The Chemistry, Biochemistry and Pharmacology of Marine Natural Products from Leptolyngbya, a Chemically Endowed Genus of Cyanobacteria

marine drugs

Review The Chemistry, Biochemistry and Pharmacology of Marine Natural Products from Leptolyngbya, a Chemically Endowed Genus of Cyanobacteria

Yueying Li 1,2, C. Benjamin Naman 2,3 , Kelsey L. Alexander 2,4 , Huashi Guan 1,* and William H. Gerwick 2,5,*

1 Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; [email protected] 2 Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, CA 92093, USA; [email protected] (C.B.N.); [email protected] (K.L.A.) 3 Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Department of Marine Pharmacy, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315800, China 4 Department of Chemistry and Biochemistry, University of California, San Diego, CA 92093, USA 5 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA 92093, USA * Correspondence: [email protected] (H.G.); [email protected] (W.H.G.)

Received: 5 September 2020; Accepted: 2 October 2020; Published: 6 October 2020

Abstract: Leptolyngbya, a well-known genus of cyanobacteria, is found in various ecological habitats including marine, fresh water, swamps, and rice ﬁelds. Species of this genus are associated with many ecological phenomena such as nitrogen ﬁxation, primary productivity through photosynthesis and algal blooms. As a result, there have been a number of investigations of the ecology, natural product chemistry, and biological characteristics of members of this genus. In general, the secondary metabolites of cyanobacteria are considered to be rich sources for drug discovery and development. In this review, the secondary metabolites reported in marine Leptolyngbya with their associated biological activities or interesting biosynthetic pathways are reviewed, and new insights and perspectives on their metabolic capacities are gained.

Keywords: Leptolyngbya; cyanobacteria; secondary metabolites

1. Introduction Cyanobacteria, also known as ‘blue-green algae’, rank among the oldest prokaryotes found on Earth and evolved to possess remarkable capabilities of oxygenic photosynthesis and nitrogen fixation, allowing them to both adapt to and affect the planet’s environmental conditions for over two billion years [1–4]. In traditional classification schema, filamentous cyanobacteria without akinetes, heterocysts or true-branching were generally assigned to the order Oscillatoriales (Section III) [5]. However, one specific genus of filamentous, nonheterocystous cyanobacteria with granular surface ornamentation was ultimately named Leptolyngbya in 1988, and placed in the order Pseudanabaenales [6–8]. The identification of this genus was supported by phylogenetic analysis, including 16S rRNA gene sequences, which at the time showed no clear relationship with any other defined genus of cyanobacteria [9]. Leptolyngbya originate from a diverse range of ecological habitats, including marine, fresh water, swamps, forests, rice fields, alkaline lakes, and even the polar desert or hot desert environments [10–13]. Members of this genus possess a variety of thin (0.5–3.5 µm), long filaments, and can grow solitarily or coiled into clusters and fine mats [14–16]. Heterocysts and akinetes are both absent in these organisms [17].

Mar. Drugs 2020, 18, 508; doi:10.3390/md18100508 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 508 2 of 29

In the taxonomic databases Algaebase [18] and CyanoDB [19] there are 138 taxonomically accepted species of Leptolyngbya listed. Marine collections of Leptolyngbya have been reported in Japan, Panama, Gulf of Thailand, the Red Sea, Hawaii, America Samoa, and others (Table1), which illustrates that this genus is distributed widely in the world. A breadth of scientific studies have been conducted on these samples in recent decades, including morphological and phylogenetic characterization, photosynthesis and nitrogen fixation research, examination of the associated microbial community, growth modulation and natural product chemistry research [4,9,17,20–22]. It is well known that cyanobacteria produce many secondary metabolites, especially when grown in different environments and conditions. Variations such as temperature, pH, dissolved phosphorous levels, nitrogen content or associated symbionts can influence the expression of different natural products [22,23]. The metabolites of cyanobacteria are known to possess potent biological activity in areas such as cytotoxicity, anti-inflammation, neuromodulatory, antibacterial, and brine shrimp toxicity [24,25]. It is possible that these observed biological activities match or mimic some of the natural ecological roles of these secondary metabolites. A limited number of Leptolyngbya field collections have been propagated in laboratory cultures [26–28]. However, they grow slowly in this environment, making for additional challenges in secondary metabolite discovery from this resource [29]. This review summarizes recent research on the secondary metabolites found in the genus Leptolyngbya, and covers chemical, pharmacological, and biosynthetic aspects. Mar. Drugs 2020, 18, 508 3 of 29

Table 1. Secondary metabolites from Leptolyngbya and the reported bioactivities from each.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference

Cytotoxicity MDA-MB-231 IC50 3.9 nM N a N a Y b Cytotoxicity A549 IC50 3.6 nM [30]

Cytotoxicity MCF-7 IC50 35.7 nM

Cytotoxicity MDA-MB-231 GI50 5.0 nM a a b N N Y Cytotoxicity A549 GI50 5.4 nM [31] Cytotoxicity PANC-1 GI50 3.1 nM

Cytotoxicity H460 LC50 < 23 nM Cytotoxicity Mouse neuro2a LC < 23 nM Coibamide A (1) 50 Cytotoxicity MDA-MB-231 GI50 2.8 nM Coiba National Park, Cytotoxicity LOX IMVI GI 7.4 nM N a N a 50 [32] Panama Cytotoxicity HL-60(TB) GI50 7.4 nM Cytotoxicity SNB-75 GI50 7.6 nM Histological Breast, CNS, colon, Good Selectivity and ovarian cancer cells Cytotoxicity U87-MG EC 28.8 nM Coiba National Park, 50 N a N a Cytotoxicity SF-295 glioblastoma cell EC 96.2 nM [33] Panama 50 Cytotoxicity MEFs EC50 96.2 nM Cytotoxicity COLO205 IC 11.5 µM 50 [33] Cytotoxicity H460 45% inhibition at 20 µM

Cytotoxicity MDA-MB-231 IC50 17.98 µM Cytotoxicity MCF-7 IC50 11.77 µM [34] N a N a Y b Cytotoxicity A549 IC50 22.80 µM

Cytotoxicity MDA-MB-231 GI50 > 16000 nM Synthetic l-HVA, l-MeAla-Coibamide A (2) Cytotoxicity A549 GI50 22800 nM [31] Cytotoxicity PANC-1 ND c

Cytotoxicity H292 IC50 124 nM Cytotoxicity MDA-MB-231 IC 66 nM N a N a N a 50 [35] Cytotoxicity PC-3 IC50 80 nM Cytotoxicity SF-295 IC50 219 nM Synthetic O-Desmethyl, l-HVA, Cytotoxicity COLO205 IC 13 µM N a N a b 50 [33] l-MeAla-Coibamide A (3) Y Cytotoxicity H460 36% inhibition at 20 µM Mar. Drugs 2020, 18, 508 4 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference

Cytotoxicity A549 IC50 19.0 nM Cytotoxicity HCT116 IC50 44.6 nM Cytotoxicity MCF-7 IC50 48.6 nM Cytotoxicity B16 IC50 54.4 nM Cytotoxicity H292 IC50 610 nM [35] Cytotoxicity MDA-MB-231 IC50 545 nM l d a a b Synthetic -HVA, -MeAla-Coibamide A (4) N N Y Cytotoxicity PC-3 IC50 424 nM Cytotoxicity SF-295 IC50 816 nM

Cytotoxicity MDA-MB-231 GI50 545 nM

Cytotoxicity A549 GI50 19 nM [31] Cytotoxicity PANC-1 ND c

Cytotoxicity MDA-MB-231 GI50 7518 nM a a b Synthetic Coibamide A-1c (5) N N Y Cytotoxicity A549 GI50 20091 nM [31] Cytotoxicity PANC-1 GI50 12417 nM

Cytotoxicity MDA-MB-231 GI50 10809 nM Synthetic Coibamide A-1d (6) N a N a Y b Cytotoxicity A549 ND c [31] Cytotoxicity PANC-1 ND c

Cytotoxicity MDA-MB-231 GI50 2662 nM a a b Synthetic Coibamide A-1e (7) N N Y Cytotoxicity A549 GI50 1995 nM [31] Cytotoxicity PANC-1 GI50 1906 nM

Cytotoxicity MDA-MB-231 GI50 5.1 nM a a b Synthetic MeAla3-MeAla6-Coibamide A-1f (8) N N Y Cytotoxicity A549 GI50 7.3 nM [31] Cytotoxicity PANC-1 GI50 7.0 nM

Cytotoxicity MDA-MB-231 GI50 5.3 nM a a b Synthetic Coibamide A-1g (9) N N Y Cytotoxicity A549 GI50 12.4 nM [31] Cytotoxicity PANC-1 GI50 32.9 nM

Cytotoxicity MDA-MB-231 GI50 61.6 nM a a b Synthetic Coibamide A-1h (10) N N Y Cytotoxicity A549 GI50 81.7 nM [31] Cytotoxicity PANC-1 GI50 124 nM Mar. Drugs 2020, 18, 508 5 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference

Cytotoxicity MDA-MB-231 GI50 20.8 nM a a b Synthetic Coibamide A-1i (11) N N Y Cytotoxicity A549 GI50 194 nM [31] Cytotoxicity PANC-1 GI50 46.3 nM

Cytotoxicity MDA-MB-231 GI50 2056 nM Synthetic Coibamide A-1j (12) N a N a Y b Cytotoxicity A549 ND c [31] Cytotoxicity PANC-1 GI50 2178 nM

Cytotoxicity MDA-MB-231 GI50 183 nM a a b Synthetic Coibamide A-1k (13) N N Y Cytotoxicity A549 GI50 222 nM [31] Cytotoxicity PANC-1 GI50 277 nM

Cytotoxicity MDA-MB-231 GI50 450 nM a a b Synthetic Coibamide A-1l (14) N N Y Cytotoxicity A549 GI50 473 nM [31] Cytotoxicity PANC-1 GI50 601 nM

Cytotoxicity MDA-MB-231 GI50 415 nM a a b Synthetic Coibamide A-1m (15) N N Y Cytotoxicity A549 GI50 511 nM [31] Cytotoxicity PANC-1 GI50 723 nM

Cytotoxicity MDA-MB-231 GI50 >16000nM Synthetic Coibamide A-1n (16) N a N a Y b Cytotoxicity A549 ND c [31] Cytotoxicity PANC-1 ND c

Cytotoxicity MDA-MB-231 GI50 470 nM a a b Synthetic Coibamide A-1o (17) N N Y Cytotoxicity A549 GI50 733 nM [31] Cytotoxicity PANC-1 GI50 828 nM

Cytotoxicity MDA-MB-231 GI50 236 nM a a b Synthetic Coibamide A-1p (18) N N Y Cytotoxicity A549 GI50 360 nM [31] Cytotoxicity PANC-1 GI50 204 nM

Cytotoxicity MDA-MB-231 GI50 239 nM a a b Synthetic Coibamide A-1q (19) N N Y Cytotoxicity A549 GI50 443 nM [31] Cytotoxicity PANC-1 GI50 415 nM

Cytotoxicity MDA-MB-231 GI50 >16000 nM Synthetic Coibamide A-1r (20) N a N a Y b Cytotoxicity A549 ND c [31] Cytotoxicity PANC-1 ND c Mar. Drugs 2020, 18, 508 6 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference

