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Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase

Abstract

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of TH17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway. Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS), inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural product derivatives. This work both explains the molecular mechanism of a promising family of therapeutics and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

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Figure 1: Chemical structures of studied compounds.
Figure 2: HF and febrifugine inhibit prolyl-tRNA synthetase activity in vitro.
Figure 3: EPRS binds to HF and determines sensitivity to HF in cells.
Figure 4: HFol binds to the active site of EPRS in an ATP-dependent manner.
Figure 5: Proline supplementation prevents activation of the AAR by HF.
Figure 6: Proline supplementation prevents the biological effects of HF.

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References

  1. Clardy, J. & Walsh, C. Lessons from natural molecules. Nature 432, 829–837 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Howitz, K.T. & Sinclair, D.A. Xenohormesis: sensing the chemical cues of other species. Cell 133, 387–391 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Coatney, G.R., Cooper, W.C., Culwell, W.B., White, W.C. & Imboden, C.A. Jr. Studies in human malaria. XXV. Trial of febrifugine, an alkaloid obtained from Dichroa febrifuga Lour., against the Chesson strain of Plasmodium vivax. J. Natl. Malar. Soc. 9, 183–186 (1950).

    CAS  PubMed  Google Scholar 

  4. Ryley, J.F. & Betts, M.J. Chemotherapy of chicken coccidiosis. Adv. Pharmacol. Chemother. 11, 221–293 (1973).

    Article  CAS  PubMed  Google Scholar 

  5. Pines, M. & Nagler, A. Halofuginone: a novel antifibrotic therapy. Gen. Pharmacol. 30, 445–450 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Elkin, M. et al. Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone. Cancer Res. 59, 4111–4118 (1999).

    CAS  PubMed  Google Scholar 

  7. McGaha, T. et al. Effect of halofuginone on the development of tight skin (TSK) syndrome. Autoimmunity 35, 277–282 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Pines, M., Snyder, D., Yarkoni, S. & Nagler, A. Halofuginone to treat fibrosis in chronic graft-versus-host disease and scleroderma. Biol. Blood Marrow Transplant. 9, 417–425 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Koon, H.B. et al. Phase II AIDS Malignancy Consortium trial of topical halofuginone in AIDS-related Kaposi sarcoma. J. Acquir. Immune Defic. Syndr. 56, 64–68 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sundrud, M.S. et al. Halofuginone inhibits TH17 cell differentiation by activating the amino acid starvation response. Science 324, 1334–1338 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Grohmann, U. & Bronte, V. Control of immune response by amino acid metabolism. Immunol. Rev. 236, 243–264 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Cobbold, S.P. et al. Infectious tolerance via the consumption of essential amino acids and mTOR signaling. Proc. Natl. Acad. Sci. USA 106, 12055–12060 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Finlay, D. & Cantrell, D.A. Metabolism, migration and memory in cytotoxic T cells. Nat. Rev. Immunol. 11, 109–117 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kilberg, M.S., Pan, Y.X., Chen, H. & Leung-Pineda, V. Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. Annu. Rev. Nutr. 25, 59–85 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Harding, H.P. et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11, 619–633 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Harding, H.P. et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099–1108 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Peng, T., Golub, T.R. & Sabatini, D.M. The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation. Mol. Cell. Biol. 22, 5575–5584 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Deval, C. et al. Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways. FEBS J. 276, 707–718 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Palii, S.S. et al. Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation. Amino Acids 37, 79–88 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zoncu, R., Efeyan, A. & Sabatini, D.M. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21–35 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Powell, J.D. & Delgoffe, G.M. The mammalian target of rapamycin: linking T cell differentiation, function, and metabolism. Immunity 33, 301–311 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hotamisligil, G.S. & Erbay, E. Nutrient sensing and inflammation in metabolic diseases. Nat. Rev. Immunol. 8, 923–934 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Esposito, M. et al. Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation. J. Neuroimmunol. 220, 52–63 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Nath, N. et al. Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis. J. Immunol. 182, 8005–8014 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Kamberov, Y.G., Kim, J., Mazitschek, R., Kuo, W.P. & Whitman, M. Microarray profiling reveals the integrated stress response is activated by halofuginone in mammary epithelial cells. BMC Res. Notes 4, 381 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kobayashi, S. et al. Catalytic asymmetric synthesis of antimalarial alkaloids febrifugine and isofebrifugine and their biological activity. J. Org. Chem. 64, 6833–6841 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Ruan, B. et al. A unique hydrophobic cluster near the active site contributes to differences in borrelidin inhibition among threonyl-tRNA synthetases. J. Biol. Chem. 280, 571–577 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Copeland, R.A. Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 46, 1–265 (2005).

    PubMed  Google Scholar 

  30. Kikuchi, H. et al. Potent antimalarial febrifugine analogues against the plasmodium malaria parasite. J. Med. Chem. 45, 2563–2570 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Heacock, D., Forsyth, C., Shiba, K. & Musier-Forsyth, K. Synthesis and aminoacyl-tRNA synthetase inhibitory activity of prolyl adenylate analogs. Bioorg. Chem. 24, 273–289 (1996).

