Structural Diversity and Biological Activity of Cyanopeptolins Produced by Nostoc edaphicum CCNP1411.

Cyanopeptolins (CPs) are one of the most commonly occurring class of cyanobacterial nonribosomal peptides. For the majority of these compounds, protease inhibition has been reported. In the current work, the structural diversity of cyanopeptolins produced by Nostoc edaphicum CCNP1411 was explored. As a result, 93 CPs, including 79 new variants, were detected and structurally characterized based on their mass fragmentation spectra. CPs isolated in higher amounts were additionally characterized by NMR. To the best of our knowledge, this is the highest number of cyanopeptides found in one strain. The biological assays performed with the 34 isolated CPs confirmed the significance of the amino acid located between Thr and the unique 3-amino-6-hydroxy-2-piperidone (Ahp) on the activity of the compounds against serine protease and HeLa cancer cells.

Biological Activity and Stability of Aeruginosamides from Cyanobacteria.

Aeruginosamides (AEGs) are classified as cyanobactins, ribosomally synthesized peptides with post-translational modifications. They have been identified in cyanobacteria of genera Microcystis, Oscillatoria, and Limnoraphis. In this work, the new data on the in vitro activities of three AEG variants, AEG A, AEG625 and AEG657, and their interactions with metabolic enzymes are reported. Two aeruginosamides, AEG625 and AEG657, decreased the viability of human breast cancer cell line T47D, but neither of the peptides was active against human liver cancer cell line Huh7. AEGs also did not change the expression of MIR92b-3p, but for AEG625, the induction of oxidative stress was observed. In the presence of a liver S9 fraction containing microsomal and cytosolic enzymes, AEG625 and AEG657 showed high stability. In the same assays, quick removal of AEG A was recorded. The peptides had mild activity against three cytochrome P450 enzymes, CYP2C9, CYP2D6 and CYP3A4, but only at the highest concentration used in the study (60 µM). The properties of AEGs, i.e., cytotoxic activity and in vitro interactions with important metabolic enzymes, form a good basis for further studies on their pharmacological potential.

Eighteen New Aeruginosamide Variants Produced by the Baltic Cyanobacterium Limnoraphis CCNP1324.

Cyanobactins are a large family of ribosomally synthesized and post-translationally modified cyanopeptides (RiPPs). Thus far, over a hundred cyanobactins have been detected in different free-living and symbiotic cyanobacteria. The majority of these peptides have a cyclic structure. The occurrence of linear cyanobactins, aeruginosamides and virenamide, has been reported sporadically and in few cyanobacterial taxa. In the current work, the production of cyanobactins by Limnoraphis sp. CCNP1324, isolated from the brackish water Baltic Sea, has been studied for the first time. In the strain, eighteen new aeruginosamide (AEG) variants have been detected. These compounds are characterized by the presence of prenyl and thiazole groups. A common element of AEGs produced by Limnoraphis sp. CCNP1324 is the sequence of the three C-terminal residues containing proline, pyrrolidine and methyl ester of thiazolidyne-4-carboxylic acid (Pro-Pyr-TzlCOOMe) or thiazolidyne-4-carboxylic acid (Pro-Pyr-TzlCOOH). The aeruginosamides with methylhomotyrosine (MeHTyr1) and with the unidentified N-terminal amino acids showed strong cytotoxic activity against human breast cancer cells (T47D).

Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea.

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed.

Cyanopeptolins with Trypsin and Chymotrypsin Inhibitory Activity from the Cyanobacterium Nostoc edaphicum CCNP1411.

Cyanopeptolins (CPs) are one of the most frequently occurring cyanobacterial peptides, many of which are inhibitors of serine proteases. Some CP variants are also acutely toxic to aquatic organisms, especially small crustaceans. In this study, thirteen CPs, including twelve new variants, were detected in the cyanobacterium Nostoc edaphicum CCNP1411 isolated from the Gulf of Gdansk (southern Baltic Sea). Structural elucidation was performed by tandem mass spectrometry with verification by NMR for CP962 and CP985. Trypsin and chymotrypsin inhibition assays confirmed the significance of the residue adjacent to 3-amino-6-hydroxy-2-piperidone (Ahp) for the activity of the peptides. Arginine-containing CPs (CPs-Arg²) inhibited trypsin at low IC50 values (0.24⁻0.26 µM) and showed mild activity against chymotrypsin (IC50 3.1⁻3.8 µM), while tyrosine-containing CPs (CPs-Tyr²) were selectively and potently active against chymotrypsin (IC50 0.26 µM). No degradation of the peptides was observed during the enzyme assays. Neither of the CPs were active against thrombin, elastase or protein phosphatase 1. Two CPs (CP962 and CP985) had no cytotoxic effects on MCF-7 breast cancer cells. Strong and selective activity of the new cyanopeptolin variants makes them potential candidates for the development of drugs against metabolic disorders and other diseases.

Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria.

Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.

Phylogenetic and molecular characteristics of two Aphanizomenon strains from the Curonian Lagoon, Southeastern Baltic Sea and their biological activities.

Polyphasic approach has become a generally accepted method for the classification of cyanobacteria. In this study, we present a detailed characterisation of two strains, KUCC C1 and KUCC C2, isolated from the Curonian Lagoon and classified to the Aphanizomenon genus. Despite phylogenetic similarity, the strains differ with respect to morphology, ultrastructure characteristics, and the metabolite profile. In the KUCC C1 extract, three unknown peptides and eight anabaenopeptins were detected, while KUCC C2 produced one unknown peptide and one aeruginosin. In both strains, a total of eleven pigments were detected. The production of myxoxantophyll, chlorophyll-a, chlorophylide-a, and zeaxanthin was higher in KUCC C2 than in KUCC C1. Extracts from both strains of Aphanizomenon also had different effects in antibacterial, anticancer and enzyme inhibition assays. Comprehensive analyses of Aphanizomenon strains performed in this study showed significant diversity between the isolates from the same bloom sample. These differences should be considered when exploring the ecological significance and biotechnological potential of a given population.

Genome Mining of Pseudanabaena galeata CCNP1313 Indicates a New Scope in the Search for Antiproliferative and Antiviral Agents.

Compounds derived from natural sources pave the way for novel drug development. Cyanobacteria is an ubiquitous phylum found in various habitats. The fitness of those microorganisms, within different biotopes, is partially dependent on secondary metabolite production. Their enhanced production under biotic/abiotic stress factors accounts for better survival rates of cells, and thereby cyanobacteria are as an enticing source of bioactive compounds. Previous studies have shown the potent activity of extracts and fractions from Pseudanabaena galeata (Böcher 1949) strain CCNP1313 against cancer cells and viruses. However, active agents remain unknown, as the selected peptides had no effect on the tested cell lines. Here, we present a bottom-up approach, pinpointing key structures involved in secondary metabolite production. Consisting of six replicons, a complete genome sequence of P. galeata strain CCNP1313 was found to carry genes for non-ribosomal peptide/polyketide synthetases embedded within chromosome spans (4.9 Mbp) and for a ribosomally synthesized peptide located on one of the plasmids (0.2 Mbp). Elucidation of metabolite synthesis pathways led to prediction of their structure. While none of the synthesis-predicted products were found in mass spectrometry analysis, unexplored synthetases are characterized by structural similarities to those producing potent bioactive compounds.

Anabaenopeptins from Nostoc edaphicum CCNP1411.

Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins.

Pseudanabaena galeata CCNP1313-Biological Activity and Peptides Production.

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549ACE2/TMPRSS2) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts.

Antiviral Cyanometabolites-A Review.

Global processes, such as climate change, frequent and distant travelling and population growth, increase the risk of viral infection spread. Unfortunately, the number of effective and accessible medicines for the prevention and treatment of these infections is limited. Therefore, in recent years, efforts have been intensified to develop new antiviral medicines or vaccines. In this review article, the structure and activity of the most promising antiviral cyanobacterial products are presented. The antiviral cyanometabolites are mainly active against the human immunodeficiency virus (HIV) and other enveloped viruses such as herpes simplex virus (HSV), Ebola or the influenza viruses. The majority of the metabolites are classified as lectins, monomeric or dimeric proteins with unique amino acid sequences. They all show activity at the nanomolar range but differ in carbohydrate specificity and recognize a different epitope on high mannose oligosaccharides. The cyanobacterial lectins include cyanovirin-N (CV-N), scytovirin (SVN), microvirin (MVN), Microcystisviridis lectin (MVL), and Oscillatoria agardhii agglutinin (OAA). Cyanobacterial polysaccharides, peptides, and other metabolites also have potential to be used as antiviral drugs. The sulfated polysaccharide, calcium spirulan (CA-SP), inhibited infection by enveloped viruses, stimulated the immune system's response, and showed antitumor activity. Microginins, the linear peptides, inhibit angiotensin-converting enzyme (ACE), therefore, their use in the treatment of COVID-19 patients with injury of the ACE2 expressing organs is considered. In addition, many cyanobacterial extracts were revealed to have antiviral activities, but the active agents have not been identified. This fact provides a good basis for further studies on the therapeutic potential of these microorganisms.