HeLa cells IC50 > 1 µM[36] KB (human nasopharyngeal MICs <0.05 µg/mL carcinoma cell line) [37] Dolastatin 12 (21) The Red Sea Y b N a Cytotoxicity LoVo (a human colon adenocarcinoma 0.08 µg/mL cell line) Murine P388 ED 7.5 10 2 µg/mL [38,39] lymphocytic leukemia 50 × − b a Ibu-Epidemethoxylyngbyastatin 3 (22) The Red Sea Y N Cytotoxicity HeLa cells IC50 > 10 µM[36]

Cytotoxicity HeLa cells IC50 335 nM Grassypeptolide D (23) Mouse neuro2a Cytotoxicity IC 599 nM [36] blastoma cells 50 The Red Sea Y b N a Cytotoxicity HeLa cells IC50 192 nM Grassypeptolide E (24) Mouse neuro2a Cytotoxicity IC 407 nM [36] blastoma cells 50 Bovine pancreatic IC 0.24 µM chymotrypsin 50 Antiproteolytic Porcine pancreatic Loggerpeptin A (25) Florida, USA a a IC 0.24 µM [40] N N Activity elastase 50 Human neutrophil IC 0.29 µM elastase 50 Bovine pancreatic IC 0.22 µM chymotrypsin 50 Antiproteolytic Porcine pancreatic Loggerpeptin B (26) Florida, USA a a IC 0.28 µM [40] N N Activity elastase 50 Human neutrophil IC 0.89 µM elastase 50 Bovine pancreatic IC 0.35 µM chymotrypsin 50 Antiproteolytic Porcine pancreatic Loggerpeptin C (27) Florida, USA a a IC 0.54 µM [40] N N Activity elastase 50 Human neutrophil IC 0.62 µM elastase 50 Mar. Drugs 2020, 18, 508 7 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference Bovine pancreatic IC 0.24 µM chymotrypsin 50 Antiproteolytic Porcine pancreatic Molassamide (28) Florida, USA a a IC 0.05 µM [40] N N Activity elastase 50 Human neutrophil IC 0.11 µM elastase 50 Antibacterial Vibrio harveyi MIC 250–1000 µg/mL Activities 2-Hydroxyethyl-11-Hydroxyhexadec-9-Enoate b a Gulf of Thailand Y N Antibacterial [41] (29) Vibrio parahaemolyticus MIC 350–1000 µg/mL Activities LPS-stimulated Anti-Inflammatory RAW264.7 murine IC 4.0 µM Activity 50 macrophages Antioxidant Radical Oxygen No activity at 146 µM Activity Scavenging [42] QS-Inhibitory V. harveyi BB120 IC50 5.6 µM Honaucin A (30) Hawaii, USA N a N a activities QS-Inhibitory E. coli JB525 IC 38.5 µM activities 50 Cytotoxicity RAW264.7 cells No activity at 1 µg/mL RANKL-induced Cellular TRAP [43] osteoclastogenesis in IC 0.63 µg/mL Activity 50 RAW264.7 cells LPS-stimulated Anti-Inflammatory RAW264.7 murine IC 4.5 µM Activity 50 macrophages Honaucin B (31) Hawaii, USA N a N a QS-Inhibitory [42] V. harveyi BB120 IC 17.6 µM activities 50 QS-Inhibitory E. coli JB525 IC > 500 µM activities 50 LPS-stimulated Anti-Inflammatory RAW264.7 murine IC 7.8 µM Activity 50 macrophages Honaucin C (32) Hawaii, USA N a N a QS-Inhibitory [42] V. harveyi BB120 IC 14.6 µM activities 50 QS-Inhibitory E. coli JB525 IC > 500 µM activities 50 Mar. Drugs 2020, 18, 508 8 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference Cytotoxicity RAW264.7 cells No activity at 1 µg/mL RANKL-induced Synthetic Br-Honaucin A (33) N a N a b Cellular TRAP [43] Y osteoclastogenesis in IC 0.54 µg/mL Activity 50 RAW264.7 cells 71.6% cell viability at Cytotoxicity RAW264.7 cells 1 µg/mL Synthetic Hex-Honaucin A (34) N a N a b RANKL-induced [43] Y Cellular TRAP osteoclastogenesis in IC50 0.68 µg/mL Activity RAW264.7 cells Cytotoxicity RAW264.7 cells No activity at 1 µg/mL RANKL-induced Synthetic I-Honaucin A (35) N a N a b Cellular TRAP [43] Y osteoclastogenesis in IC 0.61 µg/mL Activity 50 RAW264.7 cells

Cytotoxicity HeLa S3 cell IC50 0.099 µM a b Actin-Depolymerizing Leptolyngbyolide A (36) Okinawa, Japan N Y F-actin EC 12.6 µM [44] activity 50

Cytotoxicity HeLa S3 cell IC50 0.16 µM a b Actin-Depolymerizing Leptolyngbyolide B (37) Okinawa, Japan N Y F-actin EC 11.6 µM [44] activity 50

Cytotoxicity HeLa S3 cell IC50 0.64 µM a b Actin-Depolymerizing Leptolyngbyolide C (38) Okinawa, Japan N Y F-actin EC 26.9 µM [44] activity 50

Cytotoxicity HeLa S3 cell IC50 0.15 µM a b Actin-Depolymerizing Leptolyngbyolide D (39) Okinawa, Japan N Y F-actin EC 21.5 µM [44] activity 50 2+ Ca Inﬂux Murine cerebrocortical IC50 3.70 µM (Inhibition) neurons (2.29–5.98 µM, 95% CI) + a b Na Channel Mouse neuroblastoma Palmyrolide A (40) Palmyra Atoll N Y IC 5.2 µM [45] Blocking Activity (neuro2a) 50 Cytotoxicity H460 No activity at 20 µM Brine Shrimp Phormidolide (42) The Red Sea Y b N a LC 1.5 µM[46] Toxicity 50 Mar. Drugs 2020, 18, 508 9 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference

Cytotoxicity H460 cells EC50 0.9 µM Saccharomyces cerevisiae Cytotoxicity IC 14.6 µM [47] ABC16-monster 50 Brine Shrimp Brine shrimp LD 1 µg/mL Toxicity (Artemia salina) 50 Kalkipyrone A (43) a a America Samoa N N Goldﬁsh Carassius Ichthyotoxicity LD 2 µg/mL auratus 50 [48] Modestly inhibitory to NCI’s 60 human tumor Cytotoxicity several renal and cell line melanoma cell lines

Cytotoxicity H460 cells EC50 9.0 µM Kalkipyrone B (44) a a Saccharomyces cerevisiae [47] America Samoa N N Cytotoxicity IC 13.4 µM ABC16-monster 50 Adipogenic 3T3-L1 cells EC 420 nM Differentiation 50 [49] Cytotoxicity 3T3-L1 cells IC50 > 50 µM Cytotoxicity HeLa IC50 > 50 µM Yoshinone A (45) Ishigaki island, Japan N a Y b Saccharomyces cerevisiae Cytotoxicity IC 63.8 µM ABC16-monster 50 [47] Cytotoxicity H460 cells EC50 > 10 µM Adipogenic Yoshinone B1 (46) Ishigaki island, Japan N a N a 3T3-L1 cells <50% inhibition at 5 µM[49] Differentiation Adipogenic Yoshinone B2 (47) Ishigaki island, Japan N a N a 3T3-L1 cells <50% inhibition at 5 µM[49] Differentiation H460 human lung cancer Cytotoxicity IC 30 µg/ mL cells 50 Na+ Influx Mouse neuroblastoma IC (Activation and 50 (neuro2a) 20 µg/mL(Activation) Inhibition) Crossbyanol A (48) Hawaii, USA N a N a [50] Methicillin-resistant Antibacterial Staphylococcus aureus No activity at 125 µg/mL Activity (MRSA) Brine Shrimp Brine shrimp No activity at 25 µg/mL Toxicity (Artemia salina) Mar. Drugs 2020, 18, 508 10 of 29

Table 1. Cont.

Name Geographic Location Culture Total Synthesis Bioactivity Cell Line Activity Reference H460 human lung Cytotoxicity No activity at 20 µg/mL cancer cells Na+ Influx Mouse neuroblastoma (Activation and No activity at 20 µg/mL (neuro2a) Inhibition) Crossbyanol B (49) Hawaii, USA N a N a [50] Methicillin-resistant Antibacterial Staphylococcus aureus MIC 2.0-3.9 µg/mL activity (MRSA) Brine Shrimp Brine shrimp IC 2.8 µg/mL Toxicity (Artemia salina) 50 H460 human lung Cytotoxicity No activity at 20 µg/mL cancer cells Crossbyanol C (50) Hawaii, USA N a N a Na+ Influx [50] Mouse neuroblastoma (Activation and No activity at 20 µg/mL (neuro2a) Inhibition) H460 human lung Cytotoxicity No activity at 20 µg/mL cancer cells Crossbyanol D (51) Hawaii, USA N a N a Na+ Influx [50] Mouse neuroblastoma (Activation and No activity at 20 µg/mL (neuro2a) Inhibition) Leptazoline A (52) Honolulu Y b N a Cytotoxicity PANC-1 No significant activity [51] b a Leptazoline B (53) Honolulu Y N Cytotoxicity PANC-1 GI50 10 µM a Not found in literature. b Found in literature. c Not determined. Mar. Drugs 2020, 18, 508 11 of 29

