Abstract
The rise of drug resistance and limitations of current antifungal treatments highlight the urgent need for innovative antifungal strategies. Here we present the development of cis-fumaramidmycin-derived analogs inhibiting the interactions of ribosome assembly factor Mrt4 with rRNA to combat fungal infections. Through antifungal screening, we identified a promising lead 20 with strong efficacy against various drug-resistant fungi, including notorious super-fungus Candida auris. A comprehensive approach combining active-and-inactive-based protein profiling (AIBPP), chemical-genetic profiling, and fluorescence polarization revealed that the antifungal activity of 20 is primarily due to selectively inhibiting essential CaMrt4-rRNA interaction by conjointly covalent engaging C96&C189 on CaMrt4 but inactive for HuMrt4-rRNA interaction, thereby disrupting fungal ribosomal assembly. Therapeutic efficacy of 20 in both Galleria mellonella larvae and murine candidiasis models validate this antifungal strategy. Collectively, our studies provide a potential and much needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infections.
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Data availability
The X-ray crystallographic coordinates and structure factors are available from the Protein Data Bank with accession numbers 9IWD and 9XB4, respectively. The RNA-seq data for the transcriptomic analysis generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE302228. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD068797 and PXD073550. The data that support the findings of this study are available within the paper and its Supplementary Information files. Source data are provided with this paper.
Code availability
The molecular dynamics simulations in this work utilized the following publicly available software packages: GROMACS, Plumed (https://www.plumed.org), gmx_MMPBSA (https://valdes-tresancoms.github.io/gmx_MMPBSA), and Qbics (https://www.qbics.info/).
References
Denning, D. W. Global incidence and mortality of severe fungal disease. Lancet Infect. Dis. 24, e428–e438 (2024).
Say, R. F. & Fuchs, G. Fructose 1,6-bisphosphate aldolase/phosphatase may be an ancestral gluconeogenic enzyme. Nature 464, 1077–1081 (2010).
Cowen, L. E. The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nat. Rev. Microbiol. 6, 187–198 (2008).
Sarma, S. & Upadhyay, S. Current perspective on emergence, diagnosis and drug resistance in Candida auris. Infect. Drug Resist. 10, 155–165 (2017).
Hoenigl, M. et al. COVID-19-associated fungal infections. Nat. Microbiol. 7, 1127–1140 (2022).
Perfect, J. R. The antifungal pipeline: a reality check. Nat. Rev. Drug Discov. 16, 603–616 (2017).
Liu, N., Tu, J., Dong, G., Wang, Y. & Sheng, C. Emerging new targets for the treatment of resistant fungal infections. J. Med. Chem. 61, 5484–5511 (2018).
Fisher, M. C., Hawkins, N. J., Sanglard, D. & Gurr, S. J. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science 360, 739–742 (2018).
Lee, Y., Puumala, E., Robbins, N. & Cowen, L. E. Antifungal drug resistance: molecular mechanisms in candida albicans and beyond. Chem. Rev. 121, 3390–3411 (2021).
Noda, S., Shirai, T., Mori, Y., Oyama, S. & Kondo, A. Engineering a synthetic pathway for maleate in Escherichia coli. Nat. Commun. 8, 1153 (2017).
Zaro, B. W., Whitby, L. R., Lum, K. M. & Cravatt, B. F. Metabolically labile fumarate esters impart kinetic selectivity to irreversible inhibitors. J. Am. Chem. Soc. 138, 15841–15844 (2016).
Becker, D. et al. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments. Nat. Commun. 7, 12761 (2016).
Kornberg, M. D. et al. Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity. Science 360, 449–453 (2018).
Blair, H. A. Dimethyl fumarate: a review in relapsing-remitting MS. Drugs 79, 1965–1976 (2019).
Bharate, S. S. Recent developments in pharmaceutical salts: FDA approvals from 2015 to 2019. Drug Discov. Today 26, 384–398 (2021).
Maruyama, H. B. et al. New antibiotic, fumaramidmycin .1. Production, biological properties and characterization of producer strain. J. Antibiot. 28, 636–647 (1975).
Bello, I. A., Ndukwe, G. I., Amupitan, J. O., Ayo, R. G. & Shode, F. O. Syntheses and biological activity of some derivatives of C-9154 antibiotic. Int. J. Med. Chem. 2012, 148235 (2012).
