium*, *Metarrhizium*, and *Zygosporium*), peptaibols in *Trichoderma* and N-methylated peptides from *Acremonium*, *Stachylidium*, and *Talaromyces* are also characterized. It is clearly noticed that genus *Aspergillus* was the most extensively studied and it might represent a rich source of peptides with promising biological activity. However, there is no evidence about the chemotaxonomic relation between the production of a certain class of peptides in a specified genus. That could be explained based on the fact that the number of the isolated compounds is not enough to make an in-depth study about this relation. Besides, peptides from marine sources possess strongly modulated structures either in its backbone or side chain comparable to peptides of plant or human origin, which undoubtedly is due to the harsh requirements of the environment in which they live. It worthy to mention that although most of these peptides contain many functional groups, such as carbonyl and amide groups, beside esters as in the case of depsipeptides which gave them the ability to interact with many molecular targets in the cells, most of these peptides are either inactive or showed weak activity. That could be explained by the fact that most of these peptides are weakly soluble in physiological fluids that limit most of their in vivo activity [56].

It was found that about 53% of the isolated peptides exhibited various biological activities represented mainly by cytotoxic, antimicrobial, and antiviral activity, while few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity (Table 1).

**Table 1.** Bioactive peptides isolated from marine associated fungi, their sources, and biological activities.

**Table 1.** *Cont*.

**Table 1.** *Cont*.

Regarding the cytotoxic effect of the previously mentioned peptides, about 35 bioactive peptides revealed cytotoxic activities against a panel of cancer cells. Twenty five peptides showed potent cytotoxic activity however the other ten peptides exerted weak activity. This was represented by cordyheptapeptides C (**1**) and E (**3**), which exerted notable activity on MCF-7, SF-268, and NCI-H460 cancer cells with IC50 ranging between 2.5 and 12.1 μM. Besides, pronounced cytotoxic potential was exerted by efrapeptins Eα (**4**), F (**6**), and G (**7**) against H125 cells with IC50 values of about 1.3 nM. Efrapeptin G (**7**) also showed potent cytotoxic activity versus murine cancer cells. Moreover, aspergillicins A–E (**17**–**21**) exerted substantial activity with LD99 values ranging between 0.03 and 0.07 μM. Additionally, effective stimulation to HT-29 cancer cells differentiation was exerted by cyclo-(l-Trp- l-Tyr) (**30**)**.** Furthermore, asperterrestide A (**38**) showed a potent cytotoxic effect versus U937 and MOLT4 human cancer; meanwhile, psychrophilin E (**50**), selectivity prohibited the proliferation of HCT116 (colon) cell line with an IC50 value of 28.5 mM. In addition, the cytotoxic effect of dictyonamide A (**70**) can be explained in virtue of its inhibitory effect on cyclin-dependent kinase 4 that showed IC50 value equals to 0.01 μM. Rostratins A–D (**77**–**80**) showed pronounced cytotoxic activity versus (HCT-116) the human colon carcinoma showing 20, 4.48, 1.6, and 34 nM as IC50 values, respectively. Moreover, microsporins A and B (**81**–**82**) exerted an effective cytotoxic effect versus human colon adenocarcinoma (HCT-116). Additionally, *Penicillium citrinum* represents a source of cytotoxic peptides in which compounds (**86**–**87**) exerted significant cytotoxic activity on MCF-7 and HCT115 cell lines showing IC50s equal to 38 and 37 nM, respectively. Penicimutide (**88**) also showed an effective inhibitory effect on HeLa cells displaying 39.4% as an inhibition rate at 100 μg/mL (0.01 μM) approaching that of the positive control 5-fluorouracil which showed 41.4% at 100 μg/mL. Furthermore, gliocladine C (**97**) effectively inhibited HepG2 cells growth displaying IC50 value of 19.6 μM whereas, zygosporamide (**131**) which showed potent cytotoxic activity. Weak cytotoxic activities were exerted by sclerotide A (**31**), sclerotide B (**32**), cotteslosins A (**57**), and B (**58**), in addition to similanamide (**33**) which displayed weak cytotoxic activity versus MCF-7, A373, and NCI-H460 cancer cells. In addition, aspersymmetide A (**55**) showed weak cytotoxicity versus NCI-H292 as well as A431 cell lines at 10 μM; meanwhile, simplicilliumtide A (**107**), E (**111**), G (**113**), and H (**114**) exhibited weak cytotoxic effect versus either human leukemia HL-60 or K562 cell line.

