{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,20]],"date-time":"2026-04-20T21:47:31Z","timestamp":1776721651349,"version":"3.51.2"},"reference-count":43,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,11,12]],"date-time":"2021-11-12T00:00:00Z","timestamp":1636675200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["PTDC\/MED-QUI\/30944\/2017"],"award-info":[{"award-number":["PTDC\/MED-QUI\/30944\/2017"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Marine Drugs"],"abstract":"<jats:p>The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) holds a vast number of cyanobacteria whose chemical richness is still largely unknown. To expedite its bioactivity screening we developed a natural products library. Sixty strains and four environmental samples were chromatographed, using a semiautomatic HPLC system, yielding 512 fractions that were tested for their cytotoxic activity against 2D and 3D models of human colon carcinoma (HCT 116), and non-cancerous cell line hCMEC\/D3. Six fractions showed high cytotoxicity against 2D and 3D cell models (group A), and six other fractions were selected by their effects on 3D cells (group B). The metabolome of each group was organized and characterized using the MolNetEnhancer workflow, and its processing with MetaboAnalyst allowed discrimination of the mass features with the highest fold change, and thus the ones that might be bioactive. Of those, mass features without precedented identification were mostly found in group A, indicating seven possible novel bioactive molecules, alongside in silico putative annotation of five cytotoxic compounds. Manual dereplication of group B tentatively identified nine pheophytin and pheophorbide derivatives. Our approach enabled the selection of 7 out of 60 cyanobacterial strains for anticancer drug discovery, providing new data concerning the chemical composition of these cyanobacteria.<\/jats:p>","DOI":"10.3390\/md19110633","type":"journal-article","created":{"date-parts":[[2021,11,12]],"date-time":"2021-11-12T08:10:22Z","timestamp":1636704622000},"page":"633","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Uncovering the Bioactive Potential of a Cyanobacterial Natural Products Library Aided by Untargeted Metabolomics"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2337-3152","authenticated-orcid":false,"given":"Leonor","family":"Ferreira","sequence":"first","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4190-0959","authenticated-orcid":false,"given":"Jo\u00e3o","family":"Morais","sequence":"additional","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"},{"name":"Departamento de Biologia, Faculdade de Ci\u00eancias, Universidade do Porto, Rua do Campo Alegre, Edif\u00edcio FC4, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9697-5579","authenticated-orcid":false,"given":"Marco","family":"Preto","sequence":"additional","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"}]},{"given":"Raquel","family":"Silva","sequence":"additional","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7476-9195","authenticated-orcid":false,"given":"Ralph","family":"Urbatzka","sequence":"additional","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3585-2417","authenticated-orcid":false,"given":"Vitor","family":"Vasconcelos","sequence":"additional","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"},{"name":"Departamento de Biologia, Faculdade de Ci\u00eancias, Universidade do Porto, Rua do Campo Alegre, Edif\u00edcio FC4, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8737-2138","authenticated-orcid":false,"given":"Mariana","family":"Reis","sequence":"additional","affiliation":[{"name":"CIIMAR\/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leix\u00f5es, University of Porto, 4450-208 Matosinhos, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1021\/acs.jnatprod.9b01285","article-title":"Natural Products as Sources of New Drugs over the Nearly Four Decades from 01\/1981 to 09\/2019","volume":"83","author":"Newman","year":"2020","journal-title":"J. Nat. Prod."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Khalifa, S.A.M., Shedid, E.S., Saied, E.M., Jassbi, A.R., Jamebozorgi, F.H., Rateb, M.E., Du, M., Abdel-Daim, M.M., Kai, G.Y., and Al-Hammady, M.A.M. (2021). Cyanobacteria\u2014from the Oceans to the Potential Biotechnological and Biomedical Applications. Mar. Drugs, 19.","DOI":"10.3390\/md19050241"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Demay, J., Bernard, C., Reinhardt, A., and Marie, B. (2019). Natural Products from Cyanobacteria: Focus on Beneficial Activities. Mar. Drugs, 17.","DOI":"10.20944\/preprints201904.0192.v1"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Nandagopal, P., Steven, A.N., Chan, L.