Chemodiversity in Freshwater and Terrestrial Cyanobacteria – a Source for Drug Discovery

Abstract

Cyanobacteria are considered a promising source for new pharmaceutical lead compounds and a large number of chemically diverse and bioactive metabolites have been obtained from cyanobacteria over the last few decades. This review highlights the structural diversity of natural products from freshwater and terrestrial cyanobacteria. The review is divided into three areas: cytotoxic metabolites, protease inhibitors, and antimicrobial metabolites. The first section discusses the potent cytotoxins cryptophycin and tolytoxin. The second section covers protease inhibitors from freshwater and terrestrial cyanobacteria and is divided in five subsections according to structural class: aeruginosins, cyanopeptolins, microviridins, anabaenopeptins, and microginins. Structure activity relationships are discussed within each protease inhibitor class. The third section, antimicrobial metabolites from freshwater and terrestrial cyanobacteria, is divided by chemical class in three subsections: alkaloids, peptides and terpenoids. These examples emphasize the structural diversity and drug development potential of natural products from freshwater and terrestrial cyanobacteria.

1. INTRODUCTION

Cyanobacteria (also known as blue-green algae) are Gram-negative bacteria and represent the only group of prokaryotes that are able to perform oxygenic photosynthesis similar to plants. Fossil evidence indicates that cyanobacteria have populated the earth for around 3.5 billion years and ancient cyanobacteria are believed to have been instrumental in the creation of the Earth’s oxygen rich atmosphere [1,2]. Today, cyanobacteria can be found worldwide in most in terrestrial, marine, brackish and fresh water environments. This includes many extreme environments such as hot springs, arid desert soils, the Arctic, and areas of high salinity. Some authors attribute this adaptability, in part, to the ability of cyanobacteria to produce diverse secondary metabolites. Cyanobacteria are considered a promising source for new pharmaceutical lead compounds and a large number of chemically diverse metabolites have been obtained from cyanobacteria.

Many strains of cyanobacteria are known to produce toxins (hepatotoxins and neurotoxins) associated with toxic water-blooms and represent a public health hazard due to their presence in water reservoirs for drinking water supplies. Many recent and excellent reviews covering the various aspects of cyanobacterial metabolites, especially toxins, have been published [3–9]. Multiple reviews have also detailed the numerous biologically active compounds isolated from marine cyanobacteria [10–17], however, freshwater and terrestrial cyanobacteria have also contributed molecules with significant pharmacological activities, e.g. cryptophycins and tolytoxin. This review will focus on biologically active compounds isolated from freshwater and terrestrial cyanobacteria (Table 1) but, will exclude compounds that have shown either potent hepatotoxicity and/or neurotoxicity, e.g. microcystins, cylindrospermopsin, and saxitoxins. In addition, compounds from freshwater or terrestrial cyanobacteria that are members of the peptide classes listed below were also included regardless of biological activity reported.

Table 1.

