WO2013042120A2 - Synthetic ubiquitins and use thereof in drug screening assays - Google Patents

Abstract

Synthetic ubiquitins are provided in which at least one amino acid residue is replaced with a residue of an alpha-amino acid substituted by a detectable moiety, useful in various high throughput screening (HTS) methods for identifying potential drug candidates as inhibitors of ubiquitin-mediated proteolysis.

Description

SYNTHETIC UBIQUITINS AND USE THEREOF IN DRUG SCREENING ASSAYS

FIELD OF THE INVENTION

The present invention in is the fields of protein synthesis and high throughput screening assays for drug discovery.

BACKGROUND OF THE INVENTION

Ubiquitin modification of proteins

The ubiquitin-mediated proteolysis system is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell is important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. Therefore, the ubiquitin system has been implicated in the pathogenesis of numerous disease states, including oncogenesis, inflammation, viral infection, CNS disorders, and metabolic dysfunction.

One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle. Targeted proteins undergoing selective degradation, through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific ε-amino of a lysyl residue or the N-terminus of the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (El) and a ubiquitin-conjugating enzyme (E2), and may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the already conjugated ubiquitin moiety by isopeptides bond, thereby forming multi-ubiquitin chains. Ubiquitin (Ub) is able to form chains by self-conjugation onto any of its seven lysine residues (K6, Kl l, K33, K27, K29, K48, and K63).

Thus, conjugation of the 76-amino acid ubiquitin to protein substrates is a multi-step process. In an initial ATP-dependent step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an El enzyme. Activated ubiquitin is then transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates. Substrates are recognized either directly by the ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases.

In addition to the 76-amino acid Ub, there is a family of ubiquitin-like modifier proteins that are low molecular weight polypeptides (76-165 amino acids) and share between 10% and 55% sequence identity to ubiquitin (Wong et al., 2003; Schwartz & Hochstrasser, 2003). A family of small ubiquitin-like protein modifiers called SUMO comprises small proteins that covalently attach to lysine residues of another protein in a reversible fashion. SUMO attachment to its substrate proteins causes changes in the localization, activity, or binding partners of the substrate. SUMO has been shown to play a role in a multitude of processes; these include chromosome segregation, cell cycle progression, and DNA damage recovery. Defects in the SUMO pathway have been demonstrated to affect tumorigenesis and the inflammatory response as well as other human diseases.

Although ubiquitin and each ubiquitin-like protein modifier direct distinct sets of biological consequences and each requires distinct conjugation and deconjugation machinery, they share a similar cascade mechanism involving an activating enzyme (El), a conjugating enzyme (E2), and perhaps an auxiliary substrate recognition protein (E3).

Genome mining efforts have identified at least 530 human genes that encode enzymes responsible for conjugation and deconjugation of ubiquitin or ubiquitin-like protein modifiers (Wong et al., 2003). A multitude of E3s reflect their roles as specificity determinants. The human genome encodes 391 potential E3s, as defined by the presence of HECT, RING finger, PHD or U-box domains (Wong et al., 2003). The domains mediate the interaction of the E3 with the E2. As a modular system, each E2-E3 pair appears to recognize a distinct set of cellular substrates. For example, the same E2 in conjunction with different E3s may recognize distinct substrates. E3s encompass a broad spectrum of molecular architectures ranging from large multimeric complexes (e.g., anaphase promoting complex or APC), in which E2 binding, substrate recognition, and regulatory functions reside in separate subunits, to relatively simple single component enzyme (e.g., murine double minute or MDM2) in which all necessary functions are incorporated into one polypeptide. High throughput screening methods for assessment of ubiquitination activity

A summary of current high throughput screening (HTS) methods for drug discovery screening can be found in Methods in Enzymology 399 Part B, p.654 (Yi Sun). The main techniques applicable for monitoring the ubiquitin-mediated proteolysis include:

(i) Fluorescence intensity (FI) assay. Fluorescent probes are used in biochemistry to study the various binding sites in large macromolecules through the difference of the quenching rates of the bound verses free probe. Fluorescence intensity has been widely applied over the last two decades due to the vast development of new fluorophores. Typically, an optical system illuminates and excites the sample at a specific wavelength selected by a high performance optical filter. As a result, the sample emits light and a second optical system collects the emitted light. Usually, the emitted light is of lower energy and thus is composed of a longer wavelength than the excitation light.

(ii) Fluorescence Polarization (FP). Fluorescence polarization (or fluorescence anisotropy) measurements provide information on molecular orientation and mobility and processes that modulate them, including receptor-ligand interactions, protein-DNA interactions, and proteolysis. Because polarization is a general property of fluorescent molecules (with certain exceptions such as lanthanide chelates), polarization-based readouts are somewhat less dye dependent and less susceptible to environmental interferences such as pH changes than assays based on fluorescence intensity measurements. Experimentally, the degree of polarization is determined from measurements of fluorescence intensities parallel and perpendicular with respect to the plane of linearly polarized excitation light, and is expressed in terms of fluorescence polarization (P) or anisotropy (r).

(Hi) Time Resolved Fluorescence (TRF). TRF detection differs from fluorescence intensity (FI) in the timing of the excitation/emission (measurement) process. In case of standard FI the excitation and emission processes are within a time frame of nanoseconds: namely, the light emitted by the sample is measured right after the excitation. Every fluorophore has a fluorescence lifetime and the decay curve of the excitation wavelength energy will contribute differently to the background activity of the emission wavelength being measured. The use of long-lifetime fluorophores such as rare earth elements called lanthanides, particularly uropium, Gadolinium, Terbium and Samarium, minimizes the problem of background fluorescence since lanthanides have an unusual property of emitting light over long periods of time after excitation - up to milliseconds rather than nanoseconds as in standard FI. Complexes of the rare earth ions with macromolecules are preferably used in TRF since they have large Stoke's shifts and extremely long emission half-lives when compared to more traditional fluorophores.

(iv) Fluorescence Resonance Energy Transfer (FRET). FRET is a distance- dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. The efficiency of FRET is dependent on the inverse sixth power of the interaiolecular separation, making it useful over distances comparable to the dimensions of biological macromolecules. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. FRET is particularly advantageous as it is performed in a homogeneous format that is particularly amenable to HTS, since acceptor emissions, as a measure of energy transfer, can be detected without the need to separate bound from unbound assay components. When FRET is used as a contrast mechanism, colocalization of proteins and other molecules can be imaged with spatial resolution beyond the limits of conventional optical microscopy. The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined as the Forster radius (R0). The magnitude of R0 is dependent on the spectral properties of the donor and acceptor dyes and their spatial arrangement.

Donor and acceptor fluorophores have been conjugated to a variety of biomolecules creating functional assays such as: protein-protein binding, antigen-antibody binding, ligand-receptor binding, DNA hybridization and DNA-protein binding.

(v) Time Resolved-FRET (TR-FRET). TR-FRET, known also as homogeneous time-resolved fluorescence (HTRF®), unites TRF (time-resolved fluorescence) and FRET (fluorescence resonance energy transfer) principles. This combination brings together the low background benefits of TRF with the homogeneous assay format of FRET. This powerful combination provides significant benefits to drug discovery researches including assay flexibility, reliability, increased assay sensitivity, higher throughput and fewer false positive/false negative results. For screening libraries of compounds, TR-FRET is a recognized method for overcoming interference from compound autofluorescence or light scatter from precipitated compounds.

The premise of a TR-FRET assay is the same as that of a standard FRET assay: when a suitable pair of fluorophores are brought within close proximity of one another, excitation of the first fluorophore (the donor) can result in energy transfer to the second fluorophore (the acceptor). This energy transfer is detected by an increase in the fluorescence emission of the acceptor, and a decrease in the fluorescence emission of the donor. In high-throughput screening (HTS) assays, FRET is often expressed as a ratio of the intensities of the acceptor and donor fluorophores. The ratiometric nature of such a value corrects for differences in assay volumes between wells, and corrects for quenching effects due to colored compounds.

In contrast to standard FRET assays, TR-FRET assays use a long-lifetime lanthanide ions chelate or cryptates as the donor species, thereby achieving particularly extended duration - in the order of milliseconds or longer of the average time that the donor molecule spends in the excited state after accepting a photon. This is in sharp contrast to the lifetime of common fluorophores used in standard FRET assays, which are typically in the nanosecond range. Because interference from autofluorescent compounds or scattered light is also on the nanosecond timescale, these factors can negatively impact standard FRET assays. To overcome these interferences, TR-FRET assays are performed by measuring FRET after a suitable delay, typically 50 to 100 microseconds after excitation. This delay not only overcomes interference from background fluorescence or light scatter, but also avoids interference from direct excitation due to the non-instantaneous nature of the flash lamp excitation source.

TR-FRET platforms have been used for detection of E3 autoubiquitination. For example, Tularik, Inc. developed a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization (Hong, et al., 2003). Similarly, Roche developed an assay for P53 ubiquitination using the same technique (Yabuki, et al., 1999).

The rare earth chelates and cryptates commonly used as light-harvesting devices in TRF and TR-FRET and as labels in biological assays possess specific properties including stability, high light yield, ability to be linked to biomolecules, and insensitivity to fluorescence quenching. However, rare earth chelates have certain disadvantages as compared to rare earth cryptates, for example, lower stability, susceptibility to competition with other chelating compounds, and sensitivity in FRET analysis.

(vi) ELISA. Enzyme linked immunosorbent assay (ELISA) method was used for an E3 autoubiquitination assay e.g., by Rigel Pharmaceuticals (US 6,740,495 and US

6,737,244).

(vii) DELFIA. Dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a robust, high-performance immunodetection platform that provides a combination of benefits that make it the superior alternative to conventional ELISA. DELFIA utilizes the unique chemical properties of the long-lived lanthanide chelates as the tracer, mostly europium chelate fluorophores, in concert with time-resolved fluorescence (TRF) detection to create an assay that may significantly increase the signal window when compared to ELISA.

(viii) SPA. Scintillation Proximity Assay (SPA), is performed using low energy radioisotopes ( 3 H and 125 I) as labels due to their short range electron emission, and microscopic beads containing a scintillant which emits light when it is stimulated. Stimulation occurs when radio-labeled molecules interact and bind to the surface of the bead. This interaction will trigger the bead to emit light photons, which can be detected using a scintillation counting. Electrons emitted from labeled molecules not close to the surface of the beads dissipate their energy and are not detected. This binding assay has the advantage of avoiding the usual filtration or washing procedures.

SPA is employed in heterogeneous ubiquitination assays, which use a radio-labeled ubiquitin that acts as a source of low-energy elections for scintillation excitation of photon- emitting beads. The beads are coated with a separation agent such as streptavidin or an antibody. Although the signaling complex originates from a ubiquitinated protein that has been localized to the beads, there is no need to separate the soluble elements of the reaction before reading.

(ix) ECL. Electrochemiluminescence (ECL), a further heterogeneous ubiquitination assays technique, involves a specific antibody that is linked to a molecule (ORTTAG) that emits light following excitation by an electrode. This detection system requires separation of the ubiquitinated molecule in a format analogous to DELPHIA. Chemical synthesis of Ubiquitin

The ability to generate Ub polymers biochemically was initially limited to the generation of Kl l, K48, and K63 topoisomers (Matsumoto et al., 2010), a strategy that requires prior identification and production of specific E2 enzymes.

