SCREENING ASSAY FOR PROTEOLYSIS INHIBITORS

Abstract: The invention concerns a method and assay (including a gel-based assay) for specifically identifying inhibitors of muscle protein degradation, comprising using (i) ubiquitin, (ii) a human ubiquitin-carrier E1 protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle (for example, E217k) and means for determining the extent of inhibition of ubiquitin transfer (for example by scintillation proximity assay (SPA)) to a target protein (for example biotinylated histone 2A).

Description: SCREENING ASSAY FOR PROTEOLYSIS INHIBITORS Technical field The present invention relates to assays suitable for screening inhibitors of muscle protein degradation, in particular to assays for screening inhibitors of proteins involved in ubiquitin-dependent proteolysis in eukaryotic cells. Such inhibitors may have utility in the treatment of disease states associated with proteolysis. Background Eukaryotic cells contain three major proteolytic pathways namely the lysosomal, the calcium dependent and the ATP dependent pathways. The lysosomal pathway mainly degrades extracellular proteins which have entered the cells via receptor mediated endocytosis or pinocytosis. The calcium dependent proteases (the calpains) at present have no clear physiological role. The ATP dependent pathway has long been known to play a role in the degradation of misfolded or mutant proteins, however it has only recently become clear that it selectively degrades intact cellular proteins, and thereby fulfills important roles in cellular regulation. The ATP pathway is shown schematically in Fig. 1 which is explained as follows. The protease activity for the ATP dependent pathway lies in a large multi-catalytic complex known as 26S proteosome. This consists of three multi-subunit complexes, namely a 700kDa 20S proteosome core particle and two 19S cap structures (reviewed by Peters J.M., Trends Biochem. Sci. (1994), 19, 377-382). Proteins destined for 26S proteosome degradation are usually modified by addition of ubiquitin (a 76 amino acid peptide) to specific lysine residues. This ubiquitinylation reaction requires three classes of proteins called El, E2 and E3 (reviewed by Ciechanover A., Biol.Chem.Hoppe Seyler (1994), 375, 565-581). El proteins catalyze the activation of ubiquitin to produce a thiol intermediate. The E2 ubiquitin-carrier (or ubiquitin-conjugating) proteins transfer ubiquitin from El proteins to the E3 ubiquitin- protein ligase. Finally, the E3 ligase catalyzes the formation of an isopeptide bond between the ubiquitin polypeptide and the protein substrate (although some E2 proteins can directly ubiquitinylate protein substrates). Protein-bound ubiquitin may itself be ubiquitinylated, resulting in the formation of large protein-ubiquitin conjugates which are targets for destruction by the 26S proteosome (although such poly-ubiquitinylation is not essential for protein destruction to occur). Most cells contain a single El protein, and there have been two classes of E3 enzyme identified, but there are at least 15 genes in yeast alone that encode E2 enzymes (in many cases the human homologue of these has been identified). As already stated, protein degradation is now emerging as an important mechanism of cellular regulation. For instance, it is needed by cells to adapt to a changing environment, as well as for the control of time-dependent cellular programs. Proteolysis offers advantages over other possible control mechanisms because it is fast, and thereby enables the cell to rapidly reduce the level of a defined protein. It is also irreversible, so ensuring the complete loss of function of the degraded protein. However, the danger for the cell of unspecific degradation of proteins means that proteolysis has to be very selective. The role of ubiquitin-dependent proteolysis in the degradation of oncoproteins and tumour suppressors, in cell cycle control, and in stress response and the immune system has become increasingly clear over the last few years (reviewed in Hochstrasser M., Current Biology (1992), 4, 1024-1031 ; Deshaies R.J., Trends Cell Biol. (1995), 5, 428-434; Hilt W. & Wolf D., Trends Biol.Sci. (1996), 21 , 96-102). What is less clear, however, is the role of ATP ubiquitin-dependent proteolysis in the skeletal muscle loss that occurs during cachexia (i.e. the condition describing the severe weight loss and emaciation that may accompany trauma, and that accompanies many serious and often fatal diseases, and indeed may participate itself in early death in such diseases as cancer and AIDS-wasting). Ubiquitin-dependent proteolysis may also be implicated in the down regulation of signal transducing receptors. In particular, the