Compounds from Freshwater and Terrestrial Cyanobacteria

Compound	Source	Activity1	Compound Class2	Reference
1,8-dihydroxy-4-methylanthraquinone	Nostoc commune	antibacterial	polyketide	[143]
20-nor-3a-acetoxy-12-hydroxy-abieta-5,7,9,11,13-pentaene	Microcoleous lacustris	antibacterial	terpenoid	[134]
20-nor-3a-acetoxyabieta-5,7,9,11,13-pentaene	Microcoleous lacustris	antibacterial	terpenoid	[134]
4,4′-dihydroxybiphenyl	Nostoc insulare	anticyanobacterial, antibacterial, antifungal	phenolic	[144]
4-hydroxy-7-methylindan-1-one	Nostoc commune	antibacterial	indane	[143]
8-[(5-carboxy-2,9-epoxy)benzyl]-2,5-dihydroxy-1,1,4a,7,8-pentamethyl-1,2,3,4,4a,6,7,8,9,10,10-adodecahydrophenanthrene	Nostoc commune	antibacterial	terpenoid	[143]
9-deazaadenosine	Anabaena affinis (VS-1)	cytotoxic	nucleoside	[145]
A90720A	Microchaete loktakensis	protease inhibitor	peptide (cyan)	[146]
aerucyclamides A & B	Microcystis aeruginosa (PCC 7806)	cytotoxic	peptide (cyca)	[147]
aeruginoguanidines 98-A - 98-C	Microsystis aeruginosa (NIES-98)	cytotoxic	peptide (lin)	[148]
aeruginopeptin 917S-A to 917S-C	Microcystis aeruginosa	protease inhibitor, cytotoxic	peptide (cyan)	[149]
aeruginopeptin 95-A, 95-B, 228-A, 228-B	Microcystis aeruginosa	protease inhibitor	peptide (cyan)	[150]
aeruginosin 101	Microcystis aeruginosa (NIES-101)	protease inhibitor	peptide (aeru)	[151]
aeruginosin 102A & 102B	Microcystis viridis (NIES-102)	protease inhibitor	peptide (aeru)	[152]
aeruginosin 103A	Microcystis viridis (NIES-103)	protease inhibitor	peptide (aeru)	[153]
aeruginosin 205A & 205B	Oscillatoria agardhii (NIES-205)	protease inhibitor	peptide (aeru)	[154]
aeruginosin 298A & 298B	Microcystis aeruginosa (NIES-298)	protease inhibitor	peptide (aeru)	[56]
aeruginosin 89A & 89B	Microcystis aeruginosa (NIES-89)	protease inhibitor	peptide (aeru)	[151]
aeruginosin 98A – 98C	Microcystis aeruginosa (NIES-98)	protease inhibitor	peptide (aeru)	[155]
aeruginosin EI461	Microcystis aeruginosa (IL-217 and IL-231)	protease inhibitor	peptide (aeru)	[156]
aeruginosin KY608 & KY642	Microcystis sp. (IL-323)	protease inhibitor	peptide (aeru)	[157]
agardhipeptins A & B	Oscillatoria agardhii (NIES-204)	protease inhibitor	peptide (cycl)	[158]
ambigols A-C	Fischerella ambigua	antibacterial, antifungal	aromatic	[117,159,160]
ambiguine isonitriles	Fischerella ambigua, Fischerella sp.	antibacterial, antifungal, cytotoxic	alkaloid	[110,111]
anabaenopeptilide 90-A, 90-B, 202-A, 202-B	Anabaena spp.	n.r.	peptide (cyan)	[161,162]
anabaenopeptin 908 & 915	Plantothrix agardhii (CYA 126/8)	CPA inhibitor	peptide (ana)	[163]
anabaenopeptin A	Anabanea flos-aquae (NCR 525-17)	relax rat aorta	peptide (ana)	[164]
anabaenopeptin B	Anabanea flos-aquae (NCR 525-17), Oscillatoria agardhii	relax rat aorta	peptide (ana)	[164,165]
anabaenopeptin C & D	Anabaena sp.	n.r.	peptide (ana)	[161]
anabaenopeptin E-F	Oscillatoria agardhii (NIES-204)	n.r.	peptide (ana)	[166]
anabaenopeptin G-H	Oscillatoria agardhii (NIES-595)	CPA inhibitor	peptide (ana)	[167]
anabaenopeptin HU892	Microcystis aeruginosa	n.r.	peptide (ana)	[168]
anabaenopeptin I-J	Aphanzomenon flos-aquae	CPA inhibitor	peptide (ana)	[169]
anabaenopeptin T	Bloom	CPA inhibitor	peptide (ana)	[93]
anabaenopeptins NZ825, NZ841, and NZ857	Anabaena sp.	n.r.	peptide (ana)	[170]
borophycin	Nostoc linckia, Nostoc spongiaeforme	cytotoxic, antibacterial	polyketide	[171,172]
brunsvicamide A-C	Tychonema sp.	protease inhibitor	peptide (ana)	[173]
calophycin	Calothrix fusca	antifungal, cytotoxic	peptide (lipo)	[127]
calothrixin A & B	Calothrix sp.	cytotoxic, antimalarial	alkaloid	[46]
carbamidocyclophanes A-E	Nostoc sp. (CAVN 10)	cytotoxic	polyketide	[174]
comnostins A-E	Nostoc commune	antibacterial, cytotoxic	terpenoid	[133]
coriolic acid	Oscillatoria redekei	antibacterial	fatty acid	[175]
cryptophycins	Nostoc sp. (ATCC 53789)	cytotoxic, tubulin inhibitor	peptide (cryp)	[18,21–23,176]
cyanobacterin	Scytonema hofmannii	antifungal	aromatic	[177,178]
cyanopeptolin 954	Microcystis aeruginosa (NIVA CYA 43)	protease inhibitor	peptide (cyan)	[179]
cyanopeptolin 963	Microcystis sp.	protease inhibitor	peptide (cyan)	[180]
cyanopeptolin 975, 1009, 1006, 1014, 1016, 1020, 1034, 1048, and 1053	Microcystis sp.	n.r.	peptide (cyan)	[181]
cyanopeptolin CB071	Aphanocapsa sp.	protease inhibitor	peptide (cyan)	[182]
cyanopeptolin S	Microcystis sp. (Bloom)	protease inhibitor	peptide (cyan)	[183]
cyanopeptolins 880 & 960	Planktothrix agardhii	n.r.	peptide (cyan)	[163]
cyanopeptolins A-D	Microcystis sp. PCC 7806	n.r.	peptide (cyan)	[184]
cyanostatin A & B	Bloom	leucine aminopeptidase M inhibitor	peptide (micg)	[185]
cylindrocyclophanes A-F	Cylindrospermum lichenforme	cytotoxic	polyketide	[186,187]
dehydroradiosumin	Anabaena cylindrica	protease inhibitor	peptide (lin)	[188]
dendroamides A-C	Stigonema dendroideum	cytotoxic	peptide (cyca)	[44]
α-dimorphecolic acid	Oscillatoria redekei	antibacterial	fatty acid	[175]
dodecahyrdophenanthrene	Nostoc commune	antibacterial	terpenoid	[143]
ferintoic Acids A-B	Microcystis aeruginosa	n.r.	peptide (ana)	[189]
fischerellins A and B	Fischerella muscicola	antifungal, anticyanobacterial	alkaloid
fischerindoles	Fischerella muscicola	antifungal	alkaloid	[107]
hapalindoles	Hapalosiphon fontinalis, Fischerella sp.	antibacterial, antimycotic	alkaloid	[99,104,105,115]
hassallidin A & B	Hassallia sp.	antifungal	peptide (lipo)	[128,129]
hofmannolin	Scytonema hofmanni	n.r.	peptide (cyan)	[190]
ichtyopeptins A-B	Microcystis ichtyoblabe	antivirial	peptide (cyan)	[191]
indolecarbazole	Nostoc sphaericum	cytotoxic, antiviral	alkaloid	[119]
insulapeptolide A – H	Nostoc insulare	protease inhibitor	peptide (cyan)	[65]
kawaguchipeptins A & B	Microcystis aeruginosa	antibacterial	peptide (cycl)	[192]
laxaphycins A & B	Lyngbya majuscula	antifungal, cytotoxic	peptide (cycl)	[193–195]
micrococin SF608	Microcystis sp.	protease inhibitor	peptide (aeru)	[57]
microcyclamide	Microcystis aeruginosa (NIES-298)	cytotoxic	peptide (cyca)	[42]
microcystilide A	Microcystis aeruginosa (NO-15-1840)	cytotoxic	peptide (cyan)	[196]
microginin	Microcystis aeruginosa	ACE inhibitor	peptide (micg)	[91]
microginin 711, FR5, FR3, FR6, FR9, 755, 757, 764, 767, 478	Microcystis sp.	n.r.	peptide (micg)	[197]
microginin AL584	Microcystis sp.	yeast toxin	peptide (micg)	[198]
microginin FR1	Microcystis sp.	ACE inhibitor	peptide (micg)	[92]
microginin SD755	Microcystis aeruginosa	bovine amino peptidase inhibitor	peptide (micg)	[199]