Mar. Drugs 2020, 18, x FOR PEER REVIEW 9 of 27 2. Chemical Diversity of the Secondary Metabolites Isolated from Leptolyngbya 2. Chemical Diversity of the Secondary Metabolites Isolated from Leptolyngbya 2.1. Polypeptides 2.1. InPolypeptides 2008, McPhail and co-workers reported the discovery of coibamide A (1), a cyclic depsipeptide with aIn high 2008, degreeMcPhail of andN -co-workers and O-methylation reported the from discovery a Leptolyngbya of coibamidespecies A (1), a collected cyclic depsipeptide in Panama (Figurewith a1 high)[ 32 degree]. The of absolute N- and O configuration-methylation from was a determined Leptolyngbya by species a series collected of chiral in Panama HPLC (Figure analyses of1) the [32]. amino The absolute acids resulting configuration from the was hydrolyzed determined peptide by a series as well of chiral as some HPLC computational analyses of the modeling. amino Interestacids resulting in coibamide from Athe (1 hydrolyzed) mounted duepeptide to its as exquisitely well as some potent computational low-nanomolar modeling.in vitro Interestinhibition in activitycoibamide against A (1 multiple) mounted cancer due to cell its lines, exquisitely including potent human low-nanomolar NCI-H460, in MDA-MB-231, vitro inhibition H292, activity PC-3, SF-295,against mouse multiple neuro2a, cancer LOXcell lines, IMVI, including HL-60(TB), human and NCI-H460, SNB-75 cell MDA-MB-231, lines [32]. The H292, number PC-3, of cellSF-295, lines wasmouse expanded neuro2a, to includeLOX IMVI, human HL-60(TB), U87-MG, and SF-295 SNB-75 glioblastoma cell lines cells [32]. and The mouse number embryonic of cell lines fibroblasts was (MEFs),expanded and to itinclude was further human revealed U87-MG, that SF-295 the glioblastoma cyclized structure cells and was mouse crucial embryonic for potent fibroblasts biological activity(MEFs), [ 52and]. Severalit was studiesfurther subsequentlyrevealed that the reported cyclized the structure total synthesis was crucial of coibamide for potent A ( 1biological). Yao and Limactivity et al. [52]. completed Several the studies total synthesissubsequently of the re structureported the originally total synthesis reported of forcoibamide coibamide A (1 A). (Yao2), as and well asLim a synthetic et al. completedO-desmethyl the total analogue. synthesis However, of the structure both the originally1H and reported13C NMR for data coibamide for the A synthetic (2), as productwell as 2a disyntheticffered from O-desmethyl those of theanalogue. natural However, product, whichboth the indicated 1H and 13 thatC NMR the absolutedata for the configuration synthetic ofproduct this compound 2 differed required from those revision of the [ 33natural,34]. Additionally,product, which Oishi indicated and Fujii that synthesized the absolute the configurationd-N-Me-Ala epimerof this ofcompound coibamide required A (4) due revision to an epimerization [33,34]. Additionally, of this residue Oishi and during Fujii the synthesized macrocyclization the D-N process-Me- (FigureAla epimer1)[ 35 of]. Incoibamide 2015, Fang A ( and4) due Su to were an epimerization able to assign of the this correct residue configuration during the ofmacrocyclization coibamide A (1 ) withprocess the (Figure revision 1) of [35]. the Inl -HVA2015, Fang and andl-N -Me-AlaSu were able residues to assign to the thed correct-HVA configuration and d-N-Me-Ala of coibamide after total synthesisA (1) with of the this revision alternative of the along L-HVA with and its L- diastereomericN-Me-Ala residues analogues to the D (Figure-HVA and1)[ D30-N].-Me-Ala The absolute after configurationtotal synthesis of of coibamide this alternative A (1) along proposed with its by diastereomeric Fang and Su was analogues further (Figure confirmed 1) [30]. by The McPhail absolute and Cheongconfiguration using computationalof coibamide A methods (1) proposed to calculate by Fang NMR and dataSu was for further the conformational confirmed by space McPhail occupied and byCheong several using possible computational diastereomers methods and comparisonto calculate NMR with experimentaldata for the conformational values [53]. This space considerable occupied effbyort several to resolve possible the diastereomers structure of coibamide and comparison A (1) waswith largely experimental motivated values by [53]. the potentThis considerable cytotoxicity ofeffort this to molecule, resolve the and structure as a byproduct, of coibamide provided A (1) was a number largely ofmotivated new analogues by the potent for mechanistic cytotoxicity andof pharmacologicalthis molecule, and studies. as a Morebyproduct, recently, provided Su and a Fang number went of on new to synthesize analogues 18 for new mechanistic analogues and (2, 4, pharmacological studies. More recently, Su and Fang went on to synthesize 18 new analogues (2, 4, 5–20) including the originally proposed structure of coibamide A (2) as well as the revised correct 5–20) including the originally proposed structure of coibamide A (2) as well as the revised correct structure (1) to perform a structure–activity relationship study (Figure2)[ 31]. However, none of structure (1) to perform a structure–activity relationship study (Figure 2) [31]. However, none of the the coibamide A analogues were more potent cytotoxins than the natural product, indicating the coibamide A analogues were more potent cytotoxins than the natural product, indicating the strong strong correlation between the observed activity, the core molecular structure and optimization of this correlation between the observed activity, the core molecular structure and optimization of this structure through natural evolutionary processes. The only analogue that exhibited similar inhibition structure through natural evolutionary processes. The only analogue that exhibited similar inhibition as natural coibamide A was the [MeAla3-MeAla6]-coibamide (8), which significantly suppressed tumor as natural coibamide A was the [MeAla3-MeAla6]-coibamide (8), which significantly suppressed growth in vivo [31,33,35]. tumor growth in vivo [31,33,35].

FigureFigure 1. 1.Structure Structure ofof coibamidecoibamide AA (2)) from Leptolyngbya sp.sp. and and some some synthetic synthetic analogues analogues generated generated toto validate validate its its absolute absolute conﬁguration. configuration.

Mar. Drugs 2020, 18, 508 12 of 29

Mar. Drugs 2020, 18, x FOR PEER REVIEW 10 of 27

FigureFigure 2. Synthetic 2. Synthetic analogues analogues of of coibamide coibamide AA producedproduced to to explore explore structure–activity structure–activity relationships relationships (SAR)(SAR) in this in this molecular molecular class. class.

TheThe dolastatins dolastatins are are an an expansive expansive and and well-knownwell-known series series of of peptidic peptidic compounds. compounds. These These were were namednamed after after ﬁrst first being being discovered discovered from from the the sea sea hare hare DolabellaDolabella auricularia auricularia, but it, butwas itlater was found later that found thatthe the mollusk mollusk accumulates accumulates these these natural natural products products from from cyanobacteria cyanobacteria in their in their diets diets [37,39,54,55]. [37,39,54 ,55]. Dolastatin 12 (21) is one such compound, and has interesting 4-amino-2,2-dimethyl-3-oxopentanoic Dolastatin 12 (21) is one such compound, and has interesting 4-amino-2,2-dimethyl-3-oxopentanoic acid (Ibu) and (2S,3R)-3-amino-2-methylpentanoic acid (MAP) residues in its cyclic structure (Figure acid (Ibu) and (2S,3R)-3-amino-2-methylpentanoic acid (MAP) residues in its cyclic structure (Figure3). The macrocycle also includes an ester linkage across a 2-hydroxy-3-methylpentanoate (Hmp) residue, Mar. Drugs 2020, 18, 508 13 of 29

Mar. Drugs 2020, 18, x FOR PEER REVIEW 11 of 27 and isMar. thus Drugs a 2020 depsipeptide., 18, x FOR PEER While REVIEW this compound was originally isolated from D. auricularia 11 of ,27 it was noted3). to The resemble macrocycle the Lyngbyaalso includes majuscula an estermetabolite linkage across majusculamide a 2-hydroxy-3-methylpentanoate C. Dolastatin 12 (21 (Hmp)) was later residue, and is thus a depsipeptide. While this compound was originally isolated from D. auricularia, re-discovered3). The macrocycle from a mixed also includes cyanobacterial an ester assemblagelinkage across of aL. 2-hydroxy-3-methylpentanoate majuscula and Schizothrix calcicola (Hmp) from it was noted to resemble the Lyngbya majuscula metabolite majusculamide C. Dolastatin 12 (21) was Guamresidue, [36,39 and,56, 57is thus] as wella depsipeptide. as from a WhileLeptolyngbya this compoundsp. RS03 was collected originally in isolated the Red from Sea D. [ 36auricularia]. Dolastatin, later re-discovered from a mixed cyanobacterial assemblage of L. majuscula and Schizothrix calcicola 12 (21it) was may noted be present to resemble as a mixturethe Lyngbya of diastereomersmajuscula metabolite arising majusculamide from epimerization C. Dolastatin of the 12 acid-sensitive (21) was fromlater re-discoveredGuam [36,39,56,57] from a asmixed well cyanobacterialas from a Leptolyngbya assemblage sp. of RS03 L. majuscula collected and in Schizothrixthe Red Sea calcicola [36]. Ibu unitDolastatin during 12 the(21) isolationmay be present process as [a57 mixture]. The of configuration diastereomers ofarising this Ibufrom residue epimerization was determined of the from Guam1 [36,39,56,57] as well as from a Leptolyngbya sp. RS03 collected in the Red Sea [36]. by CDacid-sensitiveDolastatin and H 12 NMR (Ibu21) unit analysismay duringbe present after the hydrolysisisolationas a mixture process and of diastereomers purification[57]. The configuration [arising39]. The from of correct thisepimerization Ibu configuration residue of was the was 1 determineddeterminedacid-sensitive to beby IbuR CDby unit comparingand during H NMR the the isolation analysis free Ibu process after unit hydrolysis[57]. to Adhpa The configuration syntheticand purification standards of this [39]. Ibu [57residueThe]. Incorrect thewas early studies,configurationdetermined dolastatin by was 12CD determined (21 and) was 1H shown NMRto be Ranalysis to by inhibit comparing after actin hydrolysis the polymerization free Ibu and unit purification to [Adhpa37,58]. synthetic Dolastatin[39]. The standards correct 12 (21 ) has in vitro[57].configurationMICs In the< 0.05early wasµ gdeterminedstudies,/mL against dolastatin to be human R 12by comparing( nasopharyngeal21) was shown the free to carcinomaIbu inhibit unit toactin Adhpa cells polymerization and synthetic 0.08 µ standardsg /[37,58].mL against humanDolastatin[57]. colon In the adenocarcinoma 12 early (21) hasstudies, in vitro dolastatin cells MICs [37 <0.05]. 12 Further μ(g/mL21) was against cytotoxicity shown human to characterizationinhibit nasopharyngeal actin polymerization carcinoma showed that cells [37,58]. dolastatin and 0.08 μg/mL against human colon adenocarcinoma cells [37]. Further cytotoxicity characterization 12 hasDolastatin an in vitro 12 (IC21) has> 1 inµ vitroM against MICs <0.05 HeLa μ cellsg/mL [ 36against,56] and human a ED nasopharyngealof 7.5 10 2carcinomaµg/mL against cells and murine 50 50 − - showed0.08 μg/mL that againstdolastatin human 12 has colon an in adenocarcinoma vitro IC50 > 1 μM cellsagainst [37]. HeLa Further cells cytotoxicity[36,56]× and a characterization ED50 of 7.5 x 10 P3882 lymphocytic leukemia [38,39]. μg/mL against murine P388 lymphocytic leukemia [38,39]. - showed that dolastatin 12 has an in vitro IC50 > 1 μM against HeLa cells [36,56] and a ED50 of 7.5 x 10 2 μg/mL against murine P388 lymphocytic leukemia [38,39].

FigureFigure 3. Dolastatin 3. Dolastatin 12, 12, previously previously isolated isolated fromfrom Leptolyngbya sp.sp. as aswell well as several as several other other sources. sources. See See text fortextFigure discussion for 3.discussion Dolastatin of theof 12, the unusual previously unusual MAP MAP isolated and and from IbuIbu moieties Leptolyngbya in in dolastatin dolastatin sp. as well 12. 12.as several other sources. See text for discussion of the unusual MAP and Ibu moieties in dolastatin 12. Ibu-epidemethoxylyngbyastatinIbu-epidemethoxylyngbyastatin 3 3 ( 22(22)) has has a similar structural structural backbone backbone to dolastatin to dolastatin 12 (21 12) and (21 ) and possesses the same Ibu residue (Figure 4). However, rather than containing the MAP as in dolastatin possessesIbu-epidemethoxylyngbyastatin the same Ibu residue (Figure 34 ).(22 However,) has a similar rather structural than containing backbone to the dolastatin MAP as 12 in (21 dolastatin) and 12, Ibu-epidemethoxylyngbyastatin 3 has a molecular mass comparatively increased by 14 Da, which 12, Ibu-epidemethoxylyngbyastatinpossesses the same Ibu residue (Figure 3 has 4). a However, molecular rather mass than comparatively containing the increased MAP as in by dolastatin 14 Da, which results12, Ibu-epidemethoxylyngbyastatin from the presence of a 3-amino-2-methylhexanoic 3 has a molecular mass acidcomparatively (Amha) moiety increased instead by 14 of Da, MAP. which In results from the presence of a 3-amino-2-methylhexanoic acid (Amha) moiety instead of MAP. In vitro vitroresults cytotoxicity from the presence testing showed of a 3-amino-2-methylhexanoic that Ibu-epidemethoxylyngbyastatin acid (Amha) 3 moiety (22) is insteadat least 10-foldof MAP. less In cytotoxicity testing showed that Ibu-epidemethoxylyngbyastatin 3 (22) is at least 10-fold less toxic than toxicvitro thancytotoxicity dolastatin testing 12 (IC showed50 > 1 μ M)that against Ibu-epidemethoxylyngbyastatin HeLa cells [36,56]. 3 (22) is at least 10-fold less dolastatin 12 (IC > 1 µM) against HeLa cells [36,56]. toxic than dolastatin50 12 (IC50 > 1 μM) against HeLa cells [36,56].