Nostocyclopeptides as New Inhibitors of 20S Proteasome.

Nostocyclopeptides (Ncps) are a small class of bioactive nonribosomal peptides produced solely by cyanobacteria of the genus Nostoc. In the current work, six Ncps were isolated from Nostoc edaphicum strain CCNP1411. The bioactivity of these compounds was tested in vitro against 20S proteasome, a proteolytic complex that plays an important role in maintaining cellular proteostasis. Dysfunction of the complex leads to many pathological disorders. The assays indicated selective activity of specific Ncp variants. For two linear peptide aldehydes, Ncp-A2-L and Ncp-E2-L, the inhibitory effects on chymotrypsin-like activity were revealed, while the cyclic variant, Ncp-A2, inactivated the trypsin-like site of this enzymatic complex. The aldehyde group was confirmed to be an important element of the chymotrypsin-like activity inhibitors. The nostocyclopeptides, as novel inhibitors of 20S proteasome, increased the number of natural products that can be considered potential regulators of cellular processes.

Phytoplankton of the Curonian Lagoon as a New Interesting Source for Bioactive Natural Products. Special Impact on Cyanobacterial Metabolites.

The bioprospecting of marine and brackish water systems has increased during the last decades. In this respect, microalgae, including cyanobacteria, and their metabolites are one of the most widely explored resources. Most of the bioactive compounds are isolated from ex situ cultures of microorganisms; however, analysis of field samples could also supply valuable information about the metabolic and biotechnological potential of microalgae communities. In this work, the activity of phytoplankton samples from the Curonian Lagoon was studied. The samples were active against antibiotic resistant clinical and environmental bacterial strains as well as against serine proteases and T47D human breast adenocarcinoma cells. No significant effect was found on Daphnia magna. In addition, using LC-MS/MS, we documented the diversity of metabolites present in field samples. A list of 117 detected cyanopeptides was presented. Cyanopeptolins constituted the largest class of cyanopeptides. As complex bloom samples were analyzed, no link between the observed activity and a specific sample component can be established. However, the results of the study showed a biotechnological potential of natural products from the Curonian Lagoon.

Blooms of Toxic Cyanobacterium Nodularia spumigena in Norwegian Fjords During Holocene Warm Periods.

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level.

Limited Microcystin, Anatoxin and Cylindrospermopsin Production by Cyanobacteria from Microbial Mats in Cold Deserts.

Toxic metabolites are produced by many cyanobacterial species. There are limited data on toxigenic benthic, mat-forming cyanobacteria, and information on toxic cyanobacteria from Central Asia is even more scarce. In the present study, we examined cyanobacterial diversity and community structure, the presence of genes involved in toxin production and the occurrence of cyanotoxins in cyanobacterial mats from small water bodies in a cold high-mountain desert of Eastern Pamir. Diversity was explored using amplicon-based sequencing targeting the V3-V4 region of the 16S rRNA gene, toxin potential using PCR-based methods (mcy, nda, ana, sxt), and toxins by enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular identification of cyanobacteria showed a high similarity of abundant taxa to Nostoc PCC-73102, Nostoc PCC-7524, Nodularia PCC-935 and Leptolyngbya CYN68. The PCRs revealed the presence of mcyE and/or ndaF genes in 11 samples and mcyD in six. The partial sequences of the mcyE gene showed high sequence similarity to Nostoc, Planktothrix and uncultured cyanobacteria. LC-MS/MS analysis identified six microcystin congeners in two samples and unknown peptides in one. These results suggest that, in this extreme environment, cyanobacteria do not commonly produce microcystins, anatoxins and cylindrospermopsins, despite the high diversity and widespread occurrence of potentially toxic taxa.