involvement of the ubiquitin conjugation system in the ligand-induced endocytosis and degradation of the growth hormone receptor may be of relevance in cachetic situations. A role for increased activity of the ATP ubiquitin-dependent proteolysis system in muscle breakdown was first suggested by Medina R., Wing S., Haas A. & Goldberg A. (Biomed. Biocim Acta (1991), 50, 4-6) who demonstrated using animal models that in atrophying muscle the ATP ubiquitin-dependent proteolysis system played an important role in the degradation of the bulk of cell proteins. The authors later demonstrated increased expression of poly-ubiquitin and proteosome mRNA's in the same model (Medina R., Wing S. & Goldberg A., Biochem. J. (1995), 307, 631-637). Similar results have been observed in the muscles isolated from tumour bearing rat models (Temparis S., Aseni M., Taillandier D., Aurousseau E., Larbaud D., Obled A., Bechet D., Ferrara M., Estrela J. & Attaix D., Cancer Research (1994), 54, 5568-5573; Llovera M., Garcia-Martinez C, Agell N., Marzabal M., Lopez-Soriano F. & Argiles J., Febs lett. (1994), 338, 311-318; Baracos V., deVivo C, Hoyle D. & Goldgerg A., Am J.Physiol (1995), 268, E996-E1006) and in sepsis models (Tiao G et al, J.Clin. Invest. (1994), 94, 2255-2264; Voisin L. et al, J.Clin. Invest. (1996), 97, 1610-1617). Recent studies have confirmed that the ATP ubiquitin- dependent proteolysis system is involved in the degradation of specific soluble muscle proteins and the major myofibrillar components including Myosin, Actin, Troponin and Tropomysin (Solomeon V. & Goldberg A.L., J.Biol.Chem. (1996), 271, 26690-26697). As already stated, there are many E2 enzymes, each with a very specific role in determining the precise destiny of ubiquitin. For example, the yeast gene RAD6 encodes for E2 which appears essential for the repair of DNA caused by ultra-violet induced damage. The RAD6 yeast enzymes are homologous to the rabbit E2i4k and the human ubiquitin-carrier E2i7k proteins (Schneider R., Eckerskorn C, Lottspeich F. & Schweiger, EMBO J. (1990), 9, 1431-1435; Wing S., Dumas F. & Banville D., J. Biol. Chem. (1992), 267, 6495-6501). The mRNA encoding the rat homologue of human E2i7k protein has been shown to be raised 3-fold in atrophying muscles obtained from starved (Wing S. & Banville D., Am. J. Physiol. (1994), 267, E39-E48) and hindlimb suspension (Taillandier D et al, Biochem. J. (1996), 316, 65-72) rat models of cachexia. However, no work to date has shown whether such raised levels occur specifically in certain tissue types, or are more generally raised throughout many tissue types during muscle wasting. Work using the rat ascites hepatoma Yoshida AH- 130 model of rapid & progressive weight loss and muscle wasting has demonstrated that the β2-adrenergic agonist, Clenbuterol, prevented enhanced muscle protein degradation, and that this normalization of protein breakdown was achieved through a decrease in hyperactivation of the ATP ubiquitin- dependent proteolysis system (Costelli P., J.Clin. Invest. (1995), 95, 2367-2372). The mechanism by which Clenbuterol achieved these effects is unknown. Furthermore, at present there is no means of determining de novo which other substances possess the ability to modulate the ATP ubiquitin-dependent proteolysis system, and prevent or reduce muscle protein degradation. A method or assay which will rapidly and conveniently determine inhibitors of ATP ubiquitin-dependent proteolysis will have utility in research and development, particularly in the development of pharmaceutical substances and compositions for the treatment of diseases such as cachexia. More particularly, a valuable assay would be able to identify those inhibitors which are specifically able to prevent or reduce only muscle protein degradation. The plurality of E2 proteins in particular, and their expression in a range of tissue types (Schneider R., Eckerskorn C, Lottspeich F. & Schweiger, EMBO J. (1990), 9, 1431-1435) makes difficult the development of an assay which is largely specific for inhibitors of muscle protein degradation. It will be appreciated that although an assay may highlight inhibitors which prevent muscle protein degradation, if the inhibitors are unspecific they may also interfere with protein regulation in other tissues. As can be appreciated from the description above, the ATP ubiquitin-dependent proteolysis pathway is complex, and a suitable method or assay based on this pathway must overcome a number of problems before rapid and convenient screening for specific inhibitors of muscle protein degradation is possible. It is the aim of the present invention to deliver a practical method and assay which can identify inhibitors which are