Figure 4. Ibu-epidemethoxylyngbyastatin 3 from Leptolyngbya sp.

FigureFigure 4. 4.Ibu-epidemethoxylyngbyastatin Ibu-epidemethoxylyngbyastatin 3 3from from LeptolyngbyaLeptolyngbya sp. sp.

Mar. Drugs 2020, 18, 508 14 of 29

Mar. Drugs 2020, 18, x FOR PEER REVIEW 12 of 27 Grassypeptolides are a series of cyclic depsipeptides ﬁrst isolated from the marine cyanobacterium LyngbyaGrassypeptolides confervoides. These are peptidesa series containof cyclicd-amino depsipeptides acids, thiazoline first isolated rings, andfrom a βthe-amino marine acid (Figurecyanobacterium5)[ 59,60]. FurtherLyngbya researchconfervoides led. toThese theisolation peptides and contain description D-amino of acids, grassypeptolides thiazoline rings, D (23 and) and a E β-amino acid (Figure 5) [59,60]. Further research led to the isolation and description of (24) from a Red Sea Leptolyngbya sp. RS03, and grassypeptolides F and G from Lyngbya majuscula [61,62]. grassypeptolides D (23) and E (24) from a Red Sea Leptolyngbya sp. RS03, and grassypeptolides F and Grassypeptolides D (23) and E (24) showed cytotoxic activity to both HeLa cells (IC50 335 and 192 G from Lyngbya majuscula [61,62]. Grassypeptolides D (23) and E (24) showed cytotoxic activity to nM, respectively) and neuro2a cells (IC50 599 and 407 nM, respectively) [36,56,63]. By comparing with both HeLa cells (IC50 335 and 192 nM, respectively) and neuro2a cells (IC50 599 and 407 nM, other grassypeptolide structures and their biological activities, an initial structure–activity relationship respectively) [36,56,63]. By comparing with other grassypeptolide structures and their biological was deduced that indicated the N-Me-Phe-thn-ca-Aba-thn-ca tetrapeptide motif could be the key activities, an initial structure–activity relationship was deduced that indicated the N-Me-Phe-thn-ca- pharmacophore of the grassypeptolides [36,56]. Aba-thn-ca tetrapeptide motif could be the key pharmacophore of the grassypeptolides [36,56].

FigureFigure 5.5. GrassypeptolidesGrassypeptolides from LeptolyngbyaLeptolyngbya sp.sp. RS03. RS03.

LoggerpeptinsLoggerpeptins A–C A–C (25–27 (25–27) are) are cyclic cyclic depsipeptides depsipeptides with with 3-amino-6-hydroxy-2-piperidone 3-amino-6-hydroxy-2-piperidone (Ahp) residues(Ahp) residues (Figure6 )(Figure that were 6) isolatedthat were from isolated a Florida from cyanobacterial a Florida cyanobacterial collection identified collection morphologically identified asmorphologicallyLeptolyngbya sp. as [40 Leptolyngbya]. These compounds sp. [40]. wereThese screened compounds for serine were proteasescreened inhibitory for serine activities protease to assessinhibitory their antimetastaticactivities to assess effect their against antimetastatic breast cancer effect cells. against Loggerpeptin breast cancer A (25 cells.) and Loggerpeptin B (26) were more A potent(25) and than B loggerpeptin(26) were more C ( 27potent) with than IC50 sloggerpeptin of 0.24 and0.22 C (27µM) with against IC50 bovines of 0.24 pancreatic and 0.22 chymotrypsinμM against andbovine 0.24 andpancreatic 0.28 µ Mchymotrypsin against porcine and pancreatic 0.24 and elastase0.28 μM [ 40against]. Loggerpeptin porcine pancreatic A (25) was elastase 2- and 3-fold[40]. moreLoggerpeptin potent than A loggerpeptin(25) was 2- and B (3-fold26) and more C (27 potent) against than human loggerpeptin neutrophil B (26 elastase) and C (HNE)(27) against [40]. human All three compoundsneutrophil exhibitedelastase (HNE) antiproteolytic [40]. All activities,three compound with ICs50 exhibitedvalues under antiproteolytic 1 µM. As theactivities, major componentwith IC50 invalues the collection, under 1 loggerpeptinμM. As the major C (27 )component was subject in to the a detailedcollection, molecular loggerpeptin study C [40 (27].) Molecular was subject docking to a showeddetailed that molecular the Leu study and N-terminal [40]. Molecular Thr-1, docking Abu and showed Ala residues that the of loggerpeptinLeu and N-terminal C (27) wereThr-1, binding Abu toand the Ala subsites residues S1-S4 of of loggerpeptin HNE and porcine C (27) pancreatic were binding elastase to the [40 subsites]. S1-S4 of HNE and porcine pancreaticMolassamide elastase ([40].28) is another cyclic depsipeptide (Figure7) that was isolated along with loggerpeptins A–C (25–27)[40]. Compared to loggerpeptins (25–27), molassamide (28) exhibited much more potent inhibition activity against porcine pancreatic elastase, with an IC50 value of 50 nM, indicating the Abu residue between Ahp and Thr-1 is important to the antiproteolytic selectivity [40]. Molassamide presents similar binding patterns in molecular docking as loggerpeptin C (27), with the Abu and N-terminal Thr-1, Thr-2 and Ala binding in subsites S1–S4 of HNE and porcine pancreatic elastase [40]. Molassamide also inhibited elastase from cleaving the substrate CD40 in both biochemical and cellular assays, and it also inhibited ICAM-1 cleavage and downregulated elastase-induced ICAM-1 gene expression. Overall, this profile of activity is indicative of molassamide being a promising candidate for potential treatment of breast cancer [40].

Mar. Drugs 2020, 18, x FOR PEER REVIEW 13 of 27

Mar. Drugs 2020, 18, 508 15 of 29 Mar. Drugs 2020, 18, x FOR PEER REVIEW 13 of 27

Figure 6. Loggerpeptins from Leptolyngbya sp.

Molassamide (28) is another cyclic depsipeptide (Figure 7) that was isolated along with loggerpeptins A–C (25–27) [40]. Compared to loggerpeptins (25–27), molassamide (28) exhibited much more potent inhibition activity against porcine pancreatic elastase, with an IC50 value of 50 nM, indicating the Abu residue between Ahp and Thr-1 is important to the antiproteolytic selectivity [40]. Molassamide presents similar binding patterns in molecular docking as loggerpeptin C (27), with the Abu and N-terminal Thr-1, Thr-2 and Ala binding in subsites S1–S4 of HNE and porcine pancreatic elastase [40]. Molassamide also inhibited elastase from cleaving the substrate CD40 in both biochemical and cellular assays, and it also inhibited ICAM-1 cleavage and downregulated elastase- induced ICAM-1 gene expression. Overall, this profile of activity is indicative of molassamide being a promising candidate for potentialFigureFigure 6.treatment Loggerpeptins of breast from from cancer LeptolyngbyaLeptolyngbya [40]. sp.sp.

FigureFigure 7.7. Molassamide from Leptolyngbya sp.sp.

2.2. Simple Esters Lumyong et al. isolated an antibacterial compound, 2-hydroxyethyl-11-hydroxyhexadec-9-enoate (29) (Figure8), from Leptolyngbya sp. LT19 [41], as a result of screening for antibacterial activities. They showed compound 25 to be active against Vibrio harveyi and V. parahaemolyticus, with MIC values of 250–1000 and 350–1000 µg/mL. This antibacterial activity could potentially be useful to the shrimp aquaculture industry that is often burdened by the highly damaging Vibrio spp. pathogens [41]. However, the absolute conﬁguration of the single stereocenter in compound 25 has not yet been determined.

Figure 7. Molassamide from Leptolyngbya sp.

Mar. Drugs 2020, 18, x FOR PEER REVIEW 14 of 27

2.2. Simple Esters Mar. Drugs 2020, 18, x FOR PEER REVIEW 14 of 27 Lumyong et al. isolated an antibacterial compound, 2-hydroxyethyl-11-hydroxyhexadec-9- enoate2.2. (29 Simple) (Figure Esters 8), from Leptolyngbya sp. LT19 [41], as a result of screening for antibacterial activities. LumyongThey showed et al. compound isolated an 25 antibacterial to be active co againstmpound, Vibrio 2-hydroxyethyl-11-hydroxyhexadec-9- harveyi and V. parahaemolyticus, with MIC valuesenoate of(29 250–1000) (Figure 8), and from 350–1000 Leptolyngbya μg/mL. sp. ThisLT19 antibacterial[41], as a result activity of screening could potentiallyfor antibacterial be useful to theactivities. shrimp Theyaquaculture showed compound industry 25that to be is active often against burdened Vibrio harveyiby the and highly V. parahaemolyticus damaging Vibrio, with spp. pathogensMIC values [41]. However,of 250–1000 the and absolute 350–1000 configuratμg/mL. Thision antibacterial of the single activity stereocenter could potentially in compound be useful 25 has Mar.to Drugsthe shrimp2020, 18 ,aquaculture 508 industry that is often burdened by the highly damaging Vibrio 16spp. of 29 not yet been determined. pathogens [41]. However, the absolute configuration of the single stereocenter in compound 25 has not yet been determined.

FigureFigure 8. 2-Hydroxyethyl-11-hydroxyhexadec-9-enoate 8. 2-Hydroxyethyl-11-hydroxyhexadec-9-enoate from fromLeptolyngbya Leptolyngbyasp. sp. LT19. LT19. Figure 8. 2-Hydroxyethyl-11-hydroxyhexadec-9-enoate from Leptolyngbya sp. LT19. ChoiChoi et al. et isolated al. isolated honaucins honaucins A–C A–C (30 (30-32-32)) from from a HawaiianHawaiian collection collection of ofLeptolyngbya Leptolyngbya crossbyana crossbyana whichChoi possess et al. potent isolated anti-inflammatory honaucins A–C (30 and-32 quorum-sensing) from a Hawaiian (QS) collection inhibitory of Leptolyngbya activity (Figure crossbyana9)[ 42 ]. whichwhich possess possess potent potent anti-inflammato anti-inflammatoryry and and quorum-sensing quorum-sensing (QS) (QS) inhibi inhibitorytory activity activity (Figure (Figure 9) [42]. 9) [42]. Chemical synthesis of honaucin and a number of analogs (30–32) revealed that the each of the ChemicalChemical synthesis synthesis of honaucinof honaucin and and a anumber number of analogsanalogs ( 30(30–32–32) revealed) revealed that that the eachthe eachof the of the functional groups is critical for both of these biological activities. Further, synthetic honaucin analogues functionalfunctional groups groups is criticalis critical for for both both of of thesethese biological activities. activities. Further, Further, synthetic synthetic honaucin honaucin 4-bromo-honaucin A (33) and 40-iodohonaucin A (35) were discovered to have slightly more potent analoguesanalogues 4-bromo-honaucin 4-bromo-honaucin A A(33 (33) )and and 4 4′-iodohonaucin′-iodohonaucin A A ( 35(35) were) were discovered discovered to have to haveslightly slightly activity in cellular TRAP activity than honaucin A itself (30), with IC50 values of 0.54 and 0.61 µg/mL more potent activity in cellular TRAP activity than honaucin A itself (30), with IC50 values of 0.54 and morecompared potent activity to 0.63 µ ing/ mLcellular for 30 TRAP(Figure activity9), [ 43]. than Mechanistic honaucin pharmacological A itself (30), investigations with IC50 values of honaucin of 0.54 and 0.61 μg/mL compared to 0.63 μg/mL for 30 (Figure 9), [43]. Mechanistic pharmacological 0.61 A(μg/mL30) indicated compared that the to molecular 0.63 μg/mL target(s) for involves 30 (Figure the Nrf2-ARE 9), [43]. (Antioxidant Mechanistic Response pharmacological Element) investigations of honaucin A (30) indicated that the molecular target(s) involves the Nrf2-ARE investigationspathway, and of specificallyhonaucin involvesA (30) interactionindicated withthat Cysthe residuesmolecular on Keap1 target(s) when involves it is complexed the Nrf2-ARE with (Antioxidant Response Element) pathway, and specifically involves interaction with Cys residues on Nrf2. This allows Nrf2 to be transported to the nucleus, where it activates cytoprotective genes and (AntioxidantKeap1 when Response it is complexed Element) with pathway, Nrf2. This and allows specif Nrf2ically to involves be transported interaction to the nucleus,with Cys where residues it on Keap1generatesactivates when it ancytoprotective is anti-inflammatory complexed genes with response andNrf2. generates This [64]. allows Additionally, an anti-inflammatory Nrf2 to further be transported investigation response to [64]. ofthe bromo-honaucin Additionally,nucleus, where it activatesA(further33 )cytoprotective revealed investigation that itofgenes providesbromo-honaucin and a generates protective A (33 ) ean ffrevealedect anti-inflammatory against that boneit provides loss in responsea protective RANKL-treated [64]. effect Additionally, against murine furthermonocytebone investigation loss/ macrophagein RANKL-treated of bromo-honaucin RAW264.7 murine cells monocyte/macrophage [43 A]. (33) revealed that RAW264.7 it provides cells [43]. a protective effect against bone loss in RANKL-treated murine monocyte/macrophage RAW264.7 cells [43].

