Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis

Abstract

The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.

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Fig. 1: Compounds synthesized via ring system distortion of pleuromutilin using the CtD strategy.
Fig. 2: Synthesis and evaluation of ferroptocide.
Fig. 3: Ferroptocide induces rapid non-apoptotic cell death.
Fig. 4: Ferroptocide kills cancer cells through ferroptosis.
Fig. 5: Ferroptocide selectively and covalently modifies its target in cells.
Fig. 6: Ferroptocide modulates active-site cysteines of thioredoxin.
Fig. 7: Ferroptocide modulates the immune system.

Data availability

The data supporting the findings of this study are available within the paper and Supplementary Information and are available from the corresponding author upon request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD012805 (www.ebi.ac.uk/pride/archive/projects/PXD012805). The RNA sequencing data have been deposited to the GEO repository with accession number GSE126868 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126868). The X-ray crystallography data have been deposited in the Cambridge Crystallographic Data Centre (CCDC) using the following identifiers (www. ccdc.cam.ac.uk/structures/): 1851845 (compound P1) and 1849494 (Ferroptocide).

References

  1. 1.

    Lovering, F., Bikker, J. & Humblet, C. Escape from flatland: increasing saturation as an approach to improving clinical success. J. Med. Chem. 52, 6752–6756 (2009).

    CAS  Article  Google Scholar 

  2. 2.

    Swinney, D. C. & Anthony, J. How were new medicines discovered? Nat. Rev. Drug Discov. 10, 507–519 (2011).

    CAS  Article  Google Scholar 

  3. 3.

    Galloway, W. R. J. D., Isidro-Llobet, A. & Spring, D. R. Diversity-oriented synthesis as a tool for the discovery of novel biologically active small molecules. Nat. Commun. 1, 80–93 (2010).

    Article  Google Scholar 

  4. 4.

    Gerry, C. J. & Schreiber, S. L. Chemical probes and drug leads from advances in synthetic planning and methodology. Nat. Rev. Drug Discov. 17, 333–352 (2018).

    CAS  Article  Google Scholar 

  5. 5.

    Morrison, K. C. & Hergenrother, P. J. Natural products as starting points for the synthesis of complex and diverse compounds. Nat. Prod. Rep. 31, 6–14 (2014).

    CAS  Article  Google Scholar 

  6. 6.

    Huigens, R. W. III et al. A ring-distortion strategy to construct stereochemically complex and structurally diverse compounds from natural products. Nat. Chem. 5, 195–202 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Ciardiello, J. J. et al. A novel complexity-to-diversity strategy for the diversity-oriented synthesis of structurally diverse and complex macrocycles from quinine. Bioorg. Med. Chem. 25, 2825–2843 (2017).

    CAS  Article  Google Scholar 

  8. 8.

    Rafferty, R. J., Hicklin, R. W., Maloof, K. A. & Hergenrother, P. J. Synthesis of complex and diverse compounds through ring distortion of abietic acid. Angew. Chem. Int. Ed. 53, 220–224 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Garcia, A., Drown, B. S. & Hergenrother, P. J. Access to a structurally complex compound collection via ring distortion of the alkaloid sinomenine. Org. Lett. 18, 4852–4855 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Tasker, S. Z., Cowfer, A. E. & Hergenrother, P. J. Preparation of structurally diverse compounds from the natural product lycorine. Org. Lett. 20, 5894–5898 (2018).

    CAS  Article  Google Scholar 

  11. 11.

    Paciaroni, N. G. et al. A tryptoline ring‐distortion strategy leads to complex and diverse biologically active molecules from the indole alkaloid yohimbine. Chem. Eur. J. 23, 4327–4335 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    Govindaraju, K. et al. Novel topologically complex scaffold derived from alkaloid haemanthamine. Molecules 23, 255–263 (2018).

    Article  Google Scholar 

  13. 13.

    Charaschanya, M. & Aubé, J. Reagent-controlled regiodivergent ring expansions of steroids. Nat. Commun. 9, 934–942 (2018).

    Article  Google Scholar 

  14. 14.

    Laurent, E. et al. A ring‐distortion strategy from marine natural product ilimaquinone leads to quorum sensing modulators. Eur. J. Org. Chem. 2018, 2486–2497 (2018).