largely specific for muscle protein degradation. Disclosure of invention We have recognised that certain human ubiquitin-carrier E2 proteins have especially increased abundance in skeletal and cardiac muscle. We have also recognised that some E2 proteins can directly ubiquitinylate protein substrates, in the absence of an E3 protein. We have utilised both of these facts in the method and assay of our invention. Without the need for an E3 protein the method and assay of our invention are simpler and more convenient to use, allowing a larger number of compounds to be screened in a given time. The E3 protein is large and insoluble, making it unsuitable for production in pure form by recombinant means, and unsuitable for use in an assay such as the present invention describes. Additionally, because our method and assay do not involve E3 protein, inhibitors which are identified using our method or assay will be inhibitors of the human ubiquitin- carrier El protein, or of the specific human E2 ubiquitin-carrier protein expressed in skeletal and/or cardiac muscle which is used in the method or assay. The presence of a target protein in the method and assay of the invention provides for convenient measuring of the extent of ubiquitin transfer. Accordingly, the present invention provides a method for specifically identifying inhibitors of muscle protein degradation, comprising contacting the inhibitor, in the absence of an E3 protein, with (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and then determining the extent of inhibition of ubiquitin transfer to a target protein. It will be appreciated that ATP is necessary to initiate the ubiquitinylation reaction shown in Fig. 2. By a human ubiquitin-carrier E2 protein we mean, hereinabove in this disclosure and hereinafter, one with levels raised above those present in non-muscle cells and tissue. Reference Example 2, for example, illustrates examples of raised levels compared to a number of different non-muscle tissues. It can be seen, for example, in Reference Example 2 that levels in skeletal and/or cardiac muscle are raised by approximately two to three-fold compared to pancreas tissue, and by three to six-fold compared to placental tissue. The present invention also provides a method for specifically identifying inhibitors of muscle protein degradation, as described hereinafter for the assay of the invention. The present invention also provides an assay for specifically identifying inhibitors of muscle protein degradation, comprising (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and means for determining the extent of inhibition of ubiquitin transfer to a target protein. The present invention also provides an assay for specifically identifying inhibitors of muscle protein degradation, comprising, in the absence of an E3 protein, (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and means for determining the extent of inhibition of ubiquitin transfer to a target protein. As stated above, rat models of cachexia show raised levels of mRNA's coding for the rat homologue of human E2i7k. We have confirmed this result in the denervated model of muscle atrophy - see Reference Example 1. We have additionally demonstrated by Northern blotting that in normal tissues human ubiquitin-carrier protein E2i7k has especially increased abundance in skeletal and cardiac muscle - see Reference Example 2. Similar especially increased abundance is not seen however for the rat homologue E2 protein in skeletal and cardiac rat muscle (where levels of the E2 protein are more generally raised throughout tissue types). Based on these findings the method and assay specifically described in our invention have been developed using the human ubiquitin-carrier protein E2i7k , which can identify inhibitors which are substantially specific for muscle protein degradation. It will be appreciated that the method and assay described specifically in the present invention can also be used with E2 proteins other than E2i7k which display an increased abundance in skeletal and cardiac muscle. The human E2i7k ubiquitin-carrier protein is able to directly ubiquitinylate protein substrates. We have utilised this fact in the method and assay specifically described in our invention. It will be appreciated that the method and assay described specifically in the present invention can also be used with E2 proteins other than E2i7k which can directly ubiquitinylate protein substrates. Accordingly, the present invention preferably provides a method for specifically identifying inhibitors of muscle protein degradation, as described above and hereinafter, wherein the human ubiquitin-carrier E2 protein is E2i7k . The present invention also preferably