microginin T1 & T2	Bloom	leucine aminopeptidate and ACE inhibitor	peptide (micg)	[93]
microginins 299-A to 299-B	Microcystis aeruginosa (NIES-299)	leucine aminopeptidase inhibitor	peptide (micg)	[200]
microginins 299-C to 299-D	Microcystis aeruginosa (NIES-299)	leucine aminopeptidase inhibitor	peptide (micg)	[201]
microginins 478, 51-A, 51-B, 91-A - 91-E	Microcystis aeruginosa	aminopeptidase M and ACE inhibitor	peptide (micg)	[94]
microginins 99-A & 99-B	Microcystis aeruginosa (NIES-299)	n.r.	peptide (micg)	[201]
micropeptin 103	Microcystis viridis (NIES-103)	protease inhibitor	peptide (cyan)	[202]
micropeptin 478A & 478B	Microcystis aeruginosa	protease inhibitor	peptide (cyan)	[203]
micropeptin 88-A – 88-F	Microcystis aeruginosa (NIES-88)	protease inhibitor	peptide (cyan)	[204]
micropeptin 88-N & 88-Y	Microcystis aeruginosa (NIES-88)	protease inhibitor	peptide (cyan)	[205]
micropeptin 90	Microcystis aeruginosa (NIES-90)	protease inhibitor	peptide (cyan)	[206]
micropeptin A & B	Microcystis aeruginosa	protease inhibitor	peptide (cyan)	[207]
micropeptin C – F	Microcystis aeruginosa (NIES-100)	protease inhibitor	peptide (cyan)	[208]
micropeptin EI964 & EI992	Microcystis aeruginosa (Bloom)	protease inhibitor	peptide (cyan)	[156]
micropeptin MZ1019	Microcystis sp.	protease inhibitor	peptide (cyan)
micropeptin MZ771	Microcystis sp.	protease inhibitor	peptide (cyan)
micropeptin MZ845, MZ859, MZ925, MZ939A, MZ939B	Microcystis sp.	protease inhibitor	peptide (cyan)	[209]
micropeptin SD1002, SD944, SD979, SD999	Microcystis sp. (Bloom)	protease inhibitor	peptide (cyan)	[199]
micropeptin SF909 & SF995	Microcystis sp. (Bloom)	protease inhibitor	peptide (cyan)	[57]
micropeptin T1 & T2	Bloom	protease inhibitor	peptide (cyan)	[93]
micropeptin T-20	Microcystis aeruginosa	protease inhibitor	peptide (cyan)	[210]
micropeptins HU1069, HU989, HU1021, HU1041, HU975, HU895A, HU909, HU895B, 478-A, 478-B	Microcystis aeruginosa	protease inhibitor	peptide (cyan)	[168]
microviridin A	Microcystis viridis (NIES-102)	protease inhibitor	peptide (micv)	[75]
microviridin B & C	Microcystis aeruginosa (NIES-298)	protease inhibitor	peptide (micv)	[76]
microviridin D – F	Oscillatoria agardhii (NIES-204)	protease inhibitor	peptide (micv)	[79]
microviridin G & H	Nostoc minutum (NIES-26)	protease inhibitor	peptide (micv)	[78]
microviridin I	Oscillatoria agardhii	protease inhibitor	peptide (micv)	[77]
microviridin J	Microcystis aeruginosa (UWOCC CBS)	protease inhibitor	peptide (micv)	[211]
microviridin SD1634, SD1652, SD1684	Microcystis aeruginosa	protease inhibitor	peptide (micv)	[212]
mirabilene isonitriles A-F	Scytonema hofmannii	antifungal	polyketide	[213]
mirabimides A-E	Scytonema mirabile	cytotoxic	peptide (lin)	[214,215]
muscoride A	Nostoc muscorum	antibacterial	peptide (lin)	[216]
nodulapeptins A-B	Nodularia spumigena (AV1)	n.r.	peptide (ana)	[217]
norharmane	Nodularia harveyana	anticyanobacterial, antibacterial, antifungal	alkaloid	[144]
noscomin	Nostoc commune	antibacterial	terpenoid	[132]
nostocarboline	Nostoc sp.	trypsin & BChE inhibitor, anticyanobacterial, antialgal	alkaloid	[121,218]
nostocine A	Nostoc spongiaeforme	antialgal, toxic	purine	[219]
nostocyclin	Nostoc sp.	toxicity (brine shrimp, mouse)	peptide (cyan)	[220]
nostocyclopeptide A1 & A2	Nostoc sp. (ATCC 53789)	cytotoxic	peptide (cycl)	[221]
nostocyclophanes A-D	Nostoc linka	cytotoxic	polyketide	[222]
nostocyclyne A	Nostoc sp.	antibacterial	polyketide	[223]
nostofungicidine	Nostoc commune	antifungal, cytotoxic	peptide (lipo)	[126]
nostoginin BN741 & BN578	Nostoc sp.	bovine amino peptidase inhibitor	peptide (micg)	[224]
nostopeptin A & B	Nostoc minutum	protease inhibitor	peptide (cyan)	[66]
nostopeptin BN290	Nostoc sp.	protease inhibitor	peptide (cyan)	[224]
nostopeptin BN920	Nostoc sp. (TAU IL-235)	protease inhibitor	peptide (cyan)	[179,224]
oscillaginin A & B	Oscillatoria agardhii	n.r.	peptide (micg)	[225]
oscillamide B-C	Planktothrix spp.	protein phosphotase inhibitor	peptide (ana)	[64]
oscillamide Y	Oscillatoria agardhii	protease inhibitor	peptide (ana)	[226]
oscillapeptilide 97-A & 97-B	Oscillatoria agardhii (strain 97)	protease inhibitor	peptide (cyan)	[77]
oscillapeptin	Oscillatoria agardhii (NIES-204)	protease inhibitor	peptide (cyan)	[227]
oscillapeptin A – C, DYI, E & F	Oscillatoria agardhii	protease inhibitor	peptide (cyan)	[228]
oscillapeptin DTS	Oscillatoria agardhii	protease inhibitor	peptide (cyan)	[229]
oscillapeptin G	Oscillatoria agardhii (CYA128)	protease inhibitor	peptide (cyan)	[77]
oscillapeptin J	Planktothrix rubescens	toxic (crusteacean)	peptide (cyan)	[230]
pahayokolides A-B	Lyngbya sp.	cytotoxic	peptide (cycl)	[231,232]
plaktopeptin BL1125, BL1061, BL843	Planktothrix rubescens	protease inhibitor	peptide (cyan)	[233]
planktocyclin	Planktothrix rubescens	protease inhibitor	peptide (cyca)	[234]
schizopeptin 791	Schizotrix sp.	protease inhibitor	peptide (ana)	[235]
schizotrin A	Schizotrix sp.	antibacterial, antifungal	peptide (lipo)	[236]
scyptolin A & B	Scytonema hofmanni	protease inhibitor	peptide (cyan)	[237]
scytonemin	Stigonema sp.	cytotoxic	alkaloid	[238]
scytonemin A	Scytonema sp.	antibacterial, antifungal	peptide (lipo)	[239]
scytophycin A-E	Scytonema spp., Cylindrospermum muscicola, Scytonema pseudohomanni	cytotoxic, actin inhibitor	polyketide	[31,32]
spiroidesin	Anabaena spiroides	protease inhibitor, anti-cyanobacterial	peptide (micg)	[240]
spiroidesin	Anabaena spiroides	anticyanobacterial	peptide (lin)	[240]
tenuecyclamides A-D	Nostoc spongiaeforme var. tenue (TAU strain IL-184-6)	cytotoxic	peptide (cyca)	[171]
tetradecane, heptadecane et al.	Spirulina platensis	antibacterial, antifungal	volatile alkane	[241]
tjipanazoles	Tolypothrix tjipanasensis	antifungal, antibacterial	alkaloid	[116,160]
tolybyssidins A & B	Tolypothrix byssoidea	antifungal	peptide (cycl)	[242]
tolyporphin A	Tolypothrix nodosa Bharadwaja (UH HT-58-2)	cytotoxic	porphinoid	[243]
tolyporphin B-K	Tolypothrix nodosa Bharadwaja (UH HT-58-2)	cytotoxic	porphinoid	[244,245]
tolytoxin	Tolypothrix conglutinata var. colorata	cytotoxic, actin inhibitor	polyketide	[31]
toyocamycin, tubercidin and the corresponding 5′-α-D-glucopyranose derivatives	Cyanobacteria belonging to Scytonemataceae	antifungal, cytotoxic	nucleoside	[246]
tychonemamides A-B	Tychonema sp.	cytotoxic	peptide (cyca)	[247]
welwitindolinones	Hapalosiphon welwitschii, Westiella intricate	antifungal, anticancer	alkaloid	[109]
westellamide	Westielliopsis prolifica	cytotoxic	peptide (cyca)	[43]

