WO2011161545A2 - Non-hydrolyzable protein conjugates, methods and compositions related thereto - Google Patents

Abstract

The invention provides compositions and methods relating to non-hydrolyzable post- translational protein modifications, such as, for example, non-hydrolyzable ubiquitin and ubiquitin-like protein conjugates. In some embodiments, the invention relates to antibodies, such as linkage-specific antibodies, directed against a protein conjugate of the invention.

Description

TITLE OF THE INVENTION

NON-HYDROLYZABLE PROTEIN CONJUGATES, METHODS AND

COMPOSITION S RELATED THERE TO

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Application Serial No. 61 /351,659 filed June 4, 2010 and U.S. Provisional Application Serial No. 61/353,780, filed June 1 1 , 2010, which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of protein modification and signal transduction. More particularly, the invention provides compositions and methods relating to non- hydrolyzable post-translational protein modifications, such as, for example, non-hydrolyzable ubiquitin and ubiquitin-like protein conjugates.

BACKGROUND OF THE INVENTION

Ubiquitin (Ub) is a conserved 76 amino acid protein that is post-translationally conjugated onto target proteins' by a concerted action of ubiquitinating enzymes termed El, E2 and E3. First, the enzyme El activates Ub, at the expense of ATP, by forming a thioester with the C-terminal glycine residue of Ub. Ub is then transferred to E2/E3 enzymes, which subsequently ligate Ub onto its target substrate. It is the combination of E2/E3 enzymes that dictates the substrate specificity of ubiquitination. The Ub C-terminal carboxylate residue is conjugated to a target protein primarily to the ε-amino group of lysine residues or their N- termini. Since Ub itself contains seven lysines (K6, Kl 1, K27, K29, K33, K48 and K63), Ub polymers of various linkages with different sizes and shapes can be generated² and all possible linkages have been observed in vivo^3, that target distinct substrates for different cellular fates, regulating many cellular processes including proteasomal degradation, cell cycle progression and signal transduction.⁴'⁵ Ubiquitination can be reversed by the action of deubiquitinating enzymes (DUBs),⁶ which hydrolyze the (iso)peptide bond between the C- terminal Gly and acceptor amine moiety. Nearly 100 DUBs are encoded by the human genome but understanding of their function and mechanism of action is still limited. The chain topology, sequence and structure of the ubiquitinated target protein are believed to determine DUB specificity. Several DUBs are known to hydrolyze specific Ub-Ub linkages such as the K48 linked topology , a linkage that targets proteins for proteasomal recognition and ensuing degradation. The K63-linkage has been associated mainly with the regulation of non-proteolytic processes.² Because distinct linkages target proteins for specific cellular fates, it is important to know the determinants that allow DUBs to discriminate between them. Since there are at least a few dozen E2 enzymes and several hundred E3 enzymes, the enzymatic construction of well-defined conjugates is hampered by the lack of available E2 and E3 enzymes. To date, several synthetic methods have been developed to create (native) hydrolyzable²'³ and non-hydrolyzable ubiquitin conjugates.^4,5 As native isopeptide linked substrates are cleavable, they cannot be used for detailed affinity measurements.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

Described herein is a ubiquitin-based tool that enables systematic investigation of linkage selectivity dictated by the local peptide sequence flanking Ub-branched lysine residues in target proteins, a determinant previously described for ubiquitin E3 ligases.⁷ In certain embodiments, the invention provides methods for the facile synthesis of ubiquitin- isopeptide isosteres using copper(I)-catalyzed azide-alkyne cycloaddition chemistry, oxime formation, thioether or selenoether linked conjugates, and/or proteolytically targeted amide bonds replaced by alkyl linkers. In other embodiments, the invention provides reagents for the generation of antibodies and specific inhibitors of deubiquitinating enzymes.

In certain embodiments, the invention provides a ubiquitinated peptide comprising a ubiquitin-peptide isostere, wherein said peptide is synthesized by (a) converting ubiquitin to a protected derivative thereof using an alkylamino group containing a masked aldehyde; (b) generating a terminally conjugated ubiquitin by converting the masked aldehyde to an aldehyde; and (c) converting the aldehyde to an oxime.

In other embodiments, the invention provides a ubiquitinated peptide comprising a ubiquitin-peptide isostere, wherein said peptide is synthesized by (a) converting ubiquitin to a derivative thereof, wherein said derivative contains a terminal acetylene; and (b) generating a conjugated ubiquitin by reacting said acetylene with an azide derivative in order to generate a triazole.

In yet other embodiments, the invention provides a ubiquitinated peptide comprising a ubiquitin-peptide isostere, wherein said peptide is synthesized by (a) converting ubiquitin to a derivative thereof, wherein said derivative contains a terminal alkyl halide; and (b) generating a conjugated ubiquitin by reacting said alkyl halide with a thiol or selenol derivative in order to generate a thio- or seleno-ether containing conjugate.

In some embodiments, a ubiquitinated peptide of the invention comprises an amino acid sequence selected from Table 1 or Table 2.

In certain embodiments, the invention relates to a linkage-specific antibody directed against a ubiqutinated peptide of the invention. In some embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody reacts with a peptide conjugate comprising a native, hydrolyzable peptide bond.

In other embodiments, the invention relates to a method employing a ubiquitinated peptide of the invention in the generation of an antibody. In certain embodiments, the antibody generated is a polyclonal antibody. In other embodiments, the antibody generated is a monoclonal antibody.

In yet other embodiments, the invention relates to a method employing a ubiquitinated peptide of the invention in the selection of an antibody.

In some embodiments, a ubiquitinated peptide of the invention comprises an amino acid

sequence selected from Table 1. In certain embodiments, the peptide comprises the amino acid sequence QRLIFAG4QLEDGR, wherein 4 is azidonorvaline. In certain embodiments, the peptide comprises the amino acid sequence LSDYNIQ4ESTLHL, wherein 4 is azidonorvaline. In some embodiments, the peptide comprises an amino acid sequence selected from Table 2. In a particular embodiment, the peptide comprises the amino acid sequence EGTKAVTKYTSSK.

In yet other embodiments, the invention relates to a host cell for the production of an antibody according to the invention. In a particular embodiment, the host cell is a hybridoma cell.

In some embodiments, the invention provides a ubiquitinated peptide that inhibits the activity of at least one deubiquitinating enzyme. In certain embodiments, the at least one deubiquitinating enzyme is USP2a, USP4, USP7, USP16, or USP21.

In some embodiments, the invention provides a method employing a ubiquitinated peptide to determine the affinity of one or more deubiquitinating enzymes for lysine topoisomer mimics.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic of oxime-linked ubiquitinated peptides (D) as non- hydrolyzable Ub branched target sequences.

Figure 2 is a schematic overview of (A) native ubiquitination using the El, E2 and E3-enzyme cascade, and (B) Method using click chemistry as described herein. /^') Trypsin- mediated transpeptidation, pH = 8. ii) CuBr / tris(triazolyl) amine ligand, pH = 8.5 (Zhou et al. J. Am. Chem. Soc, 2004, 126, 8862).

Figure 3 depicts (A) total ion current (TIC) of purified ubiquitin-PA, (B) spectrum of Ubiquitin-PA peak, (C) deconvoluted mass of Ub-PA using Maxentl, (D) TIC of purified Ub-click-peptide 15 conjugate; (E) spectrum of Ub-click-peptide conjugate (F) deconvoluted mass of Ub-click-peptide conjugate using Maxentl .

Figure 4 depicts (A, C) ES+-MS and (B, D) deconvoluted MS spectrum of Ub-K48 and Ub-K48 isopeptide isosteres (biotinylated), respectively.

Figure 5 depicts a Ub-K48 isopeptide isostere resistant to hydrolysis by USP2a CD which can cleave K48 linked di-Ub.

Figure 6 depicts the specific inhibition of DUBs (USP7 and USP16) by Ub isopeptide isosteres.

Figure 7 depicts (A) the mass spectrum and (B) the deconvoluted mass spectrum of normal isotopic abundance corresponding to a Ub-K561 FANCD2 isopeptide isostere.

Figure 8 depicts binding of USP7 CD to Ub isopeptide isosteres. SPR response curves for the binding of USP7 CD (concentrations ranging from 0.39 to 50 μΜ, bottom curve to top curve respectively) to (A) biotinlylated Ub-K48 isopeptide isostere, no interaction was observed for the K63 isostere (B) The maximum RUs for every curve is plotted against the corresponding concentrations of USP7 CD used for binding measurements. Employing nonlinear regression analysis on the plotted curves, the K_Ds for binding of USP7 CD to rescpective Ub isopeptide isosteres are calculated.

Figure 9 depicts binding of USP4 CD to Ub isopeptide isosteres. SPR response curves for the binding of USP4 CD (concentrations ranging from 0.15 to 12.1 μΜ, bottom curve to top curve respectively) to (A) biotinylated Ub, (B) biotinylated Ub-K48 isopeptide isostere and (C) biotinylated Ub-K63 isopeptide isostere, all immobilized to a SA chip on separate lanes. (D) The maximum RUs for every curve in figure 9A, 9B and 9C are plotted against the corresponding concentrations of USP4 CD used for binding measurements. Employing nonlinear regression analysis on the plotted curves in D, the K_Ds for binding of USP4 CD to Ub and Ub isopeptide isosteres are calculated. Figure 10 depicts binding of USP21 CD to Ub isopeptide isosteres. SPR response curves for the binding of USP4 CD (concentrations ranging from 0.01 to 24.3 μΜ, bottom curve to top curve respectively) to (A) biotinylated Ub, (B) biotinylated Ub-K48 isopeptide isostere and (C) biotinylated Ub-K63 isopeptide isostere, all immobilized to a SA chip on separate lanes. (D) The maximum RUs for every curve in figure 10A, 10B and IOC are plotted against the corresponding concentrations of USP21 CD used for binding

measurements. Employing non-linear regression analysis on the plotted curves in D, the Kds for binding of USP21 CD to Ub and Ub isopeptide isosteres are calculated.

