Abstract
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new pharmaceuticals, yet the therapeutic potential of fungal RiPPs remains largely underexplored. Here we report asperigimycins as a distinct class of fungal RiPPs, featuring a unique heptacyclic scaffold consisting of a benzofuranoindoline core and three additional macrocycles, primarily assembled by six distinct fungi-specific DUF3328 oxidases. Inspired by the enhancement of anticancer activity through the N-terminal pyroglutamate in naturally occurring asperigimycins C and D, we chemically modify the inactive asperigimycin B with a series of lipid substitutions at its N-terminus. A derivative with a C-11 linear fatty acid, 2-L6, achieves nanomolar anticancer potency comparable to that of clinically approved antileukemia drugs. High-throughput CRISPR screening identifies the SLC46A3 transporter as a critical factor mediating 2-L6 cellular uptake into human cells. Our findings highlight the promise of engineering asperigimycins as therapeutic leads for cancer treatment.
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Data availability
All data are available in the Article or its Supplementary Information. Coordinates and associated structure factors of ApgG have been deposited in the PDB database (PDB ID 8VPL). High-throughput DNA sequencing data in this study have been deposited at the NCBI Sequence Read Archive (PRJNA1113705). The molecular network can be found at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c896cd91d51d4bf7bfb45484ada8d04f. All primers used in this Article are listed in Supplementary Table 3. Data are available from the corresponding authors upon request. Source data are provided with this paper.
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Acknowledgements
This work was supported by National Institute of Health (NIH) grants (R35GM138207 to X.G., R35CA274234 to J.C. and R35GM128779 to P.L.), the startup fund provided by the University of Pennsylvania to X.G., Welch Foundation (grant number C-2033-20200401) to Y.G., a predoctoral fellowship from the Houston Area Molecular Biophysics Program (NIH grant number T32 GM008280, Program Director Theodore Wensel, to C.C.), and Cancer Prevention and Research Institute of Texas grants (RR220087 to H.R. and RR210029 to D.G.). DFT calculations were carried out at the University of Pittsburgh Center for Research Computing and the Advanced Cyberinfrastructure Coordination Ecosystem: Services and Support (ACCESS) program, supported by NSF award numbers OAC-2117681, OAC-1928147 and OAC-1928224. B.K. was supported in part by a NLM Training Program in Biomedical Informatics and Data Science fellowship (T15LM007093-31). T.T. was supported in part by NSF EF-2126387.
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Q.N. and F.Z. performed gene knockout and biochemical assays. Q.N., F.Z., C.S., K.Y., A.Y.D., S.L. and Z.H. performed compound purification and identification, and X.Y. performed chemical synthesis. Q.N., X.Y. and D.Z. performed cytotoxicity assays. M.C.M. performed conformational sampling and DFT calculations. P.L. supervised the computational calculation. Q.N., C.C. and R.S. performed the crystallization and data analysis. Q.N., S.L., R.T., A.X. and H.Z. performed the CRISPR screening and data analysis. S.R.C., S.A.C.F. and Q.N. performed mass analysis. B.K., T.T. and Q.N. performed bioinformatic analysis. D.G. and J.W. designed the cytotoxicity assays. Y.G. designed the protein crystallization and analyzed data. H.R., Q.N. and X.G. designed the chemical synthesis. Q.N. and X.G. conceived the study and wrote the paper. X.G. supervised the study. Q.N., M.Z., P.N.L., P.L., J.W., Y.G., J.C., H.R. and X.G. reviewed and edited the paper.
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Based on the results presented herein, a provisional patent application (RICE.P0154US.P1) has been filed through Rice University.
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Extended data
Extended Data Fig. 1 Chemical structures of representative fungal RiPPs.
α-amanitin and omphalotin A from basidiomycetes (left panel) and ustiloxin B, epichloëcyclin B, victorin C, phomopsin A, and asperipin-2a from ascomycetes (right panel). The structure of epichloëcyclin B was proposed by MS/MS data. The structure of asperipin-2a was revised in 2020 by chemical synthesis72.