¹Abbreviations used: ACE: angiotensin converting enzyme; AChE: actyl Co-A esterase; BChE: butyl Co-A esterase; CPA: carboxypeptidase A; n.r.: none reported;

²Abbreviations for peptide subclasses: ana: anabaenopeptin; aeru: aeruginosin; cryp: cryptophycin; cyan: cyanopeptolin; cyca: cyclamide; cycl: other cyclic; lin: other linear; lipo; lipopeptide; micg: microginin; micv: microviridin

Each compound in Table 1 was assigned into one of the following classes: alkaloid, peptide, polyketide, porphinoid, terpenoid, volatile alkane, or other. When the compounds were grouped by these classes Fig. (1), it was revealed that the largest group was peptides, which accounted for 64.7% of the compounds reviewed. The second largest group was alkaloids (18.0%) followed by polyketides (6.3%). In this review, peptides were further categorized into the following peptide families, which are discussed later in this review: anabaenopeptin, aeruginosin, cryptophycin, cyanopeptolin, cyclamide, microginin, and microviridin. These seven classed accounted for nearly 90% of all peptides reviewed. For the other peptides (10.5%) the following families were also included: cyclic, linear, and lipopeptide. When the peptides were grouped by these families Fig. (2), the largest group was cyanopeptolin (40.1%) with the microginins as the second largest peptide family (13.4%).

Figure 1.

Compounds reviewed grouped by structural class.

Figure 2.

Peptides grouped by peptide family.

2. CYTOTOXIC METABOLITES FROM CYANOBACTERIA

Structural diversity is important when assessing the pharmaceutical potential of a group of secondary metabolites; however a more important attribute is the biological activities associated with those compounds. Many of the compounds isolated from cyanobacteria have shown significant cytotoxicity. Some of these cytotoxic compounds isolated from cyanobacteria have also shown significant antifungal activity, such as the cryptophycins as well as tolytoxin and the related scytophycins. However, many of these cyanobacterial metabolites were primarily evaluated for their cytotoxic potential. One compound in particular, cryptophycin 1 (1), resulted in the development of a clinical candidate for the treatment of cancer.

2.1. Cryptophycins: A Family of Cytotoxic Depsipeptides

Cryptophycin 1 (1) was first isolated from a cultured Nostoc sp. (ATCC 53789) as an antifungal lead by a group from Merck [18]. This depsipeptide was later found to display potent cytotoxicity against the human nasopharynx (KB, IC₅₀ 9.2 pM), colon (LoVo, IC₅₀ 10 pM), and ovarian (SKOV3, IC₅₀ 20 pM) carcinoma cell lines. The mechanism of action was determined to be inhibition of microtubule polymerization and cryptophycin 1 was shown to also be effective in drug-resistant cancer cell lines [19,20]. Cryptophycin 1 and more than 25 additional cryptophycins were isolated from a second Nostoc sp. (GSV 224) [21–23]. The structural diversity of these natural cryptophycins included differing patterns of chlorination (non-, mono-, and di-) on the tyrosine ring (residue 2), the absolute configuration of the α-carbon of the tyrosine residue, presence or absence of the 2-methyl on the 3-amino-propionic acid residue (residue 3), and absolute configuration of the epoxide as well as its substitution with a double bond (residue 1). The cryptophycins have shown varied cytotoxic potencies, however cryptophycin 1 still remains the most potent, naturally occurring cryptophycin reported.

The potent cytotoxicity of the cryptophycins led to a drug development program that ultimately resulted in the clinical evaluation of the semi-synthetic product, cryptophycin 52 (2). Cryptophycin 52 displayed similar potency as cryptophycin 1 [24] and was evaluated in phase II clinical trials for the treatment of platinum-resistant ovarian cancer and advanced lung cancer [25,26]. Cryptophycin 52 displayed modest activity, in the ovarian cancer trial, with 10 of 24 patients experiencing partial remission or a stable disease state. In this trial, there were five episodes of grade 3 or higher laboratory toxicity, which included anemia, thrombocytopenia, increased creatinine, and hyperbilirubinema. The most common side-effects were fatigue, nausea, constipation, and neuropathy; however D’Agostino et al. suggested that the compound should be further evaluated [26].

The results from the lung cancer trial showed cryptophycin 52 to have significant levels of toxicity with only limited activity. In this trial, the most common side effects were neuropathy and pain. The authors attributed the neurotoxicity to the peak dose of cryptophycin 52 and modified the dosing schedule, which helped to reduce the toxic side effects. Based upon these results, Edelman et al. suggested further evaluation using alternate dosing schedules as well as the possibility of use in combination therapy [25].

Ultimately, these disappointing results lead to the termination of clinical trials, however cryptophycin 52 may not have been the best drug candidate. In particular, chlorohydrin analogs of cryptophycin 1 and 52, named cryptophycin 8 (3) and 55 (4), showed improved efficacy, but were not stable in solution. Thus, cryptophycin 52 became the lead clinical candidate. Eventually, it was found that glycinate derivatives of the chlorohydrin cryptophycins (5–6) displayed improved stability, as compared to cryptophycin 8 and 55, and maintained the increased efficacy in vivo, as compared to cryptophycin 1 and 52 [27]. However, to date, no clinical evaluations of these glycinate derivatives have been reported.

Recent studies of the cryptophycin biosynthetic pathway have opened more avenues to create novel cryptophycin analogs. A report by Magarvet et al. showed that the substrate tolerance of the cryptophycin synthases could be used to produce new cryptophycin analogs, as well as the semi-synthetic cryptophycin 52, by introducing specific precursors to cultures of Nostoc sp. (GSV 224) [28]. The same research group also reported that the thioesterase portion of the crpD synthase could be expressed in a heterologous host, purified, and then reacted in vitro to produce cryptophycin analogs from synthetic, linear precursors [29]. This chemoenzymatic approach was further improved by the identification and purification of the cryptophycin P450 epoxidase, which allowed the chemoenzymatic formation of the benzylic epoxide from styrene-containing cryptophycin precursors [30]. Ultimately, the results from these synthetic and biosynthetic studies could be used to develop a second generation of cryptophycins with improved efficacy with lower toxicity than the first clinical candidate, cryptophycin 52.

2.2. Tolytoxin and Scytophycins: Inhibitors of Actin Polymerization

Tolytoxin and scytophycins (7–9) are structurally related macrocyclic lactones isolated from cyanobacteria belonging to the genera Scytonema, Tolypothrix, [31] and Cylindrospermum [32]. This class of compounds displayed potent cytotoxic and antifungal activities. Scytophycin A and B displayed antiproliferative activity in a human nasopharyngeal carcinoma (KB) cell line with an IC₅₀ of 1.2 nM. Scytophycins C-E displayed levels of cytotoxicity that were 10- to 100-fold less potent [31]. In addition, MDR cell lines, with a P-glycoprotein pump, were as sensitive as non-MDR cell lines [33,34] Tolytoxin also inhibited the growth of a wide array of fungi with MIC values from 0.25–8 nM [35–37]. Tolytoxin and scytophycins, as well as structurally related marine macrolides, such as swinholides and sphinxolides, have been found to inhibit the polymerization of actin microfilaments within the cell. [33,38–40] Ultimately, the potent activity of these compounds was not selective enough for the development as a drug candidate, however, these compounds may have value as agricultural antifungal agents [37].