Ma, T. et al. Low-dose metformin targets the lysosomal AMPK pathway through PEN2. Nature 603, 159–165 (2022).
Bird, R. E., Lemmel, S. A., Yu, X. & Zhou, Q. A. Bioorthogonal chemistry and its applications. Bioconjug. Chem. 32, 2457–2479 (2021).
Johansson, H. et al. Fragment-based covalent ligand screening enables rapid discovery of inhibitors for the RBR E3 ubiquitin ligase HOIP. J. Am. Chem. Soc. 141, 2703–2712 (2019).
Borsari, C. et al. Covalent proximity scanning of a distal cysteine to target PI3Kalpha. J. Am. Chem. Soc. 144, 6326–6342 (2022).
Abdeldayem, A., Raouf, Y. S., Constantinescu, S. N., Moriggl, R. & Gunning, P. T. Advances in covalent kinase inhibitors. Chem. Soc. Rev. 49, 2617–2687 (2020).
Wen, W. et al. N-acylamino saccharin as an emerging cysteine-directed covalent warhead and its application in the identification of novel fbpase inhibitors toward glucose reduction. J. Med. Chem. 65, 9126–9143 (2022).
Owen, D. R. et al. An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586–1593 (2021).
Lanning, B. R. et al. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nat. Chem. Biol. 10, 760–767 (2014).
Wen, W. et al. Structure-guided discovery of the novel covalent allosteric site and covalent inhibitors of fructose-1,6-bisphosphate aldolase to overcome the azole resistance of candidiasis. J. Med. Chem. 65, 2656–2674 (2022).
Han, X. et al. Structure-based rational design of novel inhibitors against fructose-1,6-bisphosphate aldolase from Candida albicans. J. Chem. Inf. Model. 57, 1426–1438 (2017).
Chen, X. et al. Potential antifungal targets based on glucose metabolism pathways of Candida albicans. Front. Microbiol. 11, 296 (2020).
Wei, L. et al. Cov_DOX: a method for structure prediction of covalent protein-ligand bindings. J. Med. Chem. 65, 5528–5538 (2022).
Hoepfner, D. et al. High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions. Microbiol. Res. 169, 107–120 (2014).
Baetz, K. et al. Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action. Proc. Natl. Acad Sci. USA 101, 4525–4530 (2004).
McLellan, C. A. et al. Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy. Nat. Chem. Biol. 14, 135–141 (2018).
Li, Z. et al. Nuclear export of pre-60S particles through the nuclear pore complex. Nature 618, 411–418 (2023).
Sarkar, A., Pech, M., Thoms, M., Beckmann, R. & Hurt, E. Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit. Nat. Struct. Mol. Biol. 23, 1074–1082 (2016).
Lo, K. Y., Li, Z., Wang, F., Marcotte, E. M. & Johnson, A. W. Ribosome stalk assembly requires the dual-specificity phosphatase Yvh1 for the exchange of Mrt4 with P0. J. Cell Biol. 186, 849–862 (2009).
Laserna, V. et al. Dichloro butenediamides as irreversible site-selective protein conjugation reagent. Angew. Chem. Int. Ed. Engl. 60, 23750–23755 (2021).
Fuchs, B. B., O’Brien, E., El Khoury, J. B. & Mylonakis, E. Methods for usingGalleria mellonellaas a model host to study fungal pathogenesis. Virulence 1, 475–482 (2014).
Galbiati, A., Zana, A. & Conti, P. Covalent inhibitors of GAPDH: From unspecific warheads to selective compounds. Eur. J. Med. Chem. 207, 112740 (2020).
Odds, F. C., Brown, A. J. P. & Gow, N. A. R. Antifungal agents: mechanisms of action. Trends Microbiol. 11, 272–279 (2003).
Shastry, M. et al. Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2The GenBank accession numbers for the sequences reported in this manuscript are AF107286–AF107291, AF292693 and AF248644. Microbiology 147, 383–390 (2001).
Ruggero, D. et al. Dyskeratosis congenita and cancer in mice deficient in ribosomal RNA modification. Science 299, 259–262 (2003).