DETAILED DESCRIPTION

Ubiquitin conjugation or ubiquitination is a process, occurring naturally in all eukaryotes, that has been implicated in a wide variety of critical cellular processes including protein stability, cell cycle progression, transcriptional control, receptor transport and the immune response. Ubiquitin and ubiquitin-like proteins are ~ 100 amino acid polypeptides. To date, approximately 12 different ubiquitin like proteins have been identified, including NEDD8, ISG15, FUB1, FAT 10, UBL5, SUMO-1, SUMO-2, SUMO-3, UFM1, MLP3A- LC3, ATG12 and URMl . As described herein, ligation of certain peptides with ubiquitin and ubiquitin-like proteins is disclosed (see, e.g., Jeram et al Proteomics 2009, 922-934, incorporated herein by reference, which discusses the function and structure of ubquitin and ubquitin-like proteins).

Applicants linked Ub in a non-hydrolyzable and isosteric manner with native Ub conjugates through an oxime linkage (D, Figure 1). Oxime formation is a chemoselective condensation reaction between an aminoxy and aldehyde moiety,⁹ yielding a linkage that is stable under physiological conditions.^10,11 Formation of the oxime linkage may be performed under a variety of conditions available to the ordinarily skilled artisan, including, by way of non-limiting example, HC1 or other acids. Other methods of oxime formation may also be used such as, but not limited to, those described in Larock: Comprehensive Organic

Transformations, VCH Publishers, New York, 1989.

As described herein, Ub is functionalized at the C-terminus with an aldehyde (B, Figure 1 ), that can be generated in situ from an acetal (A, Figure 1), and subsequently ligated with an aminoxy modified peptide (C, Figure 1). It will be noted that the protecting group for the aldehyde is not limited to diethyl acetal, and may be selected from any variety of suitable protecting groups for masking aldehydes. Suitable acetals, for example, may include alkyl, benzylidene or the like. The spacer between the peptide chain and Ub lacks the scissile isopeptide bond (D, Figure 1 ) and is designed to isosterically mimic the native isopeptide linkage (E, Figure 1). Structural studies of native poly-Ub chains have revealed that the isopeptide linkage region is disordered,¹²' ¹³ and it is therefore not likely that the introduction of an oxime bond will change spatial demands. Using solid phase peptide synthesis (SPPS), Applicants synthesized peptides (C, Figure 1) in which lysine residues targeted for ubiquitination were replaced by an (aminoxy acetyl)-L-diaminopropionic acid (hereafter termed X). Next, Applicants generated the required aldehyde derivative of Ub (B, Figure 1) masked as a diethyl acetal (A, Figure 1). This acetal is converted into an aldehyde in situ by treatment with aqueous HCl. The Ub diethyl acetal (A, Figure 1) is prepared conveniently by reversed trypsinolysis of Ub and aminobutyraldehyde diethyl acetal (Figure I).¹⁴ Thus, when Ub was incubated at 37°C (pH 7.5) with trypsin and 25% aq. aminobutyraldehyde diethyl acetal, over 50% conversion of Ub into A was observed within 3 hours. Following workup and purification by cation-exchange chromatography, A was isolated in >95% purity and 30% overall yield under optimized conditions.

Click chemistry also provides a straightforward way to create non-hydrolyzable ubiquitin-peptide isopeptide isosteres. Click chemistry is a modular approach to chemical synthesis that utilizes only the most practical and reliable chemical transformations. Click chemistry techniques are described, for example, in the following references, which are incorporated herein by reference in their entirety: Kolb, H. C, Finn, M. G., Sharpless, . B. Angewandte Chemie, International Edition 2001, 40, 2004; Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. Angew. Chem. Int. Ed. Engl. 2002, 41, 2596-2599; Tornoe, C. W., Christensen, C. Meldal, M. J. Org. Chem. 2002, 67, 3057; Lewis, W. G., Green, L. G, Grynszpan, F., Radic, Z., Carlier, P. R., Taylor, P., Finn, M. G., Barry, K. Angew. Chem. Int. Ed. 2002, 41, 1053; Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless, K. B., Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192: Lee, L. V., Mitchell, M. L., Huang, S.-J., Fokin, V. V., Sharpless, K. B., Wong, C.-H. J. Am. Chem. Soc. 2003, 125, 9588; Kolb, H. C, Sharpless, K. B. Drag Discovery Today 2003, 8, 1128; Manetsch, R., Krasinski, A., Radic, Z„ Raushel, J., Taylor, P, Sharpless, K. B., Kolb, H. C. J. Am. Chem. Soc. 2004, 126, 12809; Mocharla, V. P., Colasson, B., Lee, L. V., Roeper, S., Sharpless, K. B., Wong, C.-H., Kolb, H. C. Angew. Chem. Int. Ed. 2005, 44, 1 16; Whiting, M., Muldoon, J., Lin, Y.C., Silverman, S. M., Lindstrom, W., Olson, A. J., Kolb, H. C, Finn, M. G., Sharpless, K. B, Elder, J. H., Fokin, V. V. Angew. Chem. Int. Ed. Engl. 2006, 45, 1435; and Meldal, M., Tornoe, C. W. Chem. Rev. 2008, 108, 2952.

Although other click chemistry functional groups may be utilized, such as those described in the above references, the use of cycloaddition reactions is preferred, particularly the reaction of azides with alkynyl groups. Alkynes, such as terminal alkynes, and azides undergo 1,3- dipolar cycloaddition forming 1 ,4-disubstituted 1,2,3-triazoles. Alternatively, a 1,5- disubstituted 1 ,2,3-triazole can be formed using azide and alkynyl reagents (Krasinski, A., Fokin, V.V., Sharpless, K.B. Org. Lett. 2004, 6, 1237 and (Tam, A., Arnold, U., Soellner, M.B., Raines, R.T. J. Am.. Chem. Soc. 2007, 129, 12670).

Hetero-Diels-Alder reactions or 1,3-dipolar cycloaddition reactions could also be used (see Huisgen 1,3-Dipolar Cycloaddition Chemistry (Vol. 1) (Padwa, A., ed.), pp. 1 -176, Wiley; Jorgensen Angew. Chem. Int. Ed. Engl. 2000, 39, 3558-3588; Tietze, L.F. and Kettschau, G. Top. Curr. Chem. 1997, 189, 1-120).

Incorporation of azide and alkynyl moieties into amino acids or amino acid derivatives may be performed via a variety of methods known to the ordinarily skilled artisan. In addition to the examples and methods described herein, azide and alkynyl functionality may also be incorporated via methods such as, by way of non-limiting example, those described in Kiick, K.L., Saxon, E., Tirrell, D.A., Bertozzi, C.R. PNAS, USA 2002, 99, 19 and Chin, J.W., Santoro, S.W., Martin, A.B., King, D.S., Wang, L., Schultz, P.G. J. Am. Chem.. Soc. 2002, 124, 9026; Dieters, A., Schultz, P.G. Bioorg. Med. Chem. Lett. 2005, 15, 1521 .

In one embodiment, an exemplary reaction that has proven useful in this regard is the Sharpless modified Huisgen cyclization of an alkyne and an azide. In the presence of catalytic amounts of Cu(I), for example, this cyclization occurs chemoselectively to produce a 1 ,4-disubstituted triazole moiety. Cross-reactivity with common biological functional groups is not seen for either the alkyne or the azide, and both groups are stable under biological conditions. Note that the 1, 5 -di substituted regioisomer is also useful as a proteolytically stable amide isostere. Thus, the instant invention provides a practical click chemistry-based Ub-peptide ligation methodology. In certain embodiments, these conjugates can be used for screening of specific DUBs as well as binding to ubiquitinated substrate peptides.

The instant invention also provides a practical oxime-based Ub-peptide ligation methodology. Ub-isopeptide isosteres obtained according to the invention can be used to determine DUB affinities for lysine topoisomer mimics. For example, as described herein, Ub-isopeptide isosteres were used to determine DUB affinities for K48 and K63 topoisomer mimics. Applicants then determined that the sequences flanking the Ub conjugation site have a dramatic and direct effect on the affinity for DUBs. In still further embodiments, the instant invention provides Ub-peptide ligation methodology incorporating thioether or selenoether linked conjugates, and/or proteolytically targeted amide bonds replaced by alkyl linkers.

The generated proteolytically stable Ub-isopeptides of the invention are useful for a number of purposes. For example, Applicants show that the sequence surrounding the isopeptide linkage is an important determinant for substrate recognition by DUBs. In certain embodiments, proteolytically stable Ub-isopeptides of the invention can act as specific DUB inhibitors,¹⁸ which may help in the understanding of DUB-specific biochemical events. In other embodiments, as stable non-hydrolyzable ligands they can freeze DUBs in molecular transition states, capturing them in action following substrate binding but before cleavage in crystallographic studies. This will aid in understanding mechanistic aspects of DUB action. In yet other embodiments, proteolytically-stable Ub-isopeptide conjugates are useful as affinity supports for mass spectrometry-based proteomics approaches and for the generation of linkage-specific antibodies that recognize specific Ub modifications in a target protein or Ub polymers in a linkage-specific manner.