2.3. Cyclamides: Ribosomal Cyclic Hexapeptides

The cyclamides (10) are a class of cyclic hexapeptides that contain three azole or azoline rings. These compounds have been isolated from freshwater/terrestrial cyanobacteria of the orders Chroococcales, Nostocales, and Stigonematales, as well as marine organisms. A notable example of these peptides is bistramide A, which was originally isolated from Lissoclinum bistratum, a marine ascidian [41]. These compounds have a repeating motif of a nitrogen and oxygen/sulfur containing five-membered ring and an amino acid, typically with a small non-polar side chain. The five-membered heterocycles are present in both the mono-unsaturated oxazoline/thiazoline and the aromatic oxazole/thiazole form.

Many of the cyclamides have displayed cytotoxicity in a variety of cancer cell lines (IC₅₀ 2 – 3 μM) [42,43]. Dendroamide A (10, R₁= D-Val, R₂=R₄=thiazole, R₃=R₅= D-Ala, R₆=oxazole) has not been reported as cytotoxic however it was shown to reverse multi-drug resistance in MCF-7/ADR (breast cancer) cells through the inhibition of the P-glycoprotein drug efflux pump [44].

It has been shown that these compounds, in particular Microcylamide, are the post-translational products of a ribosomal precursor peptide [45]. The ribosomal nature of this biosynthetic pathway could simplify future combinatorial biosynthesis attempts of these molecules, given that introduced modified precursor peptides could be used to produce a library of cyclamide analogs.

2.4. Calothrixins: Topoisomerase I Inhibitors from Cyanobacteria

Calothrixin A (11) and B, which were isolated from a cultured Calothrix sp. (CAN 95/2), displayed antiproliferative activity against a human cervical cancer cell line (HeLa) with IC₅₀ values of 40 and 350 nM, respectively [46]. Recent studies revealed that these compound inhibited DNA topoisomerase I and did arrest the cell cycle at the S and G₂/M phases [47].

3. PROTEASE INHIBITORS FROM CYANOBACTERIA

Many cyanobacterial secondary metabolites have shown protease inhibitory activities. The role of these compounds in nature is not well understood. Various proteases, e.g. elastase, trypsin and chymotrypsin, are involved with digestion in animals, including arthropods that may graze on freshwater cyanobacteria, e.g. Daphnia magna, and researchers have reported that compounds from cyanobacteria are able to inhibit these digestive enzymes found in Daphnia [48–50]. It is possible that the protease inhibitors produced by cyanobacteria act as anti-grazing compounds however, additional research is required to substantiate this theory.

The overwhelming majority of protease inhibitors isolated from cyanobacteria are peptides, typically of non-ribosomal biosynthetic origin. Many of these peptides share common structural features that can be used to group cyanobacterial peptides into classes. For this review, we will be using the peptide classes defined by Welker and von Döhren [16]. These classes have been recognized, at least informally, by the literature both prior to as well as after this publication, which indicates wide support by the research community for these definitions. More importantly, there are good correlations between the peptide class and the type of biological activity observed. For example, cyanopeptolins are typically serine protease inhibitors, while anabaenopeptins typically lack this activity but are frequently found to inhibit exopeptidases, such as carboxypeptidase A. Furthermore, the hepatotoxic microcystins, although not discussed in this review, are strong inhibitors of protein phosphotases.

From a medicinal standpoint, inhibitors of generic or digestive proteases can be utilized in the development of therapeutic agents, since many disease processes involve proteases, e.g. viral proteases and the 20S proteasome, [51,52] that have substrate specificities similar to digestive proteases, e.g. elastase, trypsin, and chymotrypsin. For example, thrombin, which is a key enzyme in clotting and platelet formation, has been characterized as having a trypsin-like activity [53]. The 20S proteasome, an important target for cancer chemotherapy, has both trypsin- and chymotrypsin-like activities [54,55]. Thus, digestive proteases can be used as model systems for many medicinally important proteases.

3.1. Aeruginosins: A Family of Thrombin Inhibitors

The first aeruginosin, aeruginosin 298A (12, X₁=X₂=H, R₁=R₃=OH, R₂=L-Leu, R₄=argininol), was isolated from a cultured Microcystis aeruginosa (NIES-298) and selectively inhibited trypsin (IC₅₀ 0.5 μM) and thrombin (IC₅₀ 1.7 μM) [56]. Additional aeruginosins have been isolated from both cultured and field collected Microcystis species as well a cultured Oscillatoria agardhii (NIES-205). This family of linear peptides (12) contains a 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety and has a fairly conserved tetrapeptide structure. Residue 1 is a 2-hydroxy-3-phenylpropionic acid with an aromatic ring that is typically substituted with either phenolic hydroxy or halogen moieties. Residue 2 is typically a small non-polar amino acid, e.g. leucine or allo-isoleucine, but there have been a few reports of tyrosine and phenylalanine. Residue 3 is the Choi moiety, which can be hydroxylated, sulfated, or chlorinated at position 6 (12, R₃). The fourth residue of the peptide is typically a modified arginine, although two aeruginosins (298B and EI461) lack the fourth residue. It is likely that this modified arginine is important for biological activity, since the loss of this moiety correlates with a loss of protease inhibition.

Other members of the aeruginosin family include Oscillarin, which was isolated from Oscillatoria agardhii, microcin SF608, isolated from a bloom of Microcystis sp. [57], and the dysinosins, which were obtained from marine sponges [58,59]. Considering the structural similarity of the dysinosins and the aeruginosins, it is likely that the dysinosins are produced by cyanobacteria living within the sponge. It also interesting to note that the dysinosins represent the only members of the aeruginosin family obtained from a marine source.

Most members of the aeruginosin family have been shown to inhibit thrombin and plasmin, both enzymes have trypsin-like substrate specificities. In particular, chlorodysinosin A displayed the most potent inhibition of thrombin (IC₅₀ 5.7 nM) [60]. Based upon these activities, researchers have utilized members of the aeruginosin family to create synthetic thrombin inhibitors. In 2007, Hanessian et al. reported a structure-based approach that produced a set of molecules with thrombin IC₅₀ values less than 10 nM and the most potent compound displaying an IC₅₀ of 1.6 nM. [61] Further investigation of the aeruginosin family could yield new thrombin inhibitors, which could be used to treat deep vein thrombosis, myocardial infarction, and stroke [62,63].