-W., Rahmat, Z., Jamaluddin, H., and Mohd Noh, N.I. (2021). Bioactive Metabolites Produced by Cyanobacteria for Growth Adaptation and Their Pharmacological Properties. Biology, 10.","DOI":"10.3390\/biology10101061"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Tan, L.T., and Phyo, M.Y. (2020). Marine Cyanobacteria: A Source of Lead Compounds and Their Clinically-Relevant Molecular Targets. Molecules, 25.","DOI":"10.3390\/molecules25092197"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Dyshlovoy, S.A., and Honecker, F. (2020). Marine Compounds and Cancer: Updates 2020. Mar. Drugs, 18.","DOI":"10.3390\/md18120643"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1437","DOI":"10.1007\/s10811-017-1369-y","article-title":"Cyanobacterial Diversity Held in Microbial Biological Resource Centers as a Biotechnological Asset: The Case Study of the Newly Established LEGE Culture Collection","volume":"30","author":"Ramos","year":"2018","journal-title":"J. Appl. Phycol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1885","DOI":"10.1021\/acs.jnatprod.0c00072","article-title":"Chlorosphaerolactylates A\u2013D: Natural Lactylates of Chlorinated Fatty Acids Isolated from the Cyanobacterium Sphaerospermopsis sp. LEGE 00249","volume":"83","author":"Oliveira","year":"2020","journal-title":"J. Nat. Prod."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Freitas, S., Silva, N.G., Sousa, M.L., Ribeiro, T., Rosa, F., Le\u00e3o, P.N., Vasconcelos, V., Reis, M.A., and Urbatzka, R. (2019). Chlorophyll Derivatives from Marine Cyanobacteria with Lipid-Reducing Activities. Mar. Drugs, 17.","DOI":"10.3390\/md17040229"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"224","DOI":"10.3389\/fonc.2019.00224","article-title":"Antiproliferative Effects of the Natural Oxadiazine Nocuolin a Are Associated with Impairment of Mitochondrial Oxidative Phosphorylation","volume":"9","author":"Sousa","year":"2019","journal-title":"Front. Oncol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Freitas, S., Martins, R., Costa, M., Le\u00e3o, P., Vitorino, R., Vasconcelos, V., and Urbatzka, R. (2016). Hierridin B Isolated from a Marine Cyanobacterium Alters VDAC1, Mitochondrial Activity, and Cell Cycle Genes on HT-29 Colon Adenocarcinoma Cells. Mar. Drugs, 14.","DOI":"10.3390\/md14090158"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Ribeiro, T., Lemos, F., Preto, M., Azevedo, J., Sousa, M.L., Le\u00e3o, P.N., Campos, A., Linder, S., Vitorino, R., and Vasconcelos, V. (2017). Cytotoxicity of Portoamides in Human Cancer Cells and Analysis of the Molecular Mechanisms of Action. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0188817"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Antunes, J., Pereira, S., Ribeiro, T., Plowman, J.E., Thomas, A., Clerens, S., Campos, A., Vasconcelos, V., and Almeida, J.R. (2019). A Multi-Bioassay Integrated Approach to Assess the Antifouling Potential of the Cyanobacterial Metabolites Portoamides. Mar. Drugs, 17.","DOI":"10.3390\/md17020111"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1080\/09670262.2016.1163738","article-title":"A Polyphasic Approach for the Taxonomy of Cyanobacteria: Principles and Applications","volume":"51","year":"2016","journal-title":"Eur. J. Phycol."},{"key":"ref_15","first-page":"277","article-title":"Giant Marine Cyanobacteria Produce Exciting Potential Pharmaceuticals","volume":"3","author":"Gerwick","year":"2008","journal-title":"Microbe"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2484","DOI":"10.1021\/acschembio.8b00389","article-title":"NCI Program for Natural Product Discovery: A Publicly-Accessible Library of Natural Product Fractions for High-Throughput Screening","volume":"13","author":"Thornburg","year":"2018","journal-title":"ACS Chem. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"105876","DOI":"10.1016\/j.ejps.2021.105876","article-title":"Generation and Analysis of 3D Cell Culture Models for Drug Discovery","volume":"163","author":"Belfiore","year":"2021","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.toxicon.2019.12.159","article-title":"Portoamides A and B Are Mitochondrial Toxins and Induce Cytotoxicity on the Proliferative Cell Layer of in Vitro Microtumours","volume":"175","author":"Sousa","year":"2020","journal-title":"Toxicon"},{"key":"ref_19","unstructured":"Kijanska, M., and Kelm, J. (2016). In Vitro 3D Spheroids and Microtissues: ATP-Based Cell Viability and Toxicity Assays. Assay Guidance Manual [Internet], Eli Lilly& Company and the National Center for Advancing Translational Sciences."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1038\/s41592-020-0933-6","article-title":"Feature-Based Molecular Networking in the GNPS Analysis Environment","volume":"17","author":"Nothias","year":"2020","journal-title":"Nat. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4035","DOI":"10.1038\/s41467-018-06082-8","article-title":"Dereplication of Microbial Metabolites through Database Search of Mass Spectra","volume":"9","author":"Mohimani","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1093\/bioinformatics\/btx582","article-title":"Ms2lda.Org: Web-Based Topic Modelling for Substructure Discovery in Mass Spectrometry","volume":"34","author":"Wandy","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Da Silva, R.R., Wang, M., Nothias, L.F., van der Hooft, J.J.J., Caraballo-Rodr\u00edguez, A.M., Fox, E., Balunas, M.J., Klassen, J.L., Lopes, N.P., and Dorrestein, P.C. (2018). Propagating Annotations of Molecular Networks Using in Silico Fragmentation. PLoS Comput. Biol., 14.","DOI":"10.1371\/journal.pcbi.1006089"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Ernst, M., Kang, K.B., Caraballo-Rodr\u00edguez, A.M., Nothias, L.F., Wandy, J., Chen, C., Wang, M., Rogers, S., Medema, M.H., and Dorrestein, P.C. (2019). Molnetenhancer: Enhanced Molecular Networks by Integrating Metabolome Mining and Annotation Tools. Metabolites, 9.","DOI":"10.1101\/654459"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1186\/s13321-016-0174-y","article-title":"ClassyFire: Automated Chemical Classification with a Comprehensive, Computable Taxonomy","volume":"8","author":"Eisner","year":"2016","journal-title":"J. Cheminform."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"11183","DOI":"10.1073\/pnas.0914343107","article-title":"Synergistic Allelochemicals from a Freshwater Cyanobacterium","volume":"107","author":"Leao","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1021\/np2002226","article-title":"Minutissamides A-D, Antiproliferative Cyclic Decapeptides from the Cultured Cyanobacterium Anabaena Minutissima","volume":"74","author":"Kang","year":"2011","journal-title":"J. Nat. Prod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"117017","DOI":"10.1016\/j.watres.2021.117017","article-title":"CyanoMetDB, a Comprehensive Public Database of Secondary Metabolites from Cyanobacteria","volume":"196","author":"Jones","year":"2021","journal-title":"Water Res."},{"key":"ref_30","unstructured":"Vulpanovici, F.A. (2004). Biosynthesis, Production and Structural Studies of Secondary Metabolites in Cultured Marine Cyanobacteria. [Ph.D. Thesis, Oregon State University]."},{"key":"ref_31","unstructured":"Thornburg, C.C. (2013). Investigation of Unique Marine Environments for Microbial Natural Products. [Ph.D. Thesis, Oregon State University]."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Saide, A., Lauritano, C., and Ianora, A. (2020). Pheophorbide A: State of the Art. Mar. Drugs, 18.","DOI":"10.3390\/md18050257"},{"key":"ref_33","unstructured":"Lane, D.J. (1991). 16S\/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics, John Wiley and Sons."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"275","DOI":"10.2307\/3668646","article-title":"Molecular Diversity of Microcystis Strains (Cyanophyceae, Chroococcales) Based on 16S RDNA Sequences","volume":"70","author":"Wilmotte","year":"2000","journal-title":"Syst. Geogr. Plants"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3327","DOI":"10.1128\/aem.63.8.3327-3332.1997","article-title":"PCR Primers to Amplify 16S RRNA Genes from Cyanobacteria","volume":"63","author":"Muyzer","year":"1997","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1128\/AEM.06516-11","article-title":"DECIPHER, a Search-Based Approach to Chimera Identification for 16S RRNA Sequences","volume":"78","author":"Wright","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4673","DOI":"10.1093\/nar\/22.22.4673","article-title":"CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice","volume":"22","author":"Thompson","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"W293","DOI":"10.1093\/bioinformatics\/btl529","article-title":"Interactive Tree of Life (ITOL): An Online Tool for Phylogenetic Tree Display and Annotation","volume":"23","author":"Letunic","year":"2007","journal-title":"Bioinformatics"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-3-642-82340-4_1","article-title":"Liquid-Overlay Culture of Cellular Spheroids","volume":"Volume 95","author":"Carlsson","year":"1984","journal-title":"Spheroids in Cancer Research"},{"key":"ref_41","unstructured":"Plotly Technologies Inc. (2021, March 18). Collaborative Data Science. Available online: https:\/\/Chart-Studio.Plotly.Com\/."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"683","DOI":"10.3389\/fmicb.2019.00683","article-title":"Actinobacteria Isolated from Laminaria Ochroleuca: A Source of New Bioactive Compounds","volume":"10","author":"Ribeiro","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1954","DOI":"10.1038\/s41596-020-0317-5","article-title":"Reproducible Molecular Networking of Untargeted Mass Spectrometry Data Using GNPS","volume":"15","author":"Aron","year":"2020","journal-title":"Nat. Protoc."}],"container-title":["Marine Drugs"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1660-3397\/19\/11\/633\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:29:05Z","timestamp":1760167745000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1660-3397\/19\/11\/633"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,12]]},"references-count":43,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["md19110633"],"URL":"https:\/\/doi.org\/10.3390\/md19110633","relation":{},"ISSN":["1660-3397"],"issn-type":[{"value":"1660-3397","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,12]]}}}