In still other embodiments, the invention further concerns methods of synthesizing and modifying selected Ub-peptides to obtain modified ubiquitin isosteres.

The term "amine protecting group" refers to any organic moiety which is readily attached to an amine nitrogen atom, which, when bound to the amine nitrogen, renders the resulting protected amine group inert to the reaction conditions to be conducted on other portions of the compound and which, at the appropriate time, can be removed to regenerate the amine group. Examples of such amine protecting groups are known to one of ordinary skill in the art and include, but are not limited to: acyl types such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; aromatic carbamate types such as benzyloxycarbonyl (Cbz) and substituted benzyloxy-carbonyls, l -(p-biphenyl)-l-methylethoxy-carbonyl, and 9- fluorenylmethyloxycarbonyl (Fmoc); aliphatic carbamate types such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; alkyl types such as trityl and benzyl; trialkylsilane such as trimethylsilane; and thiol containing types such as phenylthiocarbonyl and dithiasuccinoyl. Preferably the amine protecting group in accordance with the present invention is selected from the group consisting of Cbz; p- methoxybenzyl carbonyl; Boc; Fmoc; Benzyl; /?-methoxybenzyl; 3,4-dimethoxybenzyl; p- methoxy phenyl; tosyl; sulfonamides; allyloxycarbonyl; trityl and methoxytrityl, all of which are well-known in the art. Due to their wide-spread application, the exact chemistry involved in the use of these groups in peptide synthesis has been described extensively and is part of the skilled person's common general knowledge. Amine protecting groups and protected amine groups are described in, e.g., C. B. Reese and E. Haslam, "Protective Groups in Organic Chemistry," J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4, respectively, and T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis," Second Edition, John Wiley and Sons, New York, N.Y., 1991, Chapters 2 and 3.

The procedures described herein may, by way of non-limiting example, be employed for the preparation of the compound s of the present invention. The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well known to a person of ordinary skill in the art, following procedures described in such references including, but not limited to Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991 ; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions, vols. 1 -40, John Wiley and Sons, New York, N.Y., 1991 ; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock: Comprehensive Organic

Transformations, VCH Publishers, New York, 1989.

Standard organic chemical reactions may be achieved using a number of different reagents including, but not limited to those described in Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

Within the context of the present invention, the meaning of the term "ligand" as such will be clear to one of ordinary skill in the art. In particular, since the present invention aims at providing a new method of chemo- and site- selective modification of proteins or peptides it will be clear that said term is meant to include e.g., any agent the conjugation of which to a selected peptide or protein has been described or suggested in the art for a certain purpose. The introduction of the ligand typically introduces or affects a particular functionality of said peptide or protein (which may therefore also be referred to herein as "functional agent" or the like). Particularly preferred examples of such ligands or functional agents include dyes, probes, labels, tags, solubility-modifying agents, enzyme targets, receptor ligands, immunomodulatory agents, co-factors, and cross-linking agents. In one embodiment the ligand is a therapeutic agent. In certain embodiments, the ligand is ubiquitin or a ubiquitin- like protein. The present invention encompasses (the use of) any isomerically pure compound as well as any racemic or non-racemic mixture. In embodiments involving application of a lysine compound of the invention, the natural L-amino acid configuration is particularly preferred. Thus, an embodiment of the invention concerns a lysine derivative as defined herein wherein the a-carbon atom has the L-configuration.

In one embodiment, the compounds of the invention, or a pharmaceutically acceptable salt thereof, may exist in the form of a single stereoisomer or mixture of stereoisomers thereof.

In another embodiment, the compounds of the invention, or a pharmaceutically acceptable salt thereof, may exist in the form of a mixture of stereoisomers.

Unless specifically noted otherwise herein, the definitions of the terms used are standard definitions used in the art of organic synthesis and pharmaceutical sciences.

Exemplary embodiments, aspects and variations are illustrated in the figures and drawings, and it is intended that the embodiments, aspects and variations, and the figures and drawings disclosed herein are to be considered illustrative and not limiting.

Peptide fragments, peptide substrates, or derivatives thereof may be synthesized by methods known to the skilled artisan, non-limiting examples of such are solution-phase chemistry and solid-phase chemistry incorporating a resin. Additionally, automated peptide synthesis may also be used. Various techniques for the synthesis of peptides are described, for example, by Lloyd- Williams, P., Albericio, F., and Girald, E. in "Chemical Approaches to the Synthesis of Peptides and Proteins," CRC Press, 1997; also see Barany, et al, Int. J. Pep. Prot. Res., 1987, 30, 705.

A functional variant of the naturally occurring polypeptide or protein may differ from said naturally occurring polypeptide or protein by minor modifications, such as, for example, substitutions, insertions, deletions, additional N- or C- terminal amino acids, and/or additional chemical moieties, but maintains the basic polypeptide and side chain structure of the naturally occurring form as well as the ability to elicit a certain biological function or activity in vitro and/or in vivo. Typically, when optimally aligned, such as by the programs GAP or BESTFIT using default parameters, a naturally occurring polypeptide and a homologue thereof share at least a certain percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 8 and gap extension penalty = 2. For proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, PNAS 1992, 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA.

Alternatively percent similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc. A "functional variant thereof herein is understood to comprise a polypeptide or protein having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99% amino acid sequence identity with the selected naturally occurring polypeptide of interest mentioned above and is still capable, under the proper conditions, of eliciting its normal functions to a significant extent.

As will be understood by one of ordinary skill in the art, modifications that may in particular be employed in accordance with the invention comprise inclusion of further ligation handles, such as those in accordance with the present invention.

Chemical peptide synthesis methods are well known to the person ordinarily skilled in the art. In accordance with the present invention the peptides are typically chemically synthesized, preferably using solid phase synthesis. In accordance with the present invention, it is not critical whether the entire sequence is synthesized through stepwise elongation only or whether the process involves ligation of two or more separately obtained fragments.

Typically, if the sequence length exceeds 100 amino acids, it may be preferable to produce separate fragments and ligate them through processes known as fragment condensation and/or chemical ligation. As will be explained in more detail hereafter, the present invention also provides a particularly advantageous chemical ligation process for the purpose of peptide synthesis. Alternatively, embodiments involving a compound as presented herein may be incorporated into a protein using orthogonal tRNA/aminoacyl-tRNA synthetase pairs, which incorporates the unnatural building block in response to a nonsense or four-base codon in the gene of the protein of interest. To date, this technology has allowed the incorporation of approximately 50 unnatural amino acids (see Xie, J. M., Schultz, P. G. Nat. Rev. Mol. Cell Biol. 2006, 7, 775).

The synthesis of the peptide is preferably performed on a solid phase substrate, yielding a peptide that is covalently attached to said substrate. One or more of the subsequent steps of the present method may be performed before or after release of the peptide from said solid phase substrate. In one embodiment of the invention, conjugation of a ubiquitin or ubiquitin-like protein to the ligation building block is performed on the solid phase as well. This strategy allows for the modified protein to be obtained directly in high purity, potentially rendering any or all further purification steps superfluous. Alternatively, the method of the invention may concern solid phase synthesis without release from the solid phase substrate prior to or after the subsequent steps of the invention, e.g., in the case of protein microarrays, where the protein can be synthesized directly on the microarray surface.

A further aspect of the invention concerns the use of the peptides or proteins of the invention as a therapeutic or diagnostic agent. Chemo- and site-selectively modified peptides may find applications in diagnostic and therapeutic methods. For example, endogenous proteins and/or peptides labelled with certain tags, probes or markers may provide useful tools as diagnostic agents. Furthermore, proteins currently known as (potential) therapeutic or diagnostic agents may be modified in accordance with the invention, such as to improve their clinical performance, e.g., by including solubility modifying ligands. Peptidic as well as non- peptidic therapeutic or diagnostic agents may be ligated to a specific protein as the ligand, using the technique of the present invention for targeting of said agent to a specific site of action.

In yet other embodiments, proteolytically stable ubiquitin- and ubiquitin-like- isopeptide conjugates are employed in the generation of linkage-specific antibodies, which recognize specific ubiquitin and/or ubiquitin-like modifications in a target protein or ubiquitin polymers in a linkage-specific manner.

Linkage-specific antibodies of the invention encompass antibodies that recognize epitopes on both the target peptide and the linked moiety (e.g., ubiquitin, SUMO). In certain embodiments, linkage-specific antibodies of the invention do not react or only weakly react with either the unmodified target peptide or with the linked moiety alone, such as ubiquitin or a ubiquitin-like peptide. In certain embodiments, the linkage-specific antibodies of the invention do not recognize the non-hydrolyzable bond itself in a non-hydrolyzable peptide conjugate of the invention. Typically, the linkage-specific antibodies of the invention will react with peptide conjugates that have a native, hydrolyzable isopeptide bond.

The basic immunoglobulin (Ig) structural unit in vertebrate systems is composed of two identical light ("L") polypeptide chains (approximately 23 kDa), and two identical heavy ("H") chains (approximately 53 to 70 kDa). The four chains are joined by disulfide bonds in a "Y" configuration. At the base of the Y, the two H chains are bound by covalent disulfide linkages.