FigureFigure 9. 9.Honaucins Honaucins A–CA–C fromfrom Leptolyngbya crossbyana andand some some synthetic synthetic derivatives. derivatives.

2.3.2.3. Macrolides Macrolides LeptolyngbyolidesLeptolyngbyolidesFigure 9. Honaucins A–DA–D A–C ((3636 from––3939),), Leptolyngbya a seriesseries of crossbyana 22-membered and somemacrolides, macrolides, synthetic were were derivatives. isolated isolated from from LeptolyngbyaLeptolyngbyasp. sp. collectedcollected inin Okinawa,Okinawa, Japan. The The absolute absolute configuration conﬁguration of of each each was was assigned assigned 2.3. Macrolidesfollowingfollowing an an asymmetric asymmetric total synthesistotal synthesis of leptolyngbyolide of leptolyngbyolide C (38) (Figure C 10(38)[44) ].(Figure Leptolyngbyolides 10) [44]. A–DLeptolyngbyolides (36–39) were screened A–D (36 for–39 cytotoxic) were screened activity for against cytotoxic HeLa activity S3 cells againstin vitro HeLaand S3 cells were in found vitro and to be Leptolyngbyolides A–D (36–39), a series of 22-membered macrolides, were isolated from moderatelywere found active, to be moderately with IC50 values active, of with 0.99, IC 0.16,50 values 0.64 and of 0.99, 0.15 0.16,µM, respectively0.64 and 0.15 [ 44μM,]. Furthermore,respectively [44]. these Leptolyngbya sp. collected in Okinawa, Japan. The absolute configuration of each was assigned metabolitesFurthermore, possessed these metabolites actin-depolymerizing possessed activityactin-depolymerizing with an EC50 of activity 12.6, 11.6, with 26.9 an and EC 21.550 ofµ 12.6,M, and 11.6, this followingmay represent an asymmetric the mechanism total for thesynthesis observed cellularof leptolyngbyolide apoptosis caused byC these(38)compounds (Figure [10)44]. [44]. Leptolyngbyolides A–D (36–39) were screened for cytotoxic activity against HeLa S3 cells in vitro and were found to be moderately active, with IC50 values of 0.99, 0.16, 0.64 and 0.15 μM, respectively [44]. Furthermore, these metabolites possessed actin-depolymerizing activity with an EC50 of 12.6, 11.6,

Mar. Drugs 2020, 18, x FOR PEER REVIEW 15 of 27 Mar. Drugs 2020, 18, x FOR PEER REVIEW 15 of 27

Mar.26.9 Drugs and2020 21.5, 18 μ,M, 508 and this may represent the mechanism for the observed cellular apoptosis caused17 of 29 26.9 and 21.5 μM, and this may represent the mechanism for the observed cellular apoptosis caused by these compounds [44]. by these compounds [44].

FigureFigure 10.10. Leptolyngbyolides from from LeptolyngbyaLeptolyngbya sp.sp. Figure 10. Leptolyngbyolides from Leptolyngbya sp. AA new new macrolide, macrolide, palmyrolidepalmyrolide AA ((4040),), was isolated from from an an environmental environmental assemblage assemblage of of a a A new macrolide, palmyrolide A (40), was isolated from an environmental assemblage of a LeptolyngbyaLeptolyngbya cf.cf. andand OscillatoriaOscillatoria sppspp.. collected collected from from Palmyra Palmyra Atoll in Atoll the Central in the CentralPacific Ocean Paciﬁc (Figure Ocean Leptolyngbya cf. and Oscillatoria spp. collected from Palmyra Atoll in the Central Pacific Ocean (Figure (Figure11). Palmyrolide 11). Palmyrolide A (40) comprises A (40) comprises a rare N-methyl a rare enamideN-methyl functionality, enamide functionality, as well as an asintriguing well as t an- 11). Palmyrolide A (40) comprises a rare N-methyl enamide functionality, as well as an intriguing t- intriguingbutyl brancht-butyl that likely branch results that biosynthetically likely results biosynthetically from the incorporation from the of malonate incorporation and three of malonate methyl butyl branch that likely results biosynthetically from the incorporation of malonate and three methyl andgroups three from methyl S-adenosyl- groups fromL-methionineS-adenosyl- (SAM),l-methionine as shown (SAM), for apratoxin as shown [45,65]. for apratoxin The absolute [45,65 ]. groups from S-adenosyl-L-methionine (SAM), as shown for apratoxin [45,65]. The absolute Theconfiguration absolute conﬁguration of this molecule of was this assigned molecule by was total assigned synthesis by of totalboth the synthesis naturalof (−)-palmyrolide both the natural A configuration of this molecule was assigned by total synthesis of both the natural (−)-palmyrolide A ( (40)-palmyrolide) and its enantiomer, A (40) and (+)-ent-palmyrolide its enantiomer, A. (+ )-ent-palmyrolideThis was necessitated A. because This was the necessitated t-butyl substituent because −(40) and its enantiomer, (+)-ent-palmyrolide A. This was necessitated because the t-butyl substituent theapparentlyt-butyl substituent provides the apparently lactone ester provides bond thewith lactone resistance ester to bond hydrolysis, with resistance precluding to hydrolysis,chemical apparently provides the lactone ester bond with resistance to hydrolysis, precluding chemical precludingdegradation chemical studies [66–68]. degradation It was studies speculated [66– 68that]. Itcyanobacterial was speculated secondary that cyanobacterial metabolites possessing secondary degradation studies [66–68]. It was speculated that cyanobacterial secondary metabolites possessing this motif, a t-butyl adjacent to ant ester, might be stable under a wide variety of environmental metabolitesthis motif, possessinga t-butyl adjacent this motif, to aan-butyl ester, adjacentmight be to stable an ester, under might a wide be stable variety under of aenvironmental wide variety of conditions, similar to what was observed in the laboratory environment. Palmyrolide A (40) exhibited environmentalconditions, similar conditions, to what similar was observed to what in was the observed laboratory in environment. the laboratory Palmyrolide environment. A ( Palmyrolide40) exhibited A potent inhibition of calcium oscillations in murine cerebrocortical neurons and sodium channel- (40potent) exhibited inhibition potent of inhibitioncalcium oscillations of calcium in oscillations murine cerebrocortical in murine cerebrocortical neurons and neurons sodium and channel- sodium blocking activity in neuroblastoma (neuro2a) cells [45]. channel-blockingblocking activity activityin neuroblastoma in neuroblastoma (neuro2a) (neuro2a) cells [45]. cells [45].

Figure 11. Palmyrolide A (40) from Leptolyngbya cf. sp. FigureFigure 11.11. Palmyrolide A ( 40)) from from LeptolyngbyaLeptolyngbya cf. cf. sp.sp. Phormidolide (41) is a 16-membered macrocyclic lactone polyketide-derived metabolite that was PhormidolidePhormidolide ((41)) is is a a 16-membered 16-membered macrocyclic macrocyclic lactone lactone polyketide-derived polyketide-derived metabolite metabolite that was that discovered from an Indonesian Leptolyngbya sp. (ISB3NOV94-8A) (Figure 12). This molecule has wasdiscovered discovered from from an Indonesian an Indonesian LeptolyngbyaLeptolyngbya sp. (ISB3NOV94-8A)sp. (ISB3NOV94-8A) (Figure (Figure 12). This12). molecule This molecule has several unique structural features including a large number of hydroxy and methyl groups on the hasseveral several unique unique structural structural features features including including a large a large number number of hydroxy of hydroxy and methyl and methyl groups groups on the on carbon backbone, several points of both cis and trans unsaturation, and a vinyl bromide at one thecarbon carbon backbone, backbone, several several points points of ofboth both ciscis andand transtrans unsaturation,unsaturation, and and a avinyl vinyl bromide bromide atat one one terminus. Phormidolide (41) showed in vitro brine shrimp toxicity, with an LC50 of 1.5 μM. Multiple terminus. Phormidolide (41) showed in vitro brine shrimp toxicity, with an LC50 of 1.5 μµM. Multiple terminus.NMR experiments, Phormidolide including (41) showed GHMBC,in vitro2D INbrineADEQUATE, shrimp toxicity, and ACCORD-ADEQUATE, with an LC50 of 1.5 M. G-BIRDR, Multiple NMR experiments, including GHMBC, 2D INADEQUATE, and ACCORD-ADEQUATE, G-BIRDR, NMRX-HSQMBC experiments, NMR includingexperiments, GHMBC, enabled 2D a J INADEQUATE,-based configuration and analysis ACCORD-ADEQUATE, and deduction of G-BIRDR, relative X-HSQMBC NMR experiments, enabled a J-based configuration analysis and deduction of relative X-HSQMBCconfiguration NMR of stereocenters; experiments, a enabledvariable atemperatureJ-based configuration Mosher ester analysis analysis and was deduction used to assign of relative the configuration of stereocenters; a variable temperature Mosher ester analysis was used to assign the configurationabsolute configuration of stereocenters; [46]. An ainvestigation variable temperature of the biosynthetic Mosher esterpathway analysis for phormidolide was used to ( assign41) used the absolute configuration [46]. An investigation of the biosynthetic pathway for phormidolide (41) used absolutea genome configuration sequencing approach, [46]. An investigation and identified of the the phormidolide biosynthetic pathwaybiosynthetic for gene phormidolide cluster (phm (41).) The used a genome sequencing approach, and identified the phormidolide biosynthetic gene cluster (phm). The aphormidolide genome sequencing (41) gene approach, cluster was and found identified to be of the the phormidolide trans-AT PKS biosynthetictype, which has gene been cluster relatively (phm ). Thephormidolide phormidolide (41 ()41 gene) gene cluster cluster was was found found to tobe beof ofthe the tran trans-ATs-AT PKS PKS type, type, which which has has been been relatively relatively rarely reported in cyanobacteria. This was based on finding two discrete trans-AT open reading frames along with KS-AT adaptor regions (ATd) within the PKS megasynthase. The megasynthase Mar. Drugs 2020, 18, x FOR PEER REVIEW 16 of 27 Mar. Drugs 2020, 18, 508 18 of 29 rarely reported in cyanobacteria. This was based on finding two discrete trans-AT open reading frames along with KS-AT adaptor regions (ATd) within the PKS megasynthase. The megasynthase possesses ketosynthases, ketoreductases, KS-AT adaptor regions, dehydratases, methyltransferases, possesses ketosynthases, ketoreductases, KS-AT adaptor regions, dehydratases, methyltransferases, O-methyltransferase, enoyl-CoA hydratases, an FkbH-like domain, a pyran synthase, an NRPS-like O-methyltransferase, enoyl-CoA hydratases, an FkbH-like domain, a pyran synthase, an NRPS-like condensationcondensation domain domain and and an an acylacyl carriercarrier proteinprotein that were consistent consistent with with structure structure of of phormidolide phormidolide (41(41).). The The biosynthetic biosynthetic pathway pathway also providedalso provided further fu supportingrther supporting evidence forevidence the absolute for the configuration absolute ofconfiguration phormidolide of (41 phormidolide), due to the stereospecificity (41), due to the of stereospecificity the ketoreductases of the observed ketoreductases in phm compared observed with in knownphm compared homologues with [26 ].known However, homologues subsequent [26]. chemical However, synthesis subsequent of key chemic fragmentsal synthesis of phormidolide of key revealedfragments the of need phormidolide for a revision revealed in configuration the need for in ata leastrevision one stereocenterin configuration [69]. in Simultaneously, at least one a reanalysisstereocenter of the[69]. biosynthetic Simultaneously, gene clustera reanalysis suggested of the additional biosynthetic revisions gene incluster configuration suggested were additional possibly requiredrevisions [70 in]. Theseconfiguration findings were stimulated possibly a concerted required computational [70]. These findings and NMR-based stimulated re-investigation a concerted ofcomputational phormidolide’s and complex NMR-based 3-dimensional re-investigation structure, of phormidolide’s leading to a complex revision 3-dimensional in several stereocenters structure, (42leading)[71]. to a revision in several stereocenters (42) [71].

