Dereplication of Natural Products with Antimicrobial and Anticancer Activity from Brazilian Cyanobacteria

Cyanobacteria are photosynthetic organisms that produce a large diversity of natural products with interesting bioactivities for biotechnological and pharmaceutical applications. Cyanobacterial extracts exhibit toxicity towards other microorganisms and cancer cells and, therefore, represent a source of potentially novel natural products for drug discovery. We tested 62 cyanobacterial strains isolated from various Brazilian biomes for antileukemic and antimicrobial activities. Extracts from 39 strains induced selective apoptosis in acute myeloid leukemia (AML) cancer cell lines. Five of these extracts also exhibited antifungal and antibacterial activities. Chemical and dereplication analyses revealed the production of nine known natural products. Natural products possibly responsible for the observed bioactivities and five unknown, chemically related chlorinated compounds present only in Brazilian cyanobacteria were illustrated in a molecular network. Our results provide new information on the vast biosynthetic potential of cyanobacteria isolated from Brazilian environments.


Introduction
Cyanobacteria are photosynthetic bacteria commonly found in diverse aquatic and terrestrial environments [1]. They produce a large range of secondary metabolites (natural products) that are predominantly produced to gain evolutionary advantages, such as adaptation to the surrounding environment or as a defense mechanism, rather than being part of primary metabolism (i.e., growth, development, or reproduction) [2][3][4]. Many cyanobacterial natural products are synthesized by non-ribosomal peptide synthetases (NRPS), polyketide synthase (PKS), or hybrid NRPS-PKS (NRPS/PKS) [5]. These molecules have diverse applications in pharmacology, biotechnology, and bioenergy production [6][7][8][9].
The cyanobacterial extracts with the highest apoptosis induction of MOLM-13 cells were also tested for cytotoxicity towards the normal rat kidney epithelial NRK cell line (Table 1). By comparing the potency of normal and malignant cells, the test provided evidence for potential use as an anticancer compound. The strains Fischerella sp. CENA72, Cyanobium sp. CENA185, Limnothrix sp. CENA217, Nostoc sp. CENA296, and Aliinostoc sp. CENA524 showed particularly selective
The cyanobacterial extracts with the highest apoptosis induction of MOLM-13 cells were also tested for cytotoxicity towards the normal rat kidney epithelial NRK cell line (Table 1). By comparing the potency of normal and malignant cells, the test provided evidence for potential use as an anticancer compound. The strains Fischerella sp. CENA72, Cyanobium sp. CENA185, Limnothrix sp. CENA217, Nostoc sp. CENA296, and Aliinostoc sp. CENA524 showed particularly selective cytotoxicity towards MOLM-13 cells over NRK cells; these strains had an over nine-fold greater EC 50 value for NRK than Toxins 2020, 12, 12 4 of 17 MOLM-13 (Table 1). It is noteworthy that the organic extracts from Cyanobium sp. CENA185 and Limnothrix sp. CENA217 showed no apparent toxicity to NRK cells after 24 h ( Figure S1). Nostoc sp. CENA69, Fischerella sp. CENA161, and Aliinostoc spp. CENA513 and CENA514 showed relatively smaller differences in cytotoxicity between the two cell lines as judged by EC 50 values than the other strains.  Figure S1); * p-value < 0.0001 calculated using extra-sum-of-squares F test in GraphPad; # Cells were not affected with highest concentration tested. § Value could not be calculated due to a lack of activity in NRK cells.
Another method to evaluate drug selectivity for one cell over another is to calculate the area under the curve (AUC) of the dose-response curves (Table 1 and Figure S1). Aliinostoc sp. CENA514 has a large AUC ratio between NRK and MOLM-13 cell lines, which indicates that the concentrations needed to kill MOLM-13 AML cells will not harm nonmalignant NRK cells. The cell extract from Nostoc sp. CENA69 was toxic for both MOLM-13 and NRK cell lines ( Figure 2 and Table 1). The low value of the half-maximum effective concentration (EC 50 ) of Nostoc sp. CENA69 shows the high potency of this cell extract for both tested cell lines (Table 1).  To identify possible antibacterial and antifungal activity, organic extracts obtained from cyanobacterial freeze-dried cells were also tested against Staphylococcus aureus and Candida albicans ( Table 2, Files S1 and S2). Fischerella spp. CENA71, CENA72, CENA161, and CENA298 and Aliinostoc sp. CENA513 showed both antibacterial and antifungal activity. Aliinostoc spp. CENA514, CENA535, and CENA548 extracts had antifungal activity while CENA524 presented antibacterial activity. Table 2. Summary of the antifungal (F), antibacterial (B), and anticancer (C) activities observed in the cyanobacterial crude extracts (see also Files S1 and S2). Bioactive cell extracts (+) showed inhibition zone in the antimicrobial assay and above 70% induction of apoptosis of MOLM-13 cell lines at the concentration of 13.3 mgDW mL −1 . Previously obtained information of the compounds produced by the studied strains are presented. Cyanobacterial extracts that induced apoptosis in less than 70% of MOLM-13 cells did not present inhibition halo in the antimicrobial assays or that were not previously described to produce a natural product were not included in this table. The use of sterile water may have influenced the susceptibility of fungal and bacterial cells. CENA514 Nodularin, Anabaenopeptin, Pseudoaeruginosin [11,43] and this study Aliinostoc sp. CENA548 This study a Cytotoxic towards MOLM-13 cells (see Table 1).*Anticancer activity has also been previously observed [40].

