ES2356770T3 - SUBSTRATE FOR ENZYMATIC ACTIVITY OF RPN 11. - Google Patents

Abstract

Peptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1 and at least one ubiquitin residue that is at least 90% identical to the sequence of the first 76 amino acids of SEQ ID NO : 2, in which the peptide is a substrate for an Rpn11 metalloprotease comprising a JAMM motif.

Description

Background

Proteolysis by the 26S proteasome proceeds by binding a ubiquitinated substrate protein to the 19S regulatory particle, followed by its desubiquitination, unfolding and translocation in the light of the 20S core, where it degrades. In recent years, it has become clear that the proteasome is an attractive target for therapeutic intervention in cancer, disorders related to the immune system, inflammation, ischemic conditions, neurodegenerative disorders and other diseases. To date, the only FDA-approved proteasome inhibitor (VELCADE ™) works by inhibiting the activity of 20S core peptidases. However, inhibition of other enzymatic activities that reside within the proteasome complex could serve as an equally effective, if not better, means of controlling proteasome function.

A target candidate is the metalloprotease, Rpn11, which resides within the 19S regulatory particle. It is responsible for the initial desubiquitination of the target proteins (Eytan, et al. JBC 268 (7): 46684674 (1993); Verma, et al. Science 298: 611-615 (2002)). The human homologue of this yeast protein is POH1. To date, a recombinant form of Rpn11 has not been generated. However, the deubiquitinating activity associated with Rpn11 can be distinguished in the context of a 26S proteasome complex purified by its sensitivity to ATP-S and 1,10-phenanthroline (OPA) and its insensitivity to 20S core protease inhibitors and Classic DUB inhibitors (ubiquitin aldehyde (UB-Al) or ubiquitin vinyl sulfone (UbVS)).

In the race to learn more about Rpn11 and its desubiquitinante activity, several essays have been used. Most use extracts of purified 26S proteasome as a source of the enzyme and observe degradation in the presence or absence of a DUB inhibitor. Detection of Rpn11 activity required a ubiquitinated protein such as Ub-sic 1 (Verma et al., Cited above) and the reaction was monitored by SDS-PAGE followed by immunoblotting or with -ubiquitin (-Ub) or well with -protein such as -sic 1. The substrates that were radiolabeled

or fluorescently labeled still required a step that separated the uncleaved substrate from the cleaved product, such as PAGE (Eytan et al., cited above) or precipitation with TCA (Yao et al., Nature 419: 403-407 (2002) ), respectively.

Therefore, a high performance assay (HTP) using novel Rpn11 substrate proteins is highly desirable in order to select large numbers of molecules to identify modulators of Rpn11 activity.

Brief description of the request

Accordingly, this invention provides peptides that are substrates of the enzymatic activity of the proteasome (for example, Rpn11) and various methods that employ peptide substrates, such as assays that measure the enzymatic activities of the proteasome (eg, Rpn11) that select agents which modulate the enzymatic activities of the proteasome, said peptides and methods being defined in the claims. The terms "peptide", "polypeptide" and "proteins" are used interchangeably herein. The term "proteasome" refers to the 26S proteasome, the 19S regulatory particle, a protein complex comprising the 19S regulatory particle, or other protein complexes or components comprising an Rpn11 or an Rpn11 type protein involved in proteolysis mediated by ubiquitin The term "Rpn11" is also used herein interchangeably with "POH1" and refers to any polypeptide that confers an enzymatic activity of Rpn11, for example, deubiquitination activity.

A first aspect of the invention provides a peptide substrate for a protease of the 26S proteasome, the 20S proteasome, or the 19S regulatory particle. The peptide substrate comprises an amino acid sequence at least 70%, 80%, 90%, 95% or 100% identical to SEQ ID NO: 1 and a ubiquitin residue that is at least 90% identical, 95 % or 100% to the sequence of the first 76 amino acids of SEQ ID NO: 2. In certain embodiments, the peptide substrate comprises a ubiquitin residue that is at least 90% identical, 95% or 100% to the sequence of the first 76 amino acids of SEQ ID NO: 2, a first amino acid sequence at least 70%, 80%, 90%, 95% or 100% identical to SEQ ID NO: 1 and an additional amino acid sequence , which begins at the C-terminal end of the first amino acid sequence, which has at least 3, 5, 8, 10, 12, 15, 18, 20, 25, 30, 40, 50, 75, 100, 200, 300, 500 or more amino acids.

Also described are peptide substrates comprising an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identical to any of SEQ ID NO: 3-15 and a residue of ubiquitin that is at least 70%, 80%, 90%, 95% or 100% identical to the sequence of the first 76 amino acids of SEQ ID NO: 2.

In certain embodiments, the peptide substrate further comprises one or more ubiquitin residues at the N-terminal end of the peptide. In certain embodiments, one or more ubiquitin residues may be linked to the peptide through the K (s) of the peptide.

In certain embodiments, a ubiquitin residue comprises an amino acid sequence that is 90% or 100% identical to the sequence of the first 76 amino acids of SEQ ID NO: 2. The 76 amino acid ubiquitin repeat unit of the SEQ ID NO: 2 is also called Ub in this document. In certain embodiments, a ubiquitin residue comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100 or more repetitions of Ub. In certain embodiments, a ubiquitin residue comprises one or more repetitions of Ub, except that the 75th amino acid of at least one repetition of Ub is A. In certain embodiments, a ubiquitin residue comprises one or more repetitions of Ub, except that the 76th amino acid of at least one repetition of Ub is A. In certain embodiments in which a ubiquitin residue comprises one or more repetitions of Ub, the 76th amino acid of at least one repetition of Ub is G or A.

In certain embodiments, the peptide substrate further comprises a detectable agent bound to the peptide. The detectable agent can be attached to the peptide using different methods, which may depend on the identity of the detectable agent and / or the part of the peptide that is to be bound with the detectable agent. For example, the detectable agent can be linked to the peptide by C residues (s) of the peptide. The detectable agent can be a fluorescent label, which can be attached to the peptide substrate by covalent or non-covalent bonding. A fluorescent label can comprise a fluorescent peptide that can be attached to the peptide by covalent or non-covalent bond, for example, a peptide bond. Alternatively, the detectable agent may be a radioactive label, for example, a radiolabeled amino acid such as 35S-C or 35S-M.

In other embodiments, the peptide substrate may comprise a target part, that is, an N-terminal or C-terminal part, which specifically binds to a selection agent. The selection agent may be an antibody, which recognizes the target part of the peptide substrate. Alternatively, the selection agent may be a divalent metal ion, which specifically associates or interacts with the target part of the peptide substrate.

Nucleic acids encoding the various proteins and peptide substrates described herein are also described.

Certain embodiments provide a method as defined in the claims for selecting an agent that modulates proteasome activity, for example, the enzymatic activity of the 26S proteasome or the 19S regulatory particle. The method includes providing a peptide substrate of the application, for example, a peptide substrate comprising an amino acid sequence of SEQ ID NO: 1 and one or more ubiquitin residues, and combining the peptide with a reaction mixture suitable for measuring Proteasome activity in the presence of a test agent. A change in proteasome activity in the presence of the test agent compared to the activity of the proteasome in the absence of the test agent indicates that the test agent modulates the activity of the proteasome. Proteasome activity can be determined, for example, by measuring the level of cleavage of the ubiquitin residue (s) from the peptide. The level of excision can be indicated by the rate and / or the degree of excision. The method is useful for selecting an agent that potentiates or inhibits proteasome activity.

Certain embodiments provide a method as defined in the claims for selecting a proteasome inhibiting agent. The proteasome inhibiting agent may comprise a ubistatin-like moiety or molecule or a ubiquitin-binding moiety or molecule. The proteasome inhibitor that binds to ubiquitin can inhibit substrate proteolysis or can inhibit proteasome activity through other mechanisms. The inhibitory agent may or may not have intrinsic fluorescence.

The method may include providing a peptide substrate of the application having a fluorescent label, for example, a peptide substrate comprising an amino acid sequence of SEQ ID NO: 1, one or more ubiquitin residues, and a fluorescent label, and determine the fluorescence polarization of the peptide substrate in the presence compared to in the absence of a test agent. A difference in fluorescence polarization in the presence of the test agent versus in the absence of the test agent indicates that the test agent may be a proteasome inhibiting agent.

Alternatively, the method may include providing a peptide substrate of the application in a suitable reaction mixture in the presence or absence of a test agent and determining if there is any difference in the molecular weight and / or the size of the peptide substrate in presence in comparison. with in the absence of the test agent. A ubistatin-like molecule can produce aggregation or multimerization of ubiquitin residues bound to a peptide, and therefore, an increase in molecular weight and / or the size of the peptide substrate in the presence compared to in the absence of the test agent indicates that The test agent may be a ubistatin type molecule. Molecular weight and / or size can be determined by various methods, such as gel electrophoresis under native conditions, size exclusion chromatography, light scattering, etc.

The method may include providing a peptide substrate of the application, for example, a peptide substrate comprising an amino acid sequence of SEQ ID NO: 1 and one or more ubiquitin residues, and determining whether the intrinsic fluorescence of a test agent It changes when the test agent is combined with the peptide substrate in a suitable reaction mixture. A change in the intrinsic fluorescence of the test agent in the presence of the peptide substrate indicates that the test agent may be a proteasome inhibitor.

In other embodiments, a method for selecting a proteasome inhibitor agent may employ a reaction mixture comprising a ubistatin molecule (eg, ubistatin A or B), in addition to a peptide substrate of the application and with or without an inhibitory agent. test. A change in the intrinsic fluorescence of the ubistatin molecule (for example, ubistatin A or B) measured in the presence of the test inhibitor agent compared to in the absence of the test inhibitor agent indicates that the test inhibitor agent is a proteasome inhibitor Ubistatin type. The test inhibitor itself may or may not have intrinsic fluorescence. This method can be particularly useful for selecting a ubistatin-like proteasome inhibitor that can compete with ubistatin to bind with ubiquitin residues.

