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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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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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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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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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