The L and H chains are each composed of a variable (V) region at the N-terminus, and a constant (C) region at the C-terminus. In the L chain, the V region (termed "V_LJL") is composed of a V (V_T.) region connected through the joining (JL) region to the C region (CL). In the H chain, the V region (VHPHJH) is composed of a variable (V_H) region linked through a combination of the diversity (DH) region and the joining (¾) region to the C region (CH). The VJL and VHDHJH regions of the L and H chains, respectively, are associated at the tips of the Y to form the antigen binding portion and determine antigen binding specificity.

The (CH) region defines the isotype, i.e., the class or subclass of antibody. Antibodies of different isotypes differ significantly in their effector functions, such as the ability to activate complement, bind to specific receptors (e.g., Fc receptors) present on a wide variety of cell types, cross mucosal and placental barriers, and form polymers of the basic four-chain IgG molecule.

Antibodies are categorized into "classes" according to the CH type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, or IgA). There are at least five types of C_H genes (CT, CK, CA, CM, and CI), and some species have multiple CH subtypes (e.g., CKi, CK₂, CK₃, and CK₄, in humans). There are a total of nine C_H genes in the haploid genome of humans, eight in mouse and rat, and several fewer in many other species. In contrast, there are normally only two types of L chain C regions (C_L), kappa (P) and lambda (∑), and only one of these C regions is present in a single L chain protein (i.e., there is only one possible L chain C region for every VLJL produced). Each i i chain class can be associated with either of the L chain classes (e.g., a C_HK region can be present in the same antibody as either a P or∑ L chain), although the C regions of the H and L chains within a particular class do not vary with antigen specificity (e.g., an IgG antibody always has a CK H chain C region regardless of the antigen specificity).

Each of the V, D, J, and C regions of the H and L chains are encoded by distinct genomic sequences. Antibody diversity is generated by recombination between the different VH, DH, and JH, gene segments in the H chain, and VL and JL gene segments in the L chain. The recombination of the different V_H, D_H, and hi genes is accomplished by DNA recombination during B cell differentiation. Briefly, the H chain sequence recombines first to generate a D_HJH complex, and then a second recombinatorial event produces a V_HD_HJH complex. A functional H chain is produced upon transcription followed by splicing of the RNA transcript. Production of a functional H chain triggers recombination in the L chain sequences to produce a rearranged Vi region which in turn forms a functional V_LJ_LCL region, i.e., the functional L chain.

The value and potential of antibodies as diagnostic and therapeutic reagents has been long-recognized in the art. Unfortunately, the field has been hampered by the slow, tedious processes required to produce large quantities of an antibody of a desired specificity. The classical cell fusion techniques allowed for efficient production of Mabs by fusing the B cell producing the antibody with an immortalized cell line. The resulting cell line is a hybridoma cell line.

The term "antibody," as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target, and includes Fv, ScFv, Fab' and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR- grafted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such as Fv and ScFv, possess

advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution. Fab fragments retain an entire light chain, as well as one-half of a heavy chain, with both chains covalently linked by the carboxy terminal disulfide bond. Fab fragments are monovalent with respect to the antigen-binding site.

Antibody fragments such as, for example, Fab, F(ab')2, Fv and scFv, retain some ability to selectively bind with its antigen or receptor and include, for example:

(i) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(ii) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(iii) F(ab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(iv) scFv, including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.

General methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference). Antibodies of the invention can be obtained by any method known in the art. They are most conveniently obtained from hybridoma cells engineered to express an antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an

immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition, e.g., Pristane.

Another method of obtaining antibodies is to immunize suitable host animals with an antigen and to follow standard procedures for polyclonal or monoclonal production.

Antibodies thus produced can be humanized by methods known in the art. Examples of humanized antibodies are provided, for instance, in United States Patent Nos. 5,530, 101 and 5,585,089.

"Humanized" antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. In one version, the heavy chain and light chain C regions are replaced with human sequence. Tn another version, the complementarity-determining regions (CDR) comprise amino acid sequences for recognition of antigen of interest, while the variable framework regions have also been converted to human sequences. See, for example, EP 0329400. In a third version, variable regions are humanized by designing consensus sequences of human and mouse variable regions, and converting residues outside the CDR that are different between the consensus sequences.

Antibodies described herein may be used for the detection of the relevant protein, for example, within the context of a cell. Accordingly, they may be altered antibodies comprising an effector protein such as a label. For example, in certain embodiments, the label is one that allows the imaging of the distribution of the antibody in vivo or in vitro. Such labels may be radioactive labels or radio-opaque labels, such as metal particles, which are readily visualizable within an embryo or a cell mass. Moreover, they may be fluorescent labels or other labels that are visualizable on tissue samples.

Recombinant genetic techniques have allowed cloning and expression of antibodies, functional fragments thereof and the antigens recognized. These engineered antibodies provide novel methods of production and treatment modalities. For instance, functional immunoglobulin fragments have been expressed in bacteria and transgenic tobacco seeds and plants. Skerra Curr. Opin. Immunol. 1993, 5, 256; Fiedler and Conrad Bio/Technology 1995, 73, 1090; Zhang et al. (1993) Cancer Res. 1993, 55, 3384; Ma et al. Science 1995, 268, 916; and, for a review of synthetic antibodies, see Barbas Nature Med. 1995, 7, 836. In addition, a phage display approach was used to engineer linkage-specific antibodies that recognize certain polyubiquitin chains (Newton, K. et al. Cell 2008, 134, 668).

Recombinant DNA technology may be used to modify the antibodies as described herein. For example, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimized by humanizing the antibodies by CDR grafting (see, e.g., European Patent Application 0 239 400 (Winter) and, optionally, framework modification (see, e.g., EP 0 239 400).

Antibodies of the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. In certain embodiments, the selected cell culture system secretes the antibody product.

In certain embodiments, the invention relates to a process for the production of an antibody comprising culturing a host, e.g., E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said antibody protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example

Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. fetal calf serum, or trace elements and growth sustaining supplements, e.g., feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells that are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example, for bacteria, in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast, in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g., in hollow fibers, microcapsules, on agarose microbeads, or ceramic cartridges.

Large quantities of the antibodies of the invention can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fiision of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneal ly into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, Nature 1975, 256, 495; US 4,376, 110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies, for example by immunoblotting, by an enzyme immunoassay, e.g., a sandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g., by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example, gel filtration, ion-exchange

chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g., affinity chromatography with a non-hydrolyzable protein conjugate of the invention, or with Protein-A. Hybridoma cells secreting the monoclonal antibodies are also provided. Preferred hybridoma cells are genetically stable, secrete monoclonal antibodies of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.

Also included is a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to a non-hydrolyzable protein conjugate of the invention, characterized in that a suitable mammal, for example a Balb/c mouse, is immunized with a one or more non-hydrolyzable protein conjugates, or antigenic fragments thereof; antibody- producing cells of the immunized mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells of Balb/c mice immunized with a non- hydrolyzable protein conjugate of the invention are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Agl4, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.

In certain embodiments, the invention provides a process for the preparation of a hybridoma cell line, characterized in that Balb/c mice are immunized by injecting subcutaneously and/or intraperitoneal ly between 10 and 10 and 10 cells expressing a non- hydrolyzable protein conjugate of the invention and a suitable adjuvant several times, e.g., four to six times, over several months, e.g., between two and four months, and spleen cells from the immunized mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. In certain embodiments, the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunized mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000. After the fusion, the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.

Recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to a non-hydrolyzable protein conjugate as described hereinbefore are also disclosed. Such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of

complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to a non-hydrolyzable protein conjugate of the invention can be enzymatically or chemically synthesized DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. In certain embodiments, these modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody. In some embodiments, the mutant DNA is a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence is also a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain variable domain and/or a light chain variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitro

mutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example, after incorporation into hybrid vectors.

Also disclosed are recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed to a non-hydrolyzable protein conjugate of the invention fused to a human constant domain g, for example γΐ , γ2, γ3 or γ4. Likewise recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to a non-hydrolyzable protein conjugate of the invention fused to a human constant domain κ or λ are also disclosed.

In another embodiment, the invention relates to recombinant DNAs coding for a recombinant polypeptide wherein the heavy chain variable domain and the light chain variable domain are linked by way of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a cleavage site and/or a peptide spacer and/or an effector molecule.

In some embodiments, the DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications. Thus, effector molecules that are toxins or enzymes, especially enzymes capable of catalyzing the activation of prodrugs, are particularly indicated. The DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.

Polypeptides and other compounds may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

Peptides and polypeptides, such as the antibodies described herein, nucleic acids and polynucleotides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulations should suit the mode of administration, and are well within the skill of the art.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The invention will now be further described by way of the following non-limiting examples.