3.2. Cyanopeptolins: Cyclic Serine Protease Inhibitors

The cyanopeptolins have a fairly conserved, hexadepsipeptide structure (13). In particular, residue 2 is a 3-amino-6-hydroxy-2-piperidone (Ahp) moiety, with only one report of an O-methyl Ahp [64]. This Ahp moiety, or the O-methyl analog, is a defining feature of this class of depsipeptides. In addition, residue 4 is a N-methyl aromatic amino acid, typically tyrosine or phenylalanine, although there have been reports of tryptophan and kynurenine in this position. In addition, a tyrosine at this position may be O-methylated as well as halogenated (chlorine or bromine). Residue 5 is commonly a small non-polar amino acid, i.e. leucine, isoleucine, or valine. Residue 6 is most often a threonine, with a few reports of β-hydroxy-γ-methyl-proline [65,66]. In either case, the side chain hydroxy of residue 6 is linked, via an ester bond, to carboxylate moiety of the amino acid at residue 5. All reported cyanopeptolins also have a side chain attached via the amino group on the amino acid at position 6. These side chains can be quite variable, but often include a terminal acyl moiety, e.g. acetic or hexanoic acid.

Presently, more than 120 different cyanopeptolins have been reported with more than 82% isolated from freshwater/terrestrial cyanobacteria. Analysis of the biological source of these freshwater/terrestrial cyanopeptolins (N=108) reveals that 65% were isolated from members of the order Chroococcales, typically Microcystis spp., 19% isolated from members of the order Nostocales, and 16% from the order Oscillatoriales. It is also interesting to note that of the cyanopeptolins obtained from marine and brackish cyanobacteria (N=23), 21 were isolated from members of the order Oscillatoriales [67–71] and the remaining two cyanopeptolins were isolated from cyanobacteria of the order Nostocales [72]. While the cyanopeptolins may come from a variety of biological sources, a common characteristic of most cyanopeptolins is the ability to inhibit various proteases, e.g. chymotrypsin, trypsin, and elastase.

A review of reported cyanopeptolins and their associated activity reveals that residue 1 frequently corresponds to the key residue (P1) for protease specificity. Based upon these observations, it has been suggested that P1 and P1′ are residues 1 and 2, respectively. A report by Lee et al. has confirmed the residue 1/P1 relationship using x-ray crystallographic data of A90720A bound to trypsin. That study revealed that the amide bond between arginine, residue 1, and Ahp, residue 2, was in close proximity to the catalytic serine (Ser195) of trypsin [73]. A separate study of the binding of scyptolin A to elastase revealed a similar structure, thus confirming the role of residue 1 and the neighboring Ahp moiety [74]. This study also showed that the Ahp moiety blocks the inclusion of a molecule of water needed for hydrolysis. It also revealed the importance of interactions between other members of the hexapeptide ring, which increase the rigidity of the bound structure. Furthermore, it was also shown that the threonine, position 6, and side chain of scyptolin A bound to the enzyme at subsites S2 to S4. These results could aid in both structure based drug design and in silico screening of cyanopeptolins in a protease focused drug development effort.

3.3. Microviridins: Ribosomal Serine Protease Inhibitors

The Microviridins (14) are polycyclic peptides that have been isolated from a variety of freshwater cyanobacteria. Microviridin A was first isolated from Microcystis viridis (NIES-102) [75] and additional microviridins have been isolated from other Microcystis spp. as well as the cyanobacteria Oscillatoria agardhii and Nostoc minutum [76–78].

These peptides have a primary structure that ranges from 13 to 14 amino acid residues in size. For sake of clarity, this review will number residues based on a 14-mer microviridin, given that the shorter, 13-mer, microviridins are equivalent to a deletion of residue 1. All microviridins have a conserved lysine at residue 6. This lysine residue is connected via an amide bond between its side chain amine to the side chain carboxylate of a glutamate, aspartate, or modified aspartate at position 11. Another two possible cyclizations have been reported in various members of the microviridins. The most common ester cyclization occurs between the terminal alcohol of the conserved threonine at position 4 and the conserved aspartate residue at position 10. Another ester cyclization can occur between the side chains of the conserved serine at position 9 and the conserved glutamate at position 12. In cases when Ser9 and Glu12 are not connected via an ester bond, the side chain of Glu12 has a terminal methyl ester.

The biological activity of the microviridins is quite varied with some being potent inhibitors of elastase, e.g. microviridin G (IC₅₀ 11 nM), while others are void of protease inhibitory activity, e.g. microviridins A, SD1684, and SD1652. All microviridins that displayed significant protease inhibition have an ester bond between Thr4 and Asp10. The only exception is microviridin F, however this compound was only a weak inhibitor of elastase and was 10-fold less potent than microviridin E, which shares the same primary structure as microviridin F but contained the key ester bond between Thr4 and Asp10 [79]. A comparison of microviridins with identical primary structures that differ in the presence of the ester bond between Ser9 and Glu12 (B vs. C and G vs. H) also showed a difference in activity. In both cases, B vs. C and G vs. H, the microviridin with an additional ester bond between Ser9 and Glu12 was more potent, however only by a factor of 2 or less [76,78]. Furthermore, a comparison of selectivity indices of the microviridins indicates that residue 5 is likely important for the protease selectivity of the microviridins. The only microviridin that selectively inhibited trypsin contained an arginine at position 5. Also the microviridins that selectively inhibited elastase vs. chymotrypsin contained a leucine at this position.

Reports by Ziermert et al. and Philmus et al. have revealed that the microviridins are synthesized from a ribosomally translated precursor peptide that is then modified to create the polycyclic structure [80,81]. Similar to the cyclamides, the ribosomal nature of this biosynthetic pathway would simplify the production of semi-synthetic libraries of microviridins analogs.

3.4. Anabaenapeptins: Uriedo-contaning Exopeptidase Inhibitors

The anabaenopeptins (15) are cyclic pentapeptides with a conserved uriedo moiety connecting a side chain residue to the pentapeptide ring through the D-lysine residue in position 1. This residue also forms part of the ring via its side chain amine. In addition, the residue at position 4 is N-methylated. 29 different anabaenopeptins have been isolated from cyanobacteria, however additional anabaenopeptins have been found in marine sponges [82–85]. Considering the structural similarity of the cyanobacterial anabaenopeptins and poriferal anabaenopeptins, it is likely that cyanobacteria living in the sponges are the source of the anabaenopeptins from those marine invertebrates.

While most of the anabaenopeptins have not been reported to inhibit endopeptidases, such as trypsin and chymotrypsin, many of the anabaenopeptins have been shown to inhibit carboxypeptidase A (CPA), an exopeptidase. It has been suggested that CPA can be used as a model enzyme for physiologically relevant exopeptidases, such as angiotensin converting enzyme [86]. In addition, some members of this class are inhibitors of carboxypeptidase U (CPU) [87]. Carboxypeptidase U is an important enzyme in the regulation of fibrinolysis, thus inhibition of CPU could provide the basis for treatment of cardiovascular diseases [87–89].