A further aspect of the invention provides a method as defined in the claims for selecting an agent that modulates the activity of various proteasome components or various phases of the ubiquitin-mediated proteolysis process. The various components of the proteasome and the various phases of ubiquitin-mediated proteolysis may include the entry into the proteasome of ubiquitinated proteins; the desubiquitination of proteins, for example, by Rpn11; the deployment of proteins, for example, AAA ATPase; degradation of desubiquitinated proteins by nucleus peptidase.

The method employs at least two peptide substrates, that is, a first peptide substrate comprising a ubiquitinated peptide and a first fluorescent label, and a second peptide substrate comprising a non-ubiquitinated peptide and a second fluorescent label, wherein the first and The second fluorescent markers are detectable at different wavelengths. The ubiquitinated peptide was defined above and comprises an amino acid sequence of SEQ ID NO: 1 and one or more ubiquitin residues. A non-ubiquitinated peptide can be any peptide that can serve as a substrate for a core peptidase, for example, a chemotryptic type protease, a tryptic type protease, or a PGPH (or caspase) protease. The method includes measuring the fluorescence at the wavelength of the first detectable marker in the presence of a 26S proteasome and in the presence and absence of a test agent, and measuring the fluorescence at the wavelength of the second detectable marker in the presence of the 26S proteasome. and in the presence and absence of a test agent. A change in the fluorescence of the first peptide substrate in the presence of the test agent compared to the fluorescence in the absence of the test agent indicates that the test agent modulates an activity associated with a 19S regulatory particle. A change in the fluorescence of the second peptide substrate in the presence of the test agent compared to the fluorescence in the absence of the test agent indicates that the test agent modulates a 20S core peptidase.

Methods and compositions employing an agent selected according to a method of the application are also described herein. For example, certain embodiments provide a method for treating or preventing in a subject a state associated with proteasome activity or proteolysis measured by aberrant ubiquitin which comprises administering to the subject a composition comprising an agent that modulates proteasome activity, particularly a Enzymatic activity associated with the 19S regulatory particle. An example of the enzymatic activity associated with the 19S regulatory particle is the enzymatic activity of Rpn11.

Brief description of the drawings

Figure 1 shows the change of S14 of the peptide comprising the sequence of SEQ ID NO: 3 to C14 (SEQ ID NO: 4) does not alter the ability of the substrate to be recognized and cleaved by Rpn11. Each peptide substrate further comprises an N-terminal ubiquitin residue that has 4 repetitions of Ub (Ub4). Figure 1 further shows that the proteolysis of the substrate Ub4-peptide (29) and Ub4-peptideC14 (29) by the 26S proteasome is inhibited by O-phenanthroline and ATPS, but not by ubiquitin aldehyde. 2 g of Ub4-peptide (29) or Ub4-peptide-C14 (29) was incubated with 26 n 60S 26S proteasome in buffer B [50 mM Tris pH 8.0; 10 mM MgCl2; 1 mM DTT; 1 mM ATP] and when indicated 5 mM ATP, 1 mM O-phenanthroline (OPA) and 5 M ubiquitin aldehyde (Ub-aldehyde) in a volume of 20 µl for 2 hours at 37 ° C. The reactions were stopped by adding 7.5 µl of loading dye and boiled at 100 ° C for 5 minutes. The reactions were then loaded on an SDS-PAGE gel, the reaction products were separated by electrophoresis and the bands were visualized with bright blue.

Figure 2 shows that the change of S14 of the peptide comprising the sequence of SEQ ID NO: 15 to C14 does not alter the ability of the substrate to be recognized and cleaved by Rpn11. Each peptide substrate further comprises an N-terminal ubiquitin residue that has 4 repetitions of Ub (Ub4). Figure 2 further shows that the proteolysis of the substrate Ub4-peptide-C14 (14) is inhibited by O-phenanthroline and ATPS using the 26S proteasome from source A but not from source B. The reactions were carried out such as described above for Figure 1, except for the following modifications: 2 µg of Ub4-C14-peptide (14) was incubated with 60 nM of 26S from either source A or source B.

Figure 3 is a schematic representation of a fluorescence polarization (FP) assay using a peptide substrate illustrative of the application comprising a fluorescent label, for example fluorescein. Figure 3 shows that cleavage of the C14-29 peptide (29) coupled to fluorescein from Ub4-C14-peptide (29) coupled to fluorescein results in a decrease in the fluorescence polarization signal.

Figure 4 shows the result of a FP assay using a peptide substrate of the application and the kinetics of Ub4-peptide (29) -fluorescein proteolysis by the 26S proteasome. Various concentrations (as indicated) of Ub4-peptide (29) coupled to fluorescein-5-maleimide were incubated with 10 nM 26S proteasome in 50 mM Tris pH 8.0; 10 mM MgCl2; 1 mM DTT; 1 mM ATP at 28 ° C, and the release of peptide (29) -fluorescein was measured by fluorescence polarization (excitation at 485 nm; emission at 510 nm).

Figure 5A illustrates a high performance FP (HTP) assay that detects Ofphantroline inhibition of Rpn11 activity. The results show that O-phenanthroline affects the kinetics of Ub4-peptide (29) -fluorescein proteolysis by the 26S proteasome. 100 nM Ub4-peptide coupled to fluorescein-5-maleimide was incubated with 10 nM 26S proteasome in 50 mM Tris pH 8.0; 10 mM MgCl2; 1 mM DTT; 1 mM ATP at 28 ° C and peptide release was measured by fluorescence polarization (excitation at 485 nm; emission at 510 nm) in the presence of various concentrations of O-phenanthroline (as indicated).

Figure 5B illustrates that the enzymatic activity of Rpn11 is not affected by the presence of a 20S proteasome inhibitor in the HTP FP assay using a peptide substrate of the application. The results show that the 20S proteasome inhibitor does not affect the kinetics of Ub4-peptide (29) -fluorescein proteolysis by the 26S proteasome. 100 nM Ub4-peptide coupled to fluorescein-5-maleimide was incubated with 10 nM 26S proteasome in 50 mM Tris pH 8.0; 10 mM MgCl2, 1 mM DTT; 1 mM ATP at 28 ° C and peptide release was measured by fluorescence polarization (485 nm excitation; 510 nm emission) in the presence of various concentrations of 20S proteasome inhibitor (as indicated).

Detailed description of the request

The main pathway for protein degradation in the nucleus and cytoplasm of eukaryotic cells is through the ubiquitin / proteasome 26S pathway. The 26S proteasome comprises two main subparticles: the 20S proteasome and the 19S regulatory particle. The 20S proteasome is a cylindrical structure with an internal cavity that contains the active sites of peptidase. Proteasome substrates are inserted into the cylinder, where they are susceptible to digestion by the active sites of the 20S proteasome peptidase. The entry into the 20S proteasome cylinder is governed by the 19S regulatory particle, which covers the ends of the 20S cylinder.

Regulatory particle 19S binds to ubiquitinated substrates and translocates them to the internal cavity of cylinder 20S, where they degrade. The 19S regulatory particle can be further subdivided into two multiproteic complexes: the base and the lid. The base comprises a set of six ATPases that are thought to deploy substrates and translocate them to the 20S proteasome. The lid comprises a set of eight proteins, some of which have known functions, for example, Rpn11. Biochemical data also indicates that the presence of the cap makes the proteasome selective for degrading ubiquitinated proteins.

The 26S proteasome lid subcomplex is evolutionarily related to the COP9-signalosome complex, but the importance of this similarity remains unknown. There are reports in the literature that the 26S proteasome preparations contain a variety of associated isopeptidase ubiquitin activities (Eytan et al., Cited above; Verma, et al., Mol Biol Cell 11: 3425 (2000)). A more recent report (Verma et al., Science 298: 611-615 (2002)) showed that a ubiquitinated substrate can be completely desubiquitinated by purified 26S proteasome to give an unmodified substrate in the presence of a 20S proteasome inhibitor, for example , VELCADE ™.

Proteins that are destined for degradation by the ubiquitin / 26S pathway are labeled by attaching a chain of multiubiquitin or polyubiquitin to the side chains of lysine residues in the target protein. Recent studies also show that N-terminal ubiquitination is also an important novel mode of protein modification and directs protein for proteolysis.

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using the 26S proteasome (Ciechanover and Ben-Saadon, Trends in Cell Biol. 14 (3): 103-106 (2004)). The ubiquitinated protein is then recognized by the 26S proteasome by a mechanism that remains poorly understood. Subsequently, the ubiquitinated protein is released from any closely bound component, desubiquitin, deployed and translocated to the central cavity of the 20S complex, where it is extensively degraded by the proteolytic active sites that are present in this internal cavity (nucleus peptidase) .

Rpn11, a metalloisopeptidase or metalloprotease and intrinsic subunit of the cap subcomplex of the 19S regulatory particle, contains a predicted, conserved metalloprotease (JAMM) motif that is critical for cell viability. For the purpose of this application, Rpn11 and POH1 are interchangeable. It is believed that Rpn11, by mediating or catalyzing the deubiquitination of the protein substrate, defines a key stage in protein degradation by the 26S proteasome (Verma et al., 2002, cited above). Rpn11 and its orthologs are described in U.S. Patent Application Publication No. 20030166243. Accordingly, the peptide substrates for Rpn11 are useful in the study of the activity of Rpn11 and in the identification of agents that modulate the activity of Rpn11, and therefore they modulate ubiquitin-mediated proteolysis.