Concerning the antimicrobial and antiviral activities, 23 peptides exerted pronounced activity, among which nine revealed a potent inhibitory effect on bacterial growth, three showed pronounced antifungal activity, whereas eleven exhibited promising antiviral activity. Pronounced antibacterial activity was observed for RHM (**8**) and efrapeptin G (**6**) against *Staphylococcus epidermidis* with MIC values equal to 0.015 and 0.049 μM, respectively. In addition, lajollamide A (**69**) showed a mild antimicrobial effect against Gram-positive bacteria exerting 61% and 30% inhibition in bacterial growth for *Bacillus subtilis* and *Staphylococcus epidermidis* at 100 μM. Besides, unguisin A (**42**) and emericellamide B (**76**) showed considerable antibacterial activity against MRSA (methicillin resistant *Staphylococcus aureus* strains) with MIC values equal to 3.8 and 6.0 μM, respectively, whereas compound (**85**) isolated from *Penicillium citrinum* also showed pronounced antibacterial activity against *S. aureus* displaying 0.04 μM as MIC. However, the effective antimicrobial activity of *cis*-cyclo (Leucyl-Tyrosyl) (**101**) can be explained in virtue of its effective inhibitory effect on bacterial biofilm formation, which is estimated by 85%. Additionally, talaropeptides A–B (**127**–**128**), showed notable antibacterial activity against the growth of *Bacillus subtilis*, a Gram positive bacterium, displaying IC50 values of 1.5 and 3.7 μM, respectively. Noteworthy to highlight is that *Trichoderma* strains afforded large amounts of peptaibols which are characterized by being small antimicrobial peptides (AMPs) that are expected to contribute to the antimicrobial defense of *Trichoderma.* For the antifungal activity, sclerotide A (**31**), sclerotide B (**32**), and simplicilliumtide J (**116**) showed notable antifungal activity.

A considerable number of fungal peptides showed powerful antiviral activity exemplified by asperterrestide A (**38**) which exerted potent potential antiviral activity versus an influenza virus of H1N1 as well as H3N2 strains that could be relied upon the presence of a 3-OH-N-CH3-Phe moiety which is rare in nature. Moreover, aspergillipeptides D–G (**62**–**65**), showed a pronounced antiviral effect versus herpes simplex virus type 1 (HSV-1) displaying IC50 of 9.5 and 19.8 μM, respectively. Halovirs A–E (**102**–**106**) showed considerable in vitro antiviral activity against herpes simplex viruses

of both type 1 and type 2 which is ultimately attributed to the presence of a *N*α-acyl chain, comprising of at least 14 carbons as well as the Aib-Pro dipeptide. However, simplicilliumtide J (**116**) exerted a potent anti - HSV-1 displaying IC50 value of 14.0 μM which could be interpreted in virtue of the presence of lactone linkage and a fatty acid chain moiety.

The anti-inflammatory activity of some of the isolated peptides can be explained via exerting different mechanisms where compounds (**60**–**61**) obtained from *Aspergillus* at 10 μM effectively inhibited IL-10 expression of the LPS-induced THP-1 cells showing 84.3% and 78.1% inhibition, respectively. Besides, 14-hydroxy-cyclopeptine (**66**) effectively inhibits nitric oxide production displaying IC50 of 0.14 μM in recombinant mouse interferon-γ -activated macrophage-like cell line.

Antidiabetic and lipid lowering activity was also observed for some of the isolated fungal peptides, such as terrelumamides A (**34**) and B (**35**), which displayed a promising improvement in insulin sensitivity as determined by the utilization of mesenchymal cells of a bone marrow origin obtained from human adopting an adipogenesis model. Additionally, psychrophilin G (**52**) exhibited pronounced lipid-reducing activity.

