Ribosomally synthesized and post-translationally modified peptides (RiPPs) are intriguing compounds with potential pharmacological applications. While many RiPPs are known as antimicrobial agents, a limited number of RiPPs with anti-proliferative effects in cancer cells are available. Here we report the discovery of nostatin A (NosA), a highly modified RiPP belonging among nitrile hydratase-like leader peptide RiPPs (proteusins), isolated from a terrestrial cyanobacterium Nostoc sp. Its structure was established based on the core peptide sequence encoded in the biosynthetic gene cluster recovered from the producing strain and subsequent detailed nuclear magnetic resonance and high-resolution mass spectrometry analyses. NosA, composed of a 30 amino-acid peptide core, features a unique combination of moieties previously not reported in RiPPs: the simultaneous presence of oxazole/thiazole heterocycles, dehydrobutyrine/dehydroalanine residues, and a sactionine bond. NosA includes an isobutyl-modified proline residue, highly unusual in natural products. NosA inhibits proliferation of multiple cancer cell lines at low nanomolar concentration while showing no hemolysis. It induces cell cycle arrest in S-phase followed by mitochondrial apoptosis employing a mechanism different from known tubulin binding and DNA damaging compounds. NosA also inhibits Staphylococcus strains while it exhibits no effect in other tested bacteria or yeasts. Due to its novel structure and selective bioactivity, NosA represents an excellent candidate for combinatorial chemistry approaches leading to development of novel NosA-based lead compounds.

CeMM Research Center for Molecular Medicine Austrian Academy of Sciences Lazarettgasse 14 1090 Wien Austria

Centre Algatech Institute of Microbiology Czech Academy of Sciences Novohradká 237 Centre Algatech Institute of Microbiology Czech Academy of Sciences 379 01 Třeboň Czech Republic

Department of Medical Biology Faculty of Science University of South Bohemia Branišovská 1645 31a 370 05 České Budějovice Czech Republic

Institute for Developmental Immunology Medical University of Innsbruck Biocenter Innsbruck Austria

Institute of Entomology Laboratory of Analytical Biochemistry and Metabolomics Biology Centre of the Czech Academy of Sciences Branišovská 1160 31 370 05 České Budějovice Czech Republic

Institute of Hydrobiology Biology Centre of the Czech Academy of Sciences Na Sádkách 702 7 370 05 České Budějovice Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences Vídeňská 1083 142 20 Praha

Institute of Pharmacy Freie Universität Berlin Königin Luise Str 2 4 14195 Berlin Germany

Institute of Pharmacy Martin Luther University Halle Wittenberg Hoher Weg 8 06120 Halle Germany

Laboratory of Antibiotic Resistance and Microbial Metabolomics Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 142 00 Praha 4 Czech Republic

Laboratory of Molecular Structure Characterization Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 142 00 Praha 4 Czech Republic

Laboratory of Structural Biology and Cell Signaling Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 142 00 Praha 4 Czech Republic

Zobrazit více v PubMed

Arnison P. G. Bibb M. J. Bierbaum G. Bowers A. A. Bugni T. S. Bulaj G. Camarero J. A. Campopiano D. J. Challis G. L. Clardy J. Cotter P. D. Craik D. J. Dawson M. Dittmann E. Donadio S. Dorrestein P. C. Entian K. D. Fischbach M. A. Garavelli J. S. Göransson U. Gruber C. W. Haft D. H. Hemscheidt T. K. Hertweck C. Hill C. Horswill A. R. Jaspars M. Kelly W. L. Klinman J. P. Kuipers O. P. Link A. J. Liu W. Marahiel M. A. Mitchell D. A. Moll G. N. Moore B. S. Müller R. Nair S. K. Nes I. F. Norris G. E. Olivera B. M. Onaka H. Patchett M. L. Piel J. Reaney M. J. T. Rebuffat S. Ross R. P. Sahl H. G. Schmidt E. W. Selsted M. E. Severinov K. Shen B. Sivonen K. Smith L. Stein T. Süssmuth R. D. Tagg J. R. Tang G. L. Truman A. W. Vederas J. C. Walsh C. T. Walton J. D. Wenzel S. C. Willey J. M. van der Donk W. A. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 2013;30:108–160. doi: 10.1039/C2NP20085F. PubMed DOI PMC