A representative amino acid sequence of an Rpn11 is exposed as follows:

14 gi regulatory subunit | 51701716 | sp | O00487 | PSDE_HUMAN no ATPase of the 26S proteasome (rpn11 regulatory subunit of the 26S proteasome) (PAD1 homologue 1 associated with the 26S proteasome)

image 1

In certain embodiments, an Rpn11 of the present invention comprises an amino acid sequence that is at least 60% identical, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99 % or 100% to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, an Rpn11 of the present invention comprises an amino acid sequence having amino acid substitutions, deletions or additions in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of SEQ ID NO: 16. In certain embodiments, such amino acid substitutions are conservative amino acid substitutions.

As reported by Verma et al., Cited above, a JAMM motive is required for the ability of Rpn11 to desubiquitinate and degrade Ub-Sic1. The predicted histidine mutation of the active site in alanine (rpn11AXA) was lethal and stabilized the substrates of the ubiquitin pathway in yeasts. A JAMM motif can be represented by the following amino acid sequence:

HSHPGFCWLSXVD (SEQ ID NO: 17)

wherein X is S or G, preferably G.

Alternatively, a JAMM motif can be characterized by the following sequence:

MVVGWYHSBPGFCWLSXVDINTQQSFEALSERAVA (SEQ ID NO: 18)

wherein X is S or G, preferably G; I is V or I, preferably I; Q is K or Q, preferably Q; A is Q or A, preferably A; S is N, T or S, preferably S; and E is S, P, D or E, preferably E.

In certain embodiments, an Rpn11 of the present invention comprises a JAMM motif that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID. NO: 17. In more preferred embodiments, an Rpn11 of the present invention comprises a JAMM motif that has amino acid substitutions, deletions or additions at 1, 2, 3, 4 or 5 positions of SEQ ID NO: 17. In certain embodiments , such amino acid substitutions are conservative amino acid substitutions. In specific embodiments, an Rpn11 of the present invention comprises a JAMM motif that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID. NO: 18. In more preferred embodiments, an Rpn11 of the present invention comprises a JAMM motif that has amino acid substitutions, deletions or additions at 1, 2, 3, 4, 5, 8, 10, 12 or 15 positions of the SEQ ID NO: 18. In certain embodiments, such amino acid substitutions are conservative amino acid substitutions.

In certain embodiments, an Rpn11 of the present invention comprises an amino acid sequence that is at least 60% identical, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99 % or 100% to the amino acid sequence of SEQ ID NO: 16, and the Rpn11 protein comprises a JAMM motif that is at least 80% identical, 85%, 90%, 95%, 98%, 99% or 100% to the amino acid sequence of SEQ ID NO: 17.

In certain embodiments, an Rpn11 of the present invention comprises an amino acid sequence that is at least 60% identical, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99 % or 100% to the amino acid sequence of SEQ ID NO: 16, and the Rpn11 protein comprises a JAMM motif that is at least 80% identical, 85%, 90%, 95%, 98%, 99% or 100% to the amino acid sequence of SEQ ID NO: 18.

In more preferred embodiments, an Rpn11 of the present invention comprises an amino acid sequence having amino acid substitutions, deletions or additions at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of SEQ ID NO: 16, and Rpn11 comprises a JAMM motif that has amino acid substitutions, deletions or additions at 1, 2, 3, 4 or 5 positions of SEQ ID NO: 17.

In more preferred embodiments, an Rpn11 of the present invention comprises an amino acid sequence having amino acid substitutions, deletions or additions at 1, 2, 5, 10, 15, 20, 30, 50, 60, 75 or 90 positions of the SEQ ID NO: 16, and the Rpn11 protein comprises a JAMM motif that has amino acid substitutions, deletions or additions at 1, 2, 3, 4, 5, 8, 10, 12 or 15 positions of SEQ ID NO: 18.

As used herein, "sequence identity" (or "% identical") refers to the percentage of identical amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize mating between sequences, that is, taking into account the gaps and inserts. Identity can be easily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, AM, ed, Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods have been designed to determine identity to give the greatest match between the sequences under test. In addition, methods for determining identity in publicly available software have been codified. Methods in computer programs for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (I): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol .

215: 403-410 (1990). The well-known Smith-Waterman algorithm can also be used to determine sequence identity.

The phrase "conservative amino acid substitution" refers to the grouping of amino acids based on certain common properties. A functional way to define common properties among individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure, Springer-Verlag). According to such analyzes, amino acid groups can be defined in which the amino acids within a group are preferably exchanged with each other, and therefore resemble each other more in their impact on the overall structure of the protein (Schulz, GE and RH Schirmer , Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this way include:

(i)

a charged group, consisting of Glu and Asp, Lys, Arg and His,

(ii)

a positively charged group, consisting of Lys, Arg and His,

(iii) a negatively charged group, which consists of Glu and Asp,

(iv)

an aromatic group, consisting of Phe, Tyr and Trp,

(v)

a ring group with nitrogen, which consists of His and Trp,

(saw)

a large aliphatic nonpolar group, consisting of Val, Leu and Ile,

(vii) a slightly polar group, consisting of Met and Cys,

(viii) a small residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro,

(ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and

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(x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue may form its own group, and the group consisting of an individual amino acid may simply be referred to by the abbreviation of one and / or three letters for that amino acid commonly used in the art.

Illustrative peptides of the application

Various peptides or proteins are described, which may comprise any one of the sequences of SEQ ID NO: 1-15 set forth below. In certain embodiments of the invention, a peptide substrate for proteasome enzyme activity (eg, Rpn11) comprises an amino acid sequence of any of SEQ ID NO: 1 and 3-15 and further comprises a ubiquitin residue. By "ubiquitin residue" is meant a single ubiquitin residue (Ub) of 76 amino acids or a remainder of polyubiquitin residue, such as the 9 repetition polyubiquitin (as represented by the first 684 amino acids of SEQ ID NO : 2) (also called Ub9), with repetitions of Ub (also called Ubn, where n can be any integer). In preferred embodiments, the ubiquitin moiety is at the N-terminal end of the peptide substrate, whereby the peptide substrate is considered modified by N-terminal ubiquitination or N-terminal polyubiquitination. Alternatively, the peptide substrate may have a "branched" primary structure, for example, the ubiquitin moiety is linked to K6 of SEQ ID NO: 1 in a peptide substrate comprising the sequence of SEQ ID NO: 1 and a moiety of ubiquitin, or the ubiquitin residue is linked to any one or more of K6 and K17 of SEQ ID NO: 3 in a peptide substrate comprising the sequence of SEQ ID NO: 3 and a ubiquitin residue. Among the first 14, 15 or 16 amino acids (in N-terminal) of any of SEQ ID NO: 1 and 3-15, one or more residues (eg, three) may be replaced by A. In any of SEQ ID NO: 3-15, one or more of the residues of S may be replaced by C.

In certain embodiments, the 75th amino acid of Ub as represented by the first 76 amino acids of SEQ ID NO: 2 may be replaced by A. In certain embodiments, the 76th amino acid in SEQ ID NO: 2 may be replaced by A. The 75th and 76th amino acids can both be substituted by A. In preferred embodiments, the 76th amino acid is G or A, and preferably is not V.

SEQ ID NO: 1

MQIFVKTLRANXXX

X can be S, C or A

SEQ ID NO: 2 Ubiquitin (ubiquitin C gene product (UBC) of 9 coding units: the underlined part is the individual ubiquitin of 76 amino acids)

> gi | 340068 | gb | AAA36789.1 | ubiquitin

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SEQ ID NO: 3 (peptide (29)) MQIFVKTLRANSSSVXKLAAALZHHHHHH X may be D or A; Z can be E or A SEQ ID NO: 4 (peptide-C14 (29)) MQIFVKTLRANSSCVXKLAAALZHHHHH X can be D or A; Z can be E or A SEQ ID NO: 5 (peptide-C13 (29)) MQIFVKTLRANSCSVXKLAAALZHHHHHH X can be D or A; Z can be E or A SEQ ID NO: 6 (peptide-C12 (29)) MQIFVKTLRANCSSVXKLAAALZHHHHHH X can be D or A; Z may be E or A SEQ ID NO: 7 (peptide-SC (29)) MQIFVKTLRANCCCVXKLAAALZHHHHHH X may be D or A; Z can be E or A SEQ ID NO: 8 (peptide (28)) MQIFVKTLRANSSSVXKLAAALZHHHHH X can be D or A; Z can be E or A SEQ ID NO: 9 (peptide (26)) MQIFVKTLRANSSSVXKLAAALZHHH X can be D or A; Z can be E or A SEQ ID NO: 10 (peptide (24)) MQIFVKTLRANSSSVXKLAAALZH X can be D or A; Z can be E or A SEQ ID NO: 11 (peptide (23)) MQIFVKTLRANSSSVXKLAAALZ X can be D or A; Z can be E or A SEQ ID NO: 12 (peptide (21)) MQIFVKTLRANSSSVXKLAAA X can be D or A SEQ ID NO: 13 (peptide (18)) MQIFVKTLRANSSSVXKL X can be D or A SEQ ID NO: 14 (peptide ( 16)) MQIFVKTLRANSSSVX X can be D or A SEQ ID NO: 15 (peptide (14)) MQIFVKTLRANSSS Nucleic acids encoding the various proteins and substrates are also described

peptides described herein. Nucleic acids can be useful, for example, in obtaining peptide substrates by recombinant molecular biology technology.

In certain embodiments, the peptide substrates comprise a detectable agent and / or a target part that specifically binds or interacts with a selection agent.