provides an assay for specifically identifying inhibitors of muscle protein degradation, as described above and hereinafter, wherein the human ubiquitin-carrier E2 protein is E2i7k . In another aspect, the present invention also provides an assay, as described above and hereinafter, for use in the method of the invention. In another aspect, the present invention also provides an assay, as described above and hereinafter, for use with an inhibitor of human ubiquitin-carrier El protein, and/or for use with an inhibitor of the specific human E2 ubiquitin-carrier protein expressed in skeletal and/or cardiac muscle that is used in the assay. Inhibitors identified using the method and assay of the present invention are provided as a further feature. Such inhibitors may act on one, or a number, of the components in the ubiquitinylation reaction of Fig.2, and be used as such. In another aspect, the present invention also provides an assay, as described above and hereinafter, together with an inhibitor of human ubiquitin-carrier El protein, and/or together with an inhibitor of the specific human E2 ubiquitin-carrier protein expressed in skeletal and/or cardiac muscle that is used in the assay. The means for determining the extent of inhibition of ubiquitin transfer to a target protein is determined by a scintillation proximity assay (SPA), by an enzyme linked immuno- sorbent assay (ELISA) or by a radio-immuno assay (RIA). In one aspect of the invention the target protein used is histone 2A, biotinylated histone 2A, troponin T or α actin. In a further aspect of the invention the target protein used is histone 2A. In a further aspect of the invention the target protein used is biotinylated histone 2A. Preferably the El and E2 proteins used are obtained by recombinant means. Using a gel-based version of the assay described in the present invention it is possible to determine whether inhibitors specifically block the El and/or the E2 protein. The gel-based assay thus allows identification of the stage in the ubiquitinylation reaction of Fig.2 at which the inhibitor is acting. Therefore, in a yet further aspect of the invention a gel-based version of the method and assay is used. Such an assay is run using standard reagents and conditions. An attractive stage at which to inhibit the ubiquitinylation reaction of Fig.2 is at the stage of transfer of ubiquitin to the E2 protein, or at the stage of transfer of ubiquitin to the target protein. The latter stage is particularly attractive for inhibition as an inhibitor acting at this stage is most likely to be substrate and/or enzyme specific. The presence of a target protein in our assay allows such inhibitors to be identified. A schematic representation of the method is given in Fig. 2, and a schematic representation of the assay is given in Fig. 3. A detailed description is provided hereinafter. Detailed description of invention Method or Assay for inhibitors of E2i7k Proteins Human skeletal muscle cDNA clones encoding both the human El and E2i7k 5 ubiquitin-carrier proteins were obtained by Polymerase Chain Reaction (PCR) of human skeletal muscle polyA" mRNA (Clontech. Palo Alto USA). These were expressed from the in- house vector pTB375NBSE (modified to maximise yield by incorporating tetracycline, rather than ampicillin resistance) in BL21 cells, in such a way as to produce recombinant protein containing a 6-histidine tag immediately adjacent to the N-terminal methionine. A 10 commercial alternative to the pTB375NBSE vector is pET21a, available from Novagen, R&D Systems Europe Ltd., UK (Cat. No. 69762-1). BL21 cells are also commercially available from Novagen (Cat. No. 69443-1). The 6-His tag was used to aid purification of the recombinant protein using a nickel chelating column. The El protein was obtained by a slow- growing process at 20°C as originally described by La Vallie et al (Bio/Technology 1993, 11, 15 187-193) using 0.0 ImM IPTG to induce. The E2 protein was obtained by conventional growing techniques at 37°C with 0.4mM IPTG. Further details are provided below in the section marked "Examples". Scintillation proximity assay (SPA) The recombinant El and E2i7k proteins obtained as described above were used to 0 develop a "mix and measure" 96-well SPA (scintillation proximity assay - Amersham) by incorporating 125 I labelled mono-ubiquitin (Amersham) on to a target protein in the presence of ATP and MgCl. Histone 2A, troponin T and actin were used successfully as target proteins. The ubiquitinylated target protein was detected using protein A labeled SPA beads (Amersham) and a polyclonal antibody to the target protein in question. 