EXAMPLE 1

Described herein is the use of the Cul-catalyzed Azide-Alkyne Cycloaddition (CuAAC)¹⁹'²⁰ for the construction of well-defined ubiquitin-peptide isopeptide isosteres. The CuAAC is a powerful ligation-method for a number of reasons. It has been proven to be compatible²¹ with proteins. Importantly, the [l,2,3]-triazole linkage is an effective amide surrogate²² that is resistant to DUB mediated cleavage. Finally, CuAAC reactions are known for their high reaction rates and yields. Furthermore, it has recently been shown that ubiquitin like proteins can be conjugated onto other proteins using click chemistry resulting in non- hydrolyzable conjugates²³.Applicants approach (Figure 2-B) begins with the Fmoc-based Solid Phase Peptide Synthesis (SPPS) of receiving end peptides in which an azidonorvaline residue (Figure 2, 4) is incorporated at the lysine conjugation site. Next, a Ub-click partner is constructed by replacing the C-terminal glycine of Ub (Figure 2, 1) with a propargylamine group (Figure 2, 2) , resulting in Ub-PA (Figure 2, 3). This C-terminal modification was introduced by trypsin-catalyzed transpeptidation¹⁴. Here trypsin reacts with the amide bond between Arg74 and Gly75 of Ub, thereby forming a reactive Ub74-trypsin intermediate. Subsequently, 4-amino-N-(prop-4-ynyl)acetamide 2 (Figure 2) reacts with this intermediate resulting in the desired transpeptidation product 3 (Figure 2). Alternatively, Ub-PA 3 can be synthesized by treating Ub(l-75)-thioester with propargylamine.

In a typical CuAAC reaction, Ub-PA (45 L, 1 50 μΜ in H₂0) is allowed to react with 10 eq. of peptide (6.75 10 mJVl in DMSO). The pH is adjusted to 8.5 by adding phosphate buffer (36.4 iL, 600 mM). If Ub-PA obtained by reversed trypsinolysis is used, trypsin inhibitor is added to the reaction mixture. Either phenylmethylsulfonyl fluoride (0.4 μΐν, 400 mM in 2-propanol) or trypsin inhibitor from soybean (1 mg/niL in H₂0) was used. The reaction is started by the addition of a freshly prepared equimolar solution of CuBr and Cu(I) stabilizing tris-triazole ligand²⁴ (2x2 μΐ, 57 μΜ in CH3CN). After 15 min., LC-ESI-MS analysis revealed that all reactions had proceeded to completion, as judged by the absence of starting material and formation of ligated product. The reaction mixture is then treated with 0.5 M of ethylenediaminetetraacetic acid (EDTA), which chelates the formed Cu(II) and prevents Ub aggregation²⁵. Next, the Cu(l/II) and ligand are removed by centrifugal filtration with 3kDa cut-off spin columns. In some cases this also results in removal of excess peptide. After washing the conjugates with additional 0.5M EDTA, excess EDTA is removed by washing 2* w ith water (Figure 3). If the sample purity was not sufficient, conjugates were purified by cation-exchange chromatography.

To assess the viability of the method, peptide sequences surrounding known ubiquitination sites were synthesized with lysine residue of interest replaced with

azidonorvaline building block 4 (Figure 2). Peptides were subsequently ubiquitinated using CuAAC and analyzed. Examples of peptide-Ub conjugates successfully synthesized according to the invention are provided in Table 1. Figure 3 depicts an example of a successful conjugation, with deconvoluted mass spectra of starting material and end product.

Materials and Methods. All chemicals were obtained from Sigma-Aldrich, unless otherwise noted.

CopperBromide was obtained in the highest quality available from Sigma-aldrich (99.999% trace metals basis). The triazole-based ligand used in reactions was synthesized according to a procedure by Fahmi et al J. Am. Chem. Soc. 2004, 126, 8862-8863), (S-(o-nitrobenzyl) cysteine was obtained according to method by Wang et al (J. Org. Chem. 1977, 42, 1286- 1290).

LC-MS analysis was performed on a system equipped with a Waters 2795 Seperation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190-750 nm), Waters Symmetry300™ C₄ 3.5 μΜ (2.1x100 mm) and LCT™ Orthogonal Acceleration Time of Flight Mass Spectrometer. Samples were run at 0.40 ml/min using 2 mobile phases: A= 0.1 % aq. formic acid and B= 0.1 % formic acid in CH₃CN. A linear gradient from 5% to 95% of B was used to elute Ubiquitin and its derivatives. Data acquisition and processing was performed using Waters MassLynx Mass Spectrometry Software 4.1.

Preparative HPLC

Shimadzu LC-20AD/T using a Q Vydac column (Grace Davison Discovery Sciences™), Ι Ομιη 10x250 mm. Mobile phases: A= 0.05% aq. TFA and B= 0.05% TFA in CH₃CN. Column T= 40°C. Flow rate= 7.5 niL/min. Gradient: 0 min, 5% B; 2 min, 5% B; 5 min, 35% B; 18 min, 65% B; 20 min, 95% B.

Cation-exchange purification of ubiquitin and conjugates

Preparative scale MonoS was performed on an AKTA (GE Healthcare Lifesciences) system equipped on a cation-exchange column (MonoS FIR 5/5). A gradient was run from 0% Buffer A (50 mM Sodium Acetate, pH=4.6) to 35% Buffer B (1 M NaCl in 50 mM Sodium Acetate, pH=4.6) in 20 column volumes (CV).

Small scale purifications were performed on an AKTAmicro (GE Healthcare Lifesciences) system equipped with a cation-exchange column (MonoS 1.6/5 PC) . A gradient was run from 0% Buffer A (50 mM Sodium Acetate, pH=4.6) to 35% Buffer B (1 M NaCl in 50 mM Sodium Acetate, pH=4.6) in 30 column volumes (CV).

Solid phase synthesis

Peptide synthesis reagents were purchased from Novabiochem. Peptides were synthesized on a Syro II (MultiSyntech) Automated Peptide synthesizer by standard 9- fluorenylmethoxycarbonyl (Fmoc) based solid phase peptides chemistry on a 25 or 50 μηιοΐ scale. Starting with the pre-loaded Fmoc amino acid Wang resin (0.2 mmol/g, Applied Biosystems), each successive amino acid (Novabiochem) was coupled in 4 molar excess for 45 min with PyBOP and DiPEA. Deprotection of the fmoc protecting group was achieved with 20% piperidine in NMP (3x1.2 mL, 2x2 and 1x5 min). Peptides were cleaved with TFA/iPr₃SiH/H20 (95/2.5/2.5), precipitated in cold pentane/diethyl ether and analysed by LC-MS. Where necessary, peptides were purified by RP-HPLC (C₁₈ column).

Synthesis of Ub75-propargylamine

A 25% (w/v) solution of 2-amino-N-(prop-2-ynyl)acetamide*HCl in double distilled water was adjusted to pH 8 with NaOH. To 2 ml of this solution 15 mg of ubiquitin and 0.5 mg of trypsin were added. The reaction was monitored by LC-MS and was finished, this is when no starting material is left, after 2.5 hours. After acidification of the reaction mixture to pH = 2 with HCl, to inactivate the trypsin, the sample is pre-purified on a C₈ semi-preparative column and ubiquitin peaks were collected. Positive peaks were identified by LC-MS and lyophilized. After reconstitution in 10 ml of Buffer A (50 mM Sodium Acetate, pH=4.6), the sample was run on a MonoS strong cation exchange column using an Akta system. At 25% conductivity the Ubiquitin₇5-propargylamine was eluted in 4, 0.75ml fractions. Positive fractions (as judged by LC-MS analysis) were pooled, washed and concentrated using a Centriprep column (Amicon Ultra, Ultracel-3K). 2.66 mg (18% Yield) of 95% pure product was obtained, as judged by standard BCA (Thermo Fisher Scientific Inc.) test, calibrated with ubiquitin standard curve. Deconvoluted mass spectrum of Ub₇₅-propargyl after purification using monoS column. Peak at 6508 Da results from in-source dissociation of ubiquitin at the EP bond near position 18, this is observed in all ubiquitin preparations, including commercial ones. Expected mass for uibiquitin-propargyl (8544 Da (average mass), found 8540 Da).

Synthesis of 2-amino-N-(prop-2-ynyl)acetamide'HCl

Boc-2-amino-N-(prop-2-ynyl)acetamide was prepared as described previously (J. Org. Chem. 2005, 70, 9595-9598). Boc-2-amino-N-(prop-2-ynyl)acetamide was deprotected using 4M HCl in 1,4-dioxane; The product was obtained as a white to off-white powder (71 ) yield) ; δ_Η(300 MHz; DMSO-d6) 3.21 (1H, t, CCH), 3.57 (2H, s, NHC¾C), 3.95 (2H, quintet, NH₃C¾C), 8.37 (3H, s, NH₃), 9.1 1 (1 H, t, NH); 5_C(75.6 MHz; DMSO-d6) 165.78 (C=0), 80.43 (ClLCCl l), 73.57 (CCH), 39.96 (NH₃CH₂C), 28.03 (NHCH₂C). NMR spectra were calibrated on DMSO solvent signal at 2.54 and 40.45 ppm for δ_Η and 5c respectively (J. Org. Chem. 1997, 62, 7512-7515). Typical method for pepfide-ubiquitin conjugation

In a typical reaction, 45 μΙ_, Ub-PA (150 μΜ stock) is allowed to react with 6,75 μΐ, a 10 mM DMSO-stock of peptide (10 eq). pH is adjusted to pH=8.5 by addition of 36.4 μί phosphate buffer (600 mM, pH=8.5). If Ub-PA was obtained using trypsin, trypsin inhibitor, phenyl-mehyl-sulfonyl-fluoride (0.4 \iL, 400 mM in 2-propanol) or trypsin inhibitor from soybean (xx \iL, 1 mg/ml in ddH20), was added to the reaction mixture. The reaction is started by the addition of 2x2 μΐ_^ of a freshly prepared 1 : 1 solution of CuBr (20 mg/ml) and Cu(I) stabilizing triazole ligand9 5 (50 mg/ml) in Acetonitrile. Samples are analyzed after 15 minutes using LC-ESI-MS to verify completion of the reaction. All reactions preceded to completion, judged by the absence of starting material Ub-PA and presence of the conj ugate. After verifying completion of the reaction, excess copper, ligand and if the membrane allows excess peptide are removed using centrifugal filtration. This is done using 3kDa cut-off spin columns. Before applying to the column the conjugate sample is diluted with 0.5M of ethylenediaminetetraacetic acid (EDTA) to chelate excess copper and prevent ubiquitin aggregationl . After washing the conjugates with EDTA. Excess EDTA is removed by washing twice with double distilled water (ddH20). If sample purity was not satisfactory, conjugates were acidified using 1 M Sodium Acetate purified using cation-exchange chromatography. Figure 1 gives an example of such conjugate.