Natural Products Produced by Brazilian Cyanobacteria
Cell extracts were analyzed using mass spectrometry, and a molecular network was constructed based on the MS/MS spectra of the detected compounds. Due to the size of the resulting network, only clusters containing potential new novel compounds detected exclusively in Brazilian cyanobacterial extracts and previously known natural products are presented in Figure 3. The complete network can be found in the Supplementary Materials ( Figure S2). Cyanobacterial cell extracts from strains producing known compounds were included in the analysis to facilitate the dereplication of different compounds (Table S3). Puwainaphycins produced by Aliinostoc spp. CENA535 and CENA548 were detected using a dereplication tool (GNPS) ( Figure S3; Table S4).

Natural Products Produced by Brazilian Cyanobacteria
Cell extracts were analyzed using mass spectrometry, and a molecular network was constructed based on the MS/MS spectra of the detected compounds. Due to the size of the resulting network, only clusters containing potential new novel compounds detected exclusively in Brazilian cyanobacterial extracts and previously known natural products are presented in Figure 3. The complete network can be found in the Supplementary Materials ( Figure S2). Cyanobacterial cell extracts from strains producing known compounds were included in the analysis to facilitate the dereplication of different compounds (Table S3). Puwainaphycins produced by Aliinostoc spp. CENA535 and CENA548 were detected using a dereplication tool (GNPS) ( Figure S3; Table S4).  (1)(2)(3)(4)(5)(6)(7)(8)(9). Compound mass/charge ratios (m/z) are indicated in the node labels. The complete network can be found in Figure S2.
Cyanobacterial extracts that showed potent anticancer or cytotoxic activity presented novel compounds detected only in Brazilian strains ( Figure 4). The chlorinated compounds (M1-5) could not be assigned in this study to any known molecule. Further information on these compounds are presented in the Supplementary Material ( Figure S4, File S3). Compound mass/charge ratios (m/z) are indicated in the node labels. The complete network can be found in Figure S2.
Cyanobacterial extracts that showed potent anticancer or cytotoxic activity presented novel compounds detected only in Brazilian strains ( Figure 4). The chlorinated compounds (M1-5) could not be assigned in this study to any known molecule. Further information on these compounds are presented in the Supplementary Material ( Figure S4, File S3). Compound mass/charge ratios (m/z; more than one isotope ion/compound could be present) are indicated in the node labels. See also Figure S4 and File S3.