5 Both protein A and avidin-linked SPA beads were successfully used in assays using histone2A or biotinylated histone2A as target substrates. A schematic representation of the SPA is given in Fig. 3. Enzyme linked immuno-sorbent assay (ELISA1 A cell based ELISA based largely on the method described by Takada K. et al (Eur. 0 J. Biochem. (1995), 233, 42-47) and later modified by Takada et al (Biochim Biophys Acta (1996), 1290, 282-288) was used for the development of the SPA assay. The ELISA technique was modified such that it too could be used as an alternative high throughput screen or as a secondary screen in place of SPA. Target proteins were "fixed" to the bottom of a 96-well ELISA plate in the presence of PEI (polyethylenimine). A reaction mixture comprising recombinant El and E2i7k, mono-ubiquitin, ATP and MgCl was used. Ubiquitinylated target protein was detected using a horse radish peroxidase linked polyclonal antibody to poly-ubiquitin. Horseradish peroxidase was detected using either ATBS or ECL (enhanced chemiluminesence) detection system. A schematic representation of the ELISA is given in Fig. 3. Radio-immuno assay (RIA) Using the approach described by Takada et al (Biochim Biophys Acta (1996), 1290, 282-288) the SPA or ELISA assays are adapted to a radio-immuno assay system. A schematic representation of the RIA is given in Fig. 3. Production of E2i7k antibodies Purified recombinant 6His-tagged E2l7 protein was used in conjunction with Freund's adjuvant to produce antibodies in New Zealand white rabbits. The anti-sera (ie. E2i7k antibodies or fragments thereof) was used to decrease the inappropriately enhanced degradation of normal proteins which occurs in certain pathological conditions including cachexia. Examples The present invention is illustrated by the following examples, in which the materials and techniques employed are set out below :- J Bluescript II vectors A recombinant cloning vector system similar to that employed by Yanisch-perron C. et al (Gene (1985), 33, 109-119) comprising a colEI-based replicon bearing a polylinker DNA fragment containing multiple unique restriction sites, flanked by bacteriophage T3 and T7 promoter sequences; a filamentous phage origin of replication and an ampicillin drug resistance marker gene. 2 Hvbond-N + (TM) A supported nylon-66 membrane with a pore size of 0.45 microns, used for the immobilisation of nucleic acids by either UN. cross linking or oven baking and supplied by Amersham International pic, Amersham. Bucks. U.K. 3 SSC (saline sodium citrate) 0.15M ΝaCl + 0.015M sodium citrate pH 7.0 4 Denhardt's reagent A solution containing 0.02% bovine serum albumin, o.o2% Ficol 400,000 (a non- ionic synthetic polymer of sucrose, dialysed and lyophilised and having an approximate molecular weight of 400,000) and 0.02% poly vinyl pyrrolidone. 5 Preparation of mRΝA Poly A + mRΝA was prepared directly from ~ lxl 0 8 C2C12 cells usinga FastTrack mRΝA isolation kit (Invitrogen). Rat tissue total mRΝA was prepared by polytron homogenisation in 4M guanidine isothiocyanate, 2.5mM citrate, 0.5% Sarkosyl (SDS), lOOmMβ-mercaptoethanol, followed by centrifugation through 5.7M CsCl, 25mM sodium acetate at 135,000g (max). Rat poly-A + was obtained usinga FastTrack mRΝA isolation kit (Invitrogen). 6 Transformations E coli transformations were generally carried out by electroporation. 400 ml cultures of strains DH5α or BL21(DE3) were grown in L-broth to an OD 600 of 0.5 and harvested at 2,000g. The cells were washed twice in ice-cold deionised water, resuspended in 1ml 10% glycerol and stored in aliquots at -70°C. Ligation mixes were desalted using millipore V series membranes (0.0025mm) pore size). 40μl of cells were incubated with lμl of ligation mix or plasmid DNA on ice for 10 minutes in 0.2cm electroporation cuvettes, and then pulsed using a Gene Pulser apparatus (BioRad) at 0.5kVcm " ', 25μF, 250 msec. Transformants were selected on L-agar supplemented with tetracyline at lOμg/ml or ampicillin at lOOμg/ml. 7 Northern Blots 2μg Poly A * or 20μg total RNA samples were analysed by electrophoresis on 1% denaturing formaldehyde agarose gels in MOPS buffer (Sambrook J., Fritsch E.F. & Maniatis T. in "Molecular Cloning, A laboratory manual", 2 nd Ed. (1989), Cold Spring Harbour Laboratory Press) and transferred onto hybond N * (Amersham). Probes were labelled with 2 P by random hexamer priming, and hybridisations were carried out in 0.28M sodium phosphate (pH 7.2), 5xDenhardt's solution, 10% dextran sulphate. 