To a solution of 25 μΐ. Ub₇₅-propargyl (as obtained with above method) was added 32.6 μί, of Phosphate buffer (600 mM, pH=8.6). Immediately after this 0.4 μΐ of phenylmethanesulfonylfluoride solution was added (300 mM in 2-propanol) to quench any residual trypsin activity. After briefly reacting and deactivating residual trypsin activity, peptide solution was added of either 0.5 μΕ or 2.5 μΐ, (50 mM or 10 mM in DMSO respectively). To this 2x 1 μΕ of freshly prepared CuBr/Ligand solution was added with at least 1 minute interval. The CuBr/Ligand solution (57 mM in acetonitrile) is prepared by mixing 9.6 μΐ, of freshly prepared CuBr solution in degassed, argon bubbled, acetonitrile (20 mg/ml, 139 mM) with 13.9 μΕ of Ligand in acetonitrile (50 mg/ml, 96 mM). Reactions are typically finished after 15 minutes. Always prepare fresh solutions of CuBr and prepare fresh CuBr/Ligand as soon as the solution turns slightly green.

Immediately after reaction, as judged by LC-MS analysis, 50 μΐ, of EDTA solution (0.5M in ddH₂0, adjusted to pH=8 with NaOH) was added. Samples are transferred into amicon spin filters (Ultracel YW-3 3 kDa MWCO, 300 μί) and filled to 300 μΕ mark with EDTA solution. After spinning down to the 50 μΤ mark, the conjugates were washed with EDTA solution (filling back to 300 mark and spinning back to 50 μΕ). After this, samples were washed twice with ddH₂0. If purity was insufficient, preparations were further purified using cation exchange chromatography, as described above in methods section. Before applying the samples, they were acidified using 1M sodium acetate buffer (pH=4.6).

Table SI gives an overview of the synthesized conjugates with associated purification method and purity. Purity is judged using peak integration in LC-MS TIC trace, injection peak and peak at 16 minutes, general impurity present in the system, were omitted.

Peptides used for click chemistry

- p n .

Table S 1 Peptides usee! for pept ide-ubiquftin conjugates, 4 indicating azidonorvsKne building block. SCE

Strong cation exchange on AKTA purifier as described sn methods section.

EX AMPLE 2

Materials and Methods. LC-MS analysis was performed on a system equipped with a Waters 2795 seperation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190-750 nm), Waters Alltima CI 8 (2.1x100 mm), Waters Symmetry300™ C4 3.5 μΜ (2.1x100 mm) reversed phase column and LCT™ Orthogonal Acceleration Time of Flight Mass Spectrometer. Samples were run at 0.40 ml/min using 2 mobile phases: A= 0.1 % aq. formic acid and B= 0.1 % formic acid in CH₃CN. Data processing was performed using Waters MassLynx 4.1 mass spectrometry software.

Preparative HPLC.

Preparative HPLC was performed on a Shimadzu LC-20AD/T equipped with a C4 Vydac column (Grace Davison Discovery Sciences^1M). Mobile phases: A= 0.05% aq. TFA and B= 0.05% TFA in CH₃CN. Column 20°C. Flow rate= 10.0 mL/min.

Solid phase peptide synthesis.

Peptide synthesis reagents were purchased from Novabiochem. Peptides were synthesized on a 25 or 50 μτηοΐ scale using a Syro II MultiSyntech automated peptide synthesizer and standard 9-fluorenylmethoxycarbonyl (Fmoc) based solid phase peptide chemistry. Starting with pre-loaded Fmoc amino acid Wang resin (0.2 mmol/g, Applied Biosystems), each successive amino acid was coupled in 4 molar excess for 45 min. with PyBOP and DiPEA. Deprotection of the Fmoc group was achieved with 20% piperidine in NMP (3x 1.2 mL, 2x2 and 1 x5 min). Peptides were cleaved with TFA/iPr₃SiH/H₂0

(95/2.5/2.5), precipitated in cold n-hexane/diethyl ether and purified by RP-HPLC (CI 8). All peptides were analyzed by LC-MS.

Determining protein concentration.

To measure the total protein concentration, Applicants used the "Pierce* BCA Protein Assay Kit" obtained from Thermo Scientific (Catalogue number: 23225). This assay allows the colorimetric detection and quantization of total protein using a bicinchoninic acid based reagent. The determination was performed according to the instructions of the manufacturer provided with the kit, with one modification. In place of BSA as a reference we used ubiquitin (obtained from Boston Biochem). Lyophilized Ubiquitin (2mg) was dissolved in deionized water (1 mL) to provide a stock solution from which dilutions were obtained. The colorimetric detection was performed using an Perkin Elmer Wallac Victor2 1420-014 spectrophotometer at 562 nm wave length.

Non-hydrolyzable K48- and K63-linked Ub-isopeptide isosteres were generated from aminoxy-functionalized peptides QRLIFAGXQLEDGR (Ub41-54) and

LSDYNIQXESTLHL (Ub56-69), respectively representing K48 and K63 Ub-linkages. Biotin was attached at the N-terminal position for immobilization onto streptavidin, for affinity measurements by surface plasmon resonance. Incubation of each of the peptides (1 .5 equiv) with diethyl acetal A (1 mg/ml) in 0.5 M aqueous HC1 for 30 minutes at 37 °C resulted in in situ acetal deprotection and ensuing complete ligation judged by LC-MS analysis (Figure 4). Finally, the ligation products were purified by preparative reversed phase HPLC. Surface plasmon resonance (SPR) was used to analyze the binding of various DUBs to the two different isosteres mimicking the most predominant (i.e., K48 and K63 linked) Ub-Ub linkages side-by-side.

To determine whether a Ub-isopeptide isostere can specifically bind to specific DUBs, Applicants compared the affinities of a small panel of DUBs for each of the isosteres and unconjugated Ub. All tested DUBs were shown to be active prior to SPR measurements. At the concentrations tested, the catalytic domain (CD) of USP7 (HAUSP) was found to bind the UbK48 isopeptide isostere with great selectivity over the UbK63 isostere or free Ub. A pplicants then tested the effect of the two isosteres and Ub on U SP7 catalytic activity and the UbK48 isostere was able to inhibit USP7 while Ub or UbK63 did not. Without being bound to theory, Applicants believe this implies that USP7 selects the peptide sequence flanking the UbK48 linkage over the K63 linkage or free Ub. Albeit with varying affinities, USP2a CD binds both the UbK48 and UbK63 isosteres, which is in agreement with earlier work that showed that both linkages are accepted by the DUB USP2a.8 USP21 CD shows very high affinity towards Ub (in nM range) and even greater affinity towards both the tested isoesteres. USP4 CD binds Ub and Ub isoesteres in similar affinities. Overall, the results described herein indicate that interaction with the peptide sequence flanking the conjugation site can form the basis for DUB selectivity. The linkage specificity of DUBs can be intrinsic to their catalytic core domains,¹⁵ and in accordance Applicants show herein that the catalytic core domains can display specific affinities toward Ub isopeptide isosteres.

To demonstrate the general applicability of the oxime ligation strategy, a panel of oxime-linked Ub-conjugates was synthesized, based on peptide sequences derived from FANCD2,¹⁶ Histone2A, Histone2B and PCNA (Table 2), all proteins that hold potential as diagnostic markers. Fast (<30 min.) and complete conversions were observed in all cases, without the need of a large excess of any of the reactants. As USP7 is known to act on ubiquitinated H2B,¹⁷ this isostere was then N-terminally biotinylated and used to measure USP7 affinity with UbK48 and UbK63 isosteres evidencing clear affinity of the H2B isostere for USP7 comparable to the UbK48 isostere.

Synthesis Ub₇₄-C4 aldehyde diethyl acetal (A).

A reaction mixture of 3 ml containing 20 mg ubiquitin (Boston Biochem), 25% aq. 4- aminobutyraldehyde diethyl acetal (Fluka) solution and 0.5 mg TPCK-treated trypsin (Worthington Biochemical Corporation, New Jersey) was adjusted to a pH of 7.5 with 2M HC1 and shaken at 37 °C for 4 hrs. The reaction was quenched with 10 mg/ml trypsin inhibitor (from soybean, Merck) to a final concentration of 0.5 mg/ml. The reaction mixture was dialyzed against 50 mM NaOAc buffer (pH 4.5) and purified over a Resource S column (Pharmacia) using a gradient of 0 - 1 M NaCl in 50 mM NaOAc (pH 4.5). Eluted fractions containing product (as judged by LC-MS analysis) were pooled and concentrated using a Centriprep column (Amicon Ultra, Ultracel-3 ). Using the BCA protein assay described above, the amount of protein in the concentrate was determined to be 6mg in the total volume (30% yield).