    Article  Google Scholar 

  15. 15.

    Xu, H. et al. Identification of a diverse synthetic abietane diterpenoid library for anticancer activity. Bioorg. Med. Chem. Lett. 27, 505–510 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Luca, L. et al. Discovery of novel cinchona‐alkaloid‐inspired oxazatwistane autophagy inhibitors. Angew. Chem. Int. Ed. 56, 2145–2150 (2017).

    Article  Google Scholar 

  17. 17.

    Richter, M. F. et al. Predictive compound accumulation rules yield a broad-spectrum antibiotic. Nature 545, 299–304 (2017).

    CAS  Article  Google Scholar 

  18. 18.

    Poulsen, S. M., Karlsson, M., Johansson, L. B. & Vester, B. The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome. Mol. Microbiol. 41, 1091–1099 (2001).

    CAS  Article  Google Scholar 

  19. 19.

    Ma, X. et al. Directed C–H bond oxidation of (+)-pleuromutilin. J. Org. Chem. 83, 6843–6892 (2018).

    CAS  Article  Google Scholar 

  20. 20.

    Farney, E. P., Feng, S. S., Schäfers, F. & Reisman, S. E. Total synthesis of (+)-pleuromutilin. J. Am. Chem. Soc. 140, 1267–1270 (2018).

    CAS  Article  Google Scholar 

  21. 21.

    Murphy, S. K., Zeng, M. & Herzon, S. B. A modular and enantioselective synthesis of the pleuromutilin antibiotics. Science 356, 956–959 (2017).

    CAS  Article  Google Scholar 

  22. 22.

    Thirring, K. et al. 12-epi-pleuromutilins. US patent WO2015110481A1 (2015).

  23. 23.

    Gibbons, E. G. Total synthesis of (±)-pleuromutilin. J. Am. Chem. Soc. 104, 1767–1769 (1982).

    CAS  Article  Google Scholar 

  24. 24.

    Paquette, L. A., Wiedeman, P. E. & Bulman-Page, P. C. (+)-Pleuromutilin synthetic studies. Degradative and de novo acquisition of a levorotatory tricyclic lactone subunit. J. Org. Chem. 53, 1441–1450 (1988).

    CAS  Article  Google Scholar 

  25. 25.

    Liu, J., Lotesta, S. D. & Sorensen, E. J. A concise synthesis of the molecular framework of pleuromutilin. Chem. Commun. 47, 1500–1502 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Birch, A. J., Holzapfel, C. W. & Rickards, R. W. The structure and some aspects of the biosynthesis of pleuromutilin. Tetrahedron 22, 359–387 (1966).

    Article  Google Scholar 

  27. 27.

    Arigoni, D. Some studies in the biosynthesis of terpenes and related compounds. Pure Appl. Chem. 17, 331–348 (1968).

    CAS  Article  Google Scholar 

  28. 28.

    Drews, J. et al. Antimicrobial activities of 81.723 hfu, a new pleuromutilin derivative. Antimicrob. Agents Chemother. 7, 507–516 (1975).

    CAS  Article  Google Scholar 

  29. 29.

    Yang, L. P. & Keam, S. J. Retapamulin: a review of its use in the management of impetigo and other uncomplicated superficial skin infections. Drugs 68, 855–873 (2008).

    CAS  Article  Google Scholar 

  30. 30.

    Berner, H., Schulz, G. & Schneider, H. Synthese ab-trans-anellierter derivate des tricyclischen diterpens pleuromutilin durch intramolekulare 1,5-hydrid-verschiebung. Tetrahedron 36, 1807–1811 (1980).

    CAS  Article  Google Scholar 

  31. 31.

    Andemichael, Y. et al. Process development for a novel pleuromutilin-derived antibiotic. Org. Process Res. Dev. 13, 729–738 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Uccello, D. P. et al. The synthesis of C-13 functionalized pleuromutilins via C–H amidation and subsequent novel rearrangement product. Tetrahedron Lett. 52, 4247–4251 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Hicklin, R. W., López Silva, T. L. & Hergenrother, P. J. Synthesis of bridged oxafenestranes from pleuromutilin. Angew. Chem. Int. Ed. 53, 9880–9883 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Botham, R. C. et al. Dual small-molecule targeting of procaspase-3 dramatically enhances zymogen activation and anticancer activity. J. Am. Chem. Soc. 136, 1312–1319 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    Parkinson, E. I., Bair, J. S., Cismesia, M. & Hergenrother, P. J. Efficient NQO1 substrates are potent and selective anticancer agents. ACS Chem. Biol. 8, 2173–2183 (2013).

