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Rapamycin-inspired macrocycles with new target specificity

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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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.

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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.

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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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Guo, Z., Hong, S.Y., Wang, J. et al. Rapamycin-inspired macrocycles with new target specificity. Nature Chem 11, 254–263 (2019). https://doi.org/10.1038/s41557-018-0187-4

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