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Published December 5, 2017 | Supplemental Material
Journal Article Open

Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway


The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface patch and the basic/polar residues surrounding it. Ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin receptor and shows strong preference for K48 linkages over K11 and K63. Furthermore, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S proteasome. Finally, ubistatin B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.

Additional Information

© 2017 Elsevier Ltd. Received 21 July 2017, Revised 9 September 2017, Accepted 24 October 2017, Available online 16 November 2017. We thank George H. Lorimer and Arnon Henn for access to their fluorimeters, Ananya Majumdar for help with setting up triple-resonance NMR experiments, Rajesh Singh for the Rub1 sample, Allan Weissman for RCC4 cells, and Konstantin Berlin for useful discussions. The project has been funded in part from the NCI Initiative for Chemical Genetics, NIH, under contract no. N01-CO-12400 to Stuart L. Schreiber, by NIH grants GM065334 to D.F. and GM095755 to D.F. and M.H.G., ISF grant 909-14 to M.H.G., and by NIH grants GM075061 and DK079307 and by Cystic Fibrosis Foundation Therapeutics grant BRODSK13XXO to J.L.B., and a Fulbright postdoctoral fellowship to M.A.N., and utilized NMR instrumentation supported in part by NSF grant DBI1040158 and neutron-scattering facilities supported in part by the NSF under agreement no. DMR-0944772. R.J.D. is a founder and shareholder of Cleave Biosciences, which is developing drugs that target the ubiquitin system. Author Contributions: T.A.L. synthesized ubistatin compounds. Rpn11 functional assays were performed by F.P., T.-F.C., R.J.D.; CFTR assays by J.L.B. and J.L.G.-F.; NMR measurements and analysis by M.A.N., U.K.N., O.W., D.Z., C.M.C., S.M.B., D.F.; SANS studies by C.A.C., S.K., D.F.; structure calculations by O.W. and M.A.N.; fluorescence measurements by M.A.N., W.M., A.T., O.H. and analyzed by D.F.; DUB assays by M.A.N., W.M., O.H., and cellular assays by M.A.N., A.T., and M.H.G. All authors contributed to writing the manuscript and revisions.

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August 21, 2023
October 17, 2023