General Protocol for Oxime conjugate formation.

1 niL of Ub74-C4 aldehyde diethyl acetal (1 mg/ml) is incubated with 1.5 equiv of the appropriate aminoxy functionalyzed peptide dissolved in 50 μΐ, deionized water. Aqueous HC1 was added from a 4 M stock to a final concentration of 0.5 M HC1 and the reaction mixture was incubated for 30 minutes at 37 °C. This resulted in in situ acetal deprotection and ensuing oxim formation. Full consumption of acetal and aldehyde was observed by LC-MS analysis in all cases. Excess peptide was removed by purification with preparative reversed phase HPLC. Following purification, the product yield was determined to be in the range of 80-85% of the input Ub74-C4 aldehyde diethyl acetal in all cases as determined with the BCA protein assay kit.

Biotinylation of Ubiquitin.

Ubiquitin (1 mg, Boston Biochem) was mixed with a buffer containing 25 mM Hepes, pH 7.6 and an equivalent amount of EZ-Link-Sulfo-NHS-LC-Biotin (Pierce). The mixture was incubated at r.t. for 2 hours, after which the product was purified by gel filtration using Hepes buffer (25 mM, pH 7.6). Mass spectrometry analysis of the ubiquitin fraction from the column confirmed 50% as mono-biotinylated Ub while the rest remained as free Ub. This fraction was free from traces of unreacted biotin.

DUB expression and purification.

A codon-optimized Usp7 CD (residues 208 to 560) was cloned into the pGEX-6P-l vector backbone using the BamHI and Not! restriction sites, respectively. Expression was perfomed using BL21(DE3) Tl resistant E.coli cells (Sigma). Induction was achieved by autoinduction (reference autoinduction medium) at 16°C using a 16 hour induction time. After centrifugation, cell pellets were resuspended in 50 mM Hepes (pH 7.5), 300 mM NaCl, 1 mM DTT and supplemented with Complete Protease Inhibitor-EDTA free tablets (Roche). Lysis was achieved using a high-pressure homogenizer (Emulsiflex, Avestin). After centrifugation for 30 minutes at 20.000 rpm using a 25.50 rotor (Avanti, Beckman), the supernatant was applied to GST beads (Amersham GE). After extensive washing with 50 mM Hepes pH 7.5, and elution with 50 mM reduced glutathione in Hepes pH 7.5 (Sigma), the eluate was purified by gel filtration on a S75 1660 column with a coupled GST FF column using an Akta system (Amersham GE). This typically yielded 10 mg of pure protein per liter culture. Full length USP7 (residues 1 -1 102) was a gift from Boston Biochem, Boston, MA, USA.

The E.coli host Rosetta2(DE3) was used for the large scale protein expression of His- tagged USP25. 5 mL of an overnight pre-culture was used to inoculate 500 mL autoinduction medium in 3 L baffled flasks and grown at 37°C until an OD₆oo of 2-3 units was reached. The temperature was then lowered to 21 °C for overnight induction. Cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl pH 8.0, 150 mM NaCI, 10 mM imidazole, 5 mM β-mercaptoethanol and 1 mM PMSF. The cells were broken by subjecting the cell suspension to a 10 second sonification pulse with a pause of 30 seconds after each pulse for a total of 5 minutes using the Misonics sonicator S-4000 at 80% maximum setting. The lysate was centrifuged at 20k for 30 minutes at 4°C to remove cellular debris and unbroken cells. The resulting supernatant was incubated with washed Talon metal affinity resin (Clontech, Inc., Palo Alto, CA) for 20 minutes at 4°C and the beads were then washed with lysis buffer. Protein was eluted with lysis buffer containing 400 mM imidazole and diluted 10-15 times with 50 mM BisTris pH 6.5 followed by cation exchange chromatograpy purification using an Akta FPLC system (GE Healthcare). The diluted sample was applied to a Poros S column equilibrated with buffer A (20 mM BisTris pH 6.5, 10 mM NaCl and 5 mM β-mercaptoethanol). The bound protein was eluted with buffer A containing 1 M NaCl using a 60% gradient in 20 column volumes. Peak fractions were pooled and concentrated by ultrafiltration using an Amicon Ultra centrifugal unit (Millipore) and applied to a Superdex 200 (GE Healthcare) gelfiltration column equilibrated with 25 mM Tris-HCl (pH 8.0), 150 mM NaCl and 5 mM β-mercaptoethanol. Peak fractions from the gelfiltration column were pooled and concentrated in an Amicon Ultra unit to 10 mg/ mL. The concentrated protein was flash frozen in liquid nitrogen and stored at -80°C.

The E.coli host Rosetta2(DE3) was used for the large scale protein expression of His-tagged USP4 catalytic domain and USP21 catalytic domain. 5 mL of an overnight pre-culture was used to inoculate 500 mL autoinduction medium in 3 L baffled flasks and grown at 37°C until an OD₆oo of 2-3 units was reached. The temperature was then lowered to 21 °C for overnight induction. Cells were harvested by centrifugation and resuspended in 50 mM Tris- I-1C1 pH 8.0, 150 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol and 1 mM PMSF. The cells were broken by subjecting the cell suspension to a 10 seconds sonification pulse with a pause of 30 seconds after each pulse for a total of 5 minutes using the Misonics sonicator S-4000 at 80% maximum setting. The lysate was centrifuged at 20k for 30 minutes at 4°C to remove cellular debris and unbroken cells. The resulting supernatant was incubated with washed Talon metal affinity resin (Clontech, Inc., Palo Alto, CA) for 20 minutes at 4°C and the beads were then washed with lysis buffer. Protein was eluted with lysis buffer containing 400mM imidazole followed by an anion exchange chromatography purification step using an Akta FPLC system (GE Healthcare). The eluted sample was applied to a PorosQ column equilibrated with buffer A (20mM Hepes pH7.5, lOOmM NaCl and 5mM β- mercaptoethanol). The bound protein was eluted with buffer A containing 1 M NaCl using a 60% gradient in 20 column volumes. Peak fractions were pooled and concentrated by ultrafiltration using an Amicon Ultra centrifugal unit (Millipore) and applied to a Superdex 200 (GE Healthcare) gelfiltration column equilibrated with 25 mM Hepes (pH 7.5), 150 mM NaCl and 5 mM β-mercaptoethanol. Peak fractions from the gelfiltration column were pooled and concentrated in an Amicon Ultra unit to 10 mg/mL. The concentrated protein was flash frozen in liquid nitrogen and stored at -80°C.

USP2a CD (residues 259-605) was a gift from Life Sensors, Malvern, PA, USA. DUB binding assay.

Binding of DUBs to Ub isospeptide isosteres was assessed with a Biacore Tl 00 apparatus. Biotinylated Ub and Ub isopeptide isosteres were immobilized separately (to 50 RU) onto streptavid in-coated Biacore sensor chips (SA chips) by resuspending them in 10 mM Hepes, 100 mM NaCl, 2 mM β-mercaptoethanol, pH 7.5. A streptavidin-coated sensor surface without ligand was used as a control surface in order to subtract unspecific binding. Binding experiments were performed at a flow rate of 30 μΙ7ιηίη. The DUBs were applied in concentrations ranging from 100 nM to 25 μΜ at room temperature. After each binding event, surfaces were regenerated by a short stripping pulse of 50 mM NaOH. Evaluation of the binding data was performed by steady-state analysis plotting saturated binding vs the respective analyte concentration. ¾ values were calculated using Prism (GraphPad Software, Inc., San Diego, CA, USA) employing non-linear regression analysis.

DUB activity assays.

2 nM HAUSP (Boston Biochem) or 1 0 nM USP16 were used to cleave 30 nM Ub-PLA2 (a gift from Progenra, Malvern, USA) in a reaction with or without 150 nM monoUb/Ub- isopeptide isostere. Proteolytic cleavage of Ub results in the release of PLA2 which in turn cleaves its substrate 2-(6-(7-nitrobenz-2-oxa-l ,3-diazol-4-yl)amino) hexanoyl-1- hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C6-HPC, Molecular Probes, Leiden, the Netherlands), 20 μΜ, liberating fluorescent NBD(¹²). Reactions were carried out in a volume of 100 μΐ in black-walled 96 well plates (Optiplate-96F, Perkin-Elmer) in 20 mM Tris-HCl, pH 8.0, 2 mM CaCl₂ and 2 mM β-mercaptoethanol. The increase in fluorescence intensity over time was determined using a Wallac Victor2 (Perkin-Elmer) plate reader with excitation and emission filters of 475 nm and 555 nm,respectively, at 37 °C.

HPLC profile of (A) UW 48- and (B) UbK63-isopeptide isosteres showed peaks at 7.45 min and 7.15 min, respectively. The 230 nm absorption profile of purified conjugates is analyzed by reversed phase HPLC analysis using a C4 column. A single major product peak eluting around 7 min. corresponds to the desired product.

