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Rapamycin-inspired macrocycles with new target specificity


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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Fig. 1: Design of rapafucin based on the scaffolds of rapamycin and FK506 and general synthetic route to rapafucin.
Fig. 2: Optimization of the linker on solid support and FKBDs to be used for the synthesis of rapafucins.
Fig. 3: Selection of amino-acid building blocks and synthetic strategy used for the construction of the rapafucin library.
Fig. 4: Rapadocin is a potent and subtype-selective inhibitor of hENT1.
Fig. 5: Pulldown of hENT1 from red cell membrane fractions using a biotin–rapadocin conjugate or GST–FKBP12–rapadocin complex, and the FKBP dependence of hENT1 inhibition by rapadocin in cells.
Fig. 6: Inhibition of kidney ischaemia reperfusion injury by rapadocin in vivo.

Data availability

The data that support the findings of this study are available from the authors upon reasonable request.


  1. 1.

    Sehgal, S. N., Baker, H. & Vezina, C. Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. J. Antibiot. 28, 727–732 (1975).

    CAS  Article  Google Scholar 

  2. 2.

    Tanaka, H. et al. Structure of FK506, a novel immunosuppressant isolated from Streptomyces. J. Am. Chem. Soc. 109, 5031–5033 (1987).

    CAS  Article  Google Scholar 

  3. 3.

    Harding, M. W., Galat, A., Uehling, D. E. & Schreiber, S. L. A receptor for the immunosuppressant FK506 is a cistrans peptidyl-prolyl isomerase. Nature 341, 758–760 (1989).

    CAS  Article  Google Scholar 

  4. 4.

    Siekierka, J. J., Hung, S. H., Poe, M., Lin, C. S. & Sigal, N. H. A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341, 755–757 (1989).

    CAS  Article  Google Scholar 

  5. 5.

    Heitman, J., Movva, N. R. & Hall, M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909 (1991).

    CAS  Article  Google Scholar 

  6. 6.

    Liu, J. et al. Calcineurin is a common target of cyclophilin–cyclosporin A and FKBP–FK506 complexes. Cell 66, 807–815 (1991).

    CAS  Article  Google Scholar 

  7. 7.

    Yang, H. et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Griffith, J. P. et al. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12–FK506 complex. Cell 82, 507–522 (1995).

    CAS  Article  Google Scholar 

  9. 9.

    Kissinger, C. R. et al. Crystal structures of human calcineurin and the human FKBP12–FK506–calcineurin complex. Nature 378, 641–644 (1995).

    CAS  Article  Google Scholar 

  10. 10.

    Marinec, P. S. et al. FK506-binding protein (FKBP) partitions a modified HIV protease inhibitor into blood cells and prolongs its lifetime in vivo. Proc. Natl Acad. Sci. USA 106, 1336–1341 (2009).

    CAS  Article  Google Scholar 

  11. 11.

    Klemm, J. D., Schreiber, S. L. & Crabtree, G. R. Dimerization as a regulatory mechanism in signal transduction. Annu. Rev. Immunol. 16, 569–592 (1998).

    CAS  Article  Google Scholar 

  12. 12.

    Bayle, J. H. et al. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 13, 99–107 (2006).

    CAS  Article  Google Scholar 

  13. 13.

    Guduru, S. K. R. & Arya, P. Synthesis and biological evaluation of rapamycin-derived, next generation small molecules. Med. Chem. Commun. 9, 27–43 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Chakraborty, T. K., Weber, H. P. & Nicolaou, K. C. Design and synthesis of a rapamycin-based high affinity binding FKBP12 ligand. Chem. Biol. 2, 157–161 (1995).

    CAS  Article  Google Scholar 

  15. 15.

    Wu, X. et al. Creating diverse target-binding surfaces on FKBP12: synthesis and evaluation of a rapamycin analogue library. ACS Comb. Sci. 13, 486–495 (2011).

    Article  Google Scholar 

  16. 16.

    Li, W., Bhat, S. & Liu, J. O. A simple and efficient route to the FKBP-binding domain from rapamycin. Tetrahedron Lett. 52, 5070–5072 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Furka, A., Sebestyen, F., Asgedom, M. & Dibo, G. General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Pept. Protein Res. 37, 487–493 (1991).

    CAS  Article  Google Scholar 

  18. 18.

    Houghten, R. A. et al. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354, 84–86 (1991).

    CAS  Article  Google Scholar 

  19. 19.

    Lam, K. S. et al. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354, 82–84 (1991).

    CAS  Article  Google Scholar 

  20. 20.

    Reichwein, J. F., Wels, B., Kruijtzer, J. A., Versluis, C. & Liskamp, R. M. Rolling loop scan: an approach featuring ring-closing metathesis for generating libraries of peptides with molecular shapes mimicking bioactive conformations or local folding of peptides and proteins. Angew. Chem. Int. Ed. 38, 3684–3687 (1999).