Discussion
There has been a recent increase in the number of studies on natural products synthesized by Brazilian cyanobacteria, particularly on toxins and protease inhibitors [43,44,[51][52][53]. However, the diversity of these organisms and their natural products will likely expand as these ecosystems are further explored [54][55][56]. Therefore, strains were isolated from different biomes and their biosynthetic potential in culture conditions is discussed here.
The 16S rRNA gene phylogenetic analysis demonstrated that, while some Nostoc strains were distributed in the previously described Nostoc clusters 1, 2, and 3.3 [43], the remaining strains were located in sparse and diverse clades more closely related to other Nostocalean genera.
Previous research indicated that analysis of a broad selection of taxonomical genera of cyanobacteria would increase the possibility of detecting novel bioactive compounds [21,43]. The analysis of several strains investigated in this study revealed significant antileukemic activity (≥70%). Approximately 21% of the aqueous and 61% of the organic extracts were bioactive, in which all the cyanobacterial strains that produced hydrophilic active compounds also contained a hydrophobic antileukemic extract. The analysis measured by AUC aids in evaluating the effect of the compounds based on concentration and is a reliable method to analyze dose-response curves [57].
Cell extracts of Cyanobium sp. CENA154, Nostoc spp. CENA67 and CENA69, and Oxynema sp. CENA135 have previously been described as having anticancer activity against the murine colon cancer cell line CT-26 and lung carcinoma 3LL [42]. In the present work, cell extracts of these strains were similarly found to induce apoptosis of the AML cell line MOLM-13. Their organic extract is responsible for the antileukemic activity, except for Nostoc sp. CENA69, in which the aqueous extract also exhibited antileukemic activity at 13.3 mgDW mL -1 concentration. CENA69 showed a high potency to induce cell death in MOLM-13 and was further tested using normal cells (NRK), which also showed high cytotoxicity. Fischerella spp. CENA71 and CENA72 and Aliinostoc spp. CENA513, CENA514, and CENA524 produce antileukemic compound(s) with higher potency against MOLM-13 cells than normal NRK cells. A similar study that investigated extracts from Portuguese cyanobacteria also revealed effects against cancer cells but did not reduce the viability of NRK cells [58].