The generation of Ub mutants by biochemical methods is largely limited by the repertoire of natural amino acids. Methods for the chemical synthesis of Ub are being developed to meet the need to provide reliable routes towards site-specifically labeled Ub derivatives. Modular procedures for Ub synthesis have recently been reported based on the ligation of segments (Erlich et al., 2010). Although such modular procedures provide good overall yields, the modular character introduces extensive purification procedures, making these procedure unsuitable for the automated parallel generation of synthetic Ubs. Previously reported linear Fmoc (9-fluorenylmethoxycarbonyl)-based syntheses of Ub led to very low yields (4%) and modest purity at best (Alexeev et al., 2003; Layfield et al., 1999). Several (semisynthetic) strategies towards Ub synthesis have been reported that allow the construction of isopeptide-linked Ub conjugates. The first approach (Chatterjee et al., 2007) uses a photolabile auxiliary that assists native chemical ligation of a recombinant Ub thioester (Hackenberger and Schwarzer, 2008). Other approaches rely on thiolysine-based chemical ligation (Yang et al., 2009; Haj-Yahya et al., 2010), and afford native isopeptide linkages after ligation to recombinant Ub thioester and subsequent desulfurization (Wan et al., 2007; Haase et al., 2008).

Recently, Oualid et al., (Oualid et al., 2010) disclosed a high-yielding Fmoc-based linear solid-phase peptide synthesis (SPPS) of Ub that allows the incorporation of desired tags and mutations as well as specific C-terminal modification and the construction of diUb conjugate in a straightforward manner. Oualid et al. discloses linear syntheses involving incorporation of pseudoproline building blocks and dimethoxybenzyl (DMB) dipeptides whereby the desired products are yielded directly and in parallel, and formation of folded and/or aggregated intermediates on-resin is said to be prevented. However, the only modifications to native Ub taught in Oualid et al. are C-terminal modifications which are actually C-terminal fusions that were obtained after the full length natural Ub was chemically synthesized and generated from a resin and the free C-terminal carboxylate thereof was condensed with GlyAMC and GlyRhodaminel lOGly to yield UbAMC and UbRhl lOGly (in very low yields, actually).

Oualid et al. also disclose SPPS synthesis of Ub mutants in which all seven lysine residues were mutated into δ-thiolysines, and a site- selective N-terminal modification with various labels that produced N-terminally labeled 5-carboxytetramethylrhodamine-Ub, 5(6)- carboxyfluorescein-Ub, and DOTA-Ub (DOTA=l, 4,7,10-tetraazacyclododecane- 1,4,7, 10- tetraacetic acid) conjugates.

All the synthesis strategies mentioned above, although presenting important developments, still, could not meet the need to obtain synthetic Ub or Ub-like modifiers in which one or more amino acids along the protein back-bone is replaced with a non-natural amino acid containing a detectable moiety. SUMMARY OF THE INVENTION

The present invention provides, in a main aspect, a novel synthetic ubiquitin or ubiquitin-like modifier protein in which at least one amino acid residue is replaced with a residue of an a-amino acid substituted by a detectable moiety. The detectable moiety may be a radioactively labeled moiety, a chromophore, a fluorescent moiety, an enzyme, biotin or an electron-dense reagent.

Depending on the detectable moiety attached onto one or more amino acid residues of the synthetic Ub or ubiquitin-like modifier (Ubl) protein, the novel Ub and Ubls provided herein may be used in various high throughput screening (HTS) methods for identifying potential drug candidates for inhibiting the ubiquitination-mediated proteolysis reaction.

In one preferred embodiment, the HTS method is time-resolved fluorescence resonance energy transfer (TR-FRET), and the synthetic Ub or Ubl used in accordance with the invention comprises a cryptate of a lanthanide ion, preferably europium cryptate or terbium cryptate. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the three dimensional structure of human ubiquitin. The blue sections are well exposed to the solvent, the green sections are "buried" sections, namely almost not exposed at all, and the gray sections have varying degrees of exposure.

Figs. 2A-2D are schematic description of TR-FRET assays performed with synthetic ubiquitins (Ubs): self ubiquitination assay comprising a synthetic Ub labeled with Eu- cryptate (2A); polyubiquitin chain elongation (self ubiquitination) assay using antibodies and two kinds of labeled synthetic Ubs: Ub labeled with Eu-cryptate and Ub labeled with a flag (biotin) (2B); chain-elongation self ubiquitination assay without antibodies and two kinds of labeled synthetic Ubs: Ub labeled with Eu-cryptate and Ub labeled with AlexaFluore647 fluorophore (2C); chain-elongation self ubiquitination assay without antibodies and two kinds of labeled synthetic Ubs: Ub labeled with Tb-cryptate and Ub labeled with fluorescein (2D).

MODES OF CARRING THE INVENTION

As important regulatory mechanisms underlying diverse biological pathways, ubiquitin and ubiquitin-like protein modification systems present important targets in the treatment of diseases. Accordingly, it is an object of the present invention to provide synthetic ubiquitin (Ub) and ubiquitin-like modifier (Ubl) proteins containing labeling moieties, and assay systems for measuring the attachment of Ub or Ubl to various target proteins.

The terms "ubiquitin-like modifier" or "Ubl" as used herein refer to the group of small proteins that are subject to conjugation machinery similar to that for ubiquitination. Non-limiting examples of Ubls include NEDD8, ISG15, SUMOl (also termed GMP1, Picl, SMTP3, Smt3C, sentrin), SUM02, SUM03, APG12, APG8. Other Ubls are listed in Wong et al. (Wong et al., 2007).

Thus, in one aspect, the present invention provides a synthetic Ub or a synthetic Ubl in which at least one amino acid residue is replaced with a residue of an a-amino acid substituted by (i.e., containing) a detectable moiety.

"a-amino acid substituted by a detectable moiety" as used herein refers to an a- amino acid having a side chain which is a detectable moiety or a side chain that is substituted by a detectable moiety.

The detectable moiety is designed to suit any assay applicable in monitoring ubiquitin-mediated binding and/or proteolysis. Detectable moieties used in accordance with the invention include, but are not limited to, a radioactively labeled moiety, a chromophore, a fluorescent moiety, an enzyme, an electron-dense reagent, and biotin.

The term "fluorescent moiety" or "fluorophore" as used herein refers to a component of a molecule or to a whole molecule which fluorescents. The fluorescent moiety absorbs energy of a specific wavelength and re-emits energy at a different (but equally specific) wavelength. The fluorophore is an aromatic or an otherwise conjugated moiety. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. Non-limiting examples of fluorophores include old generation or "traditional" dyes such as derivatives of xanthene (e.g., fluorescein, rhodamine and derivatives thereof e.g., X-Rhodamine, tetramethylrhodamine, or Lissamine Rhodamine B, Oregon green, eosin, Texas red, or Cal Fluor dyes), cyanine and derivatives thereof (e.g., indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, or Quasar dyes), naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarin derivatives (e.g., hydroxycoumarin, aminocoumarin or methoxycoumarin), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole or benzoxadiazole), pyrene derivatives such as cascade blue and the like, oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170 and the like), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow and the like), arylmethine derivatives (e.g., auramine, crystal violet or malachite green), tetrapyrrole derivatives (e.g., porphyrin, chlorophyll and bacteriochlorophyll derivatives, phtalocyanine or bilirubin), Pacific Blue, Pacific Orange, or Lucifer yellow.

New generation fluorophores, many of which are proprietary, include CF dye, NBD, BODIPY (BODIPY TMR, BODIPY F2), Alexa Fluors (Alexa Fluor 430, 488, 532, 546, 555, 568, 594, 633, 660, 680), DyLight Fluor, Atto, Tracy, FluoProbes, R- Phycoerythrin (PE), Cy3, Cy3.5, Cy2, Cy5, Cy7, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP-Cy5.5 conjugates, APC-Cy7 conjugates, TRUC, Red 613, TruRed, PerCP (peridinin chlorphyll protein), MegaStokes Dyes, HEX, FAM, EDANS, IAEDANS, Allophycocyanin (APC), QSy7, QSy9, XL665, or d2. These fluorophores are often more photostable, brighter, and/or less pH-sensitive than dyes with comparable excitation and emission.

An important class of fluorophores used in fluorescence detection assays is macromolecular complex of fluorescent metal ions, preferably complexes of rare earth lanthanides ions such as Europium, Terbium, Samarium ions and the like. It is difficult to generate fluorescence of lanthanide ions by direct excitation because of the ions' poor ability to absorb light. Therefore, lanthanides are often complexed with organic molecules that harvest light and transfer it to the lanthanide ion through intramolecular, non-radiative processes.

Fluorescent proteins are also contemplated herein as fluorophores. Two sub-classes of fluorescent proteins are encompassed by the present invention: (i) proteins with natural fluorescence such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), Red fluorescent protein (RFP) and other similar proteins known in the art; and (ii) tagged proteins, namely, any other proteins which were chemically modified to covalently bind a fluorescent molecule. For example, antibodies tagged with fluorescent moieties, or streptavidin tagged with fluorescent moieties. Radioactively labeled moiety as used herein refers to a moiety in which a radioactive isotope replaces the natural isotope. Non- limiting example of radioactively labeled moieties are proteins in which any of the atoms H, C, N and/or S is replaced with a radioactive isotope thereof, thereby labeling the desired protein.

Fluorescent enzymes which function as detectable moieties include Luciferase,

Horse Raddish Peroxidase (HRP), beta-galactosidase and others known in the art or to be discovered in the future. Electron-dense reagents are reagents that possess a sufficient electron density to be individually visible in transmission electron spectroscopy. They usually comprise a heavy metal such as gold which accounts for the high electron scattering properties of these reagents. Non-limiting examples of electron density reagents include tricyanoheptakis [4,4',4"-phosphinidynetris(benzomethanamine)] undecagolg and derivatives thereof.

In certain embodiments of the present invention, the detectable moiety is a fluorescent moiety, more preferably a macropolycyclic complex of a metal ion. In most preferred embodiments, the macropolycyclic complex is a macropolycyclic complex of a rare earth metal ion selected from a chelate or a cryptate of a lanthanide metal ion.

The synthetic Ubs and Ubls of the present invention may comprise any of the known cryptates or chelates used in HTS tests or any of the cryptates and chelates to be designed in the future.

In preferred embodiments, the detectable moiety of the synthetic Ub or Ubl is a cryptate of an ion of a rare earth (lanthanide) metal selected from Lanthanum (La³⁺); Cerium (Ce³⁺); Praseodymium (Pr³⁺); Neodymium (Nd³⁺); Promethium (Pm³⁺); Samarium (Sm³⁺); Europium (Eu³⁺); Gadolinium (Gd³⁺); Terbium (Tb³⁺); Dysprosium (Dy³⁺); Holmium (Ho³⁺); Erbium (Er³⁺); Thulium (Tm³⁺); Ytterbium (Yb³⁺); or Lutetium (Lu³⁺).

In more preferred embodiments, the detectable moiety is a cryptate of Eu³⁺, Tb³⁺, Gd³⁺ or Sm³⁺, most preferably it is an Europium cryptate (herein designated "Eu-cryptate") or a Terbium cryptate (herein designated "Tb-cryptate").

Cryptand is a macropolycyclic compound schematically represented herein by the formula:

wherein Z is a trivalent or tetravalent atom such as nitrogen, carbon or phosphorus, and the two Z atoms are linked via at least three hydrocrabon chains represented in the scheme above by . Each one of these hydrocarbon chains, independently, may contain one or more heteroatoms such as N, O or S, and/or be substituted by at least one moiety containing one or more N, O or S, and at least one of said hydrocarbon chain, preferably all three of them, is interrupted by or substituted with at least one donor unit, wherein "donor unit" as used herein denotes an energy donor molecular moiety that possesses a higher triplet energy level than the emission level of a lanthanide ion; and R is a pair of electrons, H, OH or an amino group.

