Small-Molecule Inhibitors and Probes for Ubiquitin- and Ubiquitin- Like-Specific Proteases Anna Borodovsky, [b] Huib Ovaa, [c] Wim J. N. Meester, [a] Emily S. Venanzi, [d] Matthew S. Bogyo, [e] Brian G. Hekking, [a] Hidde L. Ploegh,* [a] Benedikt M. Kessler,* [a] and Herman S. Overkleeft [f] The post-translational modification of proteins with ubiquitin (Ub) or ubiquitin-like (Ubl) modifiers is an important signal in the regulation of a variety of biological processes. The attach- ment of either a single Ub, a 76 amino acid polypeptide, or multiple Ub entities helps to control the targeting of sub- strates for degradation. Processes such as receptor internaliza- tion, trafficking to the lysosomal compartments, and regulation of gene expression are likewise affected by Ub conjugation. [1, 2] Ubiquitin-like modifiers also play a role in protein targeting and in the regulation of protein function. [3] For instance, the small Ubl modifier SUMO, a 97 amino acid polypeptide, func- tions as a nuclear targeting signal, amongst other functions. [4] Nedd8, another polypeptide with homology to Ub, regulates the activity of E3 ligases, thereby influencing the ubiquitylation process. [5, 6] In addition, a family of Ub related proteins, referred to as APGs, has been shown to control autophagy. [7, 8] The reversibility of protein modification with Ub/Ubl resem- bles a phosphorylation/dephosphorylation cycle, and is essen- tial to allowing control over this modification. At least 400 spe- cific Ub ligases, responsible for the attachment of Ub to pro- tein substrates, are thought to exist. [9, 10] The reverse reaction, removal of Ub moieties from substrates, is performed by the large family of Ub-specific proteases, USPs (also referred to as deubiquitinating enzymes, DUBs). [11–13] The attachment and re- moval of Ubl modifiers is carried out by a less numerous set of enzymes. [14, 15] Insight into the enzymatic function of individual DUBs has been obtained for several members, including USP7, [16, 17] USP8, [18, 19] UCH37, [20] USP14, [21, 22] IsoT, [23, 24] USP9, [25] BAP1, [26] UCH-L1, [27, 28] and UCH-L3. [29] However, the physiological role of many proteases of this enzyme class remains largely uncharac- terized. In addition, a high degree of functional redundancy, as inferred from mutation analysis in yeast, complicates the study of the biological roles for individual USPs (Casagrande and Ploegh, unpublished data). Many Ubls have a single conjuga- tion system and only a few deconjugating enzymes have been identified to date for SUMO, Nedd8, ISG15, and APG8. [15] An attractive strategy to further our understanding of pro- teolytic enzymes is the design of selective inhibitors. Indeed, this approach has been successfully applied to the caspase [30] and cathepsin [31] families as well as the individual enzymatic activities of the proteasome. [32] Despite considerable research efforts in recent years, there is still a lack of selective small- molecule synthetic inhibitors that target USPs and Ubl-specific proteases. The first generation of USP and Ubl-specific protease inhibi- tors is based on the entire Ub/Ubl protein itself modified at the C terminus with electrophilic entities capable of reacting with the active-site cysteine thiol, present in most Ub and Ubl- specific proteases. These electrophilic traps include aldehydes (Ubal), [33, 34] nitrile derivatives, [20] Michael acceptors (including vinyl sulfone, vinyl methyl ester), and alkyl halides. [35] Different C-terminally modified Ub moieties allow the profiling of enzyme activity, and show the selectivity of these probes for subsets of the corresponding proteases. [35] To date, only a few examples of small molecules with inhibitory potential toward USPs have been reported. [36–38] However, these examples exhib- it only moderate activity and selectivity. Unlike other proteolyt- ic enzymes, USPs require a considerable portion of the Ub moiety—in addition to the electrophilic trap mimicking the isopeptide linkage—for optimal recognition. [39, 40] Based on the structural similarity between Ub and Ubl proteins, this most likely holds true also for Ubl-specific proteases,. [41] Here we report the synthesis of a panel of peptide vinyl sul- fones harboring various portions of the Ub C terminus. We show that this strategy can be applied also to the synthesis of C-terminal peptide vinyl sulfones corresponding to the Ubl modifiers Nedd8, SUMO 1, FAT10, Fau, and APG12. Depending on their length, such compounds can efficiently target USPs and Ubl-specific proteases. Our synthetic strategy toward C-terminally modified Ub/Ubl peptides is based on the use of Kenner’s safety-catch linker, re- cently revitalized by Ellman and co-workers [42] and applied by us for the synthesis of peptide vinyl sulfone proteasome inhibi- tors. [43, 44] Briefly, standard Fmoc-based solid-phase peptide syn- thesis starting from sulfonamide resin 1 afforded immobilized oligopeptide 2, the N terminus of which is equipped with a biotin moiety, and 5, with a N-terminal, benzyl oxycarbonyl (Z) [a] Dr. W. J. N. Meester, + B. G. Hekking, Dr. H. L. Ploegh, Dr. B. M. Kessler Department of Pathology, Harvard Medical School 77 Avenue Louis Pasteur, Boston, MA 02115 (USA) Fax: (+ 1) 617-432-4775 E-mail : ploegh@hms.harvard.edu bkessler@hms.harvard.edu [b] Dr. A. Borodovsky + Current address: BiogenIdec Inc. 12 Cambridge Center, Cambridge, MA 02142 (USA) [c] Dr. H. Ovaa + Current address: Netherlands Cancer Institute, Division of Cellular Biochemistry Plesmanlaan 121, 1066 CX Amsterdam (The Netherlands) [d] E. S. Venanzi Current address: Section on Immunology and Immunogenetics, Joslin Diabetes Center Department of Medicine, Brigham and Women’s Hospital Harvard Medical School 1 Joslin Place, Boston, MA 02115 (USA) [e] Dr. M. S. Bogyo Current address: Department of Pathology, Stanford Medical School 300 Louis Pasteur Drive, Stanford, California 94305 (USA) [f] Dr. H. S. Overkleeft Current address: Gorlaeus Laboratories, Leiden Institute of Chemistry Einsteinweg 55, 2300 RA Leiden (The Netherlands) [ + ] These authors contributed equally to this work. ChemBioChem 2005, 6, 287 –291 DOI: 10.1002/cbic.200400236  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 287