Cao L. Do T. Link A. J. Mechanisms of action of ribosomally synthesized and posttranslationally modified peptides (RiPPs) J. Ind. Microbiol. Biotechnol. 2021;48 doi: 10.1093/jimb/kuab005. https://dx.doi.org/10.1093/jimb/kuab005 PubMed DOI PMC

Russell A. H. Truman A. W. Genome mining strategies for ribosomally synthesised and post-translationally modified peptides. Comput. Struct. Biotechnol. J. 2020;18:1838–1851. doi: 10.1016/j.csbj.2020.06.032. PubMed DOI PMC

Repka L. M. Chekan J. R. Nair S. K. van der Donka W. A. Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes. Chem. Rev. 2017;117:5457–5520. doi: 10.1021/acs.chemrev.6b00591. PubMed DOI PMC

Melby J. O. Nard N. J. Mitchell D. A. Thiazole/oxazole-modified microcins: complex natural products from ribosomal templates. Curr. Opin. Chem. Biol. 2011;15:369–378. doi: 10.1016/j.cbpa.2011.02.027. PubMed DOI PMC

Dunbar K. L. Melby J. O. Mitchell D. A. YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations. Nat. Chem. Biol. 2012;8:569–575. doi: 10.1038/nchembio.944. PubMed DOI PMC

Sivonen K. Leikoski N. Fewer D. P. Jokela J. Cyanobactins-ribosomal cyclic peptides produced by cyanobacteria. Appl. Microbiol. Biotechnol. 2010;86:1213–1225. doi: 10.1007/s00253-010-2482-x. PubMed DOI PMC

Kim M. Y. Vankayalapati H. Kazuo S. Wierzba K. Hurley L. H. Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex. J. Am. Chem. Soc. 2002;124:2098–2099. doi: 10.1021/ja017308q. PubMed DOI

Duquesne S. Petit V. Peduzzi J. Rebuffat S. Structural and functional diversity of microcins, gene-encoded antibacterial peptides from enterobacteria. J. Mol. Microbiol. Biotechnol. 2007;13:200–209. PubMed

Onaka H. Tabata H. Igarashi Y. Sato Y. Furumai T. Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes - I. Purification and characterization. J. Antibiot. 2001;54:1036–1044. doi: 10.7164/antibiotics.54.1036. PubMed DOI

Haft D. H. Basu M. K. Mitchell D. A. Expansion of ribosomally produced natural products: a nitrile hydratase-and Nif11-related precursor family. BMC Biol. 2010;8 doi: 10.1186/1741-7007-8-70. https://dx.doi.org/10.1186/1741-7007-8-70 PubMed DOI PMC

Bösch N. M. Borsa M. Greczmiel U. Morinaka B. I. Gugger M. Oxenius A. Vagstad A. L. Piel J. Landornamides: Antiviral Ornithine-Containing Ribosomal Peptides Discovered through Genome Mining. Angew. Chem., Int. Ed. 2020;59:11763–11768. doi: 10.1002/anie.201916321. PubMed DOI

Nguyen N. A. Cong Y. Hurrell R. C. Arias N. Garg N. Puri A. W. Schmidt E. W. Agarwal V. A Silent Biosynthetic Gene Cluster from a Methanotrophic Bacterium Potentiates Discovery of a Substrate Promiscuous Proteusin Cyclodehydratase. ACS Chem. Biol. 2022;17:1577–1585. doi: 10.1021/acschembio.2c00251. PubMed DOI PMC

Hamada T. Matsunaga S. Yano G. Fusetani N. Polytheonamides A and B, highly cytotoxic, linear polypeptides with unprecedented structural features, from the marine sponge, Theonella swinhoeiii. J. Am. Chem. Soc. 2005;127:110–118. doi: 10.1021/ja045749e. PubMed DOI

Freeman M. F. Vagstad A. L. Piel J. Polytheonamide biosynthesis showcasing the metabolic potential of sponge-associated uncultivated ‘Entotheonella’ bacteria. Curr. Opin. Chem. Biol. 2016;31:8–14. doi: 10.1016/j.cbpa.2015.11.002. PubMed DOI

Evan G. I. Vousden K. H. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001;411:342–348. doi: 10.1038/35077213. PubMed DOI