When the cryptand houses a rare earth ion, the macropolycyclic complex is termed "cryptate". The donor unit has an electron system capable of populating the triplet state Ύι by intersystem crossing after excitation of the singlet state S_1; following the absorption of luminous energy. This non-radiant energy is then transferred form the excited donor to the resonance level of the lanthanide ion embedded within the cryptand. In order to obtain a strong fluorescence of the lanthanide cryptate it is necessary for the triplet energy of the donor to be greater than that of the resonance level of the ion and have a sufficient life-time. Usually the donor unit is selected from phenyl, bisphenyl, triphenyl, phenanthroline, anthracene, bispyridine, pyridine, tripyridine, quinoline and the like. Examples of various cryptands and cryptates that may be used for the purpose of the invention are disclosed e.g., in US 4,927,923 and US 5,162,508.

Whereas excitation of a rare earth ion produces a very weak fluorescence because rare earth generally have low molar absorption coefficient ε, excitation of the donor unit of the cryptate makes it possible to enhance the fluorescence characteristics of the ion. Cryptates of rare earth ions are therefore most suitable fluorescent tracers for the purpose of designing detectable synthetic ubiquitins in accordance with the present invention.

In preferred embodiments of the invention, the energy donating unit of the cryptate is pyridine , optionally substituted,

and the cryptate may contain one, two, three, four, five, six or more energy donor units on the same hydrocarbon chain of the cryptand (preferably linked together), or on separate hydrocarbon chains.

The preferred cryptates used in accordance with the present invention are selected from trisbipyridine (TBP), pyridine bispyridine (PBP), trisbipyridine tetracarboxylate (TBP4COOH) and pyridine bispyridine tetracarboxylate (PBP4COOH) cryptates such as, but not limited to, TBP Eu-cryptate, TBP Tb-Cryptate TBP Sm-crypate, PBP Eu-cryptate, PBP Tb-Cryptate, PBP Sm-crypate, TBP4COOH Eu-cryptate, TBP4COOH Tb-cryptate, TBP4COOH Sm-crypate, PBP4COOH Eu-cryptate, PBP4COOH Tb-cryptate or PBP4COOH Sm-crypate.

In certain more preferred embodiments, the cryptate is a PBP cryptate having the formula I or a TBP having the formula II:

wherein

X is an ion of a lanthanide metal selected from La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺, or Lu³⁺, more preferably Eu³⁺, Tb³⁺, Gd³⁺ or Sm³⁺;

Ri-Rn each independently is selected from H, halogen, C C₂o hydrocarbyl, heteroaryl, heterocyclyl, alkoxy (OR), SR, COOR, COSR, -COR, -NRR', -NR(CH₂)_nNR', - CONRR' , -S0₂NRR', NRS0₂R'-, -C(0)-NR(CH₂)_n-NRR', cyano (-C≡N), sulfonyl (-S0₂), - S0₃R, -S0₂R, nitro, hydrazino (-NR-NRR'), aryl amino (-NR-(C₆-C₁₄)aryl), nitrobenzoyl (- C(0)-C₆H₄-N0₂), -NR-COR', =N-OR, =N-NR, -C(=NR)-NRR', -(R)N-C(=NR)-NRR\ >C=NR, -(CH₂)_n-NR-COR', -(CH₂)_n-CO-NRR', -0-(CH₂)_n-OR, -0-(CH₂)_n-0-(CH₂)_n-R, - PO₃RR', imidazolyl, succcinimido, haloacetyl, or dihalotriazinyl (selected from dihalo 1,2,3-triazinyl, dihalo 1,2,4-triazinyl or dihalo 1,3,5-triazinyl), wherein R and R' each independently is H or Q-C^ hydrocarbyl, preferably Q-C^ alkyl, or R and R' together with the nitrogen atom to which they are attached form a 5-6 membered saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom is optionally substituted by lower alkyl (namely Ci-C₆ alkyl), aralkyl selected from (C₆-C₁₄)aryl-(C₁-C₁o)alkyl, haloalkyl selected from iodo-, bromo- chloro- or fluoro-(C₁-C₁o)alkyl, or hydroxyalkyl selected from hydroxy-(C₁-C₁o)alkyl; and n is an integer of 2 to 10. The hydrocarbyl may optionally be interrupted by one or more heteroatoms selected from N, S and/or O and/or by aryl or heterocyclyl.

In certain embodiments, in the cryptate of formula I or II above X is Eu³⁺, Gd³⁺, Tb³⁺, or Sm³⁺; R_1; R₂, R₃, R5, R₆, Rs, R9, R11 , Ri₂, Ri₄, R15, Ri6 and R₁₇ each independently is selected from H, alkoxy, COOR, COSR, -COR, -NRR', -NR(CH₂)_nNR', -CONRR', - S0₂NRR', NRS0₂R'-, -C(0)-NR(CH₂)_n-NRR', -C≡N, -S0₂, -S0₃R, -S0₂R, or nitro, wherein n, R and R' are as defined above; and R₁₀ and R₁ each independently is H or COOH.

In preferred embodiments, X is Eu³⁺ or Tb³⁺; R_1; R₂, R₃, R5, R₆, Rs, R₉, R11, Ri₂, R14,

R₁₅, R₁₆ and R₁₇ each is H, and R₄, R₇, R₁₀ and R₁ each independently is H or COOH.

The a-amino acid substituted by a detectable moiety as provided by the present invention, is a compound of the formula Γ :

wherein

X is a spacer and is a linear carbon chain of 2-20 atoms optionally interrupted by one or more nitrogen, sulfur and/or phosphorous atoms, or X is a moiety -CH₂-X'- having a length corresponding to the length of a linear chain of 2-20 carbon atoms, and X' is selected from C₁-C₂o hydrocarbyl, C₃-C₁₂ heterocyclyl or C₆-C₁₄ heteroaryl;

W is -C(R)₂-, O, S or -N(R)-, wherein R is H, Ci-C₂o hydrocarbyl or C₃-C₁₂ heterocyclyl; and

Y is a detectable moiety;

or an enantiomer or a pharmaceutically acceptable salt thereof.

In certain embodiments X is a linear carbon chain interrupted by one or more oxygen atoms, preferably the chain contains one to 6 ethylene glycol -(CH₂-CH₂-0)- units.

The hydrocarbyl, heterocyclyl or heteroaryl of X' and W may be substituted by one or more radicals selected from C₁-C₂o hydrocarbyl, halogen, aryl, C₆-C₁₄ heteroaryl, C₃-C₁₂ heterocyclyl, nitro, epoxy, epithio, cyano, -SR, -COR, -COOR, -COSR, OR, -CONRR', - S0₃R, -S0₂R, -NRS0₂R', -S0₂NRR' -NR-COR', -NRR', =N-OR, =N-NRR' , -C(=NR)- NRR', -NR-NRR', -(R)N-C(=NR)-NRR\ >C=NR, -(CH₂)_n-NR-COR\ -(CH₂)_n-CO-NRR\ - 0-(CH₂)_n-OR, -0-(CH₂)_n-0-(CH₂)n-R, -P0₂HR, -P0₃RR', imidazolyl, succcinimido, haloacetyl or dihalotriazinyl, wherein R and R' each independently is H, C C₂o hydrocarbyl or C₂-C₁₂ heterocyclyl, or R and R' together with the nitrogen atom to which they are attached form a 5-6 membered saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom is optionally substituted by lower (CrC₆) alkyl, aralkyl, preferably (C6-C₁₄)aryl-(C₁-C₁o)alkyl, haloalkyl, preferably chloro-, bromo-, iodo- or fluoro-(C₁-C₁o)alkyl, or hydroxyalkyl, preferably hydroxy-(C₁-C₁o)alkyl. The hydrocarbyl is optionally further interrupted by one or more heteroatoms selected from N, S and/or O and/or by aryl or heterocyclyl.

More preferably, the labeled a-amino acid of the invention is a compound of the formula Γ a

(I'a) (I'b)

wherein

R is H or Ci-C₂o hydrocarbyl;

Y is an europium cryptate or a terbium cryptate moiety;

n is an integer of 2-10, preferably 2, 5 or 10;

m is an integer of 1 to 6, preferably 1 or 3; and

p is an integer of 1 to 5, preferably 1 or 2.

Particular examples of a-amino acids used in accordance with the invention for obtaining a-amino acids substituted by a detectable moiety include, but not limited to, 2,4- diaminobutanoic acid, 2,7-diaminoheptanoic acid, and 2,12-diaminododecanoic acid of the formula H₂N-CH(COOH)-(CH₂)_n-NH₂ wherein n is 2, 5 and 10, respectively; 2-amino-4-(2- aminoethoxy) butanoic acid and 2-amino-4-(2-(2-(aminomethoxy)ethoxy)ethoxy)butanoic acid of the formulae:

COOH COOH I I

H2N-CH-CH2CH2-O-CH2-CH2-NH2 and H₂N-CH-(CH₂CH₂-0)3-CH₂-NH₂ , respectively. In preferred embodiments, the a-amino acid of the invention of formula I'a or I'b is labeled with europium pyridine bispyridine (PBP) cryptate. In more preferred embodiments, the labeled a-amino acid is a compound of the formula I'a:

wherein n is an integer of 2-10, preferably 2, 5 or 10.

As used herein, the term "hydrocarbyl" means any straight or branched, saturated or unsaturated, acyclic or cyclic, including aromatic, hydrocarbyl radicals, of 1-20 carbon atoms, preferably of 1 to 10, more preferably 1 to 6, most preferably 2-5 carbon atoms, selected from an alkyl, alkenyl, alkynyl, carbocyclyl, aryl or an aralkyl radical.

In certain embodiments, the hydrocarbyl is a lower alkyl radical of 1-6, preferably of

2-5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl isobutyl, tert-butyl, pentyl or hexyl.

In one embodiment, the alkyl group has 10 carbon atoms or more, e.g. -CioH₂₁, - C₁₅H₃i, -C₁₆H₃₃, -C₁₇H₃₅, -Ci₈H₃₇, -C₂oH₄i, and the like.

In another embodiment, the Ci-C₂o hydrocarbyl is a straight or branched C₂-C₂o alkenyl or alkynyl radical, more preferably of 2-5 carbon atoms, e.g. vinyl, prop-2-en-l-yl, but-3-en-l-yl, pent-4-en-l-yl, hex-5-en-l-yl, ethynyl, propargyl, and the like.

The linear or branched alkyl, alkenyl or alkynyl defined above may be interrupted by one or more heteroatoms selected from O, S and/or N, and/or by an aryl e.g. phenyl, or heterocyclic ring, e.g. pyridyl, and may optionally further be substituted by one or more radicals selected from halogen, aryl, heteroaryl, heterocyclyl, nitro, epoxy, epithio, cyano, - SR, -COR, -COOR, -COSR, -OR, -CONRR', -OS0₃R, -S0₃R, -S0₂R, -NRS0₂R', - S0₂NRR' -NR-COR', -NRR', =N-OR, =N-NRR' , -C(=NR)-NRR', -NR-NRR', -(R)N- C(=NR)-NRR', >C=NR, -(CH₂)_n-NR-COR\ -(CH₂)_n-CO-NRR\ -0-(CH₂)_n-OR, -0-(CH₂)_n- 0-(CH₂)_n-R, -P0₂HR, -PO₃RR', imidazolyl, succcinimido, haloacetyl, or dihalotriazinyl, wherein R and R' each independently is H, hydrocarbyl as defined above, or heterocyclyl, or R and R' together with the nitrogen atom to which they are attached form a 5-6 membered saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom is optionally substituted by lower alkyl, aralkyl, haloalkyl or hydroxyalkyl.