Additionally, miscellaneous activities were reported for the isolated bioactive peptides in which Simplicilliumtide D (**110**) displayed a potent antifouling effect versus the larvae of *Bugula neritina*. Moreover, endolide A (**122**) revealed potent binding activity to the vasopressin receptor 1A displaying a Ki of 7.04 μM whereas endolide B (**123**) showed a pronounced binding to serotonin receptor 5HT2b evidenced by its Ki value which is 0.77 μM. Talaropeptide A (**126**) showed a high stability to various rat proteases existing in plasma, however, microsporins A and B (**81**–**82**) effectively inhibit histone deacetylase.

Noteworthy to mention is that about 47% of the isolated peptides showed no activity with respect to the examined biological activity and thus required further in depth biological assessment. A pie chart summarizing the percentages of isolated peptides with respect to their biological activity is represented in Figure 8.

**Figure 8.** Percentages of isolated peptides with respect to their biological activity represented by a pie chart.

#### **4. Conclusions**

Herein, we concluded that about 131 peptides were isolated from marine sources from seventeen fungal strains. Some of them exhibited much biological activity represented mainly by cytotoxic, antimicrobial, and antiviral activity, meanwhile, few of them showed antidiabetic, lipid lowering, and anti-inflammatory activity; however, others should be deeply assessed for their biological potential. Thus, if active natural products are needed for drug development, further investigations of marine fungi are recommended. In addition, it is a challenge to explore more peptides from fungal origin and so it is recommended to confirm their biological activities.

**Author Contributions:** F.S.Y.; collection of data and writing the manuscript, M.L.A., A.N.B.S. and M.W.; revised the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors received financial support from the Deutsche Forschungsgemeinschaft and Ruprecht-Karls-Universität Heidelberg within the funding program Open Access Publishing. Also, the authors Fadia S. Youssef and Abdel Nasser B. Singab acknowledge Egyptian Science and Technology Development Funds (STDF) for its financial support through grant No. NCP 25927 entitled "Drug discovery of new anti-infective and anticancer agents from marine organisms and their associated micro-organisms".

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

#### **References**

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Proteomic Analysis of the Venom of Jellyfishes** *Rhopilema esculentum* **and** *Sanderia malayensis*

#### **Thomas C. N. Leung 1,**†**, Zhe Qu 2,**†**, Wenyan Nong 2, Jerome H. L. Hui 2,\* and Sai Ming Ngai 1,\***

Received: 27 November 2020; Accepted: 17 December 2020; Published: 18 December 2020

**Abstract:** Venomics, the study of biological venoms, could potentially provide a new source of therapeutic compounds, yet information on the venoms from marine organisms, including cnidarians (sea anemones, corals, and jellyfish), is limited. This study identified the putative toxins of two species of jellyfish—edible jellyfish *Rhopilema esculentum* Kishinouye, 1891, also known as flame jellyfish, and Amuska jellyfish *Sanderia malayensis* Goette, 1886. Utilizing nano-flow liquid chromatography tandem mass spectrometry (nLC–MS/MS), 3000 proteins were identified from the nematocysts in each of the above two jellyfish species. Forty and fifty-one putative toxins were identified in *R. esculentum* and *S. malayensis*, respectively, which were further classified into eight toxin families according to their predicted functions. Amongst the identified putative toxins, hemostasis-impairing toxins and proteases were found to be the most dominant members (>60%). The present study demonstrates the first proteomes of nematocysts from two jellyfish species with economic and environmental importance, and expands the foundation and understanding of cnidarian toxins.