Cao, X. et al. Nascent alt-protein chemoproteomics reveals a pre-60S assembly checkpoint inhibitor. Nat. Chem. Biol. 18, 643–651 (2022).
Pelletier, J., Thomas, G. & Volarevic, S. Ribosome biogenesis in cancer: new players and therapeutic avenues. Nat. Rev. Cancer 18, 51–63 (2018).
Nyhus, C., Pihl, M., Hyttel, P. & Hall, V. J. Evidence for nucleolar dysfunction in Alzheimer’s disease. Rev. Neurosci. 30, 685–700 (2019).
Kawashima, S. A. et al. Potent, reversible, and specific chemical inhibitors of eukaryotic ribosome biogenesis. Cell 167, 512–524 e14 (2016).
Thomson, E., Ferreira-Cerca, S. & Hurt, E. Eukaryotic ribosome biogenesis at a glance. J. Cell Sci. 126, 4815–4821 (2013).
Nikolay, R., Schmidt, S., Schlömer, R., Deuerling, E. & Nierhaus, K. Ribosome assembly as antimicrobial target. Antibiotics 5, 18 (2016).
Stokes, J. M. & Brown, E. D. Chemical modulators of ribosome biogenesis as biological probes. Nat. Chem. Biol. 11, 924–932 (2015).
Kashif, M. et al. Recombinant expression and biophysical characterization of Mrt4 protein that involved in mRNA turnover and ribosome assembly from Saccharomyces cerevisiae. Bioengineered 13, 9103–9113 (2022).
Park, J. B., Park, H., Son, J., Ha, S. J. & Cho, H. S. Structural study of monomethyl fumarate-bound human GAPDH. Mol. Cells 42, 597–603 (2019).
Li, W. et al. Natural product 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase. Acta Pharmacol. Sin. B 43, 470–482 (2022).
Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Merkulova, M. et al. Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution. Am. J. Physiol. Cell Physiol. 300, C1442–C1455 (2011).
Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).
Acknowledgements
This work was supported by the National Key R&D Program of China (2023YFE0110100, Y.R.), Natural Science Foundation of China (Nos. 22377030, Y.R., 82330109, C.S., 22177036, J.W., 22373039, L.R., 92269102, Y.W. and 82273975, W.C.), the Program for the PCSIRT (No. IRT0953, J.W.). Additional support was provided by Guangdong Pearl River Talent Program (2021QN02Y618, Y.W.) and the Program of Introducing Talents of Discipline to Universities of China (111 Program, B17019, J.W.). We thank the staff members of Mass Spectrometry System at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Lab, China for providing technical support and assistance in data collection and analysis. We also acknowledge the staff of beamline BL10U2 at the Shanghai Synchrotron Radiation Facility (SSRF) and BL19U1 beamlines at NFPS, for technical support in X-ray diffraction data collection and analysis. We are grateful to ChomiX biotech (Nanjing, China) for proteomics support. Y.W. acknowledges computational works carried out at the Shenzhen Bay Laboratory Supercomputing Centre.
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Y.R., C.S., W.C., and J.W. conceived and designed the experiments. All authors contributed to this work as follows: H.C., J.T., J.C., B.C., Z.H., and Y.W. were contributed equally. Y.R. and H.C. edit the manuscript. H.C. performed the target identification, co-crystal structure determination; Z.H. and J.B. performed the synthesis of hit compounds; J.T. contributed the murine candidiasis models assays; J.C. and W.C. contributed to the chemical-genetic profiling, transcriptional analysis, and Galleria mellonella larvae assays; B.C. and Y. W. performed the molecular dynamic simulation; L.R. and Z.L. performed the binding energy calculations; J.S. contributed to the Mrt4 gene dosage and mutation assays in C. albicans; X.H. performed the Mrt4-rRNA interaction analysis assays, C.S. performed the LC/MS-MS experiments; N.P.M. and M.S.H. contributed to the review and editing of the manuscript. All authors have given approval to the final version of the manuscript.
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Cao, H., Tu, J., Chen, J. et al. Inhibiting Mrt4-rRNA interaction with fumaramidmycin-based derivatives as an antifungal strategy. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70226-4
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DOI: https://doi.org/10.1038/s41467-026-70226-4