In a further embodiment, the hydrocarbyl is a carbocyclyl derived from a C₃-C₂₀ monocyclic or polycyclic ring, saturated or partially unsaturated, preferably C₃-C₁₄, more preferably C₃-C₇ cycloalkyl, cycloalkenyl or cycloalkynyl radical containing only carbon atoms in the ring(s), such as cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl.

The hydrocarbyl may further be aryl or aralkyl, wherein the term "aryl" as used herein refers to a C₆-C₁₄ aromatic carbocyclic group having 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, consisting of a single, bicyclic or tricyclic ring, such as phenyl, naphthyl, carbazolyl, anthryl, phenanthryl and the like, and the term "aralkyl" refers to a radical derived from an arylalkyl compound wherein the aryl moiety is preferably a C₆-C₁₈, more preferably a C₆-C₁₄ aryl such as benzyl, phenanthryl and the like.

The term "heterocyclyl" or "heterocyclic moiety" means a radical derived from a saturated or partially unsaturated, monocyclic, bicyclic or tricyclic heterocycle of 3-12, preferably 5-10, more preferably 5-6 members in the ring wherein 1 to 3 of the heterocyclic ring(s) members are heteroatoms selected from O, S and/or N. Particular examples are dihydrofuryl, tetrahydrofuryl, pyrrolynyl, pyrrolydinyl, dihydrothienyl, dihydropyridyl, piperidinyl, quinolinyl, piperazinyl, morpholino or 1,3-dioxanyl.

The terms "heteroaryl" or "heteroaromatic moiety" refer to a mono- or polycyclic heteroaromatic ring that may comprise both carbocyclic and heterocyclic rings, wherein the heterocyclic ring(s) may contain 1 to 3 heteroatoms selected from O, S and/or N. Particular examples are pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,4-triazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, benzofuryl, isobenzofuryl, indolyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, benzodiazepinyl, and other radicals derived from further polycyclic heteroaromatic rings.

Any "carbocyclyl", "heterocyclyl", "aryl" or "heteroaryl" may be substituted by one or more radicals as defined above for hydrocarbyl. As defined herein, "a 3-7 membered saturated ring" formed by R and R' together with the N atom to which they are attached may be a ring containing only N atoms such as aziridino, pyrrolidino, piperidino, piperazino, azepino or diazepino, or it may contain a further heteroatom selected from O and S such as morpholino or thiomorpholino. The further N atom in the piperazine ring may be substituted by alkyl, e.g. lower C -C alkyl, that may be substituted by halogen, OH or amino, aralkyl, haloalkyl or hydroxyalkyl.

The term "halogen", as used herein, refers to fluoro, chloro, bromo or iodo.

Chemical synthesis of ubiquitin

The ubiquitin protein consists of 76 amino acids, and has a molecular mass of

8564.37 Da. The human ubiquitin sequence herein designated SEQ ID NO: 1 is:

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDG RTLSDYNIQKESTLHLVLRLRGG

A three dimensional structure of Ub is shown in Fig. 1. In solvent accessibility calculations made by the inventors, various segments of Ub were identified according to their relative exposure to the solvent. The "exposed", "buried" and "other" residues are shown in Fig.l in blue, green and gray, respectively. There are 48 residues that are well exposed to the solvent, 15 are buried, and 13 other residues have varying degrees of solvent exposure.

In preferred embodiments, the synthetic Ubs of the present invention are designed such that at least one of the solvent-exposed amino acids is replaced by a non-natural amino acid labeled with a detectable moiety as defined above.

In certain embodiments, only one of the "exposed" amino acids is replaced by a labeled amino acid. In other embodiments, two, three, four, five or more exposed amino acids are replaced with labeled amino acids.

In most preferred embodiments, in the synthetic Ub provided by the present invention only one amino acid is replaced with an a-amino acid comprising a detectable moiety. These preferred Ubs are herein identified by their sequence identification numbers

(SEQ ID NOs) 2 to 34. In the synthetic Ubs of SEQ ID NOs 2-34 presented below, the amino acid of the natural Ub that is replaced with a labeled a-amino acid is indicated in brackets: SEQ ID NO: 2 (Met 1); SEQ ID NO: 3 (Gin 2); SEQ ID NO: 4 (Thr 9); SEQ ID NO: 5 (Thr 14); SEQ ID NO: 6 (Blu 16); SEQ ID NO: 7 (Glu 18); SEQ ID NO: 8 (Pro 19); SEQ ID NO: 9 (Ser 20); SEQ ID NO: 10 (Glu 24); SEQ ID NO: 11 (Asn 25); SEQ ID NO: 12 (Ala 28); SEQ ID NO: 13 (Gin 31); SEQ ID NO: 14 (Asp 32); SEQ ID NO: 15 (Lys 33); SEQ ID NO: 16 (Glu 34); SEQ ID NO: 17 (Gly 35); SEQ ID NO: 18 (Pro 38); SEQ ID NO: 19 (Asp 39); SEQ ID NO: 20 (Ala 46); SEQ ID NO: 21 (Glu 51); SEQ ID NO: 22 (Asp 52); SEQ ID NO: 23 (Gly 53); SEQ ID NO: 24 (Ser 57); SEQ ID NO: 25 (Asp 58); SEQ ID NO: 26 (Thr 66); SEQ ID NO: 27 (His 68); SEQ ID NO: 28 (Val 70); SEQ ID NO: 29 (Leu 71); SEQ ID NO: 30 (Arg 72); SEQ ID NO: 31 (Leu 73); SEQ ID NO: 32 (Arg 74); SEQ ID NO: 33 (Gly 75); and SEQ ID NO: 34 (Gly 76).

The synthetic Ubs and Ubls of the invention may be prepared by a method based on the Fmoc-based linear solid-phase peptide synthesis (Fmoc-SPPS) disclosed in Oualid et al. (Oualid et al., 2010), using pseudoproline building blocks and dimethoxybenzyl (DMB) dipeptides incorporation in order to prevent the formation of folded and/or aggregated intermediates on-resin, events that can hamper cleavage of the Fmoc group and/or further elongation of the Ub chain. According to this method, six dipeptide building blocks are simultaneously incorporated into six positions in the Ub sequence using a Wang resin and standard coupling conditions (namely, 4 equiv Fmoc-protected amino acid, 4 equiv benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 8 equiv N,N-Diisopropylethylamine (DIPEA), and single coupling reactions).

Alternatively, the synthetic Ubs and Ubls can by prepared by a linear solid-phase peptide synthesis on a hyperacid-labile trityl resin with the N-terminal methionine residue protected with a Boc group as described in Oualid et al. Total deprotection of the product may be obtained with 95% trifluoroacetic acid (TFA), and generation of the Ub may be obtained through selective cleavage from the resin with 20% hexafluoro-2-propanol in CH₂C1₂ (e.g. as described in Bollhagen et al., 1994).

Application of the synthetic Ubs and Ubls of the invention in high throughput screening assays

The synthetic Ub or Ubl proteins of the invention comprising at least one amino acid labeled with a detectable moiety may be used in any of the assays that monitor ubiquitination of target proteins including polyubiquitination of target proteins, selfubiquitination (namely formation of di-or polyUb chains) and Ub-mediated proteolysis. These synthetic Ubs and Ubls are particularly useful for high throughput screening (HTS) methods for discovery of ubiquitination inhibitors as drug candidates.

Thus, in one aspect, the invention provides a HTS method for assessing the binding of Ub or Ubl (ubiquitination) to a target protein in the presence of a test compound.

The target protein to be ubiquitinated used in the method of the invention may be a

E3 ubiquitin ligase protein, such as, but not limited to, a POSH protein, a cbl-b protein, a PEM-3-like protein, Nedd4, WWP1, or a fragment of said E3 ligase protein that is ubiquitinated. Other target proteins are selected from: (i) proteins that contain the ubiquitin and ubiquitin-like recognition elements such as S5a, TAB2, TAB3 and the like; (ii) an E2 ubiquitin and ubiquitin-like conjugation enzymes such as UbcH5a-c, UbcH7, UbcHIO, Ubc9, Ubcl2 and the like; (iii) an El ubiquitin and ubiquitin-like activating enzyme such as Ubal, Aos/Uba2, APPBP1/Uba3 and UBE1L 4; and (iv) a ubiquitin or ubiquitin-like substrates such as p53, p27, and the like.

The drug screening methods contemplated by the present invention include:

Absorbance assays, wherein the synthetic Ub or Ubl proteins comprise a detectable moiety capable of absorbing light/energy. Proteins such as Ub and Ubl have natural absorbance at wavelengths of 260 to 280 nm therefore, any absorbing moiety with absorbance above these wavelengths can be typically used to tag the synthetic Ub or Ubl proteins. Specific example include, but not limited to coomasie blue, congo red, different tetrazolium salts with efficient absorbance etc.

Fluorescence intensity (FI) assay. Fluorescent probes are used for studying the binding of ubiquitin by assessing the difference between the quenching rate of the bound verses that of the free Ub.

For the FI assay according to the present invention, the synthetic Ub or Ubl contains one or more detectable a-amino acid wherein the detectable moiety comprises a fluorophore such as any of the fluorophores mentioned herein that is capable of absorbing light and fluorescence. Preferred fluorophores are sulfonamide derivatives such as dansyl chloride (5- (dimethylamino)naphthalene-l-sulfonyl chloride) and dansyl amide that react with aliphatic and aromatic amines in the target protein to produce stable blue- or blue-green-fluorescent sulfonamide adducts. Other suitable dyes include fluorescein, rhodamin, BODIPY TMR dye, BODIPY FL dye, Oregon Green 488 dye, Alexa Fluor 488 dye, Oregon Green 514 dye Alexa Fluor 594 dye and the like. Fluorescence Polarization (FP). Fluorescence polarization modulates the effect on orientation and mobility caused by Ub binding to a target protein. Light emitted by a fluorescently labeled Ub will be relatively anisotropic (non-polarized) when the ubiquitin has a high degree of rotational freedom. As the rotational freedom decreases due Ub conjugation with a target protein, the emitted light becomes increasingly isotropic (polarized). Thus, by detecting the polarization of light emitted by a fluorescently labeled Ub protein, it is possible to assess the degree to ubiquitination of a target protein.

In FP assay performed in accordance with the invention, a synthetic Ub or Ubl is used comprising at least one amino acid labeled with a fluorescent moiety which may be any of the fluorophores mentioned above. However, new generation fluorophores are preferred since they generally produce less perturbation of target protein-binding affinity and other activity parameters as compared to conventional (old generation) dyes such as fluorescein and rhodamine. Preferred Ubs and Ubls are those labeled with BODIPY dyes such as, but not limited to, BODIPY TMR dye and BODIPY FL dye. Other preferred Ubs and Ubls for use in FP are those labeled with Oregon Green 488 dye, Alexa Fluor 488 dye, Oregon Green 514 dye and Alexa Fluor 594 dye, lanthanides chelates, coumarin and the like.

In certain embodiments, E3-mediated ubiquitination is assayed in the absence or presence of a test compound using any of the labeled synthetic Ub or Ubls mentioned above, wherein the changes in rotational freedom of Ub or Ubl due to binding to E3 is determined by measuring the fluorescence polarization of the Ub or Ubl. If the fluorescence polarization of the labeled Ub or Ubl in the presence of the test compound is altered relative to the fluorescence polarization of the labeled Ub or Ubl in the absence of the test compound, a potential modifier of E3 ubiquitination is identified.