Discussion
There has been a recent increase in the number of studies on natural products synthesized by Brazilian cyanobacteria, particularly on toxins and protease inhibitors [43,44,[51][52][53]. However, the diversity of these organisms and their natural products will likely expand as these ecosystems are further explored [54][55][56]. Therefore, strains were isolated from different biomes and their biosynthetic potential in culture conditions is discussed here.
The 16S rRNA gene phylogenetic analysis demonstrated that, while some Nostoc strains were distributed in the previously described Nostoc clusters 1, 2, and 3.3 [43], the remaining strains were located in sparse and diverse clades more closely related to other Nostocalean genera.
Previous research indicated that analysis of a broad selection of taxonomical genera of cyanobacteria would increase the possibility of detecting novel bioactive compounds [21,43]. The analysis of several strains investigated in this study revealed significant antileukemic activity (≥70%). Approximately 21% of the aqueous and 61% of the organic extracts were bioactive, in which all the cyanobacterial strains that produced hydrophilic active compounds also contained a hydrophobic antileukemic extract. The analysis measured by AUC aids in evaluating the effect of the compounds based on concentration and is a reliable method to analyze dose-response curves [57].
Cell extracts of Cyanobium sp. CENA154, Nostoc spp. CENA67 and CENA69, and Oxynema sp. CENA135 have previously been described as having anticancer activity against the murine colon cancer cell line CT-26 and lung carcinoma 3LL [42]. In the present work, cell extracts of these strains were similarly found to induce apoptosis of the AML cell line MOLM-13. Their organic extract is responsible for the antileukemic activity, except for Nostoc sp. CENA69, in which the aqueous extract also exhibited antileukemic activity at 13.3 mgDW mL -1 concentration. CENA69 showed a high potency to induce cell death in MOLM-13 and was further tested using normal cells (NRK), which also showed high cytotoxicity. Fischerella spp. CENA71 and CENA72 and Aliinostoc spp. CENA513, CENA514, and CENA524 produce antileukemic compound(s) with higher potency against MOLM-13 cells than normal NRK cells. A similar study that investigated extracts from Portuguese cyanobacteria also revealed effects against cancer cells but did not reduce the viability of NRK cells [58].
Among the 39 strains exhibiting high antileukemic activity, 12 of them produce previously described compounds. Cyanobium spp. CENA153 and CENA185, Fischerella sp. CENA161, Oxynema sp. CENA135, and Phormidium sp. CENA270 produce microcystins. These peptides are potent hepatotoxins due to their protein serine/threonine phosphatase inhibitor activity [59]. Hepatocytes efficiently take up microcystin and are therefore the primary target of toxicity [60]. However, high doses of microcystin can also induce oxidative stress [61]. The cellular effects of protein phosphatase inhibitors, such as microcystin and nodularin, are due to differences in uptake. These toxins do not distinguish between malignant and nonmalignant cells if microinjected into the cells [62]. Nevertheless, previous studies have shown that there is no correlation between phosphatase inhibitors and apoptogenic activity against SH-SY5Y-neuroblastoma and AML cells [58,63]. The lipopeptides hassallidin and puwainaphycin have been previously shown to have cytotoxic activity [64,65]. Aeruginosin and anabaenopeptin are protease inhibitors with no reported cytotoxic effects. Lastly, saxitoxin is a neurotoxin that is associated with cytotoxic effects on mammalian cells [66][67][68]. Further analysis using isolated compounds would be necessary to show which are the antileukemic compounds produced by the tested Brazilian cyanobacteria. Three of the 62 strains are likely to produce new antibacterial, antifungal, or anticancer molecules.
The construction of a molecular network was used to explore the complex variety of natural products synthesized by the analyzed cyanobacteria. Although most of the molecules are produced by both Brazilian and control strains, approximately 20% of the resulting clusters are exclusively present in the group of Brazilian strains. Compounds that had already been identified in the strains, such as microcystin-LR (Fischerella sp. CENA161) [48], [D-Leu 1 ]microcystin-LR (Phormidium sp. CENA270) [49], anabaenopeptin, and nodularin (Aliinostoc sp. CENA543) [43], were again found here ( Figure S2 and Table S4). The molecular network also highlighted molecules that are possibly related to their bioactivities, though the mass spectrometry analysis was not sufficient for identification and further isolation and characterization are necessary.

Conclusions
Brazilian cyanobacteria belonging to 5 different orders and isolated from different biomes showed anticancer, antibacterial, and antifungal activity. The known compounds microcystin, saxitoxin, aeruginosin, hassallidin, nodularin, anabaenopeptin, pseudoaeruginosin, and puwainaphycin were detected by mass spectrometry analysis and a dereplication tool. Nonetheless, the biosynthetic potential of the strains remains mostly unknown as most of the molecular network could not be assigned to known natural products. Among these compounds, some were found only in Brazilian strains and could potentially be involved in the detected bioactivities. The unknown chlorinated molecules detected in extracts showing relatively high anticancer activity are promising candidates for isolation and characterization. We hypothesize that further studies with Brazilian cyanobacteria may reveal an even larger biosynthetic repertoire, as the tested strains are expected to represent just a small fraction of isolation sources in the ecosystems in the country.