**Keywords:** jellyfish; *Rhopilema esculentum*; *Sanderia malayensis*; proteome; venom; toxin

#### **1. Introduction**

Phylum Cnidaria Hatschek, 1888, is one of the most ancient phyla that can be traced back to Cambrian [1]. This phylum is divided into five classes: Anthozoa Ehrenberg, 1834 (corals and sea anemones), Cubozoa Werner, 1973 (box jellyfish), Hydrozoa Owen, 1843, Scyphozoa Götte, 1887 (true jellyfish), and Staurozoa Marques & Collins, 2004 (stalked jellyfish) [2]. Approximately 12,000 extant species are known from freshwater and marine habitats worldwide, from shallow coastal waters to the deep seas [3,4]. Cnidarians are characterized by the presence of nematocytes, which constitute an important specialized cell type that assists prey capture and predator deterrence. Each nematocyte houses a unique organelle called the nematocyst. This Golgi apparatus-derived organelle consists of a capsule containing an inverted tubule immersed in a mixture of venomous substances [5]. Upon mechanical or chemical stimulation, the tubule everts and injects a venomous mixture into the prey or predator. Jellyfish possess nematocytes mainly on the tentacles [6]. The composition of the venom inside the nematocyst varies between different jellyfish species and may be a mixture of proteinaceous and non-proteinaceous toxins with hemolytic, neurotoxic, cytotoxic, and dermonecrotic activities [6]. Depending on the composition of the venom, the symptoms of jellyfish envenomation vary from species to species, ranging from mild local symptoms such as pruritus, pain, and burning sensation to serious manifestations, including hypotension, cardiac failure, respiratory distress, pulmonary edema, and even death [6–9].

Despite the potential hazard to health, evidence is accumulating for jellyfish venoms as promising sources of therapeutic agents. For example, peptide fractions of *Chrysaora quinquecirrha* Desor, 1848, venom have shown anticancer activities in an Ehrlich ascites carcinoma (EAC) tumor mouse model [10]. Similarly, *Nemopilema nomurai* Kishinouye, 1922, crude venom also exhibits anticancer activities in the HepG2 liver cancer cell line and HepG2 xenograft mouse models [11]. Furthermore, the crude venom of *Pelagia noctiluca* Forsskal, 1775, and its peptide fractions not only demonstrate anticancer activities in several cancer cell lines, but also show anti-inflammatory activities [12]. The protein components of *P. noctiluca* venom also exhibit analgesic functions in mouse models, and it has also been suggested that *P. noctiluca* venom is a promising source of neuroprotective drugs due to its plasma antibutyrylcholinestrasic activities [13]. However, the individual components that exhibit the therapeutic functions are not well characterized because the compositions of jellyfish venoms are not well studied, considering that of the 7235 animal toxins and venom proteins recorded in the Tox-Prot database, only six are derived from jellyfish (as of October 2020 [14]). This knowledge gap greatly hinders the discovery of potential drug candidates in jellyfish venom.

Recently, our group reported high-quality de novo reference genomes and transcriptomes for the edible jellyfish *Rhopilema esculentum* and the Amuska jellyfish *Sanderia malayensis*, as well as their transcriptomes [15]. Herein, we extend this study to identify the putative toxins in these two jellyfish species at the protein level. In this work, the venoms were enriched by isolating the nematocytes using the density gradient centrifugation technique. The nematocyte proteomes were determined by nano-flow liquid chromatography tandem mass spectrometry (nLC–MS/MS) and the putative toxins were identified by subsequent bioinformatic analysis.

This study provides the first insights into the venom proteomes of *R. esculentum* and *S. malayensis,* not only facilitating the screening, isolation, and characterization of their novel therapeutic compounds, but also providing clues to the evolutionary and ecological role of these toxins.