By "detectable agent" herein is meant an agent that can be detected and / or "visualized" by methods known in the art. For example, a fluorescent label or a radioactive label can be used as a detectable agent, and a peptide substrate that includes such a detectable agent can be detected and / or "visualized" by known methods that measure / detect fluorescence (for example, polarization assays of fluorescence) or radioactivity (for example, autoradiography or scintillation counting). A fluorescent label may comprise a fluorophore or a fluorescent peptide (such as the green or blue fluorescent protein). Examples of fluorophores include, but are not limited to, FITC, fluorescein-5-maleimide, BODIPY 499/508 maleimide, BODIPY FL N- (2-aminoethyl) maleimide, tetramethylrodamine-6-maleimide and tetramethylrodamine-5maleimide, 5-iodoacetamidofluorescein ( -IAF), 6-iodoacetamidofluorescein (6-IAF), Oregon Green® 488 iodoacetamide, and Oregon Green® 488 maleimide. A fluorophore can be "visualized" on a gel by detection with an anti-fluorophore antibody or in a suitable reaction mixture by detection with a homogeneous fluorescence assay such as fluorescence polarization.

In certain embodiments, a detectable agent is equivalent to a target part that specifically binds to a selection agent. For example, a fluorescent marker with a fluorophore can be a target part that specifically binds to an anti-fluorophore antibody. In certain embodiments, a detectable agent is different from a target part in which the detectable agent can be directly detected, while the target part further requires a corresponding selection agent to aid in the detection and / or visualization. For example, the target part can be a protein such as GST, and a corresponding selection agent can be an anti-GST antibody that can be used to "visualize" the target part (and thereby the peptide substrate comprising the target part) by, for example, ELISA or Western blot. One skilled in the art can appreciate that various molecules, for example, proteins, DNA, RNA, peptidomimetics, small molecules, can serve as the target part. In preferred embodiments, certain well-known epitopes such as the target part may be employed, for example, HA tag (hemagglutinin), FLAG epitope, Myc epitope, His6 tag and T7 epitope, and their corresponding antibodies, well known, may be used to determine its presence or absence. For a target part such as the His6 tag, a divalent metal ion (eg, Ni2 +) can also serve as a selection agent.

In certain preferred embodiments, a target part is located at the N-terminal end of a peptide substrate of the application.

Methods of obtaining peptides

Methods of obtaining the peptide substrates described herein are also described herein.

The peptides, for example, any of SEQ ID NO: 1 and 3-15 can be manufactured by conventional chemical peptide synthesis. Alternatively, recombinant molecular biology techniques can be used. Chemical synthesis and recombinant technology can be used in combination to obtain the peptide substrates of the application.

A nucleic acid encoding a protein of the application may comprise a nucleic acid or fusion gene (for example, a fusion nucleic acid encoding a peptide substrate comprising a "fused" ubiquitin residue with a sequence of any of SEQ ID NO: 1 and 3-15), the methods for obtaining them being known in the art. For example, the binding of various gene or DNA fragments (for example, a DNA fragment comprising a nucleic acid encoding a ubiquitin residue, and a DNA fragment comprising a nucleic acid encoding a peptide having the sequence of any of SEQ ID NO: 1 and 3-15) encoding different polypeptide sequences is carried out according to conventional techniques, employing terminal ends with blunt ends or stepped ends for ligation, digestion with restriction enzymes to provide ends appropriate terminals, filling cohesive ends as appropriate, treatment with alkaline phosphatase to avoid unwanted binding and enzymatic ligation. In certain embodiments, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary projections between two consecutive gene fragments that can subsequently be hybridized to generate a chimeric or fusion gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).

The recombinant nucleic acid constructs of the description can be manufactured using conventional recombinant DNA methodologies well known and absolutely documented in the art, as well as using well known biosynthetic and chemosynthetic methodologies by using routine nucleotide or peptide chemical techniques and synthesizers of Automated peptides or nucleotides. Such routine methodologies are described for example in the following publications, Hilvert, 1 Chem.-Biol. 201-3 (1994); Muir et al., 95 Proc. Natl Acad. Sci. USA 6705-10 (1998); Wallace, 6 Cuff. Opin. Biotechnol 40310 (1995); Miranda et al., 96 Proc. Natl Acad. Sci. USA 1181-86 (1999); Liu et al., 91 Proc. Natl Acad. Sci. USA 6584-88 (1994). Suitable for use in the present description are naturally occurring amino acids and nucleotides; amino acids and nucleotides that do not occur naturally; modified or unusual amino acids; modified bases, amino acid sequences containing modified amino acids after translation and / modified links, crosslinks and busy ends, non-peptidyl bonds, etc .; and, which also include without limitation, those remains disclosed in World Intellectual Documentation. Standard St. 25 (1998) including tables 1 to 6 in Appendix 2, incorporated herein by reference. The person skilled in the art will appreciate equivalents to the above based on routine experimentation, together with knowledge of the art.

For example, the contemplated DNA constructs can be manufactured by assembling synthetic nucleotide sequences and / or binding DNA restriction fragments to produce a synthetic DNA molecule. The DNA molecules are then ligated, cloned or subcloned into an expression vehicle, for example an expression plasmid (for example, pET28a or pGEX), and transfected into an appropriate host cell, for example E. coli. The contemplated protein construct encoded by the DNA molecule is then expressed, purified, folded again, tested in vitro for the determination of certain attributes, for example, binding activity with a selection agent (for example , an anti-Myc antibody) that has binding affinity for its corresponding target part (for example, the Myc epitope) on the peptide substrates, and / or ability to serve as an enzymatic substrate to measure proteasome activity.

Methods of using peptides to select agents that modulate proteasome activity

The present invention also provides methods of using the peptide substrates described herein, for example, by selecting one or more agents that modulate proteasome activity. As described above, proteasome activity involves various components, and an agent that modulates a particular component of proteasome activity can be selected, for example, the entry of a protein selected as a target for degradation (e.g., modified by ubiquitination) in the proteasome, the Rpn11 activity or other metalloprotease activity of the 19S regulatory particle, the ATPase activity, or the peptidase activity of the core of the 20S complex.

In one embodiment, an enzyme or enzymatic preparation used in the screening assays provided by the present application is a 26S proteasome polypeptide complex or 19S regulatory particle, or a polypeptide or polypeptide complex having Rpn11 enzymatic activity. A 26S proteasome polypeptide complex or 19S regulatory particle, or a polypeptide or polypeptide complex having Rpn11 enzymatic activity can be obtained by any suitable methodology. For example, the 26S proteasome can be purified from eukaryotic tissues or cells, for example, S. cerevisiae or human.

In another embodiment, incubation in the presence and absence of a test agent, a protein

or target peptide substrate, and the 26S proteasome of the screening assays provided by the present application is optionally carried out in the presence of a 20S proteasome inhibitor. Any 20S proteasome inhibitor or degradation process inhibitor may be used for the purpose of the present application. For example, a 20S proteasome inhibitor may be MG132, lactacystin, epoxomycin, YU101, PS-349, PS-519, LLnL, or derivatives thereof. In general, such an inhibitor prevents or decreases the degradation of a target protein that is not conjugated to ubiquitin.

In yet another embodiment, the incubation is further performed in the presence of an energy source, for example, ATP. In yet another embodiment, the incubation is further carried out in the presence of a deubiquitination inhibitor by means of a conventional isopeptidase ubiquitin, for example, an isopeptidase ubiquitin other than those associated with a JAB subunit such as the 26S proteasome. An example of such an inhibitor is ubiquitin aldehyde, for example, at 2-5 µM.

In certain embodiments, the detection of the enzymatic activity of the proteasome employs a ubiquitinated protein substrate such as a peptide substrate as described herein, and the reaction was monitored by SDS-PAGE followed by immunoblotting with either anti-ubiquitin antibody. or another antibody specific for another component of the peptide substrate, for example, a target part such as Myc. Alternatively, after being exposed to the enzyme or the enzyme preparation to be tested, peptide substrates comprising a radiolabel or fluorescent label can be subjected to SDS-PAGE to separate the uncleaved peptide substrate from the cleaved product (Eytan et al. , cited above) or precipitation with TCA (Yao et al., Nature 419: 403-407 (2002)), and radioactivity or fluorescence can be detected by means known in the art.

Certain embodiments provide a method for selecting an agent that modulates the enzymatic activity of the 26S proteasome, the 19S regulatory particle, or a polypeptide or polypeptide complex that has Rpn11 enzymatic activity. The method includes providing a peptide substrate of the application, that is, a peptide substrate comprising an amino acid sequence of SEQ ID NO: 1 and one or more ubiquitin residues, and combining the peptide with a reaction mixture suitable for measuring Proteasome activity in the presence of a test agent. A change in proteasome activity in the presence of the test agent compared to the activity of the proteasome in the absence of the test agent indicates that the test agent modulates the activity of the proteasome. Proteasome activity can be determined, for example, by measuring the level of cleavage of the ubiquitin residue (s) from the peptide. The level of excision can be indicated by the rate and / or the degree of excision. The method is useful for selecting an agent that potentiates or inhibits proteasome activity.

A recent publication (Verma et al., Science 306: 117-120 (2004)) identified another type of proteasome inhibitor, called "ubistatin." Ubistatin works by binding to ubiquitin or polyubiquitin chains bound to the selected protein. as a target for degradation, and thereby preventing the peptide molecule from entering the proteasome. The peptide substrates of the present application can be used to select proteistase inhibitors of the ubistatin type. For example, a peptide substrate of the application can be used to compare an effect of the test inhibitory agent on the activity of the 26S proteasome (eg, measuring IC50) with its effect on the isopeptidase T activity. If the effects are equivalent for Different enzymes or enzymatic preparations, the test inhibitory agent is acting on the peptide substrate, not on enzymes, and therefore acts as a ubistatin molecule. It is also observed that ubistatin A, also called compound 92 (Verma et al., 2004, cited above), binds to a substrate protein, and the intrinsic fluorescence detected from compound 92 increases. This inherent characteristic, therefore, allows to select ubistatin-type molecules in an assay that measures changes, in presence compared to in the absence of a test agent, in the intrinsic fluorescence of ubistatin and / or the test agent. A competition based assay can be used in which a ubistatin molecule, a test agent and a peptide substrate are combined with each other in a test / reaction mixture suitable for measuring intrinsic fluorescence, and a change in intrinsic fluorescence of the Ubistatin in the presence compared to the absence of the test agent in the mixture indicates that the test agent may be of the ubistatin type and may compete with the ubistatin to bind to the peptide substrate. Another assay may be employed in which a test agent and a peptide substrate are combined with each other in a test / reaction mixture suitable for measuring intrinsic fluorescence, and a change in the intrinsic fluorescence of the test agent in the presence of the peptide substrate indicates that the test agent is a ubistatin-like proteasome inhibitor.