Cyanobacterial Strains
Nostoc spp. CENA67 and CENA69 and Fischerella spp. CENA71 and CENA72 (No. 3-6 in Table S1) were isolated from the anthropogenic Amazon Dark Earth soils, which are highly fertile anthropogenic soils produced by native populations of Central Amazonia [84]. Phormidium, Nodosilinea, Chlorogloea, Cyanobium, and Halotia strains (No. 7-14 in Table S1) originate from Cardoso Island on the coast of the State of São Paulo. This region is covered by the Atlantic Forest, mangroves, and restingas (herbaceous vegetation) and is part of one of the most biodiverse ecosystems in the world [85]. Caatinga, the biome in which Aulosira, Calothrix, Chroococcidiopsis, Cyanospira, Fischerella, Lyngbya, Nostoc, and Phormidium strains (No. 17-43 in Table S1) were isolated, is a semiarid region with singular adapted species that is largely neglected by conservation policies in Brazil [54]. Lastly, Alkalinema, Geminocystis, Leptolyngbya, Nodosilinea, Nostoc, Pannus, and Pantanalinema strains (No. 1 and 46-59 in Table S1) were isolated from the Pantanal, which is the largest continental wetland on the planet and is colonized by unique cyanobacteria [54]. The strains are maintained in the UHCC culture collection of the University of Helsinki, Helsinki, Finland, and the culture collection of CENA/University of São Paulo, Piracicaba, São Paulo, Brazil.

DNA Extraction and Sequencing, and Phylogenetic Analyses
Total genomic DNA of the strains was extracted from 3-weeks old cultures according to the method by Fiore et al. [86]. The 16S rRNA genes were obtained by PCR using the set primer 27F-23S30R [87]. PCR products were cloned and sequenced, and the resulting reads were assembled into contigs using the Phred/Phrap/Consed package [88][89][90].

Cyanobacterial Extracts
The Brazilian strains selected for the bioactivity assays (Table S1) were cultivated in 3 L of Z8 [94] medium (with or without 0.467 g/L of NaNO 3 as nitrogen source; see Table S5) under constant light of 10 ± 1 µmol m −2 s −1 at 20 ± 1 • C. Cells in exponential phase of growth (21-28 days of cultivation) were freeze-dried, and 100 mg was used for methanol extraction. To perform the extraction, 1 mL of methanol and glass beads (0.5-mm diameter, Scientific Industries INC, Bohemia, NY, USA) were added to the cell matter to be disrupted in a plastic tube using a FastPrep™-24 homogenizer (Fastprep ® -24, MpBiomedicals, Irvine, CA, USA) for three times of 30 s at 6.5 m/s. The sample tubes were centrifuged at 10,000× g for 5 min, and the supernatant was reserved in a new tube. The extraction was repeated. The supernatant from both extractions was combined and dried using a vacuum concentrator (RVC 2-18 CDplus, Martin Christ, Osterode, Germany). Samples were resuspended in 500 µL of methanol (Sigma-Aldrich, San Luis, MO, USA) by vortexing and pipetting. Dichloromethane (Merck) and milli-Q water were added 1:1 v/v (500 µL:500 µL), and the samples were inverted to mix the solvents. The samples were then centrifuged at 10,000× g for 5 min at 20 • C. The aqueous and organic phases were collected, placed in different tubes, and left with the lid open at room temperature in a laboratory fume hood overnight. The aqueous phase was evaporated in a speed-vac (Eppendorf) at 30 • C. Dried samples were stored at −20 • C. Organic-phase extracts were resuspended in 75 µL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, ≥99.9%, A.C.S. spectrophotometric grade), while the aqueous phase was resuspended in 300 µL of 25% DMSO (25 DMSO:75 milli-Q water). Samples were stored at −20 • C.
Frieze-dried cells (100 mg) were extracted twice with 1 mL of methanol and centrifuged as described above. The extracts (300 µL) were added to discs and evaporated overnight. The discs were used for antibacterial and antifungal assays. A volume of 40 µL of filtered (22 µm) extract was mixed with 160 µL of methanol to be analyzed by mass spectrometry and stored at −20 • C. Frieze-dried cells (10 to 24.3 mg) from 10 cyanobacteria (Table S3) were extracted once with 1 mL of methanol as described above, and 200 µL of the extract was filtered and reserved for mass spectrometry analysis.