Rayan A. Raiyn J. Falah M. Nature is the best source of anticancer drugs: Indexing natural products for their anticancer bioactivity. PLoS One. 2017;12 doi: 10.1371/journal.pone.0187925. https://dx.doi.org/10.1371/journal.pone.0187925 PubMed DOI PMC

Butler M. S. Robertson A. A. B. Cooper M. A. Natural product and natural product derived drugs in clinical trials. Nat. Prod. Rep. 2014;31:1612–1661. doi: 10.1039/C4NP00064A. PubMed DOI

Pucci B. Kasten M. Giordano A. Cell cycle and apoptosis. Neoplasia. 2000;2:291–299. doi: 10.1038/sj.neo.7900101. PubMed DOI PMC

Shi Y. G. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 2002;9:459–470. doi: 10.1016/S1097-2765(02)00482-3. PubMed DOI

Voracova K. Paichlova J. Vickova K. Hrouzek P. Screening of cyanobacterial extracts for apoptotic inducers: a combined approach of caspase-3/7 homogeneous assay and time-lapse microscopy. J. Appl. Phycol. 2017;29:1933–1943. doi: 10.1007/s10811-017-1122-6. DOI

Freeman M. F. Helf M. J. Bhushan A. Morinaka B. I. Piel J. Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium. Nat. Chem. 2017;9:387–395. doi: 10.1038/nchem.2666. PubMed DOI

Wang Z. Q. Forelli N. Hernandez Y. Ternei M. Brady S. F. Lapcin, a potent dual topoisomerase I/II inhibitor discovered by soil metagenome guided total chemical synthesis. Nat. Commun. 2022;13 doi: 10.1038/s41467-022-28292-x. PubMed DOI PMC

Opekar S. Zahradnícková H. Vodrázka P. Rimnácová L. Simek P. Moos M. A chiral GC-MS method for analysis of secondary amino acids after heptafluorobutyl chloroformate & methylamine derivatization. Amino Acids. 2021;53:347–358. doi: 10.1007/s00726-021-02949-1. PubMed DOI

Zahradnícková H. Opekar S. Rimnácová L. Simek P. Moos M. Chiral secondary amino acids, their importance, and methods of analysis. Amino Acids. 2022;54:687–719. doi: 10.1007/s00726-022-03136-6. PubMed DOI

Benjdia A. Berteau O. Radical SAM Enzymes and Ribosomally-Synthesized and Post-translationally Modified Peptides: A Growing Importance in the Microbiomes. Front. Chem. 2021;9 doi: 10.3389/fchem.2021.678068. PubMed DOI PMC

Travin D. Y. Watson Z. L. Metelev M. Ward F. R. Osterman I. A. Khven I. M. Khabibullina N. F. Serebryakova M. Mergaert P. Polikanov Y. S. Cate J. H. D. Severinov K. Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition. Nat. Commun. 2019;10 doi: 10.1038/s41467-019-12589-5. PubMed DOI PMC

Suzuki M. Komaki H. Kaweewan I. Dohra H. Hemmi H. Nakagawa H. Yamamura H. Hayakawa M. Kodani S. Isolation and structure determination of new linear azole-containing peptides spongiicolazolicins A and B from Streptomyces sp. CWH03. Appl. Microbiol. Biotechnol. 2021;105:93–104. doi: 10.1007/s00253-020-11016-w. PubMed DOI

Naidu B. N. Sorenson M. E. Connolly T. P. Ueda Y. Michael addition of amines and thiols to dehydroalanine amides: A remarkable rate acceleration in water. J. Org. Chem. 2003;68:10098–10102. doi: 10.1021/jo034762z. PubMed DOI

Chen Y. L. Wang J. X. Li G. Q. Yang Y. P. Ding W. Current Advancements in Sactipeptide Natural Products. Front. Chem. 2021;9 doi: 10.3389/fchem.2021.595991. PubMed DOI PMC

Mahanta N. Hudson G. A. Mitchell D. A. Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis. Biochemistry. 2017;56:5229–5244. doi: 10.1021/acs.biochem.7b00771. PubMed DOI PMC