Extended Data Fig. 2 Molecular network of metabolites of 12 Aspergillus sp. strains.
This figure showed major clusters in the molecular network. Node sizes represent relative precursor ion intensity. Pie charts represent relative spectral counts of defined groups with different colors. Clusters circled are labeled with the potential compound types identified by GNPS analysis for the corresponding cluster.
Extended Data Fig. 3 Key 2D NMR data and the lowest energy conformer of 1.
a. COSY and HMBC correlations. b. ROESY correlations. The relative configuration of 1 was assigned by detailed interpretation of the ROESY spectrum, in which the correlations of H-27/H-22 and H-27/H-21 revealed that these protons are cofacial. Correlations between H-4/H-6, H-6/H-10, and H-10/H-12 indicated that H-6 and H-12 are the same oriented, while the small coupling constant ( < 3 Hz) suggested that H-12 and H-13 are the same oriented. H-36, H-37, and H-38 were assigned as the same oriented since the ROESY correlations were observed between H-36/H-38 and H-37/H-38. In general, all the amino acid residues were proposed to be L-configuration, thus the absolute configuration was determined as 2S, 4S, 6S, 12R, 13S, 18S, 21S, 23S, 27R, 34R, 38S, 41S. Considering the similar chemical shifts and biosynthetic origins, the stereochemistry of 2-4, was proposed to be the same as those of 1. c. Lowest-energy conformer of compound 1.
Extended Data Fig. 4 LC-MS analysis of mutants with different truncated ApgAT mutations.
The left panel showed EIC of targeted compounds from the extract broth of ApgAT mutants. The proposed chemical structures of these derivatives are shown in the right panel.
Extended Data Fig. 5 Bioinformatic analysis of apg homologous gene clusters.
Homologous gene clusters of apg are found in diverse fungal species. Homologous enzymes of ApgYa and ApgYe are highlighted. The putative core peptides encoded by corresponding homologous BGCs indicated the close relationship between ApgYa and ApgYe with the modification of Leu, Trp, and Tyr.
Extended Data Fig. 6 Sequence similarity network analysis of DUF3328 enzymes.
It contains selected sequences of DUF3328 enzyme family (PF11807) using an alignment score of 52 and visualized using Cytoscape 3.10.073. This sequence similarity network was performed by EFI-EST74 (https://efi.igb.illinois.edu/efi-est/).
Extended Data Fig. 7 HPLC analysis of gene deletion mutants.
(i) ΔapgB, (ii) ΔapgC, (iii) ΔapgD, (iv) ΔapgQ, (v) ΔapgE, (vi) ΔapgF, (vii) ΔapgH, (viii) ΔapgI. (UV 210 nm).
Extended Data Fig. 8 Biochemical characterization of ApgG.
a. Overall structure of ApgG (green) and comparison with human glutaminyl cyclotransferase (PDB ID 3PBE) (purple). b, c. Molecular docking of ApgG and 15 was performed using AutoDock Vina. d. Relative production ratio of ApgG mutants with 15. All assays in this figure are presented as mean ± s.d.; n = 3 biologically independent samples. e. Proposed enzymatic mechanism of ApgG.
Supplementary information
Supplementary Information
Supplementary Methods, Tables 1–23 and Figs. 1–137.
Supplementary Data 1
Cartesian coordinate for the computational analysis in Supplementary Figs. 9 and 10.
Supplementary Data 2
Source data for HRMS of 2-L1–2-L7 in Supplementary Figs. 131–137.
Supplementary Data 3
Source data for cytotoxicity assays of asperigimycins in Supplementary Fig. 16.
Source data
Source Data Fig. 3
Source data for dose-dependent curve.
Source Data Fig. 4
Source data for cell viability tests and cellular uptake experiments.
Source Data Extended Data Fig. 8
Source Data for reaction efficiency of ApgG mutants.
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Nie, Q., Zhao, F., Yu, X. et al. A class of benzofuranoindoline-bearing heptacyclic fungal RiPPs with anticancer activities. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01946-9
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DOI: https://doi.org/10.1038/s41589-025-01946-9