A selection method of the invention includes providing a peptide substrate of the application having a fluorescent label, for example, a peptide substrate comprising an amino acid sequence of SEQ ID NO: 1, one or more ubiquitin residues and a label. fluorescent, and determine the fluorescence polarization of the peptide substrate in the presence compared to in the absence of a test agent. A difference in fluorescence polarization in the presence of the test agent versus in the absence of the test agent indicates that the test agent may be a proteasome inhibiting agent. Without wishing to restrict itself to any theory, the test agent can function in a ubistatin-like manner, for example, producing aggregation or multimerization of ubiquitin residues of the peptide substrates. Such aggregation or multimerization can be detected by any suitable methodology to detect changes in the molecular weight and / or the size of the molecules, for example, of the peptide substrates. A suitable methodology may be gel electrophoresis under native conditions, size exclusion chromatography (or gel filtration), fluorescence polarization, light scattering, or any other suitable means.

Certain embodiments provide a method for selecting a proteasome inhibitor agent. The proteasome inhibitor may comprise a ubistatin-like molecule or a ubiquitin-like binding molecule. The proteasome inhibitor that binds to ubiquitin can inhibit substrate proteolysis or can inhibit proteasome activity through other mechanisms. The inhibitory agent may or may not have intrinsic fluorescence.

Certain embodiments also provide a method for selecting a proteasome inhibitor agent that modulates AAA ATPase activity. Without wishing to restrict themselves to any particular theory, it has been observed that the AAA ATPase activity is coupled with the enzymatic activity of Rpn11. Consequently, a specific inhibitory agent for the enzymatic activity of Rpn11 can also modulate AAA ATPase activity.

A further aspect of the invention provides a method for selecting an agent that modulates the activity of various proteasome components or various phases of the ubiquitin-mediated proteolysis process. The various components of the proteasome and the various phases of ubiquitin-mediated proteolysis may include the entry into the proteasome of ubiquitinated proteins; the desubiquitination of proteins, for example, by Rpn11; the deployment of proteins, for example, AAA ATPase; degradation of desubiquitinated proteins by a core peptidase.

The method employs at least two peptide substrates, that is, a first peptide substrate comprising a ubiquitinated peptide and a first fluorescent label, and a second peptide substrate comprising a non-ubiquitinated peptide and a second fluorescent label, wherein the first and The second fluorescent markers are detectable at different wavelengths. A ubiquitinated peptide was defined above and comprises an amino acid sequence of SEQ ID NO: 1 and ubiquitin residues. A non-ubiquitinated peptide can be any peptide that can serve as a substrate for a core peptidase, for example, a chemotryptic type protease, a tryptic type protease, or a PGPH (or caspase) protease. The method includes measuring the fluorescence at the wavelength of the first detectable label in the presence and absence of a test agent, and measuring the fluorescence at the wavelength of the second detectable marker in the presence and absence of a test agent. A change in the fluorescence of the first peptide substrate in the presence of the test agent compared to the fluorescence in the absence of the test agent indicates that the test agent modulates an activity associated with a 19S regulatory particle. A change in the fluorescence of the second peptide substrate in the presence of the test agent compared to the fluorescence in the absence of the test agent indicates that the test agent modulates a 20S core peptidase.

In addition, high performance assays employing certain peptide substrates (eg, a peptide substrate comprising a fluorescent label) described herein are described, assays that can be designed based on the methods described herein. High performance assays are particularly useful for selecting and identifying agents that can modulate proteasome activity and thereby ubiquitin-mediated protein degradation.

A high performance assay may employ a selection method that determines whether a test agent produces changes in the size and / or molecular weight of a peptide substrate in a suitable reaction mixture. Such a selection method may employ a fluorescence polarization assay to measure changes in the size and / or molecular weight of the peptide substrate.

The test agent used for the methods of selection of the present application can be any agent of any library of compounds or molecules. In one embodiment, the test agent is selected or derived from compounds that are likely to inhibit metalloproteinase activity, for example, compounds that have zinc binding functionality. For example, the test agent can be any compound having a hydroxamate moiety or a member of a hydroxamate compound library, reverse hydroxamate compound library, thiol compound library, carboxylate compound library, or compound library of phosphonic acid.

The agents tested in the screening methods to identify agents that modulate proteasome activity provided herein may be of any physical type. Examples of agents include, but are not limited to, biomolecules, including, but not limited to, amino acids, peptides, polypeptides, peptidomimetics, nucleotides, nucleic acids (including DNA, cDNA, RNA, antisense RNA and any single or double stranded form of acids nucleic and structural derivatives and analogs thereof), polynucleotides, saccharides, fatty acids, steroids, carbohydrates, lipids, lipoproteins and glycoproteins. Such biomolecules can be substantially purified, or they can be present in a mixture, such as a cell extract or supernatant. Test agents also include natural or synthetic chemical compounds, such as simple or complex organic molecules, compounds containing metal and inorganic ions (including, for example, Gd3 +, lead and lanthanum). Also included are pharmacological compounds, which may optionally be subjected to random or directed chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogues.

Uses of selected agents

Methods and compositions are also described which employ an agent selected according to a method of the application. For example, certain embodiments provide a method for treating or preventing in a subject a condition associated with ubiquitin-mediated proteolysis or proteasome activity that comprises administering to the subject a composition comprising an agent that modulates proteasome activity, particularly, the enzymatic activity of Rpn11.

The term "prevent" is recognized in the art, and when used in relation to a condition, such as a local recurrence (eg, pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical status, is well understood in the art, and includes the administration of a composition that reduces the frequency of, or delays the onset of, the symptoms of a medical condition in a subject in relation to a subject that does not receive the composition. Therefore, cancer prevention includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment in relation to an untreated control population, and / or delaying the onset of detectable cancerous growths in a treated population versus a non-treated control population, for example, in a statistically and / or clinically significant amount. The prevention of an infection includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and / or delaying the onset of infection symptoms in a treated population versus a population untreated control. Pain prevention includes, for example, reducing the magnitude of, or alternatively delaying, the pain sensations experienced by the subjects in a treated population versus an untreated control population.

The term "treat" includes prophylactic and / or therapeutic treatments. The term "prophylactic or therapeutic" treatment is recognized in the art and includes administration to the host of one or more of the subject compositions. If they are administered before the clinical manifestation of the unwanted state (for example, disease or other unwanted state of the host animal) then the treatment is prophylactic, (that is, it protects the host against the development of the unwanted state), while if it is administered after the manifestation of the unwanted state, the treatment is therapeutic, (that is, it is intended to decrease, improve or stabilize the existing unwanted state or its side effects).

Any agent that can inhibit or decrease proteasome activity, for example, by modulating the deconjugation, elimination or separation of a ubiquitin molecule from a target protein, can be used prophylactically or therapeutically to treat or prevent various conditions associated with protein regulation by degradation or proteolysis. For example, any agent identified by the methods of selection of the present application that may decrease the decongestion of ubiquitin of a target protein, for example, an inhibitor of the isopeptidase activity of the 26S proteasome, can be used therapeutically to treat neoplastic growth, angiogenesis, infection, inflammation, diseases related to the immune system ischemia and reperfusion injury, multiple sclerosis, rheumatoid arthritis, neurodegenerative states and psoriasis. Certain conditions or disorders associated with proteasome activity or aberrant ubiquitin-mediated protein degradation can be characterized by the accumulation of ubiquitinated proteins.

Many diseases and conditions are associated with protein regulation through proteolysis or proteasome activity. Such diseases and conditions include restenosis, inflammation, rheumatoid arthritis, tissue injury due to inflammation, hyperproliferative diseases, severe or arthritic psoriasis, progressive muscular atrophy diseases, chronic infectious diseases, abnormal immune response, states involving vulnerable plaques, state-related lesions. ischemic, and viral infection and proliferation. Agents identified or selected by the present application may be useful for the treatment of conditions associated with chronic inflammation, including, but not limited to COPD, psoriasis, bronchitis, emphysema and cystic fibrosis.

The agents identified herein can be used to treat conditions mediated directly by the proteolytic function of the proteasome such as progressive muscular atrophy, or indirectly mediated by proteins that are processed by the proteasome, such as NF-B. The proteasome participates in the rapid elimination and processing after translation of the proteins (for example, enzymes) involved in cell regulation (e.g., cell cycle, gene transcription and matabolic pathways), intercellular communication, and immune response ( for example, presentation of antigens). Specific examples discussed below include the -amyloid protein and regulatory proteins such as cyclin, TGF- and the transcription factor NF-B.

The use of agents identified herein for the treatment of diseases and neurodegenerative conditions, including, but not limited to, stroke, ischemic injury to the nervous system, neural trauma (e.g., percussion brain damage, injury) is also described. in the spinal cord and damage from trauma to the nervous system), multiple sclerosis and other neuropathies mediated by the immune system (for example, Guillain-Barre syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy and Fisher syndrome ), complex of dementia associated with HIV / AIDS, axonomy, diabetic neuropathy, Parkinson's disease, Huntington's disease, multiple sclerosis, bacterial, parasitic, fungal and viral meningitis, encephalitis, vascular dementia, multiple infarction dementia, body dementia Lewy, frontal lobe dementia such as Pick's disease, demenc Subcortical ias (such as Huntington's disease or progressive supranuclear paralysis), focal cortical atrophy syndromes (such as primary aphasia), toxic metabolic dementias (such as chronic hypothyroidism or B12 deficiency), and dementias caused by infections (such as chronic meningitis or by syphilis).