In certain other embodiments, FP assay is employed in order to evaluate ubiquitination of El, E2 or other Ub binding proteins such as S5a, TAB2, or TAB3, or substrate proteins such as p53 and p27.

Time Resolved Fluorescence (TRF). TRF takes advantage of the unique properties of the rare earth elements lanthanides. Thus, the synthetic Ub or Ubl proteins for a TRF assay will comprise, in accordance with the present invention, at least one amino acid tagged or labeled with a macropolycyclic complex of a lanthanide ion such as Eu³⁺, Tb³⁺,

Dy³⁺ or Sm³⁺, more preferably a cryptate of one of these ions. Fluorescence Resonance Energy Transfer (FRET). FRET is employed successfully as a platform where a signal may be produced by the proximity of two fluorescent dyes. FRET uses two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source triggers an energy transfer to the acceptor provided they are within a given proximity to each other. The acceptor in turn emits light at its given wavelength.

Any couple of fluorescence moieties wherein the emission of one of them (the donor) overlaps the absorbance of the second (the acceptor) may be used in accordance with the invention. In most applications, the donor and acceptor dyes are different, in which case FRET can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. When the donor and acceptor are the same, FRET can be detected by the resulting fluorescence depolarization.

In certain ubiquitin FRET assays, a target protein is the acceptor and is directly or indirectly labeled with a dye, and a FRET signal is produced upon ligation of a labeled synthetic ubiquitin thereto. In other ubiquitin FRET assays, a synthetic Ub of the invention is labeled with two dyes and a mixture of the labeled Ubs is used in a reaction where the FRET signal originates from the ubiquitin chain formation. A much greater signal to noise ratio may be achieved with this method, as numerous ubiquitin-ubiquitin signaling units are present in one chain as compared to any given target protein.

Non-limiting examples for pairs of fluorophores that can be used in accordance with the invention, wherein each dye in a pair may be attached as a detectable moiety to one or more amino acids in the synthetic Ub or Ubl of the invention, include (the typical values of R0 for some of these pairs are indicated in brackets): Fluorescein - Tetramethylrhodamine (R0=55); IAEDANS- Fluorescein (R0=46); EDANS - EDANS (R0=33); Fluorescein - Fluorescein (R0=44); BODIPY FL - BODIPY FL (R0=57); and Fluorescein - QSY 7 and QSY 9 dyes (R0=61).

Time Resolved-FRET (TR-FRET). TR-FRET unites TRF and FRET principles, and brings together the low background fluorescence benefits of TRF with the homogeneous assay format of FRET. This method is also known as HTRF (homogeneous time resolved fluorescence).

The life time of common fluorophores used in standard FRET is in the nanosecond range. Therefore, interference from autofluorescent compounds or scattered light which is also on the nanosecond timescale, these factors can negatively impact standard FRET assays.

TR-FRET assays use a long-lifetime lanthanide chelate or cryptate as the donor species. Lanthanide chelates and cryptates are unique in that their excited state lifetime (the average time that the molecule spends in the excited state after accepting a photon) can be on the order of milliseconds or even longer. This is in sharp contrast to the lifetime of common fluorophores used in standard FRET assays.

TR-FRET assays overcome interferences from scattered light and non-specific fluorescence of nanoseconds life time by measuring FRET after a suitable delay of typically 50 to 100 microseconds post excitation. This delay also avoids interference from direct excitation due to the non-instantaneous nature of the flash lamp excitation source.

Any of the known lanthanide chelates or cryptates may be used in TR-FRET; however, for HTS assays terbium and europium chelates and cryptates are preferred, more preferably terbium or europium cryptates. The unique advantages offered by terbium over europium when used as the donor species is that terbium-based TR-FRET assays can use common fluorophores such as fluorescein or fluorescein-labeled reagents as the acceptor, whereas europium-based systems employ allophycocyanins (APC) or biotinylated molecules as acceptors, that must then be indirectly labeled via streptavidin-mediated recruitment of APC.

In accordance with preferred embodiments of the present invention, the synthetic ubiquitin or ubiquitin-like protein modifier will contain a cryptated lanthanide ion, preferably Eu³⁺, Sm³⁺ or Tb³⁺ as a moiety covalently attached to the side chain of one or more amino acids in its sequence. The acceptor may be any fluorophore or dye that absorbs at the emission wave length of the donor. For example, acceptors suitable for the donor europium must absorb around 620 nm. Non-limiting examples of acceptors useful in TR- FRET assays include fluorescein, XL665, a modified allophycocyanin or d2.

Bioluminescence Resonance Energy Transfer (BRET). BRET assay is based on the efficient resonance energy transfer (RET) between a bioluminescent donor moiety and a fluorescent acceptor moiety. The donor is a fusion protein usually containing Renilla luciferase (Rluc) which catalyzes, in the presence of oxygen, the transformation of the coelenterazine substrate DeepBlueC (DBC) into coelenteramide with concomitant light emission peaking at 395 nm (blue light). When a suitable acceptor is in close proximity, the blue light is captured by RET. Any synthetic Ub or Ubl comprising at least one amino acid substituted by a fluorescent acceptor moiety that can efficiently absorb the luminescence energy emitted by the Rluc/DBC reaction is suitable for BRET assessment of ubiquitination of a target protein attached to DeepBlueC (DPC).

In alternative embodiments, in BRET assays performed in accordance with the invention, the synthetic Ub or Ubl contains at least one amino acid substituted by DBC moiety and ubiquitination of a target protein is assessed in the presence of Rluc or the fusion protein, wherein said target protein contains the acceptor of blue light emitted by the Ub or Ubl.

Scintillation Proximity assay (SPA). A radio assay in which radioactively labeled moieties such as Tritium-( 3 H), 125 I, 33 P or 14 C-labeled moieties are used to label the ubiquitin. In this assay, the ubiquitination target bind the SPA beads containing a scintillant through suitable antibodies or other means bringing the ubiquitin to a close proximity with the SPA beads such that it can stimulate the scintillant contained within the beads to emit light.

Alpha Screen. Amplified Luminescent Proximity Homogeneous Assay, or Alpha

Technology (AlphaScreen®, AlphaLISA® and Surefire®), is a bead-based chemistry used to study biomolecular interactions. This homogeneous assay measures the interaction of two molecules e.g., ubiquitin and E3 or a substrate protein, bioconjugated to "donor" and "acceptor" polystyrene beads. When interactions occur between these molecules, the beads are brought close enough to cause a chemical reaction leading to energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal, which is directly proportional to the amount of binding.

Alpha screen employs oxygen channeling chemistry and exploits the short diffusion distance of singlet oxygen to initiate a chemiluminescent reaction near the site where it was formed. Because the lifetime of the singlet oxygen reactive species in water is very short (approximately four microseconds), the donor and acceptor beads need to be bound to one another to generate a signal. Beads that do not bind exhibit a very low singlet oxygen concentration that contributes minimally to the background signal.

Thus, in another aspect, the present invention provides a method for identifying an inhibitor of ubiquitination of a target protein or of Ub-mediated proteolysis in which a synthetic Ub or Ubl of the invention is used.

In certain embodiments, the method for identifying an inhibitor of ubiquitin- mediated proteolysis comprises the steps of: (i) measuring binding of a synthetic ubiquitin or ubiquitin-like modifier protein in which at least one amino acid residue is replaced with a residue of an a-amino acid substituted by a detectable moiety, to a target protein in the presence and absence of a test compound; and

(ii) identifying the test compound as an inhibitor of ubiquitination when a decreased signal is detected in the presence of the test compound compared to the signal detected in the absence of the test compound.

In preferred embodiments, the HTS method provided by the invention is TR-FRET or HTRF employing a synthetic Ubs or Ubls of the invention.

For a typical TR-FRET ubiquitination assay, a synthetic ubiquitin or ubiquitin-like modifier proteins comprising a fluorescent moiety and a target protein to be ubiquitinated comprising a second fluorescent moiety, are incubated with ubiquitin-conjugating enzymes (El, E2, and E3), and ATP. The enzymes conjugate the labeled ubiquitins onto the target protein, resulting in mono- or polyubiquitination. One of the fluorophores, the donor, has a shorter excitation wavelength than the excitation wavelength of the second fluorophore, the acceptor. Preferably, the difference in excitation wavelengths of the two fluorophores is 100-200 nm.

At a time interval of 50-100 microseconds after the donor fluorophore has been excited, the emission spectra of both the donor and acceptor is measured. Because it is important to measure energy transfer to the acceptor without interference from the emitting donor, the acceptor emission is collected as far as possible from emission peaks of the donor. In addition, a filter is most often used when measuring emitted light from the acceptor, centered at the peak-emission wavelength and having a bandwidth of 10-30 nm. This filter screens out small overlapping contributions of donor emitted fluorescence.

The first emission peak of the donor is measured, preferably using a suitable bandwidth filter that isolates this peak from acceptor emission that "bleeds through". Then, the emission of the acceptor due to FRET is referenced or "ratioed" to the emission of the donor. The ratiometric nature of such a value corrects for differences in assay volumes between wells, and corrects for quenching effects due to colored compounds. Since the TR- FRET value is a unitless ratio derived from the underlying donor and acceptor signals which depend on instrument settings (such as instrument gain), this value is not dependent on the signal-to-noise (S/N), signal-to-background (S/B), and the resulting "top" and "bottom" of an assay window of the assay settings and instrument used. The extent of target protein ubiquitination is directly related to the TR-FRET signal. In general, an increase in the TR FRET signal signifies the ubiquitination of the target protein, whereas no increase in the TR-FRET signal would suggest that the target protein is not ubiquitinated.

In HTS applications, a test compound is introduced to measure the effectiveness of the compound in inhibiting or promoting ubiquitination of the target protein. If the compound inhibits the ubiquitination reaction, a decrease in the TR-FRET signal (compared to control) would be observed due to a decrease in the ubiquitination of the target protein. Conversely, an increase in the TR-FRET signal would be observed if the compound promotes the ubiquitination of the target protein.

Several formats of HTS HTRF ubiquitination assays are provided by the present invention. In certain embodiments, the HTRF method employs epitope tags. The Anti- Epitope ubiquitination assay uses a lanthanide chelate or cryptate (preferably Eu³⁺-cryptate or Tb³⁺-cryptate) labeled anti epitope antibody as the donor, and a synthetic Ub or Ubl of the invention tagged with a dye, for example fluorescein. The Ub target protein, tagged with an epitope, is mixed with the labeled Ub in the presence of El, E2, E3 and ATP, in the absence or the presence of a test compound. The detection reagent, a labeled anti-epitope antibody is then added to the ubiquitination reaction to complete the TR-FRET pairing. The addition of dithiothreitol (DTT) is optional, and may be required to activate some ubiquitin- conjugating enzymes. To stop the ubiquitination reaction, EDTA can be added at a concentration equal to the Mg²⁺ concentration within the reaction to prevent ATP hydrolysis.

An example for a TR-FRET pair that can be used in accordance with this embodiment is a synthetic ubiquitin comprising an amino acid substituted by a fluorescein moiety, and the protein tagged with an epitope is GST-UbcHl (E2-25K). The anti-epitope antibody is Tb-anti-GST. Other types of ubiquitination targets may be E3 ubiquitin ligases, Ub/Ubl binding proteins, ubiquitination substrates and other. The tags may be any other tag beside GST and it may also be antibodies for the ubiquitination target itself. The anti- epitope antibody may be labeled with other lanthanide ions such as Eu³⁺, Dy³⁺ or Sm³⁺, more preferably a cryptate of one of these ions.