    Article  CAS  Google Scholar 

  32. Splan, K.E., Ignatov, M.E. & Musier-Forsyth, K. Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase. J. Biol. Chem. 283, 7128–7134 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Yaremchuk, A., Tukalo, M., Grotli, M. & Cusack, S. A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase. J. Mol. Biol. 309, 989–1002 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Ibba, M. & Soll, D. Aminoacyl-tRNA synthesis. Annu. Rev. Biochem. 69, 617–650 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Boye, K. & Maelandsmo, G.M. S100A4 and metastasis: a small actor playing many roles. Am. J. Pathol. 176, 528–535 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Oslejsková, L. et al. Metastasis-inducing S100A4 protein is associated with the disease activity of rheumatoid arthritis. Rheumatology (Oxford) 48, 1590–1594 (2009).

    Article  Google Scholar 

  37. Pleiss, J.A., Whitworth, G.B., Bergkessel, M. & Guthrie, C. Rapid, transcript-specific changes in splicing in response to environmental stress. Mol. Cell 27, 928–937 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mukhopadhyay, R., Jia, J., Arif, A., Ray, P.S. & Fox, P.L. The GAIT system: a gatekeeper of inflammatory gene expression. Trends Biochem. Sci. 34, 324–331 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Fontana, L., Partridge, L. & Longo, V.D. Extending healthy life span–from yeast to humans. Science 328, 321–326 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Anderson, R.M. & Weindruch, R. Metabolic reprogramming, caloric restriction and aging. Trends Endocrinol. Metab. 21, 134–141 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Haigis, M.C. & Yankner, B.A. The aging stress response. Mol. Cell 40, 333–344 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Caro, P. et al. Effect of 40% restriction of dietary amino acids (except methionine) on mitochondrial oxidative stress and biogenesis, AIF and SIRT1 in rat liver. Biogerontology 10, 579–592 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Xiao, F. et al. Leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways. Diabetes (2011).

  44. Zelante, T. et al. IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance. Eur. J. Immunol. 37, 2695–2706 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Huang, L., Baban, B., Johnson, B.A. III & Mellor, A.L. Dendritic cells, indoleamine 2,3 dioxygenase and acquired immune privilege. Int. Rev. Immunol. 29, 133–155 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. von Bubnoff, D. et al. Indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells and macrophages in infectious and noninfectious cutaneous granulomas. J. Am. Acad. Dermatol. 65, 819–832 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Bronte, V. & Zanovello, P. Regulation of immune responses by L-arginine metabolism. Nat. Rev. Immunol. 5, 641–654 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Plouffe, D. et al. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc. Natl. Acad. Sci. USA 105, 9059–9064 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ting, S.M., Bogner, P. & Dignam, J.D. Isolation of prolyl-tRNA synthetase as a free form and as a form associated with glutamyl-tRNA synthetase. J. Biol. Chem. 267, 17701–17709 (1992).

    CAS  PubMed  Google Scholar 

  50. Jahn, M.J., Jahn, D., Kumar, A.M. & Soll, D. Mono Q chromatography permits recycling of DNA template and purification of RNA transcripts after T7 RNA polymerase reaction. Nucleic Acids Res. 19, 2786 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to thank R. Copeland (Epizyme) for advice on the execution of tight-binding analysis, W. Kuo Harvard Catalyst Laboratory for Innovative Translational Technologies for assistance with the establishment of qPCR assays, and C. Walsh (Harvard), T. Roberts (Dana-Farber Cancer Institute) and S. Thomas (National Institute of Environmental Health Science, USA) for their valuable comments on the manuscript. This work was supported by US National Institutes of Health (NIH) grant GM089885 and a Harvard Technology Accelerator Award (to M.W.); by grants PJ00812701 and PJ008196 from The Next Generation BioGreen 21 Program, Rural Development Administration, Republic of Korea (to C.Y.Y. and H.K.L.); and by NIH grants AI40127 and AI48213 and Juvenile Diabetes Research Foundation 17-2010-421 (to A.R.).

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Contributions

T.L.K. and M.W. conceived of the idea for the study; designed, directed and interpreted experiments; performed the experiments central to target identification and enzymological characterization; and wrote the manuscript. R.M. conceived of, designed and synthesized critical chemical compounds for use in these studies; performed and interpreted experiments; and helped prepare the manuscript. C.-Y.Y. designed, directed and interpreted experiments performed in Korea; performed and interpreted the experiments involving proline rescue of HF-mediated effects on the AAR pathway and antifibrotic effects; and edited the manuscript. D.Z. performed and interpreted experiments central to EPRS knockdown and HF sensitization as well as proline rescue of HF antifibrotic effects and edited the manuscript. M.S.S. planned and performed the immunology experiments; analyzed and interpreted data; and edited the manuscript. M.H., M.E., J.Y., Y.-J.K. and H.-k.L. performed experiments, M.H. contributed Figure 2c, and J.F.C. carried out the experiment in Supplementary Figure 17. D.F.W. supervised the malaria experiments. J.D.D. provided vital reagents, direction and technical expertise for the EPRS enzyme assays and edited the manuscript. A.R. designed, directed and interpreted the immunology experiments and edited the manuscript.

Corresponding authors

Correspondence to Tracy L Keller, Chang-Yeol Yeo, Ralph Mazitschek or Malcolm Whitman.

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Competing interests

T.K., M.W., R.M., M.S. and A.R. have patent applications pending at the US patent office related to the potential therapeutic use of halofuginone and its derivatives.

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Keller, T., Zocco, D., Sundrud, M. et al. Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol 8, 311–317 (2012). https://doi.org/10.1038/nchembio.790

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