    CAS  Article  Google Scholar 

  36. 36.

    Palchaudhuri, R. et al. A small molecule that induces intrinsic pathway apoptosis with unparalleled speed. Cell Rep. 13, 2027–2036 (2015).

    CAS  Article  Google Scholar 

  37. 37.

    Linkermann, A., Stockwell, B. R., Krautwald, S. & Anders, H.-J. Regulated cell death and inflammation: an auto-amplification loop causes organ failure. Nat. Rev. Immunol. 14, 759–767 (2014).

    CAS  Article  Google Scholar 

  38. 38.

    Galluzzi, L., Senovilla, L., Zitvogel, L. & Kroemer, G. The secret ally: immunostimulation by anticancer drugs. Nat. Rev. Drug Discov. 11, 215–233 (2012).

    CAS  Article  Google Scholar 

  39. 39.

    Lee, H. Y. et al. Reactive oxygen species synergize to potently and selectively induce cancer cell death. ACS Chem. Biol. 12, 1416–1424 (2017).

    CAS  Article  Google Scholar 

  40. 40.

    Stockwell, B. R. et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology and disease. Cell 171, 273–285 (2017).

    CAS  Article  Google Scholar 

  41. 41.

    Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

    CAS  Article  Google Scholar 

  42. 42.

    Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).

    CAS  Article  Google Scholar 

  43. 43.

    Shimada, K. et al. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis. Nat. Chem. Biol. 12, 497–503 (2016).

    CAS  Article  Google Scholar 

  44. 44.

    Gaschler, M. M. et al. FINO2 initiates ferroptosis through GPX4 inactivation and iron oxidation. Nat. Chem. Biol. 14, 507–515 (2018).

    CAS  Article  Google Scholar 

  45. 45.

    Lewerenz, J. et al. Oxytosis/ferroptosis—(re-)emerging roles for oxidative stress-dependent non-apoptotic cell death in diseases of the central nervous system. Front. Neurosci. 12, 214 (2018).

    Article  Google Scholar 

  46. 46.

    Murphy, T. H. et al. Calcium-dependent glutamate cytotoxicity in a neuronal cell line. Brain Res. 444, 325–332 (1988).

    CAS  Article  Google Scholar 

  47. 47.

    Miyamoto, M., Murphy, T. H., Schnaar, R. L. & Coyle, J. T. Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line. J. Pharmacol. Exp. Ther. 250, 1132–1140 (1989).

    CAS  PubMed  Google Scholar 

  48. 48.

    Seiler, A. et al. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metabol. 8, 237–248 (2008).

    CAS  Article  Google Scholar 

  49. 49.

    Yang, W. S. & Stockwell, B. R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15, 234–245 (2008).

    CAS  Article  Google Scholar 

  50. 50.

    Subramanian, A. et al. A next generation connectivity map: L1000 platform and the first 1,000,000 profiles. Cell 171, 1437–1452 (2017).

    CAS  Article  Google Scholar 

  51. 51.

    Dixon, S. J. et al. Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosis. eLife 3, e02523 (2014).

    Article  Google Scholar 

  52. 52.

    Banerjee, R., Pace, N. J., Brown, D. R. & Weerapana, E. 1,3,5-Triazine as a modular scaffold for covalent inhibitors with streamlined target identification. J. Am. Chem. Soc. 135, 2497–2500 (2013).

    CAS  Article  Google Scholar 

  53. 53.

    Holmgren, A. Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3, 239–243 (1995).

    CAS  Article  Google Scholar 

  54. 54.

    Nordberg, J. & Arnér, E. S. J. Reactive oxygen species, antioxidants and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287–1312 (2001).

    CAS  Article  Google Scholar 

  55. 55.