The Anti-Epitope ubiquitination assay can be used when the target protein contains an epitope tag. Since the TR-FRET donor is located on the introduced antibody, this assay cannot be used for the detection of inhibitors of mono- or polyubiquitination because ubiquitin chain formation is not required to complete the TR-FRET pairings.

In certain embodiments, the HTRF assay is an intra-chain ubiquitination assay. According to these embodiments, a mixture of synthetic Ub and/or Ubl is used wherein some of the Ubs or Ubls in the mixture comprise one or more amino acid substituted by a fluorescent moiety which serves as the donor, and the other Ubs or Ubls comprise one or more amino acid substituted by a fluorescent moiety which is the acceptor. In the ubiquitination reaction, these two kinds of labeled Ubs or Ubls are mixed with the protein to be ubiquitinated, El, E2 and E3, as well as ATP, in the absence or presence of a test compound.

The intra-chain ubiquitination assay is used for detecting the polyubiquitination of a target protein. Since both the TR-FRET donor and acceptor are located on ubiquitin itself, no development step or reagent addition step is required. This allows the intra-chain ubiquitination reaction to be used for real-time ubiquitination readout (or ubiquitination kinetics) or as an endpoint assay. An example for TR-FRET donor in accordance with this embodiment is Tb-cryptate tagged ubiquitin, and the acceptor may be fluorescein-ubiquitin, both of which are synthetic Ubs of the invention. Another example is the pair Eu-tagged ubiquitin as the donor and AlexaFluor647 -ubiquitin as the acceptor.

In certain other embodiment, the HTRF method provided by the invention is a biotin/streptavidin ubiquitination assay. According to these embodiment, a mixture of synthetic Ubs or Ubls of the invention comprising one or more amino acids tagged with biotin, and synthetic Ubs or Ubls comprising one or more amino acids substituted with a fluorescent moiety which is the acceptor, are mixed in the ubiquitination assay with a target protein to be ubiquitinated, El, E2, E3 and ATP in the presence of absence of a test compound. Then, the TR-FRET donor, which is streptavidine tagged with a fluorescent donor moiety is added. The formation of polyubiquitinated target protein is assessed in the presence of a test compound. If a polyubiquitin chain is formed, fluorescently-labeled streptavidine attached to biotin on a Ub or Ubl will donate, following its excitation, excitation energy to an acceptor fluorescent moiety on an adjacent Ub or Ubl and a TR- FRET signal will be generated, indicating that the test compound did not inhibit polyubiquitin chain formation. An example of a synthetic Ub comprising a fluorescence- acceptor moiety is Ub-fluorescein. In preferred embodiments, the method of the invention employs synthetic Ubs and Ubls that comprise one or more a-amino acids of formula Γ which is substituted by any of the detectable moieties mentioned herein. In more preferred embodiments, the detectable moiety is Eu cryptate or Tb cryptate, more preferably a pyridine bispyridine (PBP) or tri- bispyridine (TBP) cryptate of the formula I or II. Most preferably, the amino acid is a compound of the formula I'a or I'b.

In more preferred embodiments of the invention, the synthetic Ubs of SEQ ID NOs: 2-34 are used for screening inhibitors of target proteins ubiquitination by any of the methods mentioned above.

In a further aspect, the present invention provides kits for assessing ubiquitination and/or ubiquitin-mediated proteolysis, comprising one or more synthetic Ub or Ubl of the invention. In certain embodiments, the kits of the invention comprises a synthetic Ub or Ubl labeled with a detectable moiety in accordance with the invention, and buffers and reagents suitable for carrying out the methods of the invention. In other embodiments, the kits comprise a synthetic Ub or Ubl labeled with a detectable moiety in accordance with the invention, and one or more target proteins or recognition element, and buffers suitable for carrying out the methods of the invention. The recognition element may be an antibody, such as a monoclonal antibody, S5a, TAB2, and TAB3. The kits may further comprise El, E2, E3 and ATP.

EXAMPLES

Example 1. Synthesis of a-amino acids of the formula I'a substituted with Eu-cryptate

(i) Synthesis of 2,7-diaminoheptanoic acid

The a-amino acids substituted by a detectable moiety useful for the purpose of the invention are those represented herein by formula I wherein W is H. Particularly, a-amino acids of the formula I'a and I'b were used. Most of these amino acids are commercially available. These a-amino acids can also be synthesized according to the procedure disclosed in Tetrahedron Letters, 33(50), 7725-6, 1992. The general synthesis of a-amino acids of the formula I'a is presented in Scheme 1. 2,7-Diaminoheptanoic acid was synthesized as follows: (a) Lithium bis(trimethylsilyl)amide ([(CH₃)₃Si]₂Nli; a strong non-nucleophilic base also commonly abbreviated as Lithium HexaMethylDiSilazide (LiHMDS)) (1.2eq) was added to a solution of (2R,3S)-benzyl 6-oxo-2,3-diphenylmorpholine-4-carboxylate in dry tetrahydrofuran (THF; (CH₂)₄0)/hexamethylphosphoramide (HMPA; [(CH₃)₂N]₃PO) under Argon at -78°C, for deprotonation. The mixture was stirred for 30 min at -78°C. Then 1,5- diiodopentane (1.2eq) was added to the solution mixture. The reaction was warmed slowly to 0°C, and monitored by TLC until completion. The reaction mixture was worked up with an aqueous solution of NH₄C1 and extracted with ethyl acetate. The crude was then purified by column chromatography.

(b) Sodium azide (NaN₃) was added dropwise to the compound obtained in (a) in

DMF at RT. Then, the reaction mixture was warmed to 90°C for 1 H. The reaction mixture was cooled down and worked up with brine. The crude product was taken for the next step.

(c) The crude compound obtained in (b) was added to a solution of MeOH/THF/AcOH/H₂0 followed by addition of PdCl₂. The reaction mixture was stirred for 10 min and then put under H₂ atmosphere. After completion of the reaction, 2N HC1 solution was added at 0°C and the mixture was stirred for 10 min.

The reaction was then extracted with EtOAc to give the free 2,7-diaminoheptanoic acid amino.

The amino acids 2,4-diaminobutanoic acid, 2,12-diaminododecanoic acid, 2-amino- 4-(2-aminoethoxy) butanoic acid, and 2-amino-4-(2-(2-(aminomethoxy) ethoxy)ethoxy)butanoic acid where synthesized using the method described above, wherein in step (a) the relevant dihaloalkyl (e.g., 1,2-di-iodoethane or 1, 10 di-iododecane) or dihalopolyethylene glycol (e.g., l-iodo-2-(2-iodoethoxy)ethane or l-iodo-2-(2-(2- (iodomethoxy)ethoxy)ethoxy)ethane) were used.

(ii) Synthesis of pyridine bispyridine (PBP) Eu-cryptate (compound 6)

Synthesis of 6 was based on Vila et al., 2004. Compound 6, pyridine bipyridine (PBP) europium cryptate, was synthesized as depicted in Scheme 2, via intermediate compounds 1-5 as follows:

a. Synthesis of compound 1. Bromination of diethyl 2,6-dimethyl-3,5-carboxylate pyridine (0.5g) was carried out with N-bromosuccinimide (NBS) (0.9g) dissolved in CC1₄, and then azobisisobutyronitrile (azobis) (4mg) was added to the mixture at room temperature. The reaction mixture was refluxed under tungsten lamp (100W) for 4 hours, followed by thin-layer chromatography (TLC). After completion, the solvent was evaporated by vacuum and the crude material was purified by column chromatography (eluent: CHCI₃). The dibromo derivative 1 in the scheme was obtained with 40 % yield.

b. Synthesis of compound 2. 6,6'-Dimethyl-2,2'-bispyridine (0.3 g) was dissolved in anhydrous CHCI₃ at room temperature. In parallel, meta-chloroperoxybenzoic acid (mCPBA) (0.6 g) was dissolved in CHCI₃ followed by addition of anhydrous Na₂S0₄. After 30 min, the Na₂S0₄ was filtrated and the solution of mCPBA was directly added to the bispyridine solution at room temperature. The reaction mixture was stirred for 20 h and then poured into an aqueous solution of NaHC0₃. The intermediate 6,6'-dimethyl-N,N'- dihydroxy-2,2' -bispyridine was thus obtained. The organic solution was recovered and washed 3 times by a saturated aqueous solution of NaCl and then dried over NaS0₄. After removing the solvent, the dihydroxy compound was used without further purification.

6,6' -dimethyl-Ν,Ν' -dihydroxy-2,2' -bispyridine (0.2 g) was dissolved in anhydrous CHC1₃, then trifluoroacetic anhydride was added and the reaction was stirred for 19 h. CHC1₃ was removed and replaced by anhydrous tetrahydrofuran (THF). LiBr (0.8 g) and a catalytic amount of dimethylformamide (DMF) were added to the solution. The reaction was refluxed under N₂ for 48 h. The solvent was then evaporated and the crude material was purified on silica gel chromatography (CHCl₃/MeOH 95/5) to obtain the 6,6'- dibromomethyl-2,2' -bispyridine compound 2 in Scheme 2.

c. Synthesis of compound 3. Bispyridine compound 2 was dissolved in acetonitrile followed by the addition of p-toluene sulfonamide (TosNH₂) and K₂C0₃. The reaction mixture was stirred at room temperature for 24 h. The reaction flask was put in ice bath to obtain a precipitate. The precipitate was filtered and washed with cold water, CHC1₃ and EtOH. The organic layers were collected and the solvent removed to obtain the N-Tosylated Compound 3 in Scheme 2 (Fp >300 as described in literature).

d. Synthesis of compound 4. Compound 3 was dissolved in concentrated H₂S0₄ and refluxed for 2 h. The reaction mixture was neutralized by a concentrated solution of NaOH and extracted with CHC1₃. The organic layers were combined and dried by NaS0₄. The solvent was removed by vacuum to give Compound 4 (yield 80 %)

e. Synthesis of compound 5. A 3-neck flask with a reflux system under nitrogen was loaded with Compound 4 and acetonitrile. Li₂C0₃ was added followed by a dropwise addition of an acetonitrile solution of Compound 1. The reaction was stirred at room temperature until completion of the addition, and then refluxed for 24 h. The reaction flask was cooled in an ice bath to obtain a precipitate. After removal of the solvent by vacuum, the crude material was purified by semi-preparative HPLC to give the PBP Li Compound 5 (60% yield).

f. Synthesis of compound 6. Compound 5 was dissolved in a mixture of CH₃CN and MeOH, followed by addition of EuCl₃. The reaction mixture was stirred at room temperature for 3h, and then refluxed. The reaction was monitored by HPLC. After completion, the reaction mixture was cooled to room temperature in order to obtain a precipitate. The solvent was removed and the crude material was purified by preparative HPLC to obtain PBP Eu-cryptate 6.

(Hi) Synthesis of a-amino acid substituted with Eu-cryptate

A general synthesis of a-amino acid of formula I'a substituted with PBP Eu-cryptate (compound 6) is described in Scheme 3. Conjugation of PBP Eu-cryptate obtained in (ii) above with the a-amino acid obtained in (i) was carried out as follows:

The synthetic amino acid and the Eu-cryptate were dissolved in THF and potassium carbonate was added.

After completion, the reaction was washed with brine and extracted with ethyl acetate to give the PBP Eu-cryptate substituted amino acid.