Alzheimer's disease is characterized by extracellular deposits of the amiloid (-AP) protein in senile plaques and cerebral vessels. -AP is a peptide fragment of 39 to 42 amino acids derived from an amyloid protein precursor (APP). At least three isoforms of APP (of 695, 751 and 770 amino acids) are known. The alternative splicing of mRNA generates the isoforms; Normal processing affects a part of the -AP sequence, thereby preventing the generation of -AP. It is believed that the abnormal processing of the protein by the proteasome contributes to the abundance of -AP in the brain with Alzheimer's. The enzyme that processes APP in rats contains approximately ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has an N-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the  subunit of the human macropain (Kojima, S. et al ., Fed. Eur. Biochem. Soc., (1992) 304: 57-60). The enzyme that processes the APP cleaves at the Gln15-Lys16 link; in the presence of the calcium ion, the enzyme also cleaves at the Met-1-Asp1 bond and at the Asp1-Ala2 bond to release the extracellular domain of -AP.

An example, therefore, is a method of treating Alzheimer's disease, which includes administering to an individual an effective amount of an agent or composition (eg, a pharmaceutical composition) disclosed herein. Such a treatment includes reducing the -AP processing rate, reducing the de-AP plaque formation rate, reducing the -AP generation rate and reducing the clinical signs of Alzheimer's disease.

Other examples are related to the diseases of cachexia and progressive muscular atrophy. The proteasome degrades many proteins in the maturation reticulocytes and growing fibroblasts. In insulin or serum-deprived cells, the proteolysis rate is almost doubled. Proteasome inhibition reduces proteolysis, thereby reducing both loss of muscle protein and nitrogen load in the kidneys or liver. The inhibitors of the application are useful for treating conditions such as cancer, chronic infectious diseases, fever, denervation and muscle inactivity (atrophy), nerve injury, fasting, kidney failure associated with acidosis, diabetes and liver failure. See, for example, U.S. Patent No. 5,340,736. Embodiments of the description therefore encompass methods to: reduce the rate of degradation of muscle proteins in a cell; reduce the rate of intracellular protein degradation; reduce the degradation rate of p53 protein in a cell; and inhibit the growth of p53-related cancers. Each of these methods includes contacting a cell (in vivo or in vitro, for example, a muscle in a subject) with an effective amount of an agent or composition (eg, a pharmaceutical composition) disclosed herein. document.

Fibrosis is the excessive and persistent formation of scar tissue that results from hyperproliferative growth of fibroblasts and is associated with the activation of the TGF- signaling pathway. Fibrosis involves wide deposition of the extracellular matrix and can occur within virtually any tissue or through several different tissues. Normally, the level of intracellular signaling protein (Smad) that activates transcription of the target genes with TGF- stimulation is regulated by proteasome activity. However, accelerated degradation of TGF- signaling components has been observed in cancers and other hyperproliferative states. Therefore, certain aspects of the description refer to a method for treating hyperproliferative states such as diabetic retinopathy, macular degeneration, diabetic neuropathy, glomerulosclerosis, IgA neuropathy, cirrhosis, biliary atresia, congestive heart failure, scleroderma, radiation-induced fibrosis and pulmonary fibrosis (idiomatic pulmonary fibrosis, collagen vascular disease, sarcoidosis, interstitial lung diseases and extrinsic pulmonary disorders). The treatment of burn victims is often hampered by fibrosis, therefore, a further embodiment of the application is the topical or systemic administration of the inhibitors to treat burns. Wound closure after surgery is often associated with disfiguring scars, which can be avoided by inhibiting fibrosis. Therefore, in certain aspects, the description refers to a method for the prevention or reduction of healing.

Another protein processed by the proteasome is NF-B, a member of the Rel family of proteins. The Rel family of transcription activating proteins can be divided into two groups. The first group requires proteolytic processing and includes p50 (NF-1B1, 105 kDa) and p52 (NF-2, 100 kDa). The second group does not require proteolytic processing and includes p65 (RelA, Rel (c-Rel) and RelB). Rel family members can form both homodimers and heterodimers; NF-B, for example, is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IB and p105, the two proteins are degraded and processed, respectively, to produce active NF-B that is translocated from the cytoplasm to the nucleus. Ubiquitinated p105 is also processed by purified proteasomes (Palombella et al., Cell (1994) 78: 773-785). Active NF-formaB forms a stereospecific enhancer complex with other transcription activators and, for example, HMG I (Y), which induces selective expression of a particular gene.

NF-regulaB regulates genes involved in the immune and inflammatory response, and mitotic events. For example, NF-B is required for the expression of the  light chain gel of immunoglobulins, the  chain gene of the IL-2 receptor, the major histocompatibility complex gene and several cytosine genes encoding for, for example, IL-2, IL-6, granulocyte colony stimulating factor and IFN- (Palombella et al., Cell (1994) 78: 773-785). Some embodiments of the application include methods to affect the level of expression of IL-2, MHC-I, IL-6, TNF, IFN- or any of the other proteins mentioned above, each method including administering to a subject a quantity Effective of an agent identified in this document. The complexes, including p50, are rapid mediators of acute immune and inflammatory responses (Thanos, D. and Maniatis, T., Cell (1995) 80: 529-532).

NF-tambiénB also participates in the expression of cell adhesion genes encoding Eselectin, P-selectin, ICAM and VCAM-1 (Collins, T., Lab. Invest. (1993) 68: 499-508). One embodiment of the application is a method of inhibiting cell adhesion (eg, cell adhesion mediated by Eselectin, P-selectin, ICAM or VCAM-1), which includes contacting a cell with (or administering to a subject) an effective amount of an agent (or a pharmaceutical composition) identified herein.

Ischemia and reperfusion injury result in hypoxia, a state in which there is a deficiency of oxygen that reaches the body's tissues. This state produces an increase in the degradation of I-B, thereby resulting in the activation of NF-B. It has been shown that the severity of the injury that results in hypoxia can be reduced with the administration of a proteasome inhibitor. Therefore, certain aspects of the description relate to a method of treating an ischemic state or reperfusion injury comprising administering to an individual in need of such treatment an effective amount of an agent identified herein. Examples of such conditions or injuries include, but are not limited to, acute coronary syndrome (vulnerable plaques), arterial occlusive disease (cardiac, cerebral, peripheral and vascular arterial occlusions), atherosclerosis (coronary sclerosis, coronary heart disease), heart attacks, heart failure, pancreatitis , myocardial hypertrophy, stenosis and restenosis.

NF-tambiénB also specifically binds to the HIV enhancer / promoter. When compared to mac239 Nef protein, pbj 14 HIV regulatory Nef protein differs by two amino acids in the region that controls protein kinase binding. It is believed that protein kinase signals the phosphorylation of IB, which triggers the degradation of IB through the ubiquitin proteasome pathway. After degradation, NF-B is released into the nucleus, thereby enhancing HIV transcription (Cohen, J., Science, (1995) 267: 960). Two embodiments of the application are a method to inhibit or reduce HIV infection in a subject, and a method to decrease the level of viral gene expression, including each method administering to the subject an effective amount of an agent identified herein. .

It is considered that the excess production of lipopolysaccharide-induced cytokines (LPS) such as TNF is essential for the processes associated with septic shock. In addition, it is generally accepted that the first stage in the activation of cells by LPS is the binding of LPS to specific membrane receptors. The  and  subunits of the 20S proteasome complex have been identified as proteins that bind to LPS, suggesting that LPS-induced signal transduction may be an important therapeutic target in the treatment or prevention of septicemia (Qureshi, N. et al., J. Immun. (2003) 171: 1515-1525). Therefore, in certain embodiments, the agents identified in the application for TNF inhibition can be used to prevent and / or treat septic shock.

Intracellular proteolysis generates small peptides for presentation to T lymphocytes to induce immune responses mediated by class 1 CMH. The immune system selects anthologous cells that are infected by viruses or have undergone oncogenic transformation. An embodiment is a method of inhibiting the presentation of antigens in a cell, which includes exposing the cell to an agent described herein. An agent of the application can be used to treat conditions related to the immune system, such as allergy, asthma, organ / tissue rejection (graft versus host disease), and autoimmune diseases, including, but not limited to, lupus, rheumatoid arthritis , psoriasis, multiple sclerosis and inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). Therefore, a further embodiment is a method of suppressing a subject's immune system (for example, inhibiting rejection of a transplant, allergies, autoimmune diseases and asthma), which includes administering to the subject an effective amount of an agent identified in the present document

A method to alter the repertoire of antigenic peptides produced by the proteasome or other Ntn with multicatalytic activity is also described. For example, if the PGPH activity of the 20S proteasome is selectively inhibited, the proteasome will produce a different set of antigenic peptides and will be presented in CMH molecules on the surfaces of the cells from which it would be produced and presented or without any enzymatic inhibition, or with, for example, selective inhibition of the chyotrypsin-like activity of the proteasome.

Certain proteasome inhibitors block both degradation and processing of ubiquitinated NFB in vitro and in vivo. Proteasome inhibitors also block the degradation of IB and the activation of NF-B (Palombella, et al. Cell (1994) 78: 773-785; and Traenckner, et al., EMBO J. (1994) 13: 5433-5441). An embodiment of the application is a method of inhibiting the degradation of IB-, which includes contacting the cell with an agent identified herein. A further embodiment is a method of reducing the cellular content of NF-enB in a cell, muscle, organ or subject, which includes contacting the cell, muscle, organ or subject with an agent identified herein.