    CAS  Article  Google Scholar 

  21. 21.

    Liu, J. et al. Inhibition of T cell signaling by immunophilin–ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry 31, 3896–3901 (1992).

    CAS  Article  Google Scholar 

  22. 22.

    Halt, D. A. et al. Design, synthesis and kinetic evaluation of high-affinity FKBP ligands and the X-ray crystal structures of their complexes with FKBP12. J. Am. Chem. Soc. 115, 9925–9938 (1993).

    Article  Google Scholar 

  23. 23.

    Clackson, T. et al. Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl Acad. Sci. USA 95, 10437–10442 (1998).

    CAS  Article  Google Scholar 

  24. 24.

    Sagan, S., Karoyan, P., Lequin, O., Chassaing, G. & Lavielle, S. N- and Calpha-methylation in biologically active peptides: synthesis, structural and functional aspects. Curr. Med. Chem. 11, 2799–2822 (2004).

    CAS  Article  Google Scholar 

  25. 25.

    Ahmed, S. A., Gogal, R. M. Jr & Walsh, J. E. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J. Immunol. Methods 170, 211–224 (1994).

    CAS  Article  Google Scholar 

  26. 26.

    Young, J. D., Yao, S. Y., Baldwin, J. M., Cass, C. E. & Baldwin, S. A. The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. Mol. Aspects Med. 34, 529–547 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Owen, R. P. et al. Functional characterization and haplotype analysis of polymorphisms in the human equilibrative nucleoside transporter, ENT2. Drug Metab. Dispos. 34, 12–15 (2006).

    CAS  Article  Google Scholar 

  28. 28.

    Boswell-Casteel, R. C. & Hays, F. A. Equilibrative nucleoside transporters—a review. Nucleos. Nucleot. Nucl. 36, 7–30 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Xiao, J. C., Zhang, T. P. & Zhao, Y. P. Human equilibrative nucleoside transporter 1 (hENT1) predicts the Asian patient response to gemcitabine-based chemotherapy in pancreatic cancer. Hepato-Gastroenterol. 60, 258–262 (2013).

    Google Scholar 

  30. 30.

    Meijer, L. L., Puik, J. R., Peters, G. J., Kazemier, G. & Giovannetti, E. hENT-1 Expression and localization predict outcome after adjuvant gemcitabine in resected cholangiocarcinoma patients. Oncologist 21, e4 (2016).

    Article  Google Scholar 

  31. 31.

    Jacobson, K. A. & Gao, Z. G. Adenosine receptors as therapeutic targets. Nat. Rev. Drug Discov. 5, 247–264 (2006).

    CAS  Article  Google Scholar 

  32. 32.

    Loffler, M., Morote-Garcia, J. C., Eltzschig, S. A., Coe, I. R. & Eltzschig, H. K. Physiological roles of vascular nucleoside transporters. Arterioscler. Thromb. Vasc. Biol. 27, 1004–1013 (2007).

    Article  Google Scholar 

  33. 33.

    Headrick, J. P. & Lasley, R. D. Adenosine receptors and reperfusion injury of the heart. Handb. Exp. Pharmacol. 2009, 189–214 (2009).

    Article  Google Scholar 

  34. 34.

    Cass, C. E. & Paterson, A. R. Inhibition by nitrobenzylthioinosine of uptake of adenosine, 2′-deoxyadenosine and 9-β-d-arabinofuranosyladenine by human and mouse erythrocytes. Biochem. Pharmacol. 24, 1989–1993 (1975).

    CAS  Article  Google Scholar 

  35. 35.

    Scholtissek, C. Studies on the uptake of nucleic acid precursors into cells in tissue culture. Biochim. Biophys. Acta 158, 435–447 (1968).

    CAS  Article  Google Scholar 

  36. 36.

    Ward, J. L., Sherali, A., Mo, Z. P. & Tse, C. M. Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine. J. Biol. Chem. 275, 8375–8381 (2000).

    CAS  Article  Google Scholar 

  37. 37.

    Rehan, S. & Jaakola, V. P. Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells. Protein Expr. Purif. 114, 99–107 (2015).

    CAS  Article  Google Scholar 

  38. 38.

    Rehan, S., Ashok, Y., Nanekar, R. & Jaakola, V. P. Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1. Biochem. Pharmacol. 98, 681–689 (2015).

    CAS  Article  Google Scholar 

  39. 39.

    Hammond, J. R. Interaction of a series of draflazine analogues with equilibrative nucleoside transporters: species differences and transporter subtype selectivity. Naunyn Schmiedebergs Arch. Pharmacol. 361, 373–382 (2000).