In this way, the a-amino acids 2,4-diaminobutanoic acid, 2,12-diaminododecanoic acid, 2-amino-4-(2-aminoethoxy) butanoic acid and 2-amino-4-(2-(2- (aminomethoxy)ethoxy)ethoxy)butanoic acid substituted with Eu³⁺-cryptate were obtained.

Example 2. Solvent accessibility measurements for ubiquitin

A theoretic solvent accessibility to various segments of the ubiquitin in its three dimensional structure was calculated using Accelrys Discovery Studio 3.0. The solvent accessibility calculates the solvent accessible surface of protein residues. The calculation is done for all protein residues in the context of all atoms, excluding water. The result gives the total Solvent Accessible Surface (SAS). For the analysis, the program's default parameters of 240 for the Grid points per atom, and 1.4 for the Probe radius were used.

The three dimensional structure of Ub is shown in Fig. 1. The solvent-exposed sections are colored blue, the "buried" sections are colored green and other sections are colored gray. The exposed residues have more than 25% from maximum SAS, the buried residues have less than 10% from maximum SAS, and the other residues have between 10%-25% from maximum SAS.

There are 48 "exposed" residues as follows: MET1, GLN2, LYS6, LEU8, THR9, GLY10, LYS11, THR12, THR14, GLU16, GLU18, PR019, SER20, GLU24, ASN25, ALA28, GLN31, ASP32, LYS33, GLU34, GLY35, PR037, PR038, ASP39, GLN40, ALA46, GLY47, LYS48, GLN49, GLU51, ASP52, GLY53, ARG54, SER57, ASP58, ASN60, GLN62, LYS63, GLU64, THR66, HIS68, VAL70, LEU71, ARG72, LEU73, ARG74, GLY75, GLY76

Fifteen residues are "buried" residues, as follows: ILE3, VAL5, ILE13, LEU15,

VAL17, ILE23, VAL26, LYS27, ILE30, GLN41, LEU43, LEU50, LEU56, ILE61, LEU67

The remaining 13 other residues with various exposure degrees are: PHE4, THR7, ASP21, THR22, LYS29, ILE36, ARG42, ILE44, PHE45, THR55, TYR59, SER65, LEU69.

The synthetic Ubs were designed such that one or more of the "exposed" amino acids was replaced by an a-amino acid substituted by Eu-cryptate. All lysine residues except Lys33 in the exposed section were preserved.

Example 3. Synthesis of Ubiquitin comprising an a-amino acid labeled with Eu- cryptate.

Synthesis of ubiquitin was carried out on a 0.25 mmol scale on a peptide synthesizer equipped with 757 absorbance detector linked to an integrator for on-line monitoring of the deprotection solution containing the piperidine-Fmoc adduct. All amino acids were triple- coupled with the symmetrical anhydride (2 mmol of amino acid), followed by two hydroxybenzotriazole (HOBt) active-ester (1 mmol of amino acid) couplings using diisopropylcarbodiimide (DIC) in dioxan as the activating agent. Glycine was single- coupled using 2 mmol of symmetrical anhydride, and histidine was triple-coupled using the HOBt active-ester method. Each coupling step lasted 30 min with a capping step using acetic anhydride in DMF for 6 min on the completion of the coupling step. This was followed by the removal of the Fmoc group with successive 5, 3, 3 and 1 min treatments with 20 % piperidine in DMF, with extensive washing with DMF between each deprotection.

The synthesis was monitored using real-time assessment of the deprotection solution containing the chromophoric Fmoc-piperidine adduct at 302 nm in a continuous flow mode from the synthesizer to the u.v. monitor. This provided a continuous record of every deprotection step throughout the synthesis. The final amino acid was left with the Fmoc protecting group attached so that u.v. monitoring could be carried out manually.

During synthesis of the solvent-exposed segments of Ub, amino acid labeled with Eu-cryptate is added to the reaction mixture.

The following synthetic Ubs designated by their SEQ ID NOs were prepared, in which the amino acid that is replaced with a labeled amino acid is indicated in brackets:

SEQ ID NO: 2 (Met 1); SEQ ID NO: 3 (Gin 2); SEQ ID NO: 4 (Thr 9); SEQ ID NO: 5 (Thr 14); SEQ ID NO: 6 (Blu 16); SEQ ID NO: 7 (Glu 18); SEQ ID NO: 8 (Pro 19); SEQ ID NO: 9 (Ser 20); SEQ ID NO: 10 (Glu 24); SEQ ID NO: 11 (Asn 25); SEQ ID NO: 12 (Ala 28); SEQ ID NO: 13 (Gin 31); SEQ ID NO: 14 (Asp 32); SEQ ID NO: 15 (Lys 33); SEQ ID NO: 16 (Glu 34); SEQ ID NO: 17 (Gly 35); SEQ ID NO: 18 (Pro 38); SEQ ID NO: 19 (Asp 39); SEQ ID NO: 20 (Ala 46); SEQ ID NO: 21 (Glu 51); SEQ ID NO: 22 (Asp 52); SEQ ID NO: 23 (Gly 53); SEQ ID NO: 24 (Ser 57); SEQ ID NO: 25 (Asp 58); SEQ ID NO: 26 (Thr 66); SEQ ID NO: 27 (His 68); SEQ ID NO: 28 (Val 70); SEQ ID NO: 29 (Leu 71); SEQ ID NO: 30 (Arg 72); SEQ ID NO: 31 (Leu 73); SEQ ID NO: 32 (Arg 74); SEQ ID NO: 33 (Gly 75); and SEQ ID NO: 34 (Gly 76).

Example 4. HTRF assay

Four HTRF assays using the synthetic ubiquitins of the invention are schematically described in Figs. 2A-2D.

Self ubiquitination assay comprising a synthetic Ub labeled with Eu-cryptate, E2 and

E3, is described in Fig. 2A. In this assay, E3 is tagged with an epitope onto which anti- epitope antibody labeled with the fluorophore XL655 is bound. The donor, Eu-cryptate-Ub is excited at 320 nm. The donor emits at 620 nm, the energy is absorbed by the XL655 fluorophore and emitted at 665 nm.

Polyubiquitin chain elongation (self ubiquitination) assay using antibodies is depicted in Fig. 2B. In this assay, synthetic Ub labeled with Eu-cryptate is mixed with E2,

E3 and with synthetic Ub labeled with a flag (biotin). Then, anti-flag antibody (streptavidine) labeled with the fluorophore XL655 is added. When selfubiquitination occurs, the donor, Eu-cryptate-Ub excited at 320 nm, emits light at 620 nm, which is absorbed by XL655 and emitted at 665 nm. Chain-elongation self ubiquitination assay without antibodies is depicted in Fig. 2C. In this assay, synthetic Ub labeled with Eu-cryptate and synthetic Ub labeled with AlexaFluore647 are mixed with E2 and E3. When selfubiquitination occurs, the donor, Eu- cryptate-Ub excited at 320 nm, emits light at 620 nm, which is absorbed by AlexaFluore647 and emitted at 647 nm.

A further chain-elongation self ubiquitination assay without antibodies is depicted in Fig 2D. In this assay, Ub labeled with Tb-cryptate and synthetic Ub labeled with fluorescein are mixed with E2 and E3. When selfubiquitination occurs, the donor, Tb- cryptate-Ub excited at 320 nm, emits light at 495 nm, which is absorbed by fluorescein and emitted at 520 nm.

Scheme 1



 REFERENCES

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Claims

1. A synthetic ubiquitin or ubiquitin-like modifier protein in which at least one amino acid residue is replaced with a residue of an a-amino acid substituted by a detectable moiety.

2. The synthetic protein according to claim 1, wherein said detectable moiety is a radioactively labeled moiety, a chromophore, a fluorescent moiety, an enzyme, biotin or an electron-dense reagent.

3. The synthetic protein according to claim 2, wherein said detectable moiety is a fluorescent moiety selected from a fluorescent dye, a macromolecular complex of a fluorescent metal ion or a fluorescent protein.

4. The synthetic protein according to claim 3, wherein said macromolecular complex of a fluorescent metal ion is a macropolycyclic complex of a lanthanide metal ion.

5. The synthetic protein according to claim 4, wherein said macropolycyclic complex of a lanthanide metal ion is a chelate or cryptate.

6. The synthetic protein according to claim 5, wherein said cryptate of a lanthanide metal ion is selected from La³⁺-, Ce³⁺-, Pr³⁺-, Nd³⁺-, Pm³⁺-, Sm³⁺-, Eu³⁺-, Gd³⁺-, Tb³⁺-, Dy³⁺-, Ho³⁺-, Er³⁺-, Tm³⁺-, Yb³⁺-, or Lu³⁺-cryptate, more preferably Eu³⁺-, Tb³⁺-, Gd³⁺-, or Sm³⁺-cryptate.

7. The synthetic protein according to claim 6, wherein the cryptate is a pyridine bispyridine (PBP) cryptate having the formula I or a trisbipyridine (TBP) having the formula II:

wherein

X is an ion of a lanthanide metal selected from La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺, or Lu³⁺, more preferably Eu³⁺, Tb³⁺, Gd³⁺, or Sm³⁺;

Ri-Rn each independently is selected from H, halogen, C C₂o hydrocarbyl, heteroaryl, heterocyclyl, nitro, cyano, -COR, -COOR, -COSR, OR, SR, -CONRR' , -S0₂, - S0₃R, -S0₂R, -NRS0₂R', -S0₂NRR' -NR-COR', -NRR', -NR(CH₂)_n-NR' , =N-OR, =N- NRR', -NR-(C₆-C₁₄)aryl, (-C(0)-C₆H₄-N0₂), -C(=NR)-NRR', -NR-NRR' , -C(O)- NR(CH₂)_n-NRR', -(R)N-C(=NR)-NRR\ >C=NR, -(CH₂)_n-NR-COR', -(CH₂)_n-CO-NRR', - 0-(CH₂)_n-OR, -0-(CH₂)_n-0-(CH₂)_n-R, -PO₃RR', imidazolyl, succcinimido, haloacetyl, or dihalotriazinyl, wherein R and R' each independently is H or Q-C^ hydrocarbyl, preferably C C₂o alkyl, or R and R' together with the nitrogen atom to which they are attached form a 5-6 membered saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom is optionally substituted by Q-C₆ alkyl, aralkyl selected from (C₆-C₁₄)aryl-(C₁-C₁o)alkyl, haloalkyl selected from iodo-, bromo- chloro- or fluoro-(C₁-C₁o)alkyl, or hydroxyalkyl selected from hydroxy-(Ci- Cio)alkyl; and n is an integer of 2 to 10.

8. The cryptate of formula I or II according to claim 7, wherein X is selected from Eu³⁺, Gd³⁺, Tb³⁺, or Sm³⁺; R_1; R₂, R₃, R₅, R₆, R₈, R₉, R11 , R12, R_W, R15, R½ and R₁₇ each independently is selected from H, alkoxy, COOR, COSR, -COR, -NRR', -NR(CH₂)_n-NR', - CONRR', -S0₂NRR', NRS0₂R'-, -C(0)-NR(CH₂)_n-NRR', -C≡N, -S0₂, -S0₃R, -S0₂R, or nitro, wherein n, R and R' are as defined in claim 7; and R₁₀ and R₁₃ each independently is H or COOH.

9. The cryptate of formula I or II according to claim 8, wherein X is Eu or Tb ; Ri, R₂, R₃, R₅, Re, Rs, R9, R11, R12, Ri4, R15, R½ and R₁₇ each is H, and R₄, R₇, Rio and R_i3 each independently is H or COOH.