FigureFigure 12. 12.Phormidolide Phormidolide fromfrom LeptolyngbyaLeptolyngbya sp.sp. with the original prop proposedosed stereostructure stereostructure (41 (41) )and and revisedrevised stereostructure stereostructure ( 42(42).).

2.4.2.4. Pyrones Pyrones KalkipyroneKalkipyrone A (A43 )(43 was) was ﬁrst reportedfirst reported in a mixed in a assemblage mixed assemblage of Lyngbya of majuscula Lyngbyaand majusculaTolypothrix andsp. (FigureTolypothrix 13), and sp. (Figure was found 13), toand have was potent found brine to have shrimp potent toxicity brine shrimp (LD50 = toxicity1 µg/mL) (LD and50 = ichthyotoxicity1 μg/mL) and againstichthyotoxicityCarassius against auratus Carassiusgoldﬁsh auratus (LD50 goldfish= 2 µg /(LDmL)50 [=48 2]. μg/mL) Kalkipyrone [48]. Kalkipyrone A (43) and A its(43) analogue and its kalkipyroneanalogue kalkipyrone B (44) were B later(44) were found later as metabolites found as metabolites of a Leptolyngbya of a Leptolyngbyasp. (ASG15JUL14-6) sp. (ASG15JUL14-6) collected fromcollected America from Samoa America (Figure Samoa 12 (Figure)[47]. 12) Kalkipyrone [47]. Kalkipyrone A (43) A and (43) B and (44 )B were(44) were moderately moderately toxic toxic to a Saccharomycesto a Saccharomyces cerevisiae cerevisiaestrain strain lacking lacking 16 ATP-binding 16 ATP-binding cassette cassette transporter transporter pump genespump (ABC16-monstergenes (ABC16- 50 strain;monster IC 50strain;= 14.6 IC and = 14.6 13.4 andµM, 13.4 respectively), μM, respectively), while the whilein vitro the incytotoxicity vitro cytotoxicity of these of two these natural two natural products against H460 human lung cancer cells was somewhat more potent (EC50 = 0.9 and products against H460 human lung cancer cells was somewhat more potent (EC50 = 0.9 and 9.0 µM, respectively)9.0 μM, respectively) [47]. [47].

Mar. Drugs 2020, 18, 508 19 of 29 Mar. Drugs 2020, 18, x FOR PEER REVIEW 17 of 27 Mar. Drugs 2020, 18, x FOR PEER REVIEW 17 of 27

FigureFigure 13. Kalkpyrones from from LeptolyngbyaLeptolyngbya sp.sp. Figure 13. Kalkpyrones from Leptolyngbya sp. ThreeThree relatedrelatedγ γ-pyrone-containing-pyrone-containing polyketides polyketides described described as as yoshinones yoshinones A, A, B1, B1, and and B2 B2 (45 (45–47–47), ), werewere isolatedThreeisolated related fromfrom γ Leptolyngbya-pyrone-containing sp.sp. collected collected polyketides from from Ishigaki described Ishigaki island as island yoshinones Okinawa, Okinawa, JapanA, B1, Japan (Figure and B2 (Figure 14)(45.– The47 14), ). Theabsolutewere absolute isolated configuration configuration from Leptolyngbya of each of compound each sp. collected compound was from atte wasmpted Ishigaki attempted by islanda modified byOkinawa, a Mosher’s modified Japan method; Mosher’s (Figure however, 14) method;. The however,theseabsolute failed configuration these possibly failed as possibly of a eachresult compound as of a the result low was of amount theatte lowmpteds of amounts samples by a modified ofavailable samples Mosher’s [49]. available Thus, method; to [49 assign ].however, Thus, the to assignabsolutethese thefailed absoluteconfiguration possibly configuration as aand result provide of and the provide lowsufficient amount suffi amountscients of samples amounts for availablefurther for further [49].research, research, Thus, theto the assignabsolute absolute the stereochemistrystereochemistryabsolute configuration waswas achievedachieved and provide through sufficienttotal synt synthesis hesisamounts and and comparisonfor comparison further of ofresearch, NMR NMR and andthe chiroptical chiropticalabsolute stereochemistry was achieved through total synthesis and comparison of NMR and chiroptical propertiesproperties betweenbetween thethe natural product product an andd synthetic synthetic standards standards [72]. [72 Yoshinone]. Yoshinone A (45 A) ( 45was) was found found to inhibitproperties adipogenic between thedifferentiation natural product against and 3T3-L1synthetic cells, standards with an [72]. EC Yoshinone50 of 420 nMA (45 and) was with found little to to inhibit adipogenic differentiation against 3T3-L1 cells, with an EC50 of 420 nM and with little cytotoxicityinhibit adipogenic (IC50 = 63.8 differentiation μM to S. cerevisiae against ABC16-monster 3T3-L1 cells, cell)with [47,49]. an EC Th50 eof adipogenic 420 nM and differentiation with little cytotoxicity (IC50 = 63.8 µM to S. cerevisiae ABC16-monster cell) [47,49]. The adipogenic differentiation againstcytotoxicity 3T3-L1 (IC cells50 = 63.8 of yoshinone μM to S. cerevisiae B1 (46) and ABC16-monster B2 (47) was considerably cell) [47,49]. lessThe potent,adipogenic with differentiation less than 50% against 3T3-L1 cells of yoshinone B1 (46) and B2 (47) was considerably less potent, with less than 50% activityagainst 3T3-L1observed cells at oftested yoshinone concentrations B1 (46) and up B2to 5(47 μ)M was [48]. considerably Further examination less potent, of with structure–activity less than 50% activity observed at tested concentrations up to 5 µM[48]. Further examination of structure–activity relationshipsactivity observed in this at drugtested class concentrations indicated that up theto 5 po μMsition [48]. of Further the pyrone examination ring and ofside structure–activity chain olefin are relationships in this drug class indicated that the position of the pyrone ring and side chain olefin are importantrelationships for in the this inhibition drug class of indicatedadipogenic that differentiation. the position of Further the pyrone in vitro ring and and inside vivo chain experiments olefin are important for the inhibition of adipogenic differentiation. Further in vitro and in vivo experiments showedimportant that for yoshinone the inhibition A (45 of) stimulatesadipogenic lactate differentiation. accumulation Further deriving in vitro from and the in glycolyticvivo experiments system, showed that yoshinone A (45) stimulates lactate accumulation deriving from the glycolytic system, andshowed increases that yoshinone fat utilization A (45 to) stimulatescompensate lactate for an accumulation insufficient energy deriving supply from [73].the glycolytic These properties system, and increases fat utilization to compensate for an insufficient energy supply [73]. These properties couldand increases possibly fat support utilization the utility to compensate of yoshinone for Aan in insufficient anti-obesity energy indications. supply [73]. These properties couldcould possibly possibly support support thethe utilityutility ofof yoshinoneyoshinone A in anti-obesity indications. indications.

Figure 14. Yoshinones from Leptolyngbya sp. FigureFigure 14. Yoshinones from LeptolyngbyaLeptolyngbya sp.sp. 2.5. Polyaromatics 2.5.2.5. Polyaromatics Polyaromatics A series of brominated polyphenolics, crossbyanols A–D (48-51), were isolated from an extensive benthicAA series series Leptolyngbya of of brominatedbrominated crossbyana polyphenolics,polyphenolics, bloom in Hawaii crossbyanols (Figure A–D A–D 15) (( 4848[50].--5151 ),),In were were addition isolated isolated to fromthe from high an an extensive extensivelevel of benthicbromination,benthicLeptolyngbya Leptolyngbya crossbyanol crossbyanacrossbyana B–D ( 49bloombloom-51) also in Hawaiihave sulfated (Figure phenolic 15)15)[ [50].50 ].fu Innctionalities. In addition addition to toThese the the high highmetabolites level level of of bromination,werebromination, suggested crossbyanol crossbyanol to play a role B–DB–D in ( (49the49–- 51observed) also have coral sulfated toxicity phenolic phenoliccaused by fu functionalities. thenctionalities. overgrowing These These cyanobacteria. metabolites metabolites wereCrossbyanolwere suggested suggested A to (to48 play play) was aa rolerolefound inin theto activate observed sodium coral coral toxicity toxicityinflux causedin caused mouse by by theneuroblastoma the overgrowing overgrowing (neuro2a) cyanobacteria. cyanobacteria. cells, CrossbyanolwithCrossbyanol an EC50 A20A( μ48(48g/mL,)) waswas whereas foundfound to tocrossbyanol activateactivate sodium B (49) possessedinflux inﬂux in in mouse mouseantibiotic neuroblastoma neuroblastoma activity, with (neuro2a) an (neuro2a) MIC of cells, 2.0- cells, with3.9with μ an ang/mL ECEC50 50against 20 μµg/mL,g /mL,methicillin-resistant whereas whereas crossbyanol crossbyanol Staphylococcus B ( B49 ()49 possessed) possessed aureus antibiotic (MRSA). antibiotic activity,The activity, latter with metabolite withan MIC an of MIC also2.0- of 2.0–3.9showed3.9 μg/mLµ gmoderately/mL against against methicillin-resistantpotent methicillin-resistant brine shrimp StaphylococcustoxicityStaphylococcus (IC50 2.8aureus aureusμg/mL). (MRSA).(MRSA). Crossbyanols The The latter latter C metabolite(50 metabolite) and D also(51 also) showedwereshowed not moderately moderately observed to potentpotent have biological brinebrine shrimpshrimp activity toxicity in th ese(IC (IC 50tests,50 2.82.8 μandµg/mL).g/ mL).all four Crossbyanols Crossbyanols compounds C wereC(50 (50) and)inactive and D D(51 as (51) ) werecytotoxinswere not not observed observed to H460 to humanto have have biological lungbiological cancer activityactivity cells [50]. in ththese ese tests, tests, and and all all four four compounds compounds were were inactive inactive as as cytotoxinscytotoxins to to H460 H460 human human lunglung cancercancer cellscells [[50].50].