Golakoti T. Yoshida W. Y. Chaganty S. Moore R. E. Isolation and structure determination of nostocyclopeptides A1 and A2 from the terrestrial cyanobacterium Nostoc, sp ATCC53789. J. Nat. Prod. 2001;64:54–59. doi: 10.1021/np000316k. PubMed DOI

Fewer D. P. Jokela J. Rouhiainen L. Wahlsten M. Koskenniemi K. Stal L. J. Sivonen K. The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena. Mol. Microbiol. 2009;73:924–937. doi: 10.1111/j.1365-2958.2009.06816.x. PubMed DOI

Tomek P. Hrouzek P. Kuzma M. Sykora J. Fiser R. Cerny J. Novák P. Bártová S. Simek P. Hof M. Kavan D. Kopecky J. Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium Desmonostoc muscorum, Permeabilizes Phospholipid Membranes by Reducing Their Fluidity. Chem. Res. Toxicol. 2015;28:216–224. PubMed

Janata J. Kamenik Z. Gazak R. Kadlcik S. Najmanova L. Biosynthesis and incorporation of an alkylproline-derivative (APD) precursor into complex natural products. Nat. Prod. Rep. 2018;35:257–289. doi: 10.1039/C7NP00047B. PubMed DOI

Hoffmann D. Hevel J. M. Moore R. E. Moore B. S. Sequence analysis and biochemical characterization of the nostopeptolide A biosynthetic gene cluster from Nostoc sp GSV224. Gene. 2003;311:171–180. doi: 10.1016/S0378-1119(03)00587-0. PubMed DOI

Jiraskova P. Gazak R. Kamenik Z. Steiningerova L. Najmanova L. Kadlcik S. Novotna J. Kuzma M. Janata J. New Concept of the Biosynthesis of 4-Alkyl-L-Proline Precursors of Lincomycin, Hormaomycin, and Pyrrolobenzodiazepines: Could a γ-Glutamyltransferase Cleave the C-C Bond? Front. Microbiol. 2016;7 doi: 10.3389/fmicb.2016.00276. PubMed DOI PMC

Spaller B. L. Trieu J. M. Almeida P. F. Hemolytic Activity of Membrane-Active Peptides Correlates with the Thermodynamics of Binding to 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine Bilayers. J. Membr. Biol. 2013;246:257–262. doi: 10.1007/s00232-013-9525-z. PubMed DOI PMC

Hudson G. A. Mitchell D. A. RiPP antibiotics: biosynthesis and engineering potential. Curr. Opin. Microbiol. 2018;45:61–69. doi: 10.1016/j.mib.2018.02.010. PubMed DOI PMC

Di Cesare E. Verrico A. Miele A. Giubettini M. Rovella P. Coluccia A. Famiglini V. La Regina G. Cundari E. Silvestri R. Lavia P. Mitotic cell death induction by targeting the mitotic spindle with tubulin-inhibitory indole derivative molecules. Oncotarget. 2017;8:19738–19759. doi: 10.18632/oncotarget.14980. PubMed DOI PMC

Kingston D. G. I. Tubulin-Interactive Natural Products as Anticancer Agents. J. Nat. Prod. 2009;72:507–515. doi: 10.1021/np800568j. PubMed DOI PMC

Martano G. Delmotte N. Kiefer P. Christen P. Kentner D. Bumann D. Vorholt J. A. Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry. Nat. Protoc. 2015;10:1–11. doi: 10.1038/nprot.2014.198. PubMed DOI

Moos M. Korbelova J. Stetina T. Opekar S. Simek P. Grgac R. Kostal V. Cryoprotective Metabolites Are Sourced from Both External Diet and Internal Macromolecular Reserves during Metabolic Reprogramming for Freeze Tolerance in Drosophilid Fly, Chymomyza costata. Metabolites. 2022;12 doi: 10.3390/metabo12020163. https://dx.doi.org/10.3390/metabo12020163 PubMed DOI PMC

Nordlund N. Reichard P. Ribonucleotide reductases. Annu. Rev. Biochem. 2006;75:681–706. doi: 10.1146/annurev.biochem.75.103004.142443. PubMed DOI

Nocentini G. Ribonucleotide reductase inhibitors: New strategies for cancer chemotherapy. Crit. Rev. Oncol. Hematol. 1996;22:89–126. doi: 10.1016/1040-8428(95)00187-5. PubMed DOI