10. The synthetic protein according to any one of claims 1 to 9, wherein said a-amino acid substituted by a detectable moiety is a compound of the formula Γ :

wherein

X is a linear carbon chain of 2-20 atoms optionally interrupted by one or more nitrogen, sulfur and/or phosphorous atoms, or X is a moiety -CH₂-X' - having a length corresponding to the length of a linear chain of 2-20 carbon atoms, wherein X' is selected from Ci-C₂o hydrocarbyl, C₃-Ci₂ heterocyclyl or C₆-Ci₄ heteroaryl;

W is -C(R)₂-, O, S or -N(R)-, wherein R is H, Ci-C₂₀ hydrocarbyl or C₃-C₁₂ heterocyclyl; and

Y is a detectable moiety.

11. An a-amino acid of the formula Γ :

wherein

X is a linear carbon chain of 2-20 atoms optionally interrupted by one or more nitrogen, sulfur and/or phosphorous atoms, or X is a moiety -CH₂-X'- having a length corresponding to the length of a linear chain of 2-20 carbon atoms, wherein X' is selected from Ci-C2o hydrocarbyl, C₃-C₁₂ heterocyclyl or C₆-C₁₄ heteroaryl;

W is -C(R)₂-, O, S or NR, wherein R is H, C₁-C₂₀ hydrocarbyl or C₃-C₁₂ heterocyclyl; and

Y is a detectable moiety.

12. The synthetic protein according to claim 10, or the a-amino acid according to claim 11, wherein said hydrocarbyl is a straight or branched, acyclic or cyclic, saturated, unsaturated or aromatic, hydrocarbyl radical, of 1-20 carbon atoms, optionally of 1 to 10, 1 to 6, or of 2-5 carbon atoms, selected from an alkyl, alkenyl, alkynyl, carbocyclyl, aryl or aralkyl radical, said alkyl, alkenyl or alkynyl is optionally interrupted by one or more heteroatoms selected from O, S and/or N, and/or by C₆-C₁₄ aryl or C₃-C₁₂ heterocyclic ring, and said hydrocarbyl is optionally further substituted by one or more radicals selected from halogen, C₆-C₁₄ aryl, C₆-C₁₄ heteroaryl, C₃-C₁₂ heterocyclyl, nitro, epoxy, epithio, cyano, - SR, -COR, -COOR, -COSR, OR, -CONRR' , -S0₃R, -S0₂R, -NRS0₂R' , -S0₂NRR' -NR- COR', -NRR' , =N-OR, =N-NRR' , -C(=NR)-NRR', -NR-NRR' , -(R)N-C(=NR)-NRR\ >C=NR, -(CH₂)_n-NR-COR\ -(CH₂)_n-CO-NRR\ -0-(CH₂)_n-OR, -0-(CH₂)_n-0-(CH₂)_n-R, - P0₂HR, -P0₃RR' , imidazolyl, succcinimido, haloacetyl, or dihalotriazinyl, wherein R and R' each independently is H, C C₂o hydrocarbyl or C₂-C₁₂ heterocyclyl, or R and R' together with the nitrogen atom to which they are attached form a 5-6 membered saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom is optionally substituted by C -C alkyl, (C₆-C₁₄)aryl- (C₁-C₁o)alkyl, chloro-, bromo-, iodo- or fluoro-(C₁-C₁o)alkyl, or hydroxy-(C₁-C₁o)alkyl.

13. The synthetic protein or the a-amino acid according to claim 12, wherein said alkyl is a straight or branched lower alkyl of 1 to 6, optionally 2-5 carbon atoms selected from ethyl, propyl, isopropyl, butyl isobutyl, tert-butyl or pentyl.

14. The synthetic protein according to claim 10 or 12, or the a-amino acid according to claim 11 or 12, wherein: (i) said carbocyclyl is derived from a C₃-C₂o monocyclic or polycyclic ring, saturated or partially unsaturated, optionally C₃-C₁₄, or C₃-C₇ cycloalkyl, cycloalkenyl or cycloalkynyl radical such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl; (ii) said aryl is an aromatic carbocyclic group having 6 to 14 carbon atoms, optionally 6 to 10 carbon atoms, consisting of a single, bicyclic or tricyclic ring, such as phenyl, naphthyl, carbazolyl, anthryl, or phenanthryl; (iii) said heterocyclyl is a radical derived from a saturated, partially unsaturated, monocyclic, bicyclic or tricyclic heterocycle of 3-12, optionally 5-10, or 5-6 members in the ring containing 1 to 3 heteroatoms selected from O, S and/or N, such as dihydrofuryl, tetrahydrofuryl, pyrrolynyl, pyrrolydinyl, dihydrothienyl, dihydropyridyl, piperidinyl, quinolinyl, piperazinyl, morpholino or 1,3-dioxanyl; (iv) said heteroaryl is a mono- or polycyclic heteroaromatic ring optionally comprising both carbocyclic and heterocyclic rings, containing 1 to 3 heteroatoms selected from O, S and/or N such as pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,4-triazinyl, 1,2,3-triazinyl, 1,3,5- triazinyl, benzofuryl, isobenzofuryl, indolyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazoly and benzodiazepinyl;

said carbocyclyl, aryl, heterocyclyl, or heteroaryl may be substituted by one or more radicals selected from halogen, C₆-C₁₄ aryl, C₆-C₁₄ heteroaryl, C3-C₁₂ heterocyclyl, nitro, epoxy, epithio, cyano, -SR, -COR, -COOR, -COSR, OR, -CONRR', -S0₃R, -S0₂R, - NRS0₂R', -S0₂NRR' -NR-COR', -NRR', =N-OR, =N-NRR' , -C(=NR)-NRR', -NR-NRR' , -(R)N-C(=NR)-NRR', >C=NR, -(CH₂)_n-NR-COR', -(CH₂)_n-CO-NRR', -0-(CH₂)_n-OR, -0- (CH₂)_n-0-(CH₂)_n-R, -P0₂HR, -P0₃RR', imidazolyl, succcinimido, haloacetyl, or dihalotriazinyl, wherein R and R' each independently is H, Q-C^ hydrocarbyl or C₂-C₁₂ heterocyclyl, or R and R' together with the nitrogen atom to which they are attached form a 5-6 membered saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom is optionally substituted by Q-C₆ alkyl, (C₆-C₁₄)aryl-(C₁-C₁o)alkyl, chloro-, bromo-, iodo- or fluoro-(C₁-C₁o)alkyl, or hydroxy- (C _\ -C _\ o)alkyl ..

15. The synthetic protein according to claim 10, or the a-amino acid according to claim 10, wherein said detectable moiety is as defined in any one of claims 2 to 9.

16. The synthetic protein according to any one of claims 10 to 15, or the a-amino acid according to any one of claims 10 to 14, wherein said a-amino acid is a compound of the formula I'a or I'b:

(I'a) (I'b) wherein

R is H or Ci-C₂o hydrocarbyl;

Y is an Eu³⁺-cryptate or a Tb³⁺-cryptate moiety;

n is an integer of 2-10, optionally 2, 5 or 10;

m is an integer of 1 to 6, optionally 1 or 3; and

p is an integer of 1 to 5, optionally 1 or 2.

17. The synthetic protein or the a-amino acid according to claim 16, wherein the compound of formula I'a is:

wherein n is an integer of 2-10.

18. The synthetic protein according to any one of claims 1 to 10 or 12 to 17, selected from a ubiquitin of SEQ ID NO: 2-34.

19. A method for identifying an inhibitor of ubiquitin-mediated proteolysis comprising the steps of: (i) measuring binding of a synthetic ubiquitin or ubiquitin-like modifier protein, in which at least one amino acid residue is replaced with a residue of an a-amino acid substituted by a detectable moiety, to a target protein in the presence and absence of a test compound; and

(ii) identifying the test compound as an inhibitor of ubiquitin-mediated proteolysis when a decreased signal is detected in the presence of the test compound compared to the signal detected in the absence of the test compound.

20. The method according to claim 19, wherein said detectable moiety is as defined in any one of claims 2 to 9.

21. The method according to claim 19, wherein said a-amino acid substituted by a detectable moiety is a compound of the formula Γ as defined in any one of claims 11 to 17.

22. The method according to claim 19, selected from time-resolved fluorescence (TRF), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET), dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), scintillation proximity assay (SPA), electrochemiluminescence (ECL), fluorescence intensity (FI), fluorescence polarization (FP), bioluminescence resonance energy transfer (BRET), or AlphaScreen.

23. The method according to claim 22, which is time-resolved fluorescence resonance energy transfer (TR-FRET), wherein said synthetic ubiquitin or ubiquitin-like modifier protein comprises a first fluorescent moiety, the target protein comprises a second fluorescent moiety, and wherein in step (ii) the test compound is identified as an inhibitor of ubiquitin-mediated proteolysis when a decreased fluorescence signal of the second fluorescent moiety is detected in the presence of the test compound compared to the signal detected in the absence of the test compound.

24. The TR-FRET method according to claim 23, wherein said synthetic ubiquitin or ubiquitin-like modifier protein is a synthetic protein in which at least one amino acid residue is replaced by a residue of an a-amino acid of the formula Γ as defined in any one of claims 11 to 17.

25. The TR-FRET method according to claim 23, wherein said first fluorescent moiety is a macropolycyclic complex of a lanthanide ion selected from a chelate or cryptate.

26. The TR-FRET method according to claim 25, wherein said macropolycyclic complex of a lanthanide ion is a cryptate.

27. The TR-FRET method according to claim 26, wherein said cryptate is a compound of the formula I or II defined in of any one of claims 7 to 9.

28. The TR-FRET method according to claim 24, wherein said a-amino acid substituted by a detectable moiety is a compound of the formula I'a or I'b defined in claim 16.

29. The TR-FRET method according to claim 28, wherein said a-amino acid substituted by a detectable moiety is the compound defined in claim 17.

30. The method according to any one of claims 19 or 23 to 29, wherein said target protein is selected from: (i) an E3 ubiquitin ligase protein, such as a POSH protein, a cbl-b protein, a PEM-3-like protein, Nedd4 or WWPl, or a fragment of said E3 ubiquitin ligase protein that is ubiquitinated; (ii) a protein that contains ubiquitin and ubiquitin-like recognition elements such as S5a, TAB2 or TAB3; (iii) an E2 ubiquitin and ubiquitin-like conjugation enzymes such as UbcH5a-c, UbcH7, UbcHIO, Ubc9 or Ubcl2; (iv) an El ubiquitin and ubiquitin-like activating enzyme such as Ubal, Aos/Uba2, APPBP1/Uba3 or UBE1L 4; and (v) a substrate of ubiquitin or ubiquitin-like modifier protein such as p53 or p27.

31. The method according to any one of claims 19 or 23 to 29, wherein said second fluorescent moiety is selected from XL665, d2, allophycocyanin, Cy5, Ulight dye, SureLight APC dye, Fluorescein, GFP or AlexaFluor® 647.

32. The method according to any one of claims 19 to 31, wherein said synthetic protein is a synthetic ubiquitin of SEQ ID NO: 2-34.

33. A kit for assessing ubiquitination and/or ubiquitin-mediated proteolysis, comprising buffers and reagents suitable for carrying out ubiquitin-mediated proteolysis and one or more synthetic ubiquitin or ubiquitin-like modifier protein in which at least one amino acid residue is replaced with a residue of an a-amino acid substituted by a detectable moiety as defined in any one of claims 1 to 10 and 12 to 18.