Other eukaryotic transcription factors that require proteolytic processing include the general TFIIA transcription factor, the accessory herpes simplex virus VP16 (host cell factor) protein, the virus-inducible IFN regulatory factor 2 protein, and the binding protein 1 to the sterile regulatory element attached to the membrane.

Methods for affecting cyclin-dependent eukaryotic cell cycles, including exposing a cell (in vitro or in vivo) to an agent identified herein, are also described. Cyclines are proteins involved in the control of the cell cycle. The proteasome participates in the degradation of cyclines Examples of cyclines include mitotic cyclines, G1 cyclines and cyclin B. Degradation of cyclines allows one cell to leave one phase of the cell cycle (for example, mitosis) and enter another (by example, division). It is believed that all cyclines are associated with the protein kinase p34cdc2 or related kinases. The selection signal as the target of proteolysis is located in amino acids 42-RAALGNISEN-50 (destruction box). There is evidence that cyclin becomes a form vulnerable to a ubiquitin ligase or that a specific cyclin ligase is activated during mitosis (Ciechanover, A., Cell, (1994) 79: 13-21). Proteasome inhibition inhibits cyclin degradation, and therefore inhibits cell proliferation, for example, in cyclin-related cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87: 7071-7075 ). An embodiment of the application is a method of treating a proliferative disease in a subject (eg, cancer, psoriasis or restenosis), which includes administering to the subject an effective amount of an agent identified herein. The application also encompasses a method of treating cyclin-related inflammation in a subject, which includes administering to a subject a therapeutically effective amount of an agent identified herein.

Additional examples are methods to affect the protease-dependent regulation of oncoproteins and methods of treatment or inhibition of cancer growth, each method including exposing a cell (in vivo, for example, in a subject, or in vitro) to an identified agent in the present document. E6 proteins derived from HPV-16 and HPV-18 stimulate conjugation and degradation dependent on ATP and ubiquitin of p53 in crude reticulocyte lysates. It has been shown that the p53 recessive oncogene accumulates at the non-permissive temperature in a cell line with a mutated thermolabile E1. Elevated levels of p53 can lead to apoptosis. Examples of protooncoproteins degraded by the ubiquitin system include c-Mos, c-Fos and c-Jun. An embodiment is a method of treating apoptosis related to p53, which includes administering to an individual an effective amount of an agent identified herein.

The agents of the present application are useful for the treatment of a parasitic infection, such as infections caused by protozoan parasites. The proteasome of these parasites is considered to be mainly involved in cell differentiation and replication activities (Paugam et al., Trends Parasitol. 2003, 19 (2): 55-59). In addition, entamoeba species have been shown to lose encyst capacity when exposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res. 1997, 28, Spec NO: 139-140). In certain embodiments, the agents are useful for the treatment of parasitic infections in humans produced by a protozoan parasite selected from Plasmodium sps. (including P. falciparum, P. vivax, P. malariae and P. ovale, which produce malaria), Trypanosoma sps. (including T. cruzi, which causes Chagas disease, and T. brucei that causes African sleeping sickness), Leishmania sps. (including L. amazonensis, L. donovani, L. infantum, L. mexicana, etc.), Pneumocystis carinii (a protozoan known to cause pneumonia in patients with AIDS and other immunosuppressed patients), Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens and Giardia lamblia. In certain embodiments, the agents are useful for the treatment of parasitic infections in animals and livestock produced by a protozoan parasite selected from Plasmodium hermani, Cryptosporidium sps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona and Neurospora crassa. Other compounds useful as proteasome inhibitors in the treatment of parasitic diseases are described in WO 98/10779, which is incorporated herein in its entirety.

Inhibiting agents may inhibit proteasome activity irreversibly in a parasite. It has been shown that an irreversible inhibition of this type induces the paralysis of the enzymatic activity without recovery in red blood cells and white blood cells. In certain embodiments, the long half-life of blood cells may provide prolonged protection with respect to therapy against recurrent exposures to parasites. In certain embodiments, the long half-life of blood cells may provide prolonged protection with respect to chemoprophylaxis against future infection.

It has also been shown that inhibitors that bind to the 20S proteasome stimulate bone formation in bone organ cultures. In addition, when such inhibitors have been administered systemically to mice, certain proteasome inhibitors increased bone volume and bone formation rates by more than 70% (Garrett, IR et al., J. Clin. Invest. (2003) 111: 1771-1782), which therefore suggests that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation. Therefore, the inhibiting agents identified herein may

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be useful in the treatment and / or prevention of diseases associated with bone loss, such as osteroporosis.

Bone tissue is an excellent source of factors that have the ability to stimulate bone cells. Therefore, bovine bone tissue extracts contain not only structural proteins that are responsible for maintaining bone structural integrity, but also biologically active bone growth factors that can stimulate bone cell proliferation. Among the latter factors there is a family of recently described proteins called bone morphogenetic proteins (BMP). All these growth factors have effects on other types of cells, as well as on bone cells, including Hardy, MH, et al., Trans Genet (1992) 8: 55-61 describe the evidence that bone morphogenetic proteins (BMP ), are expressed differentially in the hair follicles during development. Harris, S. E., et al., J Bone Miner Res (1994) 9: 855-863 describe the effects of TGF on the expression of BMP-2 and other substances in bone cells. BMP-2 expression also occurs in the hair follicles during maturation and after the period of cell proliferation (Hardy, et al., Cited above). Therefore, the agents of the description can also be useful for stimulating hair follicle growth.

The agents identified herein may also be useful as diagnostic agents (for example, in diagnostic kits or for use in clinical laboratories) to select proteins (for example, enzymes, transcription factors) processed by Ntn hydrolases, including The proteasome The agents are also useful as research reagents to specifically bind the X / MB1 subunit or the  chain and inhibit the proteolytic activities associated with it. For example, the activity of (and specific inhibitors of) other proteasome subunits can be determined.

Most cellular proteins undergo proteolytic processing during maturation or activation. The inhibitors identified herein can be used to determine if a physiological, developmental or cellular process or performance is regulated by proteolytic activity. Such a method includes obtaining a microorganism, an intact cell preparation, or a cell extract; exposing the microorganism, cell preparation or cell extract to an agent identified herein; expose the microorganism, cell preparation or cell extract exposed to the agent to a signal, and monitor the process or performance.

The agents of the present description useful for therapeutic treatment can be administered alone, in a composition with a suitable pharmaceutical carrier, or in combination with other therapeutic agents. An effective amount of the agents to be administered can be determined on a case-by-case basis. Factors that should normally be considered include age, body weight, the stage of the ailment, other pathological conditions, the duration of treatment, and the response to initial treatment. Normally, the agents are prepared as injectables, or as a suspension

or as a liquid solution. However, solid forms suitable for dissolution in, or suspension in, liquid vehicles before injection can also be prepared. The agent can also be formulated in an enteric coated tablet or gelatin capsule according to methods known in the art. The agents of the present description can be administered in any way that is medically acceptable that may depend on the identity of the agent and / or the pathological state or the lesion being treated. Possible routes of administration include injections, parenteral routes such as intravascular, intravenous, intraepidural or other, as well as oral, nasal, ophthalmic, rectal, topical or pulmonary, for example, by inhalation, nebulization or aerosol treatment. The agents can also be applied directly to the tissue surfaces, for example, during surgery. The application also specifically includes the administration of sustained release, by means such as slow absorption injections or erodible implants. Examples of diseases and conditions undergoing treatment using drug-coated medical devices that release the compounds identified using the methods of the present application include atherosclerosis, acute coronary syndrome, Alzheimer's disease, cancer, fever, muscle inactivity (atrophy) , denervation, vascular occlusions, stroke, HIV infection, nerve injury, kidney failure associated with acidosis and liver failure. See, for example, U.S. Patent No. 5,340,736.

Examples

Example 1. Reagents:

1) Buffer A (50 mM Tris pH 7.4, 0.01% NP-40, 1 mM DTT; 7.5 MgM MgCl 2; 7.5 ATM ATP);

2) Ub4-pep-C14 (29) -Oregon Green prepared using Ub4-pep-C14 (29) covalently coupled to Oregon Green 488-iodoacetamide and purified without the starting product;

3) LLVY-AMC, acquired from Boston Biochem;

4) 26S proteasome purified from humans and acquired from BioMol; 5) 384-well polystyrene plates purchased from Molecular Devices;

6) Ubistatin A (Compound 92 or C92);

7) Rabbit isopeptidase T acquired from Boston Biochem

Example 2. Method:

5 µl of compound [compound 30 µM in buffer A + 1% DMSO], 5 µl of substrate [Ub4-pep-C14 (29) -Oregon Green 30 nM and LLVY-AMC 15 µM in buffer were mixed A] and 5 µl of enzyme [1.5 nM 26S proteasome] in 384 well plates and incubated at 28 ° C for 75 minutes. The reactions were stopped with the addition of 5 µl of 20S 4 µM inhibitor (PR-171) and 4 mM EDTA. The processing of the 20S LLVY-AMC substrate to release AMC was determined by measuring AMC fluorescence [excitation at 340 nm; emission at 465 nm]. The substrate processing of 19S Ub4-pep-C14 (29) -Oregon Green to release pep-C14 (29) -Oregon Green was determined by measuring the fluorescence polarization of Oregon Green [excitation at 485 nm; emission at 535 nm].

Example 3. Different categories of modulators or test agents:

Modulators of the 20S proteasome activity affect the release of AMC from LLVYAMC (second non-ubiquitinated substrate) and affect the chemotryptic type activity of the 20S proteasome.

Modulators of 19S proteasome activity affect the release of pep-C14 (29) -Oregon Green from Ub4-pep-C14 (29) -Oregon Green (first ubiquitinated substrate). These modulators can act in one of three ways:

1) Ubiquitin binding thus preventing the Ub4-pep-C14 (29) -Oregon Green substrate from being processed by the 19S proteasome.