    Mukherjee, A. et al. A cellular and molecular investigation of the action of PMX464, a putative thioredoxin inhibitor, in normal and colorectal cancer cell lines. Br. J. Pharmacol. 151, 1167–1175 (2007).

    CAS  Article  Google Scholar 

  56. 56.

    Baker, A. F. et al. The antitumor thioredoxin-1 inhibitor PX-12 (1-methylpropyl 2-imidazolyl disulfide) decreases thioredoxin-1 and VEGF levels in cancer patient plasma. J. Lab. Clin. Med. 147, 83–90 (2006).

    CAS  Article  Google Scholar 

  57. 57.

    Kirkpatrick, D. L. et al. Mechanisms of inhibition of the thioredoxin growth factor system by antitumor 2-imidazolyl disulfides. Biochem. Pharmacol. 55, 987–994 (1998).

    CAS  Article  Google Scholar 

  58. 58.

    Sexton, D. W. Targeting airway inflammation: PMX464 and the epithelial bulls eye. Br. J. Pharmacol. 155, 620–622 (2008).

    CAS  Article  Google Scholar 

  59. 59.

    Reynoso, E. et al. Thioredoxin-1 actively maintains the pseudokinase MLKL in a reduced state to suppress disulfide bond-dependent MLKL polymer formation and necroptosis. J. Biol. Chem. 292, 17514–17524 (2017).

    CAS  Article  Google Scholar 

  60. 60.

    You, B. R., Shin, H. R. & Park, W. H. PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis. Int. J. Oncol. 44, 301–308 (2014).

    CAS  Article  Google Scholar 

  61. 61.

    Li, Y., Qian, L. & Yuan, J. Small molecule probes for cellular death machines. Curr. Opin. Chem. Biol. 39, 74–82 (2017).

    CAS  Article  Google Scholar 

  62. 62.

    Galluzzi, L. et al. Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ. 25, 486–541 (2018).

    Article  Google Scholar 

  63. 63.

    Mai, T. T. et al. Salinomycin kills cancer stem cells by sequestering iron in lysosomes. Nat. Chem. 9, 1025–1033 (2017).

    CAS  Article  Google Scholar 

  64. 64.

    Bruni, A. et al. Ferroptosis-inducing agents compromise in vitro human islet viability and function. Cell Death Dis. 9, 595–605 (2018).

    Article  Google Scholar 

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Acknowledgements

The authors acknowledge support from the University of Illinois and Cancer Scholars for Translational and Applied Research (C*STAR) programme and thank the NIH (R01GM118575) for support of some of the synthetic studies. The authors thank W. Woods and P. Perez Pinera for assistance with CRISPR–Cas9 studies, D. Gray and T. Woods for X-ray analysis of compounds, L. Dirikolu for calculation of pharmacokinetic (PK) parameters, B. Drown for chemoinformatic analysis of compounds, M.E. Vinyard and A. Sowers for synthetic assistance and S. Tasker for many helpful discussions.

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P.J.H., E.L. and R.W.H. conceived this study. R.W.H. designed and synthesized all compounds. E.L. designed and performed all biological experiments and analysed data. H.Y.L. performed the mouse model studies. S.E.M. synthesized the lead compound for the mouse model. L.A.C. and E.W. collected and analysed the LC/LC–MS/MS data. P.J.H. supervised this research. P.J.H. and E.L. wrote this manuscript with the assistance of R.W.H. All authors have given their approval of the final version of the manuscript.

Corresponding author

Correspondence to Paul J. Hergenrother.

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The University of Illinois has filed patents on some compounds described in this manuscript.

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Supplementary Information

Supplementary Information

The Supplementary Information contains Supplementary Figs. 1–15, methods, chemical schemes, and NMR data

Reporting Summary

Crystallographic data

Crystallographic data for compound P1. CCDC reference 1851845

Crystallographic data

Structure factors file for compound P1. CCDC reference 1851845

Crystallographic data

Crystallographic data for Ferroptocide. CCDC reference 1849494

Crystallographic data

Structure factors file for Ferroptocide. CCDC reference 1849494

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Llabani, E., Hicklin, R.W., Lee, H.Y. et al. Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis. Nat. Chem. 11, 521–532 (2019). https://doi.org/10.1038/s41557-019-0261-6

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