Abstract

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.

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Acknowledgements

This work was made possible by the NIH Director’s Pioneer Award, the Flight Attendant Medical Research Institute and a generous gift from S. Yan and H. Mao (J.O.L.), a Damon Runyon Postdoctoral Fellowship (H.P.) and an NIH Postdoctoral Training Award (M.D.). V.O.P. is supported by the Academy of Finland (grant no. 289737) and the Sigrid Juselius Foundation. The authors thank S.A. Head for critical comments on the manuscript. Correspondence and requests for materials should be addressed to J.O.L.

Author information

Author notes

    • Sam Y. Hong

    Present address: Rapafusyn Pharmaceuticals, Baltimore, MD, USA

    • Jingxin Wang

    Present address: The Scripps Research Institute, La Jolla, CA, USA

    • Wukun Liu

    Present address: Institute of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China

    • Manisha Das

    Present address: Food and Drug Administration, Rockville, MD, USA

    • Wei Li

    Present address: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

  1. These authors contributed equally: Zufeng Guo, Sam Y. Hong, Jingxin Wang.

Affiliations

  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA

    • Zufeng Guo
    • , Sam Y. Hong
    • , Jingxin Wang
    • , Wukun Liu
    • , Hanjing Peng
    • , Manisha Das
    • , Wei Li
    • , Shridhar Bhat
    • , Brandon Peiffer
    •  & Jun O. Liu
  2. The SJ Yan and HJ Mao Laboratory of Chemical Biology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    • Zufeng Guo
    • , Sam Y. Hong
    • , Jingxin Wang
    • , Wukun Liu
    • , Hanjing Peng
    • , Manisha Das
    • , Wei Li
    • , Brandon Peiffer
    •  & Jun O. Liu
  3. HiLIFE and Institute of Biotechnology, University of Helsinki, Helsinki, Finland

    • Shahid Rehan
    •  & Ville O. Paavilainen
  4. Rapafusyn Pharmaceuticals, Baltimore, MD, USA

    • Brett R. Ullman
  5. Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    • Chung-Ming Tse
  6. Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada

    • Zlatina Tarmakova
    •  & Imogen Coe
  7. Department of Enzymology, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany

    • Cordelia Schiene-Fischer
    •  & Gunter Fischer
  8. Department of Surgery, Johns Hopkins School of Medicine, Baltimore, MD, USA

    • Zhaoli Sun

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Contributions

J.O.L. conceived the original idea. Z.G., S.Y.H., J.W., S.R., W.L., H.P., M.D., W.L., S.B., B.P., B.R.U., Z.T., C.S.-F., V.O.P. and Z.S. designed and conducted the experiments. Z.G., S.Y.H., J.W., S.R., W.L., H.P., M.D., W.L., S.B., B.P., B.R.U., Z.T., C.S.-F., C.-M.T., G.F., I.C., V.O.P., Z.S. and J.O.L. analysed the results. J.O.L., J.W., Z.G., S.Y.H. and V.O.P. co-wrote the manuscript. Z.G., S.Y.H. and J.W. contributed equally to this work. All authors reviewed and provided input into the revision of the manuscript.

Competing interests

Patent applications covering the rapafucin library and rapadocin have been filed by Johns Hopkins University and licensed to Rapafusyn Pharmaceuticals, Inc. J.O.L. is a co-founder of, as well as a Scientific Advisory Board Member for, Rapafusyn Pharmaceuticals, Inc. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies.

Corresponding author

Correspondence to Jun O. Liu.

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https://doi.org/10.1038/s41557-018-0187-4