#### **2. Results**

#### *2.1. Transcriptome and Protein Database Construction*

Next-generation sequencing (NGS) was used to construct the *R. esculentum* appendages and the *S. malayensis* tentacle transcriptome followed by gene model predictions using funannotate [15]. Based on the results of transcriptomic analysis, the*R. esculentum* and *S. malayensis* protein databases were generated with 18,923 and 26,914 protein sequences, respectively. Gene Ontology (GO) analysis was performed by the eggNOG-mapper [16] and annotations were assigned to three primary GO domains: biological process (BP), cellular component (CC), and molecular function (MF). In total, 8786 (46.43%) *R. esculentum* proteins and 9138 (33.95%) *S. malayensis* proteins were successfully annotated with 143,350 and 153,009 GO terms, respectively (Table 1 and Figure 1A,B). In addition, 4187 and 4485 enzymes were identified in *R. esculentum* and *S. malayensis*, respectively, classified according to their Enzyme Commission (EC) number. The proportional distributions of the enzymes in both species were similar, which were dominated by transferases and hydrolases (Table 1 and Figure 2). Furthermore, to identify and annotate the putative toxins, the protein sequences generated were run against the UniProt animal venom proteins and toxins database (Tox-Prot) using BLASTp [17]. Protein sequences with an e-value of <1.0 × 10−5 to entries in the database were used as input into "ToxClassifier" to exclude non-toxic homologs [18]. A total of 190 and 186 putative toxins were found in *R. esculentum* and *S. malayensis*, respectively. The toxin profiles of both species were similar in which the toxins could be classified into eight toxin families: hemostasis-impairing toxins, proteases, phospholipases, neurotoxins, cysteine-rich proteins, protease inhibitors, pore-forming toxins, and other toxins (Tables S1 and S2).

**Table 1.** Description of the analysis of the protein databases generated from the *R. esculentum* and *S. malayensis* transcriptomes.

**Figure 1.** *Cont*.

**Figure 1.** The 10 most represented Gene Oncology (GO) terms of the (**A**) *Rhopilema esculentum* and (**B**) *Sanderia malayensis* protein databases in the three domains of biological process (BP), molecular function (MF), and cellular component (CC) are presented.

#### *2.2. Identification of* R. esculentum *and* S. malayensis *Nematocyst Proteins by nano-LC-ESI MS*/*MS*

The nematocysts from *R. esculentum* and *S. malayensis* were purified and their protein profiles were revealed by proteomic analysis. A total of 3083 and 3559 proteins were identified in *R. esculentum* and *S. malayensis*, respectively (Tables S3 and S4), including 40 *R. esculentum* and 51 *S. malayensis* putative toxins. According to their predicted biological function, these toxins were classified into the eight toxin families (Table 2, Tables S5 and S6). The proportional distributions of these toxin families were also similar between the two species (Figure 3). Hemostasis-impairing toxins comprised the most abundant class of identified toxins, representing 32.5% and 39.2% of the *R. esculentum* and *S. malayensis* toxins, respectively, most of which were homologous to ryncolin, a family of proteinaceous toxins originally described from *Cerberus rynchops*. This family also includes a variety of C-type lectins (i.e., C-type lectin, C-type lectin lectoxin-Lio2, and galactose-specific lectin nattectin). In addition, other toxins such as prothrombin activator, coagulation factor V, coagulation factor X, and snaclec bothrojaracin subunit homologs were also identified in the proteome of both species.

Proteases comprised the second most predominant toxin family in both *R. esculentum* and *S. malayensis* proteome, accounting for 27.5% and 21.5% of the *R. esculentum* and *S. malayensis* putative toxins, respectively. Among this family, metalloproteinases were the most abundant. In the *R. esculentum* toxin proteome, three out of the seven metalloproteinases found were homologous to zinc metalloproteinase-disintegrin proteins. Meanwhile, a further two were neprilysin-1 homologs, another two were homologs of astacin-like metalloproteases. In the *S. malayensis* toxins, nine metalloproteinases were found, four of which were zinc metalloproteinase-disintegrin proteins. Four astacin-like metalloproteases and one neprilysin-1 were also detected.

Besides these two major classes of toxins, the *R. esculentum* and *S. malayensis* venoms also exhibited similar proportional distributions of other toxins. Meanwhile, l-amino-acid oxidase, acetylcholinesterase, and venom acid phosphatase were only found in *S. malayensis* venom, and U-actitoxin-Avd3j and calglandulin were only detected in *R. esculentum* venom.

**Figure 2.** Distribution of the enzyme predicted by eggNOG-mapper in the protein databases of the (**A**) *R. esculentum* and (**B**) *S. malayensis* jellyfishes.