Walther et al. reported the synthesis of 17 brunsvicamide A analogs and evaluated these compounds for inhibition of CPA [90]. The natural product, brunsvicamide A (15, R₂=Val, R₃=Leu, R₄=Trp, R₅=Phe, R_S=Ile) had an IC₅₀ value of 5.0 nM, with the most potent, synthetic analog (15, R₂=Val, R₃=Leu, R₄=Ala, R₅=Phe, R_S=Ile) displaying an IC₅₀ of 4.8 nM. It is important to note that the difference between these two molecules was the presence of L-N-methyl-tryptophan at residue 4 in brunsvicamide A, while residue 4 in the analog was L-N-methyl-alanine. This would indicate that CPA can accommodate a variety of amino acids at this position although, the presence of a polar residue, e.g. L-N-methyl-serine, significantly decreased the potency (IC₅₀ 28.9 nM). Ultimately, these results could provide a basis for ligand-based design of carboxypeptidase inhibitors.

3.5. Microginins: Linear Aminopeptidase Inhibitors

The microginins (16) are a class of linear lipopeptides that contain 4 to 6 amino acid residues. The first residue is a 3-amino-2-hydroxydecanoic acid. In some cases, the terminal carbon of the decanoic acid is halogenated, typically with chlorine. The second residue (R₂) varies and alanine, isoleucine, tyrosine, and threonine have been reported at this position. The third residue (R₃) is typically a small non-polar amino acid, e.g. valine, leucine, and proline, and can be N-methylated. Residue four (R₄) is typically tyrosine, often N-methylated, but there have been reports of proline and N-methyl-homotyrosine. Residue five (R₅), if present, is typically an aromatic amino acid or proline. The four known hexapeptide microginins, microginins 299A to 299D, all possess a tyrosine residue at position 6.

Members of the microginin class have been shown to inhibit exopeptidases. Nine microginins have been reported to inhibit leucine aminopeptidase (IC₅₀ 2.3 – 19.2 μM), while only four microginins have shown inhibition of angiotensin converting enzyme (ACE) (IC₅₀ 6.8 to 13.2 μM) [91–94]. All four of these ACE inhibiting microginins were five residues in length and had a tyrosine residue at position 5. Three of the four ACE inhibiting microginins were more potent inhibitors of leucine aminopeptidase than ACE, whereas microginin 478 (16, X=H, R_1a=OH, R_1b=CH₃, R₂=Val, R₃=N-methyl-valine, R₄=N-methyl-tyrosine, R₅=Tyr) was a more potent inhibitor ACE than leucine aminopeptidase by a factor of 10 [94]. The other three ACE inhibiting microginins contained an alanine at position 2 and lacked the N-methyl group on the amino-hydroxydecanoic acid residue at position 1. Currently, it is difficult to determine which feature (MeAhda1 or Val2) is important for ACE selectivity due to the lack of microginin 478 analogs that vary these features independently.

Inhibition of ACE is of particular interest since ACE is target for the treatment of hypertension and congestive heart failure [95]. Synthetic ACE inhibitors have been approved by FDA for the treatment of cardiovascular disease, thus further investigation of the microginin class of peptides, in particular microginin 478, could result in the development of additional ACE inhibitors [96,97].

4. ANTIMICROBIAL NATURAL PRODUCTS FROM CYANOBACTERIA

Several of the metabolites mentioned in the previous section also possess potent antimicrobial activity. For example, cryptophycin 1 was initially discovered as an antifungal agent [98]. The tolytoxin/scytophycins are also strong antifungal agents [35–37]. The following section will highlight the structural diversity and antimicrobial activity of selected natural products from terrestrial and freshwater cyanobacteria. This section is divided according to substance class, i.e. alkaloids, peptides, and terpenoids.

4.1. Antimicrobial alkaloids

The first hapalindole alkaloids (17–18), hapalindoles A and B, were obtained from the cyanobacterium Hapalosiphon fontinalis in 1984 [99]. Since then over 60 hapalindole type alkaloids have been identified from cyanobacteria of the family Stigonemataceae [100,101]. These indole alkaloids include the hapalindoles (17–18) [102–106], fischerindoles (19) [107], welwitindolinones (20) [108,109], and ambiguine isonitriles (21–22) [100,110,111]. These alkaloids are closely related, biosynthetically, and the polycyclic carbon skeleton is proposed to be formed by the condensation of a tryptophan derivative and geranyl pyrophosphate [99,101,109]. The ambiguine isonitriles (21–22) have an additional isoprene unit attached to C-2 of the indole moiety and this isoprene moiety is often cyclized to form an additional seven-membered ring. There is substantial flexibility in the degree of oxidation in this seven-membered ring. Most hapalindole type alkaloids contain an isonitrile or isothiocyanate moiety and, though obtained from non-marine species, many contain a chlorine atom. The co-occurrence of non-chlorinated and chlorinated hapalindole type alkaloids in cyanobacteria has been suggested to be due to some imperfection in the biosynthesis and the resulting arrays of compounds have been proposed to give ecological advantages [110].

These structurally intriguing natural products possess many biological activities. Hapalindoles (17–18) from Hapalosiphon fontinalis [99,105,112], hapalindoles and fischerindole L from Fischerella muscicola [107] and welwitindolinones from Hapalosiphon welwitschii and Westiella intricate [109] were responsible for the antifungal (MIC 2.9 – 5.5 μM) and antibacterial (MIC 0.18 – 105 μM) activities associated with these cyanobacterial strains [112–114].

Ambiguine A-G isonitriles (21–22) were first discovered as fungicidal hapalindole-type alkaloids from Fischerella ambigua [111]. More than a decade later, ambiguine H-J isonitriles were isolated from a Fischerella sp. collected in Israel [110]. Ambiguine H and I isonitriles showed strong antibacterial and antifungal activity (MIC 0.19–27 μM). We recently reported the structures of five additional ambiguine isonitriles, K-O, obtained from F. ambigua [100]. Several isolates were active against Mycobacterium tuberculosis, the most active compound was ambiguine K isonitrile (MIC 6.6 μM), while ambiguine A isonitrile was the most potent against Bacillus anthracis with a MIC of 1.0 μM. Most of the isolates also possessed strong antifungal activities, similar to levels previously reported for other ambiguine isonitriles and hapalindoles [110,115], while only being moderately toxic in the Vero cell assay (IC₅₀ 26.0 to >128 μM). The diversity of both structures and biological activity of these alkaloids could be leveraged to for further drug development. In particular, the SAR of these compounds, with the currently unidentified mechanism of action, could provide a starting point for target based drug design efforts.