Cell-Based Assay
The acute myeloid leukemia (AML) cell line MOLM-13 (ACC, 554; [95]) and rat kidney epithelial cell line NRK (ATCC, CRL-6509) were used. Aqueous (4 µL) and organic (1 µL) cyanobacterial extracts were cultured in RPMI or DMEM medium, respectively, and both were supplemented with fetal bovine serum (FBS, F7524, Sigma-Aldrich) and penicillin and streptomycin (P0781, Sigma-Aldrich). The MOLM-13 cells were maintained at concentrations between 10 5 and 8 × 10 5 cells/mL. NRK cells were detached by mild trypsin treatment at 70-90% confluence and reseeded at 30-50% confluence. The cells were incubated in a humified atmosphere at 37 • C and 5% CO 2 . To test for cytotoxicity, extracts were added in 96-well plates (Nunclon TM Delta Surface, Thermo Scientific) and MOLM-13 cell suspensions (25-40 × 10 5 cells/mL) were added to a final volume of 0.1 mL in each well. NRK cells were seeded in wells the day before the experiment (7-8 × 10 4 cells/mL) and left overnight to attach. Fresh medium containing the cyanobacterial cell extracts were added to the wells. Both cell lines were incubated at 37 • C for 24 h before the cells were fixed by adding 100 µL of 3.7% formaldehyde in phosphate-buffered saline buffer, pH 7.4. The DNA-specific dye Hoechst 33342 (0.01 mg/mL) was then added. Plates were kept at 4 • C overnight, and nuclear morphology was assessed as previously described [96] using a Zeiss Axio Vert.a1 microscope. In brief, we scrutinized Hoechst-stained nuclei and scored them as normal or abnormal. Normal nuclei were moderately stained and bean-shaped, whereas nuclei from dead (apoptotic or necrotic) cells were typically hypercondensed, fragmented, and not bean-shaped. See Figure 2 and Reference [96] for examples and details. The numbers of normal and abnormal nuclei were used to determine the percentage of cell death. At least 100 cells were counted to determine cytotoxicity.
The extracts were first screened for cytotoxic potential, and based on the cytotoxic potential of those inducing more than 50% cell death (50-70%, 70-90%, and above 90%), dilutions series were made to estimate EC 50 values. All extracts were tested in duplicates. EC 50 values were calculated, and dose-response curves were created using the nonlinear regression function of GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). The program was also employed for calculating p-value using the extra-sum-of-squares F test.

Antifungal and Antibacterial Assays
The activity of crude extracts obtained from Brazilian cyanobacteria were tested for antifungal (Candida albicans HAMBI484) and antibacterial (Staphylococcus aureus HAMBI66) activities. The antifungal test was performed as previously described [32]. PDA and BHI media were used for the growth and activity assay of Candida albicans HAMBI484 (at 35 • C) and Staphylococcus aureus HAMBI66 (at 37 • C), respectively. Microbial cells grown overnight on solid media were added to sterile water in a sterile tube, and turbidity was measured at 530 nm to approximately McFarland 0.5. The S. aureus HAMBI66 inoculum (800 µL) was spread onto BHI plates and dried for 30 min in sterile conditions. C. albicans HAMBI484 inoculum was spread using a sterile swab. The discs containing dry cyanobacterial cell extracts were added to the plates and incubated at 35 • C or 37 • C overnight, after which the inhibitions zones were measured. Nystatin (25 µg) and kanamycin (1000 µg, Abtek biological) were used as positive controls against C. albicans HAMBI484 and S. aureus HAMBI66, respectively.

Liquid Chromatography-Mass Spectrometry (LC-MS)
Samples were injected into an Acquity UPLC system (Waters, Manchester, UK) equipped with a Kinetex ® C8 100 LC Column (50 × 2.1 mm, 1.7 µm, 100 Å). The UPLC was operated with a flow-rate of 0.3 mL/min in gradient mode at a temperature of 40 • C. Solvents used in the gradient were A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile/isopropanol (1:1). The initial conditions of the linear gradient were A: 95% and B: 5% and the conditions were changed to A: 0% and B: 100% in 5 min. The injection volume was 0.5 µL. Mass spectra were recorded with a Waters Synapt G2-Si mass spectrometer (Waters, Manchester, UK). Measurements were performed using positive and negative electrospray ionization (ESI) in resolution mode. Precursor ions were scanned in the range from m/z 400 to 2000 and productions from m/z 50 to 2000. MS analyses were performed with scan times of 0.1 s and MS/MS analyses with scan times of 0.2 s. The capillary voltage was 1.5 kV (2.0 kV in negative ionization), source temperature was 120 • C, sampling cone was 40.0, source offset was 80.0, desolvation temperature was 600 • C, desolvation gas flow was 1000 L/h, and nebulizer gas flow was 6.5 Bar. Leucine-encephalin was used as a lock mass, and calibration was performed with sodium formate and Ultramark 1621.