**Table 2.** Toxin families identified in each jellyfish species.

58

Q5BLY5

 Venom acid phosphatase Acph-1 (EC <IP_ADDRESS>) (allergen Api m 3)

**Table 2.** *Cont.*

**Figure 3.** Graphical overview of the distribution of the toxin families identified in the *R. esculentum* and *S. malayensis* proteomes.

#### *2.3. Functional Analysis of the Putative Toxins*

A total of 282 and 408 GO terms were assigned to 20 (50%) *R. esculentum* and 27 (54.9%) *S. malayensis* putative toxins, respectively (Tables S3 and S4). The 10 most represented GO terms in the three domains of biological process (BP), cellular component (CC), and molecular function (MF) are shown in Figure 4. Furthermore, the presence of signal peptides was predicted by SignalP, showing that 52.5% and 29.4% of the *R. esculentum* and *S. malayensis* putative toxins contain secretory signal peptides, respectively. Moreover, DeepLoc analysis indicated that 52.5% and 54.9% of the putative toxins were located in the extracellular region. Taken together, there 75% of the putative toxins in *R. esculentum* and 62.7% in *S. malayensis* were predicted as extracellular proteins (by SignalP and/or DeepLoc). Additionally, 16 and 20 enzymes were identified in the *R. esculentum* and *S. malayensis* putative toxins. In both species, hydrolase (EC 3) was the predominant enzyme (13 and 16 of *R. esculentum* and *S. malayensis* putative toxins, respectively; Figure 5A,C), the majority of which was comprised by esterase (EC3.1) and peptidase (EC 3.4) (Figure 5B,D).

**Figure 4.** The 10 most represented GO terms of the (**A**) *R. esculentum* and (**B**) *S. malayensis* potential toxins in the three domains of biological process (BP), molecular function (MF), and cellular component (CC) are presented.

**Figure 5.** Distribution of the enzymes predicted by eggNOG-mapper in the putative toxins of (**A**) *R. esculentum* and (**B**) *S. malayensis* and the proportional distribution of the hydrolase subclass in (**C**) *R. esculentum* and (**D**) *S. malayensis*.

#### *2.4. InterProScan Analysis*

The protein domains of the putative toxins were annotated by InterProScan. A total of 47 and 55 protein domains were assigned to the *R. esculentum* and *S. malayensis* putative toxins, respectively (Tables S7 and S8). The top ten most represented domains are shown in Figure 6. In general, the results agreed with the BLASTp-ToxClassifier results. Two domains related to hemostasis were found, namely, fibrinogen, alpha/beta/gamma chain, C-terminal globular domain, and coagulation factor 5/8 C-terminal domain, the former being highly represented in both species. Although there were no predominant single protease-related domains, a variety of protease-related domains (peptidase M12B, peptidase M12A, astacin-like metallopeptidase domain, serine proteases/trypsin domain, and disintegrin domain) were found. Furthermore, the domains related to cysteine-rich proteins (CAP domain and SCP domain), the C-type lectin-like domain, and the phospholipase A2 domain were screened in both species. In addition, the ShKT domain and the Kunitz domain, the protein domains with high potential therapeutic value, were also detected in the putative toxins of both species. A total of three and six ShKT domain-containing proteins were identified in the *R. esculentum* and *S. malayensis* putative toxins, respectively, most (seven out of nine) of which were protease-type toxins. In these ShKT domain-containing proteases, the ShKT domain is either associated with the trypsin domain or the peptidase\_M12A domain (a metalloproteases domain). Although the sequence of the ShKT domain found in trypsin was slightly different form that of metalloproteases, both of them were characterized by the typical pattern of six cysteines, except the second ShKT domain of Sma\_022066-T1 (Sma\_022066-T1\_M12\_ShKT\_2), which lacked the fifth cysteine residue of the motif (Figure 7A,B). Furthermore, a total of five Kunitz domains were detected in four protease inhibitor-type putative toxins, with Sma\_015170-T1 containing two domains. Most of the detected Kunitz domains displayed the conserved motif of C-8X-C-15X-C-4X-YGGC-12X-C-3X-C. The only exception was the Kunitz domains of Sma\_021821-T1, which deviated from the conserved architecture by a Y31F substitution (Figure 7C).