Mar.Mar. Drugs Drugs 20202020, 18,,18 x ,FOR 508 PEER REVIEW 20 of 1829 of 27 Mar. Drugs 2020, 18, x FOR PEER REVIEW 18 of 27

Figure 15. Crossbyanols from Leptolyngbya crossbyana. FigureFigure 15. 15. CrossbyanolsCrossbyanols from from Leptolyngbyacrossbyana. crossbyana.

2.6.2.6. Oxazolines Oxazolines 2.6. Oxazolines A seriesA series of ofpolar polar oxazolin oxazolines,es, named named leptazolines leptazolines A–D ((52–5552–55),), werewere isolated isolated from from the the culture culture A series of polar oxazolines, named leptazolines A–D (52–55), were isolated from the culture mediamedia of ofa aLeptolyngbyaLeptolyngbya sp. (Figure(Figure 16 16))[51 [51].]. Their Their planar planar structures structures were were characterized characterized by MS andby MS NMR and mediaNMRalong alongof with a Leptolyngbya formationwith formation of sp. acetate (Figure of derivatives.acetate 16) [51]. derivatives. RelativeTheir planar conﬁguration Relative structures configuration was were determined characterized was by comparisondetermined by MS ofand by NMRcomparisoncarbon along shifts ofwith carbon with formation those shifts calculated with of thoseacetate by densitycalculated derivatives. functional by density Relative theory functional (DFT).configuration Interestingly, theory was(DFT). the determined calculationsInterestingly, by comparisonthewere calculations found of tocarbon were vary asshiftsfound a function with to vary those of theas calculated computera function operatingby of density the computer system functional (Ubuntu operating theory 16, Windows(DFT).system Interestingly, (Ubuntu 10, MAC 16, theWindowsMavericks, calculations 10, MACMAC were Mojave). Mavericks, found to Biological vary MAC as assay Mojave).a function showed Biological of that the leptazolinecomputer assay showed Boperating (53) modestlythat system leptazoline inhibited (Ubuntu B the ( 5316,) Windowsmodestlygrowth inhibited of10, PANC-1 MAC the cells,Mavericks, growth with a of GI MACPANC-150 of 10 Mojave).µ M[cells,51 ],with whereasBiological a GI50 leptazoline of assay 10 μM showed A[51], (52) whereas with that its aromaticleptazoline leptazoline chlorine BA (5352) modestlywithatom its aromatic did inhibited not show chlorine the any growth signiﬁcant atom of did PANC-1 activitynot show cells, to thisany with cellsignificant a line. GI50 of activity 10 μM [51],to this whereas cell line. leptazoline A (52) with its aromatic chlorine atom did not show any significant activity to this cell line.

FigureFigure 16. 16. LeptazolinesLeptazolines from Leptolyngbya sp. sp. Figure 16. Leptazolines from Leptolyngbya sp. 2.7. Other 2.7. Other 2.7. Other 2.7.1. Toxins 2.7.1. Toxins 2.7.1. ToxinsCyanobacteria are known to produce a variety of toxins, including those that are hepatotoxic, neurotoxic,Cyanobacteria or cardiotoxic, are known and whichto produce generally a variety increase of economic toxins, burdensincluding and those impact that public are healthhepatotoxic, [23]. Cyanobacteria are known to produce a variety of toxins, including those that are hepatotoxic, neurotoxic,Cyanobacterial or cardiotoxic, populations and are which known generally to sporadically increase grow economic excessively burdens to form and blooms, impact and public in some health neurotoxic,[23].cases Cyanobacterial these or are cardiotoxic, harmful. populations A and total which of are 34 known speciesgenerally fromto sporad increase 15 generaically economic and grow ﬁve excessively burdens families were and to screenedimpactform blooms, public for known andhealth in [23].some Cyanobacterial cases these are populationsharmful. A total are knownof 34 species to sporad fromically 15 genera grow andexcessively five families to form were blooms, screened and for in someknown cases toxins these including are harmful. the Aneurotoxic total of 34 speciessaxitoxin from (56 15) generaand the and hepatotoxic five families microcystins were screened (e.g., for knownmicrocystin-LR, toxins including 57) (Figure the 17) neurotoxic [17,23,74]. Leptolyngbyasaxitoxin (56 collections) and the from hepatotoxic the Red Seamicrocystins had on average (e.g., microcystin-LR,58.9 μg/g dry wt. 57 and) (Figure 438–489 17) μ [17,23,74].g/g dry wt. Leptolyngbya of these two collections toxin classes, from respectively. the Red Sea Toxin had productionon average 58.9 μg/g dry wt. and 438–489 μg/g dry wt. of these two toxin classes, respectively. Toxin production

Mar. DrugsMar. Drugs2020, 202018, x, 18FOR, 508 PEER REVIEW 21 of 29 19 of 27 by marine Leptolyngbya poses toxicological risks to marine organisms that may feed on them, or that toxins including the neurotoxic saxitoxin (56) and the hepatotoxic microcystins (e.g., microcystin-LR, may be exposed to the cyanotoxins present in seawater [74]. 57) (Figure 17)[17,23,74]. Leptolyngbya collections from the Red Sea had on average 58.9 µg/g dry Mar. Drugs 2020, 18, x FOR PEER REVIEW 19 of 27 wt. and 438–489 µg/g dry wt. of these two toxin classes, respectively. Toxin production by marine Leptolyngbya poses toxicological risks to marine organisms that may feed on them, or that may be by marine Leptolyngbya poses toxicological risks to marine organisms that may feed on them, or that exposedmay be toexposed the cyanotoxins to the cyanotoxins present inpresent seawater in seawater [74]. [74].

Figure 17. Two toxins, saxitoxin (56) and microcystin-LR (57), isolated from Leptolyngbya sp.

2.7.2. Non-ToxicFigureFigure Metabolites 17. 17. TwoTwo toxins,toxins, saxitoxinsaxitoxin (56) and microcystin-LR ( (5757),), isolated isolated from from LeptolyngbyaLeptolyngbya sp.sp.

Non-toxic2.7.2.2.7.2. Non-Toxic Non-Toxic secondary Metabolites Metabolites metabolites from cyanobacteria include various chemical classes such as phytohormones,Non-toxicNon-toxic siderophores, secondarysecondary metabolitesmetabolites and UV-absorbing from cyanobacte cyanobacteria compria oundsinclude include suchvarious various as chemical chemicalmycosporine classes classes suchamino such as as acids (MAAs)phytohormones,phytohormones, and scytonemin siderophores,siderophores, (58); all of andan thesed UV-absorbing have been reportedcomp compoundsounds in Leptolyngbyasuch such as as mycosporine mycosporine sp. (Figure amino amino 18) acids [23,75,76]. acids These(MAAs) latter(MAAs) two and and series scytonemin scytonemin of compounds ( (5858);); allall ofof thesethesehave have been been been shown reported reported to protect in in LeptolyngbyaLeptolyngbya photosynthetic sp.sp. (Figure (Figure cyanobacteria 18) 18 [23,75,76].)[23,75,76 ]. from solar TheseUVThese damage latter latter two two [23]. series series An of ofinvestigation compoundscompounds have of UV-B been sh shownphotoprotectiveown to to protect protect photosynthetic photosynthetic compounds cyanobacteria in cyanobacteria marine Leptolyngbya from from discoveredsolarsolar UV UVshinorine damage damage [ (23[23].59].) An(FigureAn investigationinvestigation 18), which of UV-B is now photoprotective realized to compounds compoundsbe one of thein in marine marinemost LeptolyngbyadominantLeptolyngbya MAAs discovered shinorine (59) (Figure 18), which is now realized to be one of the most dominant MAAs presentdiscovered in several shinorine species ( 59of) cyanobacteria (Figure 18), which [75] is. Scytonemin now realized has to be a onebroader of the absorption most dominant profile MAAs than the presentpresent in in several several species species ofof cyanobacteriacyanobacteria [[75]75].. Scytonemin Scytonemin has has a a broader broader absorption absorption profile proﬁle than than the the MAAs, protecting against the solar irradiance damage across the UV (UV-A, -B and -C; 250–425 nm). MAAs,MAAs, protecting protecting againstagainst thethe solarsolar irradiance damage damage across across the the UV UV (UV-A, (UV-A, -B -B and and -C; -C; 250–425 250–425 nm). nm). Interestingly, scytonemin also shows interesting anti-inflammatory activity through inhibition of Interestingly,Interestingly, scytoneminscytonemin alsoalso showsshows interestinginteresting anti-inflammatory anti-inﬂammatory activity activity through through inhibition inhibition of of polo-likepolo-likepolo-like kinase kinase kinase stimulated stimulated stimulated cell cellcell proliferation proliferationproliferation pathways pathways [77]. [[77].77]. The The The biosynthesis biosynthesis biosynthesis of of scytonemin, scytonemin,of scytonemin, deriving deriving deriving from fromthefrom assembly the the assembly assembly of tyrosine of of tyrosine tyrosine and andand tryptophan tryptophantryptophan dede derivedrived components, components,components, has has has been been been studied studied studied at at the the at genomic genomicthe genomic and mechanisticandand mechanistic mechanistic level levellevel in several inin severalseveral studies; studies;studies; however, however, all of ofof these thesethese studies studies studies have have have been been been conducted conducted conducted in in other other in other generageneragenera of cyanobacteria of of cyanobacteria cyanobacteria such suchsuch as asasNostoc NostocNostoc punctiforme punctiformepunctiforme ATCCATCC 29133 29133 and and LyngbyaLyngbya Lyngbya aestuarii aestuarii aestuarii [78–81].[78– 81[78–81].].

Figure 18. Scytonemin (58) and the MAA shinorine (59) from Leptolyngbya sp.