2) Inhibition of the AAA ATPase activity of the 19S proteasome.

3) Inhibition of the Rpn11 isopeptidase activity of the 19S proteasome.

Example 4. The binding of a test agent to ubiquitin can be determined by one or more of the following tests:

1) The IC50 for the action of a test agent in the processing of the Ub4-pepC14 (29) -Oregon Green substrate by means of the 26S proteasome enzyme is equivalent to the IC50 for the action of a test agent in the processing of the Ub4 substrate -pep-C14 (29) -Oregon Green by an unrelated deubiquitinating enzyme (eg, isopeptidase T). The test agent is acting on the substrate Ub4-pep-C14 (29) -Oregon Green and preventing its processing by deubiquitinating enzymes.

2) The test agent acts to multimerize Ub4-pep-C14 (29) -Oregon Green thereby preventing the release of pep-C14 (29) -Oregon Green. Multimerization can be detected by one of several methods, for example, gel filtration; light scattering; gel electrophoresis under native conditions; fluorescence polarization.

3) The intrinsic fluorescence of a test agent either increases or decreases with binding to Ub4-pep-C14 (29), where the test agent has an intrinsic fluorescence.

4) When 2.5 µM of ubistatin A (C92) is incubated with 2.5 µM of Ub4-pep-C14 (29), the intrinsic fluorescence of ubistatin A (C92) [excitation at 350 nm; emission at 450 nm] increases several times. A test agent that binds to ubiquitin can affect the binding of ubistatin A (C92) to Ub4-pep-C14 (29). This can be determined by measuring the intrinsic fluorescence of ubistatin A (C92) [excitation at 350 nm; emission at 450 nm] with Ub4-pep-C 14 (29) in the presence or absence of a test agent, which is a competition test between ubistatin A and the test agent for binding to ubiquitin.

Example 5. Inhibition of the AAA ATPase activity of the 19S proteasome by a test agent can be determined by one or both of the following assays:

1) The IC50 for the action of a test agent in the processing of the Ub4-pepC14 (29) -Oregon Green substrate by the 26S proteasome enzyme in buffer A is lower compared to the IC50 for the action of a test agent in the processing of the Ub4-pep-C14 (29) -Oregon Green substrate by means of the 26S proteasome enzyme in buffer A with 750 mM ATP and 750 mM MgCl2. The action of such a test agent would be competitive with ATP and the ATP sites in the AAA ATPase would thus be occupied.

2) The test agent affects the release of phosphate from ATP by the 26S proteasome. Phosphate levels can be determined by conventional methods, for example, a malachite green binding assay.

Example 6. Inhibition of Rpn11 activity of the 19S proteasome by a test agent:

The assay can be designed based on the fact that the test agent affects the release of pep-C14 (29) -Oregon Green from Ub4-pep-C14 (29) -Oregon Green but does not fall within the agent categories inhibitors identified by examples 4 and 5 as described above.

10 Example 7. Compounds identified as inhibitors:

Using the method described in Example 2, the following compounds were identified as inhibitors of 26S proteasome activity:

image2

image3

The molecules can be further classified based on which substrate was altered, as described in Example 3. Compounds 1, 2 and 3 affect the chemotyptic type activity of the 20S proteasome. Compound 4 prevents the Ub4-pep-C14 (29) -Oregon Green substrate from being processed by the 19S proteasome.

It should be recognized that these compounds may be useful for any application disclosed herein.

Claims (28)

one.

Peptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1 and at least one ubiquitin residue that is at least 90% identical to the sequence of the first 76 amino acids of SEQ ID NO : 2, in which the peptide is a substrate for an Rpn11 metalloprotease comprising a JAMM motif.

2.

Peptide according to claim 1, wherein the JAMM motif is at least 80% identical to SEQ ID NO: 17.

3.

Peptide according to claim 1, wherein the JAMM motif is at least 80% identical to SEQ ID NO: 18.

Four.

Peptide according to any one of claims 1 to 3, wherein the metalloprotease is at least 80% identical to the sequence of SEQ ID NO: 16.

5.

Peptide according to any one of claims 1 to 4, further comprising at least 3 additional amino acids at the C-terminal end of the peptide.

6.

Peptide according to claim 1, wherein the amino acid sequence has the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid other than A is replaced by A.

7.

Peptide according to claim 1, wherein the amino acid sequence has the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid other than A is replaced by A.

8.

Peptide according to any one of claims 1 to 5, further comprising one or more ubiquitin residues at the N-terminal end of the peptide.

9.

Peptide according to claim 8, wherein the ubiquitin residue consists of two or more repetitions of an amino acid sequence that is at least 90% identical to the sequence of the first 76 amino acids of SEQ ID NO: 2.

10.

Peptide according to any one of claims 1 to 9, wherein the ubiquitin moiety comprises the sequence of the first 76 amino acids of SEQ ID NO: 2 modified so that the 75th amino acid is an A.

eleven.

Peptide according to any one of claims 1 to 9, wherein the ubiquitin residue comprises the sequence of the first 76 amino acids of SEQ ID NO: 2 modified so that amino acid 76 ° is an A.

12.

Peptide according to any one of claims 1 to 11, further comprising a detectable agent bound to the peptide.

13.

Peptide according to claim 12, wherein the detectable agent is a fluorescent label.

14.

Peptide according to claim 13, wherein the fluorescent marker is a fluorescent peptide attached to the C-terminal end of the peptide by means of a peptide bond.

fifteen.

Peptide according to claim 12, wherein the detectable agent is a radioactive label.

16.

Peptide according to any one of claims 1 to 11, further comprising an N-terminal target part or C-terminal target part that specifically binds to a selection agent.

17.

Peptide according to claim 16, wherein the selection agent is an antibody.

18.

Peptide according to claim 16, wherein the selection agent is a divalent metal ion.

19.

Method for selecting an agent that modulates the proteasome activity of an Rpn11 metalloprotease, the method comprising:

(to)

providing the peptide according to claim 1; Y

(b)

combining the peptide with a test agent in a reaction mixture suitable for measuring the activity of the Rpn11 metalloprotease under conditions that allow the rest of ubiquitin to be cleaved, in which the Rpn11 metalloprotease comprises an at least identical amino acid sequence in a 80% to the sequence of SEQ ID NO: 16 and a JAMM motif at least 80% identical to the sequence of SEQ ID NO: 17;

wherein a change in the rate or degree of excision of the rest of ubiquitin from the peptide in the presence of the test agent compared to the rate or degree of excision of the rest of ubiquitin from the peptide in the absence of the agent test indicates that the test agent modulates proteasome activity.

twenty.

Method according to claim 19, wherein Rpn11 is present in a 19S regulatory particle or a 26S proteasome.

twenty-one.

Method for selecting an agent that inhibits the proteasome activity of an Rpn11 metalloprotease comprising a JAMM motif, the method comprising:

(to)

providing the peptide according to claim 1 in a mixture with ubistatin; Y

(b)

compare the intrinsic ubistatin fluorescence in the presence of a test inhibitor agent in the mixture with the intrinsic ubistatin fluorescence in the absence of the test inhibitor agent in the mixture,

wherein a difference in the intrinsic fluorescence of ubistatin in the presence of the test inhibitor agent compared to in the absence of the test inhibitor agent indicates that the test inhibitor agent inhibits proteasome activity. 22. Method for selecting an agent that inhibits the proteasome activity of an Rpn11 metalloprotease comprising a JAMM motif, the method comprising:

(to)

providing the peptide according to claim 1;

(b)

combining the peptide of (a) with a test inhibitory agent that has intrinsic fluorescence in a mixture; Y

(C)

measure the fluorescence of the intrinsic fluorescence of the test inhibitor agent in the mixture;

wherein a change in the measured fluorescence of the test inhibitor agent in the mixture with respect to intrinsic fluorescence indicates that the test inhibitor agent inhibits proteasome activity. 23. Method for selecting an agent that modulates the proteasome activity of an Rpn11 metalloprotease comprising a JAMM motif, the method comprising:

(to)

providing the peptide according to claim 1 in a reaction mixture; Y

(b)

determine if a test agent produces a change in the size and / or molecular weight of the peptide;

wherein a test agent that produces a change in the size and / or molecular weight of the peptide substrate modulates the activity of the proteasome.

24.

Method according to claim 23, wherein the peptide substrate further comprising a fluorescent label.

25.

Method according to claim 24, wherein the change in the size and / or molecular weight of the peptide substrate is determined by fluorescence polarization.

26.

Method according to claim 23, wherein the change in the size and / or molecular weight of the peptide substrate is determined by gel electrophoresis under native conditions, gel filtration chromatography, or light scattering.

27.

Method for selecting an agent that modulates the activity of various components of a 26S proteasome, comprising:

(to)

providing a first peptide substrate comprising the peptide according to claim 1 labeled with a first fluorescent label, a second peptide substrate comprising a non-ubiquitinated peptide labeled with a second fluorescent label, wherein the first and second fluorescent labels are detectable at different wavelengths;

(b)

measure the fluorescence at the wavelength of the first detectable marker in the presence of the 26S proteasome and in the presence and absence of a test agent; Y

(C)

measure the fluorescence at the wavelength of the second detectable marker in the presence of the 26S proteasome and in the presence and absence of a test agent; Y

wherein a change in the fluorescence of the first peptide substrate in the presence of the test agent compared to the fluorescence in the absence of the test agent indicates that the test agent modulates an activity associated with the 19S regulatory particle, and a change in the Fluorescence of the second peptide substrate in the presence of the test agent compared to fluorescence in the absence of the test agent indicates that the test agent modulates a peptide of the 20S core. 28. The method according to claim 27, wherein the 26S proteasome, the first peptide substrate and the second peptide substrate are combined in a single reaction mixture.