    CAS  Article  Google Scholar 

  40. 40.

    Bierer, B. E. et al. Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc. Natl Acad. Sci. USA 87, 9231–9235 (1990).

    CAS  Article  Google Scholar 

  41. 41.

    Chresta, C. M. et al. AZD8055 is a potent, selective and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 70, 288–298 (2010).

    CAS  Article  Google Scholar 

  42. 42.

    Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. & Schmid, F. X. Cyclophilin and peptidyl-prolyl cis–trans isomerase are probably identical proteins. Nature 337, 476–478 (1989).

    CAS  Article  Google Scholar 

  43. 43.

    Korchynskyi, O. & ten Dijke, P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J. Biol. Chem. 277, 4883–4891 (2002).

    CAS  Article  Google Scholar 

  44. 44.

    Kugimiya, F. et al. Mechanism of osteogenic induction by FK506 via BMP/Smad pathways. Biochem. Biophys. Res. Commun. 338, 872–879 (2005).

    CAS  Article  Google Scholar 

  45. 45.

    Spiekerkoetter, E. et al. FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. J. Clin. Invest. 123, 3600–3613 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    Day, Y. J., Huang, L., Ye, H., Linden, J. & Okusa, M. D. Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages. Am. J. Physiol. Renal Physiol. 288, F722–F731 (2005).

    CAS  Article  Google Scholar 

  47. 47.

    Lappas, C. M., Day, Y. J., Marshall, M. A., Engelhard, V. H. & Linden, J. Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1d-dependent NKT cell activation. J. Exp. Med. 203, 2639–2648 (2006).

    CAS  Article  Google Scholar 

  48. 48.

    Grenz, A. et al. The reno-vascular A2B adenosine receptor protects the kidney from ischemia. PLoS Med. 5, e137 (2008).

    Article  Google Scholar 

  49. 49.

    Liu, M. et al. Acute kidney injury leads to inflammation and functional changes in the brain. J. Am. Soc. Nephrol. 19, 1360–1370 (2008).

    CAS  Article  Google Scholar 

  50. 50.

    Yan, L. & Muller, C. E. Preparation, properties, reactions and adenosine receptor affinities of sulfophenylxanthine nitrophenyl esters: toward the development of sulfonic acid prodrugs with peroral bioavailability. J. Med. Chem. 47, 1031–1043 (2004).

    CAS  Article  Google Scholar 

  51. 51.

    Arai, T., Kouama, Y., Suenaga, T. & Honda, H. Ascomycin, an antifungal antibiotic. J. Antibiot. 15, 231–232 (1962).

    CAS  PubMed  Google Scholar 

  52. 52.

    Hatanaka, H. et al. FR-900520 and FR-900523, novel immunosuppressants isolated from a Streptomyces. II. Fermentation, isolation and physico-chemical and biological characteristics. J. Antibiot. 41, 1592–1601 (1988).

    CAS  Article  Google Scholar 

  53. 53.

    Hasko, G., Linden, J., Cronstein, B. & Pacher, P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat. Rev. Drug Discov. 7, 759–770 (2008).

    CAS  Article  Google Scholar 

  54. 54.

    Vaswani, M., Linda, F. K. & Ramesh, S. Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 85–102 (2003).

    CAS  Article  Google Scholar 

  55. 55.

    Chen, J. F., Eltzschig, H. K. & Fredholm, B. B. Adenosine receptors as drug targets—what are the challenges? Nat. Rev. Drug Discov. 12, 265–286 (2013).

    CAS  Article  Google Scholar 

  56. 56.

    Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

    CAS  Article  Google Scholar 

  57. 57.

    Dazert, E. & Hall, M. N. mTOR signaling in disease. Curr. Opin. Cell Biol. 23, 744–755 (2011).

    CAS  Article  Google Scholar 

  58. 58.

    Liu, J. O. Calmodulin-dependent phosphatase, kinases and transcriptional corepressors involved in T-cell activation. Immunol. Rev. 228, 184–198 (2009).

    CAS  Article  Google Scholar 

  59. 59.

    Schreiber, S. L. & Crabtree, G. R. The mechanism of action of cyclosporin A and FK506. Immunol. Today 13, 136–142 (1992).

    CAS  Article  Google Scholar 

  60. 60.

    Rao, A., Luo, C. & Hogan, P. G. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15, 707–747 (1997).

    CAS  Article  Google Scholar 

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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.

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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.

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Correspondence to Jun O. Liu.

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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.

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Supplementary Figures 1-16, Supplementary Tables 1-5, and Materials and Methods

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Guo, Z., Hong, S.Y., Wang, J. et al. Rapamycin-inspired macrocycles with new target specificity. Nature Chem 11, 254–263 (2019).

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