In order to test hydrolytic stability of Ub isopeptide isostere towards cleavage by a DUBs, conjugates were analyzed after cleavage reactions. The effect of 40 nM of USP2a CD

⁽12) Nicholson et al. , Protein Sci. 2008 , 17, 1035. on 15 μΜ Ub-K48 isopeptide isostere was compared with native 15 μΜ K48 linked di-Ub. USP2a CD is known to cleave K48 linked poly Ub chains {Protein Sci. 2008, 17, 1305). Following incubation in assay buffer for 2 hrs, the assay contents were analyzed on a 12% Bis-Tris gel ( uPage, Invitrogen), next to a molecular weight marker (SeaBlue plus2 , Invitrogen). Results are shown in Figure 5, native K48 linked diUb was cleaved by USP2a CD to a significant extent whereas the Ub-K48 isopeptide isostere remained stable evidencing resistance to DUB-mediated hydrolysis.

Applicants tested if the Ub isopeptide isosteres can specifically inhibit DUB action side by side with Ub as Ub is known to inhibit the activity of several DUBs ( EMBO Rep. 2009, 10, 466). The effect on the activity of the DUBs USP7/HAUSP and USP16 were compared upon treatment with monoUb and the linkage specific isostere using the in vitro Ub DUB reporter assay. As can be seen in Figure 6, the non-hydrolyzable UbK48 isopeptide isostere inhibits USP7/HAUSP potently compared to the Ub control confirming the specific interaction of USP7/HAUSP with the peptide sequence flanking the UbK48 site. In contrast, incubation with USP16 gave the opposite effect: Ub itself can inhibit USP16.

Figure 7 shows the mass spectrum and deconvolved mass spectrum of normal isotopic abundance corresponding to a Ub-K561 FANCD2 isopeptide isostere

Determination of ds for DUBs towards Ub and Ub-isosteres

Figure 8 shows the binding of USP7 CD to Ub isopeptide isosteres. SPR response curves for the binding of USP7 CD (concentrations ranging from 0.39 to 50 μΜ, bottom curve to top curve respectively) to (A) biotiniylated Ub-K48 isopeptide isostere, no interaction was observed for the K63 isostere (B) The maximum RUs for every curve is plotted against the corresponding concentrations of USP7 CD used for binding

measurements. Employing non-linear regression analysis on the plotted curves, the KDs for binding of USP7 CD to respective Ub isopeptide isosteres are calculated.

Figure 9 shows binding of USP4 CD to Ub isopeptide isosteres. SPR response curves for the binding of USP4 CD (concentrations ranging from 0.15 to 12.15 μΜ, bottom curve to top curve respectively) to (A) biotinlated Ub, (B) biotiniylated Ub-K48 isopeptide isostere and (C) biotinylated Ub-K63 isopeptide isostere, all immobilized to a SA chip on separate lanes. (D) The maximum RUs for every curve in figure A, B and C are plotted against the corresponding concentrations of USP4 CD used for binding measurements. Employing nonlinear regression analysis on the plotted curves in D, the KDs for binding of USP4 CD to Ub and Ub isopeptide isosteres are calculated.

Figure 10 shows binding of USP21 CD to Ub isopeptide isosteres. SPR response curves for the binding of USP4 CD (concentrations ranging from 0.01 to 24.3 μΜ, bottom curve to top curve respectively) to (A) biotinlated Ub, (B) biotinlylated Ub-K48 isopeptide isostere and (C) biotinylated Ub-K63 isopeptide isostere, all immobilized to a SA chip on separate lanes. (D) The maximum RUs for every curve in figure A, B and C are plotted against the corresponding concentrations of USP21 CD used for binding measurements. Employing non-linear regression analysis on the plotted curves in D, the Kds for binding of USP21 CD to Ub and Ub isopeptide isosteres are calculated.

EXAMPLE 3

Scheme 1 depicts a synthetic approach towards proteolytically resistant thioether linked Ub conjugates. Here the isopeptide bond of native Ub conjugates (1.5) is replaced by a carbon-carbon bond. First, a Ub(l-75) thioester, obtained from expressed intein-fusion constructs (see A. Borodovsky et al, Chem. Biol. 2002, 9, 1 149), is coupled to (commercially available) 5-bromo-l -pentylamine, resulting in Ub-bromide conjugate 1.1. This can then be used as ubiquitination agent for both cysteine (Cys) and selenocysteine (Sec) residues (1.3). Here the Cys/Sec residue functions as a ligation handle that can be introduced readily by site- directed mutagenesis (e.g. of a lysine residue, 1.2) or synthetically (SPPS). In general, the following Cys/Sec alkylation is performed under basic conditions (e.g. sodium phosphate buffer pH 8 - 9).²⁶ Overall, this approach gives a proteolytically stable well-defined Ub conj ugate (1.4) in which an thioether containing alkane chain, mimics the native and isopeptidic linked Gly-Lys dipeptide (1.5).

Intein mediated

expression

Ub(1-75)-MESNa

Scheme l . Approach towards DUB resistant thioether linked Ub conjugates

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Having thus described in detail embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Each patent, patent application, and publication cited or described in the present application is hereby incorporated by reference in its entirety as if each individual patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.

Table 1. Peptides used to created peptide-ubiqutin-click conjugates.4 indicates Azidonorvaline, C * is a (S-(o-nitrobenzyl) cysteine.

# Sequence Source sequence

1 MQIFV4TLTGKT1T Ub(l-14)

2 FVKTLTG4TITLEV Ub(4-17)

3 SDTIENV4AKIQDK Ub (20-33)

4 TIENVKA4IQDKEG Ub (22-35)

5 VKAKIQD4EGIPPD Ub (25-38)

6 QRLIFAG4QLEDGR Ub (41-54)

7 LSDYNIQ4ESTLIIL Ub (56-69)

8 GDAWIS,\A4DGV FSASGE PCNA (156-172), CI 63 A

9 GDAWISC*A4DGVKFSASGE PCNA (156- 172)

10 QDDMHLVIR4QLSSTVFKY FANCD2 (553 - 570)

11 PGPEETSE4VENGSLAD PTEN (281 -297), C296A

12 PGPEETSE4VENGSLC'*D PTEN(281 -297)

13 IKEIVSRN4RRYQEDGF PTEN (5 -21)

14 QAVLLP4KTESHHKA H2A(113-127)

15 QAVLLPK4TESHHKA H2A(113-127)

16 VLLP4KTSATVGPKA ΙΙ2ΛΧ(113- 127)

17 VLLPK4TSATVGPKA H2AX(113-127)

18 EGTKAVT4YTSSK H2B(113-125)

Table 2. X= conjugation site.

Protein (human) targeted lysine sequence

Claims

WHAT IS CLAIMED IS:

1 . A ubiquitinated peptide comprising a ubiquitin-peptide isostere, wherein said peptide is synthesized by:

(a) converting ubiquitin to a protected derivative thereof using an alkylamino group containing a masked aldehyde;

(b) generating a terminally conjugated ubiquitin by converting the masked aldehyde to an aldehyde; and

(c) converting the aldehyde to an oxime.

2. A ubiquitinated peptide comprising a ubiquitin-peptide isostere, wherein said peptide is synthesized by:

(a) converting ubiquitin to a derivative thereof, wherein said derivative contains a terminal acetylene; and

(b) generating a conjugated ubiquitin by reacting said acetylene with an azide derivative in order to generate a triazole.

3. A ubiquitinated peptide comprising a ubiquitin-peptide isostere, wherein said peptide is synthesized by:

(a) converting ubiquitin to a derivative thereof, wherein said derivative contains a terminal alkyl halide; and

(b) generating a conjugated ubiquitin by reacting said alkyl halide with a thiol or selenol derivative in order to generate a thio- or seleno-ether containing conjugate.

4. The ubiquitinated peptide of any of claims 1 -3, wherein the peptide comprises an amino acid sequence selected from Table 1 or Table 2.

5. A linkage-specific antibody directed against a peptide of any one of claims 1 -3.

6. The antibody of claim 5, wherein the antibody is a monoclonal antibody.

7. The antibody of claim 5, wherein the antibody reacts with a peptide conjugate

comprising a native, hydrolyzable peptide bond.

8. Use of a peptide of any one of claims 1 -3 in the generation of an antibody

9. The use of a peptide of claim 8, wherein the antibody generated is a polyclonal antibody.

10. The use of a peptide of claim 8, wherein the antibody generated is a monoclonal antibody.

11. Use of a peptide of any of claims 1-3 in the selection of an antibody.

12. Use of a peptide of any of claims 5-1 1, wherein the peptide comprises an amino acid sequence selected from Table 1 or Table 2.

13. Use of a peptide of claim 12, wherein the peptide comprises the amino acid sequence QRLIFAG4QLEDGR, wherein 4 is azidonorvaline.

14. Use of a peptide of claim 12, wherein the peptide comprises the amino acid sequence LSDYNIQ4ESTLHL, wherein 4 is azidonorvaline.

15. Use of a peptide of claim 12, wherein the peptide comprises the amino ac id sequence EGTKAVTKYTSSK.

16. A host cell for the production of an antibody according to any of claims 5-1 1 .

17. The host cell of claim 16, wherein the host cell is a hybridoma cell.

18. Use of a peptide of any of claims 5-1 1 , wherein the ubiquitinated peptide inhibits the activity of at least one deubiquitinating enzyme.

19. Use of claim 18, wherein the at least one deubiquitinating enzyme is selected from USP2a, USP4, USP7, USP16, and USP21 .

20. Use of a peptide of any of claims 1-3 in a method to determine the affinity of one or more deubiquitinating enzymes for lysine topoisomer mimics.