2.7.3. PhenolicFigure FigureCompounds 18. Scytonemin 18. Scytonemin (58 (58) and) and the the MAA MAA shinorine ( 59(59) from) fromLeptolyngbya Leptolyngbyasp. sp. Phenolic compounds, including flavonoids and lignans, are typically natural antioxidants as 2.7.3. Phenolicwell as anCompounds important group of bioactive compounds [82]. The extract from a thermophilic cyanobacterium Leptolyngbya sp. collected from northern Tunisia was screened by HPLC and showed Phenolicthe presence compounds, of 25 phenolic including compounds—gallic flavonoids acidand ( 60lignans,), hydroxytyrosol are typically (61), protocatechuicnatural antioxidants acid as well as(62 ),an vanillic important acid (63 group), isovanillic of bioactive acid (64), 3-hydroxybenzoiccompounds [82]. acid The(3-HBA) extract (65), 4-hydroxybenzoicfrom a thermophilic cyanobacteriumacid (4-HBA) Leptolyngbya (66), resorcinol sp. collected (67), naphtoresorcinol from northern (68 Tunisia), syringic was acid screened (69), catechol by HPLC (70 and), and showed the presenceoleuropein of 25(71 phenolic) (Figure compounds—gallic19); chlorogenic acid acid(72), (dihyrdro-60), hydroxytyrosolp-coumaric acid (61 (),73 protocatechuic), dihyrdro-m- acid (62), vanillic acid (63), isovanillic acid (64), 3-hydroxybenzoic acid (3-HBA) (65), 4-hydroxybenzoic acid (4-HBA) (66), resorcinol (67), naphtoresorcinol (68), syringic acid (69), catechol (70), and oleuropein (71) (Figure 19); chlorogenic acid (72), dihyrdro-p-coumaric acid (73), dihyrdro-m-

Mar. Drugs 2020, 18, 508 22 of 29

2.7.3. Phenolic Compounds Phenolic compounds, including ﬂavonoids and lignans, are typically natural antioxidants as well as an important group of bioactive compounds [82]. The extract from a thermophilic cyanobacterium Leptolyngbya sp. collected from northern Tunisia was screened by HPLC and showed the presence of 25 phenolic compounds—gallic acid (60), hydroxytyrosol (61), protocatechuic acid (62), vanillic acid (63), isovanillic acid (64), 3-hydroxybenzoic acid (3-HBA) (65), 4-hydroxybenzoic acid (4-HBA) (66), resorcinol (67), naphtoresorcinol (68), syringic acid (69), catechol (70), and oleuropein (71) (Figure 19); chlorogenicMar. Drugs 2020 acid, 18,( x72 FOR), dihyrdro- PEER REVIEWp-coumaric acid (73), dihyrdro-m-coumaric acid (74), ferulic acid 20 of ( 2775 ), and rosamerinic acids (76) (Figure 20); catechin (77), luteolin-7-glucoside (78), apigenin-7-glucoside (79), coumaric acid (74), ferulic acid (75), and rosamerinic acids (76) (Figure 20); catechin (77), luteolin-7- ﬂavone (80), naringenin (81), luteolin (82), and apigenin (83) (Figure 21); resveratrol (84) and pinoresinol glucoside (78), apigenin-7-glucoside (79), flavone (80), naringenin (81), luteolin (82), and apigenin (83) (85(Figure) (Figure 21); 22 resveratrol)—demonstrating (84) and thatpinoresinolLeptolyngbya (85) (Figuremay constitute 22)—demonstrating a rich source that of antioxidantLeptolyngbya natural may productsconstitute [83 a ,rich84]. source These of compounds antioxidant natural also have products inherent [83,84]. UV-absorbing These compounds properties, also have albeit inherent at more restrictedUV-absorbing wavelengths properties, than albeit scytonemin at more (restricted58). wavelengths than scytonemin (58).

FigureFigure 19.19. HydroxybenzoicHydroxybenzoic acids (HBAs) (HBAs) from from LeptolyngbyaLeptolyngbya sp.sp.

Figure 20. Hydroxycinnamic acids (HCAs) from Leptolyngbya sp.

Mar. Drugs 2020, 18, x FOR PEER REVIEW 20 of 27

coumaric acid (74), ferulic acid (75), and rosamerinic acids (76) (Figure 20); catechin (77), luteolin-7- glucoside (78), apigenin-7-glucoside (79), flavone (80), naringenin (81), luteolin (82), and apigenin (83) (Figure 21); resveratrol (84) and pinoresinol (85) (Figure 22)—demonstrating that Leptolyngbya may constitute a rich source of antioxidant natural products [83,84]. These compounds also have inherent UV-absorbing properties, albeit at more restricted wavelengths than scytonemin (58).

Mar. Drugs 2020, 18, 508 23 of 29 Figure 19. Hydroxybenzoic acids (HBAs) from Leptolyngbya sp.

Mar. Drugs 2020,, 18,, xx FORFORFigureFigure PEERPEER REVIEW REVIEW20.20. Hydroxycinnamic acids acids (HCAs) (HCAs) from from LeptolyngbyaLeptolyngbya sp.sp. 21 21 ofof 2727

FigureFigure 21. Flavonoids from from LeptolyngbyaLeptolyngbya sp. sp.

FigureFigure 22.22. Stilbene and and a a lignan lignan from from LeptolyngbyaLeptolyngbya sp.sp.

2.7.4.2.7.4. Odorous Odorous MetabolitesMetabolites GeosminGeosmin ((8686)) andand 2-methylisoborneol2-methylisoborneol ( (8787)) are are two two earthy-musty earthy-musty odorous odorous terpenoid terpenoid secondary secondary metabolitesmetabolites that that were were firstfirst isolatedisolated fromfrom actinomyce actinomycetestes (Figure (Figure 23). 23). Later, Later, th theseese same same molecules molecules were were foundfound to to have have a a significant significant presencepresence inin manymany cyanobacteriacyanobacteria species. species. These These simple simple terpenoids terpenoids have have eacheach been been reported reported numerous numerous timestimes asas being among the the main main causes causes for for off-flavors off-flavors in in water water and and other other productsproducts [ 85[85].]. Wang Wang et et al. al. isolated isolated86 86and andand87 87from fromfromLeptolyngbya Leptolyngbya bijugata bijugatastrains, strains, andand quantified quantified eacheach at 13.6–22.4at 13.6–22.4 and and 12.3–57.5 12.3–57.5µg /μL,g/L, respectively, respectively, demonstrating demonstrating their their production production by byLeptolyngbya Leptolyngbya[86 [86].[86].].

Figure 23. Geosmin and 2-methylisoborneol (MIB) from Leptolyngbya bijugata.

2.7.5. Pigments Phycocyanin is a pigment–protein complex found in cyanobacteria and eukaryotic algae that functions as a light-harvesting pigment. It is widelywidely usedused inin biotechnological,biotechnological, foodfood andand pharmaceutical industries [87]. Schipper et al. analyzed the phycocyanin content in Leptolyngbya sp. QUCCCM 56 from a desert environment to reveal that it possesses higher andand purerpurer phycocyaninphycocyanin compared to the current commercial source, Arthrospira platensis [87].[87]. OtherOther thanthan absorbingabsorbing light,light, phycocyanin also shows potential anti-aging and proteostasis-suppressive activities. With phycocyanin treatment, the life span of wild-type (N2) C. elegans waswas extendedextended fromfrom 14.814.8 toto 19.119.1 daysdays [88].

Mar. Drugs 2020, 18, x FOR PEER REVIEW 21 of 27

Figure 21. Flavonoids from Leptolyngbya sp.

Figure 22. Stilbene and a lignan from Leptolyngbya sp.

2.7.4. Odorous Metabolites Geosmin (86) and 2-methylisoborneol (87) are two earthy-musty odorous terpenoid secondary metabolites that were first isolated from actinomycetes (Figure 23). Later, these same molecules were found to have a significant presence in many cyanobacteria species. These simple terpenoids have each been reported numerous times as being among the main causes for off-flavors in water and other Mar.products Drugs 2020 [85]., 18 ,Wang 508 et al. isolated 86 and 87 from Leptolyngbya bijugata strains, and quantified 24each of 29 at 13.6–22.4 and 12.3–57.5 μg/L, respectively, demonstrating their production by Leptolyngbya [86].

FigureFigure 23. 23.Geosmin Geosmin andand 2-methylisoborneol2-methylisoborneol (MIB) (MIB) from from LeptolyngbyaLeptolyngbya bijugata. bijugata.

2.7.5.2.7.5. Pigments Pigments PhycocyaninPhycocyanin isis aa pigment–proteinpigment–protein complexcomplex found in cyanobacteria and and eukaryotic eukaryotic algae algae that that functionsfunctions as aas light-harvesting a light-harvesting pigment. pigment. It is widely It is usedwidely in biotechnological, used in biotechnological, food and pharmaceutical food and industriespharmaceutical [87]. Schipper industries et [87]. al. analyzed Schipper the et phycocyaninal. analyzed the content phycocyanin in Leptolyngbya contentsp. in QUCCCMLeptolyngbya 56 fromsp. QUCCCM a desert environment 56 from a desert to revealenvironment that it to possesses reveal that higher it possesses and purer higher phycocyanin and purer phycocyanin compared to thecompared current commercialto the current source, commercialArthrospira source, platensis Arthrospira[87]. platensis Other than [87]. absorbing Other than light, absorbing phycocyanin light, alsophycocyanin shows potential also anti-agingshows potential and proteostasis-suppressive anti-aging and proteostasis-suppressi activities. With phycocyaninve activities. treatment, With thephycocyanin life span of treatment, wild-type the (N2) lifeC. span elegans of wild-typewas extended (N2) C. from elegans 14.8 was to 19.1 extended days [ 88from]. 14.8 to 19.1 days [88]. 3. Laboratory Cultivation Despite many attempts, only a few environmental collections of Leptolyngbya have been propagated under laboratory culture conditions and, from these, there has been a relatively low rate of natural product isolation. Rather, most of the compounds reported in this genus have been discovered from environmental samples (Table1). Martins et al. screened ﬁve Leptolyngbya strains among 28 cyanobacteria samples collected from the Portuguese Coast and cultured in Z8 medium, and showed cytotoxic activities of the extracts against multiple human tumor cell lines [15,16,89]. The most commonly used media for cyanobacteria culture is BG11 [53], originally formulated in 1988 to possess synthetic sea salt, microelements mixture, deionized water and vitamin mixtures [28]. In most cases, even with suitable media and abiotic factors, ﬁlamentous cyanobacteria are slow-growing life forms, normally with growth rates much slower than algae and other bacteria, thereby presenting a challenge for secondary metabolite discovery due to the small quantities of biomass produced in cultures [28,90]. The 2-hydroxyethyl-11-hydroxyhexadec-9-enoate, by contrast, was able to be isolated from the laboratory culture of Leptolyngbya with the relatively small biomass of 383.6 g because of its reduced structural complexity and higher production yield [41].

4. Conclusions Leptolyngbya is a widely distributed genus of cyanobacteria that has emerged to be a very rich source of structurally novel and biologically active natural products. However, to date, this genus appears to be underexplored for its chemical, biological and biosynthetic potential when compared to some other genera of ﬁlamentous cyanobacteria, such as Moorena and Symploca [90]. Working with Leptolyngbya has been challenging due to the diﬃculty in bringing it into culture in the laboratory environment. This has impeded not only new compound discovery, but also exploration of its genomic characteristics, including those that are responsible for natural product biosynthesis. It is possible that developments with the heterologous expression of cyanobacterial natural product pathways will enable a more extensive exploration of the rich secondary metabolome of this genus in the future [91,92].

Author Contributions: Conceptualization, Y.L. and W.H.G.; formal analysis, Y.L. and W.H.G.; investigation, Y.L. and W.H.G.; resources, Y.L. and W.H.G.; data curation, Y.L. and W.H.G.; writing—original draft preparation, Y.L.; writing—review and editing, Y.L., C.B.N., K.L.A., H.G., W.H.G; visualization, Y.L. and W.H.G.; supervision, H.G. and W.H.G.; project administration, W.H.G; funding acquisition, W.H.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NIH grant GM107550 (to W.H.G). Mar. Drugs 2020, 18, 508 25 of 29

Acknowledgments: Y.L. thanks financial support provided by the fellowship from the Ocean University of China. We thank E. Glukhov for providing the photomicrographs of Leptolyngbya in culture. Conflicts of Interest: William H. Gerwick declares a competing financial interest as a cofounder of NMR Finder LLC.

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