Molecular Network
The data obtained with LC-MS was converted to mzXML using MSConvert [97]. A molecular network was obtained using the GNPS: Global Natural Products Social Molecular Network website [98]. The data was filtered, peaks with + or −17 Da of the precursor were removed, and peaks throughout the spectrum other than the top six +/−50 Da were filtered out. The data were then clustered with MS-Cluster with a parent mass tolerance of 0.2 Da and a MS/MS fragment ion tolerance of 0.2 Da to create consensus spectra. Consensus spectra that contained less than two spectra were discarded. A network was then created where edges were filtered to have a cosine score above 0.7 and more than six matched peaks. Further edges between two nodes were kept in the network only if each of the nodes appeared in each other's respective top 10 most similar nodes. The spectra in the network were then searched against the GNPS spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least six matched peaks.
The in silico peptidic natural product dereplicator tool from the GNPS website was used to search for potential known compounds produced by Brazilian cyanobacteria. The precursor and fragment ion mass tolerance selected was 0.02 Da, and a search for analogs was performed (VarQuest).

Data Availability Statement
The 16S rRNA or 16S-23S rRNA sequence accession numbers in NCBI are  Figure S1: Dose-response curves of the selected cyanobacterial extracts on NRK (Blue) and MOLM-13 (red) cell lines: These were the basis for estimating EC 50 values, Figure S2: Complete molecular networking of cyanobacterial extracts from Brazilian (purple) and control (green) strains: Known compounds are indicated (see also Tables  S3 and S4). Node numbers represent mass/charge ratios (m/z), Figure S3: MS/MS spectra of puwainaphycins identified in Aliinostoc sp: CENA535 using the dereplication tool (GNPS). See Table S4 for further information, Figure S4: Spectra of potentially new compounds produced only by Brazilian strains (see also Figure 4 and File S3), Table S1: List of Brazilian cyanobacterial isolates tested for the synthesis of anticancer and antimicrobial compounds: The accession number of their 16S rRNA sequences and the habitat, site, city, and state from where the samples were collected are indicated, Table S2: Human leukemia MOLM-13 cells apoptosis caused by crude extracts of aqueous and organic cyanobacterial cell extracts (average of two parallel measurements indicated in %). Dilutions (0.3×, 0.1×, and 0.01×) are indicated in the table, and samples not tested in different dilutions are indicated with (−). Red color indicates high amounts of apoptotic cells, and green indicates high amounts of normal cells. The error (Er) indicates the difference value of the average with one of the duplicates, Table S3: List of cyanobacteria used as a control in the LC-MS analysis to compare the previously discovered compounds with the molecules produced by Brazilian strains, Table S4: Dereplicator analysis of Brazilian cyanobacteria cell extracts. Predicted compounds are organized by p-value (cutoff of 10 −11 ); FDR cutoff of 15%. Compounds found in previous studies are in bold, File S1: Pictures of the antifungal activity essays using Candida albicans HAMBI484: The use of sterile water may have influenced the susceptibility of the fungal cells, File S2: Pictures of the antibacterial activity essays using Staphylococcus aureus HAMBI66: The use of sterile water may have influenced in the susceptibility of the bacterial cells, File S3: Retention times of chlorinated compounds detected in Fischerella sp. CENA72, Table S5: Culture media used for the cultivation of the strains: The addition of nitrogen refers to 0.467 g/L of NaNO 3 .