Many of the antifungal and antibacterial metabolites, including hapalindole-type alkaloids, found in cyanobacteria are indole alkaloids. The tjipanazoles, a family of indolo[2,3-a]carbazoles from cyanobacteria, are structurally related to the protein kinase inhibitor, staurosporine [116,117]. Tjipanazoles A1 and A2 (23–24), while devoid of protein kinase inhibition, displayed selective antifungal activity against rice blast and leaf rust wheat fungal infections. Recently, 6-cyano-5-methoxy-12-methylindolo[2,3-a]carbazole (25) was identified as a B. anthracis inhibitor [118]. This metabolite was previously reported to possess antiviral and weak cytotoxic activities [119]. Nostocarboline (26), a carbolinium alkaloid from Nostoc sp. 78-12A, was first reported as a cholinesterase inhibitor [120]. Nostocarboline was later found to be inhibit the growth of cyanobacteria and green alga with MIC values of 1 μM [121] as well as potent antiplasmodial activity against the malaria parasite, Plasmodium falciparum [122]. Together these examples highlight the antifungal/antibacterial potential of cyanobacterial indole alkaloids.

Many extracts of cyanobacteria show antialgal and anticyanobacterial activity [123] and several antialgal and anticyanobacterial metabolites have been reported. One of these metabolites, fischerellin A (27) from Fischerella muscicola, was found to be a potent inhibitor of photosystem-II leading to the growth inhibition of both cyanobacteria and higher plants. In addition, fischerellin A showed antifungal activity against several agronomically important microorganisms [124]. A minor component, fischerellin B, also displayed similar algicidal activities [125].

4.2. Antimicrobial Peptides

Many cytotoxic cyanobacterial peptides display antifungal and/or antibacterial activities, e.g. cryptophycin (1) [98], nostofungicidine [126] and calophycin [127]. Some cyanobacterial peptides have been reported with antifungal or antibacterial activities. Hassallidin A (28) and B, glycosylated lipopeptides, were isolated from Hassallia sp. [128,129]. The hassallidins displayed selective antifungal activity against Candida sp. and Cryptococcus neoformans with MIC values ranging from 3.5–11 μM. Brunsvicamides B (15, R₂=Ile, R₃=Leu, R₄=Trp, R₅=Phe, R_S=Ile) and C (15, R₂=Val, R₃=Leu, R₄=D-N′-formyl-kynurenine, R₅=Phe, R_S=Ile), anabaenopeptins obtained from Tychonema sp., were both found to inhibit the protein tyrosine phosphatase B of M. tuberculosis (MptpB) with IC₅₀ values of 7.3 and 8.0 μM, respectively [130]. These two examples demonstrate the potential cyanobacterial peptides as drug development leads for new antimicrobial agents. Specifically, the brunsvicamides and related anabaenopeptins could provide sufficient chemical diversity to perform SAR studies of MptpB and thus lead to the development of improved treatments for tuberculosis.

4.3. Antimicrobial Terpenoids

There are a few published reports of biologically active terpenoids from cyanobacteria. Tolypodiol was the first diterpenoid reported from cyanobacteria [131]. A structurally related antibacterial meroterpenoid, noscomin (29), was isolated from Nostoc commune [132]. Noscomin displayed antibacterial activity against Staphylococcus epidermidis comparable to chloramphenicol. Comnostins A-E, additional antibacterial meroterpenoids, were identified from the culture medium of the same species [133]. Two abietane diterpenoids, 20-nor-3a-acetoxyabieta-5,7,9,11,13-pentaene and its 12-hydroxy analog (30–31) were isolated from Microcoleous lacustris. Both compounds exhibited moderate antibacterial activity against S. aureus [134]. Scytoscalarol (32) is an antibacterial and antifungal guanidine-bearing sesterterpene obtained from Scytonema sp. [135]. Scytoscalarol displayed potent antibacterial and antifungal activity against several microbial strains (S. aureus MIC 1.7 μM, C. albicans MIC 3.6 μM, and B. anthracis MIC 6.0 μM), while only weak cytotoxicity was observed in a Vero cell line assay (IC₅₀ 135 μM). These compounds add to the chemodiversity of antimicrobial compounds from freshwater and terrestrial cyanobacteria. As with the previous classes of antimicrobial compounds, further investigation could lead to the development of new therapies for the treatment of infectious diseases as well as elucidate new drug targets.

5. DISCUSSION

Cyanobacteria have been cited as a promising and productive source for new pharmaceutical lead compounds [136] and a large number of chemically diverse metabolites have been obtained from cyanobacteria. Screening of cyanobactrial extracts for antineoplastic activity indicated a hit rate similar to that found for other microorganisms such as actinobacteria [137]. However, the rate of rediscovery of known bioactive compounds is significantly lower among the cyanobacteria than from other microorganisms [138]. Genetic analysis of cyanobacteria has further confirmed that these organisms contain a diversity of secondary metabolite pathways. Studies have indicated the presence of NRPS and PKS secondary metabolite pathways in a variety of cyanobacteria [139,140]. In addition, published cyanobacterial genome sequences have been used to detect potential NRPS and PKS genes using computational approaches [141]. These genetic studies further support the fact that there is a wealth of secondary metabolites to be found in cyanobacteria.

Studies of bioactive metabolites from cyanobacteria have been performed with both field-collected material and laboratory-grown cultures. Field collected material (water-blooms or cyanobacterial mats) often contains an assemblage of multiple organisms, which can raise a concern regarding the true origin of an isolated natural product. This can also make the recollection and re-isolation of a particular natural product difficult. In addition, field-collections are only feasible for cyanobacteria that naturally have dense growth. Laboratory-grown cultures, on the other hand, use unialgal strains and allow for the investigation of species that may not grow to sufficient density in the wild. Laboratory-grown cultures also afford the ability for scale up to obtain additional biomass for the re-isolation of any natural products of interest.

However, the slow growth and low biomass yield can make it a challenge to study bioactive metabolites from cultured cyanobacteria: The incubation period in 10–20 L glass vessel ranges from 1–7 weeks and the average cell mass yield is 0.3 g/L [35]. Recent advances in analytical techniques, mainly the sensitivity improvement in NMR spectroscopy, enable the structure determination to be performed on very small sample amounts. This increased NMR sensitivity, mainly obtained through microcoil and cryoprobe NMR technologies, now facilitate the discovery of natural products at the nanomole scale [142]. These sensitivity improvements will improve the discovery of bioactive secondary metabolites from cyanobacteria. It will enable studies to be performed on smaller sample amount and thus lower the initial growth volume required to study cultured cyanobacteria. It will also allow for direct investigations of less abundant field-collected cyanobacteria.

From a genetic standpoint, the secondary metabolic pathways of cyanobacteria are very similar to other bacteria, such as actinobacteria. Thus, it is possible to study cyanobacteria using the techniques developed to study and manipulate secondary metabolite production in other bacteria. The access to the genetic products of the cyanobacteria can be used to produce “semi-natural” analogs of isolated natural products. This has already been demonstrated in the case of cryptophycins [28,29]. Using these genetic techniques in combination with the highly sensitive analytical techniques mentioned above, it is now possible to tap into the great diversity of biologically active cyanobacterial metabolites that were previously unreachable due to resource limitations.

Figure 3. Cytotoxic metabolites.

Numbers in bold italics indicate residue number.

Figure 4. Protease inhibitors.

Numbers in bold italics indicate residue number.

Figure 5.

Anti-microbial alkaloids

Figure 6.

Anti-microbial peptides and terpenoids