A natural depsipeptide antibiotic binds the E-site of the bacterial ribosome

Article A natural depsipeptide antibiotic binds the E-site of the bacterial ribosome https://doi.org/10.1038/s41586-026-10589-2 Received: 18 June 2025 Accepted: 23 April 2026 Published online: xx xx xxxx Manpreet Kaur1,2,3,7, Dmitrii Y. Travin4,5,7, Max J. Berger6,7, Manoj Jangra1,2,3, Martino Morici6, Haaris A. Safdari6, Dorota Klepacki4,5, Wenliang Wang1,2,3, Michael Cook1,2,3, Sommer Chou1,2,3, Allison K. Guitor1,2,3, Kalinka Koteva1,2,3, Min Xu1,2,3, Linda Ejim1,2,3, Aline Fiebig1,2,3, Yeganeh Yousefi1,2,3, Brian K. Coombes1,2,3, Lesley Macneil1,2,3, Nora Vázquez-Laslop4,5, Alexander S. Mankin4,5 ✉, Daniel N. Wilson6 ✉ & Gerard D. Wright1,2,3 ✉ Open access Check for updates A key challenge in addressing the antibiotic resistance crisis is identifying new antimicrobial compounds1. Although natural products produced by fungi and bacteria, particularly actinomycetes, have been the source of most antibiotics discovered over the past 80 years, they have fallen out of favour owing to the frequent rediscovery of known drug scaffolds2. The current perception is that antibiotic-producing actinomycetes have been over-mined and possess little novelty left to yield. Here we demonstrate that by using improved fractionation approaches that enrich previously overlooked minor products, even well-studied strains of antibiotic-producing actinomycetes can provide new chemical scaffolds with unique modes of action. By fractionating a library of natural product extracts from soil bacteria, we show that Streptomyces rimosus, the source of the wellknown antibiotic oxytetracycline, produces a cyclic depsipeptide antibiotic that we call manikomycin. Manikomycin can kill multidrug-resistant Enterobacteriaceae and is not susceptible to resistance associated with clinically used antibiotics. Biochemical, genetic and structural analyses reveal that manikomycin binds in the E-site of the large subunit of the bacterial ribosome, preventing entry of the 3′ end of the tRNA into the E-site and effectively hindering the translocation step of protein synthesis in a sequence-context-specific manner. Manikomycin is the first antibacterial agent, to our knowledge, to target the critical but underexplored E-site in the large ribosomal subunit, highlighting its value as a lead for developing new antibiotics. Microbial natural products, particularly those derived from actinomycetes, have been a dominant source of antibacterial agents over the past 80 years. These antibiotics were identified by testing the ability of crude extracts of their producers to inhibit bacterial growth, a strategy termed the Waksman platform after microbiologist Selman Waksman, who pioneered this approach1. Despite the initial spectacular success of that approach, its utility has decreased over the years, owing to the frequent re-isolation of common chemical scaffolds and the shift toward target-based high-throughput screens of readily sourced synthetic compounds2,3. However, the rise of multidrug-resistant pathogens combined with the limited success of target-based screens and synthetic chemical libraries in antibiotic drug discovery has reignited interest in microbial natural products as sources of new antimicrobial agents. The growing realization of the untapped genomic potential in actinomycetes has fuelled a renewed interest in these microbes as sources of new drug leads4. Streptomyces genomes show an abundance of biosynthetic gene clusters (BGCs) that potentially encode antibacterial compounds. However, only a small fraction of such compounds have been isolated, owing to the often low levels of BGC expression, dominance of one of the several produced antibacterial compounds, and insufficiently discriminating analytical approaches. One strategy to tap into the cryptic antibiotic pool is the improved fractionation of natural product extracts5, enabling the separation of components with overlapping activities, such as two unrelated antibiotics6. Here we report the application of this strategy to discover a new antibiotic with a previously unknown mode of action. We identified a novel cyclic depsipeptide antibiotic, manikomycin (MKM), derived from Streptomyces rimosus, which has been known since 1950 as a producer of the well-known antibiotic oxytetracycline7. We show that MKM exhibits a unique mechanism of action: it binds to the E-site of the large subunit of the bacterial ribosome and interferes with translocation in a context-specific manner. Additionally, we found that MKM is effective against drug-resistant David Braley Centre for Antibiotics Discovery, McMaster University, Hamilton, Ontario, Canada. 2M. G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada. 3Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada. 4Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA. 5Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, USA. 6Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany. 7These authors contributed equally: Manpreet Kaur, Dmitrii Y. Travin, Max J. Berger. ✉e-mail: ; ; 1 Nature | www.nature.com | 1 Article D-Phe3 O D-Orn2 R2 R1 HN (R) N H O (R) NH O O (R) Thr4 (S) O HN D-Arg1 NH HN N H O O NH2 D-Arg7 (R) 0 +ESI EIC(592.3300) S.coelicolor pCGW7.5 man BGC 5.0 0 HO N His9 +ESI EIC(592.3300) S.coelicolor pCGW 1 O (S) HN NH2 (R) HN H N (S) HN 2 (R) O E. coli BW25113 ΔtolCΔbamB D-Arg6 N H NH c NH NH NH2 O (R) H N (R) O H 2N D-Asn5 Count (×105) a +ESI EIC(592.3300) S. rimosus WAC 7405 Thr8 7.5 5.0 2.5 R1 R2 MKM-A H -(CH2)3-NH2 [Orn] MKM-B H -(CH2)3-NH-C(NH)-NH2 [Arg] MKM-E Arg 0 0.4 0.6 0.8 1.0 -(CH2)3-NH2 [Orn] b 1.2 1.4 1.6 1.8 Acquisition time (min) manA manB manD Biosynthetic Resistance Transporter Biosynthetic additional Module 1 Arg ManA C A PCP Module 2 D-Arg C A Module 7 D -Arg ManB C A PCP PCP Module 3 C A Module 4 D-Phe D-Orn/D-Arg E C A PCP PCP E C Module 9 A C A PCP Module 5 Thr E C A PCP Module 6 D-Asn C A PCP E Module10 Thr E manR manC manE Others Module 8 D -Arg E 5 kb Regulator His PCP C A PCP TE Fig. 1 | Identification of the cyclic depsipeptide MKM produced by S. rimosus. a, Chemical structure of MKMs. The table represents the substitutions in the various isoforms. Numbering of residues reflects MKM-A structure. b, Top, man BCG, the BGC responsible for MKM production. The predicted functions of selected encoded proteins are listed. Bottom, the modular structure of the NRPSs ManA and ManB. A, adenylation domain; C, condensation domain; PCP, peptidyl carrier protein; E, epimerization domain; TE, thioesterase domain. c, Liquid chromatography–mass spectrometry analysis and bioactivity of partially purified extracts of MKM conjugants obtained from the heterologous expression of (...truncated)