0.1% SDS at 65°C. Membranes were washed to a final stringency of 0.2xSSC,0.1% SDS at 65°C. To control for loading variations, blots were stripped after autoradiography by boiling in 0.1%SDS, and then rehybridised using a probe containing 1.2kb of a rat glyceraldehyde-3 -phosphate dehydrogenase cDNA (GAPDH) (Fort P., Marty L., Pieechaczyk M., El Sabrouty S., Jeanteur P. & Blanchard J.M., Nucleic Acid Res. ( 1985), 13, 1431-1442). Analysis of poly A * RNA's from human tissues was carried out using a panel of commercially available pre-blotted RNAs (Clontech Laboratories, Palo Alto, Ca USA). 8 Ubiquitin Conjugation Assay Conjugation assays were performed at 37°C for 2 hours. The reaction mixtures contained 50mM Tris-HCl (pH 7.5), 2mM ATP, 5mM MgCl 2 , 0.5mM DTT, recombinant El, recombinant E2, 7k , lμg 125 I-Ubiquitin (Amersham) and 2μg Histone (Sigma). Following incubation, reactions were terminated by addition of sample loading buffer with or without 5% β-mercaptoethanol. Reactions containing β-mercaptoethanol were boiled for 5 mins. The reaction products were resolved by SDS-PAGE (12%) acrylamide gels, BIO-RAD) and, after drying the gel, visualised by autoradiography. 9 Biotinylation of Histone2A Histone2A was biotinylated using a kit purchased from Boehringer Mannheim (cat. no. 1418165) and was used according to the manufacturer's instructions. In summary, free amino groups of the target protein (histone2A in this case) were reacted with D-biotinyl-ε- aminocaproic acid-N-hydroxysuccinimide ester (biotin-7-NHS) by forming a stable amide bond. Non-reacted biotin-7-NHS was separated on a Sephadex G-25 column. The molar concentrations used were 4mg Histone2A in 1ml of phosphate buffered saline to which was added 20mg/ml biotin-7-NHS. The incubation was carried out at ambient temperature for 2 hours with gentle shaking. K) Scintillation proximity assay (SPA) Conjugation assays were performed at room temperature for 2 hours. The reaction mixtures contained 50mM Tris-HCl (pH 7.5), 2mM ATP, 5mM MgCl 2 , 0.5mM DTT, recombinant El. recombinant E2, 7k , lμg l25 I-Ubiquitin (Amersham) and 2μg of biotinylated Histone (Sigma) to give a final volume of lOOμl. Following incubation, reactions were terminated by addition of lOmMEDTA and O.lmg/well avidin linked SPA beads (Amersham Int) Reference Example 1 : Confirmation of denervated model of muscle atrophy. Northern blotting was carried out as described above using full length E2 l7k labelled by random priming. The blot was re-probed with GAPDH as a control for loading and a graphical representation of the ratio of intensity of E2 l7k / GAPDH after analysis on an MD phosphoimager is shown in Fig. 4 wherein :- Lane 1 is inervated rat soleus total RNA (2 μg), Lane 2 is denervated rat soleus total RNA (2 μg), Lane 3 is denervated rat soleus total (2 μg) + lmg/kg Clenbuterol and Lane 4 is C2C12 cell control mRNA (2 μg ). Series 1 is the mean of 4 determinations and Series 2 demonstrates the standard deviation in the measurements. Reference Example 2 : Northern blotting of E2 abundance levels in different tissue types. Northern blotting was carried out as described above using full length E2, 7k labelled by random priming. The human multiple (healthy) tissue blot was obtained from Clontech and contained 2μg of polyA + mRNA from each of the following tissues:- Lane 1 pancreas, Lane 2 kidney, Lane 3 skeletal muscle, Lane 4 liver, Lane 5 lung, Lane 6 placenta, Lane 7 brain and Lane 8 heart. Especially increased abundance of E2 17k was seen in lanes 3 and 8 (heart and skeletal muscle) as Fig.5 shows (prepared in a similar manner to that described for Reference Example 1 , and again showing the standard deviation in the measurements). Example 1 The method and assay described above using E2 k , and SPA means for determining the extent of ubiquitin transfer to biotinylated histone 2A, was used in the presence and absence of the competitive inhibitor Me-ubiquitin (preparation described in Hershko A. & Heller H., Biochem. Biophys. Res. Comm. (1985), 128, 1079-1086) as a standard. In the presence of Me-ubiquitin a reduced (SPA) count was observed as the Me-ubiquitin blocked transfer of labelled ubiquitin to the target protein. A dose-response curve was produced by measuring the (SPA) count at different concentrations of the Me-ubiquitin inhibitor to give an IC 50 (the concentration necessary to produce 50% inhibition of the uninhibited signal) of 3.3xlO "6 M (based on 100% chemical methylation of ubiquitin). Example 2 Compound of Example 2 The method and assay described above using E2 17k , and SPA means for determining the extent of ubiquitin transfer to biotinylated histone 2A, was used to screen a series of compounds. In the presence of the above compound of Example 2 a reduced (SPA) count was observed as the compound blocked transfer of labelled ubiquitin to the target protein. A dose- response curve was produced by measuring the (SPA) count at different concentrations of the inhibitor, giving an IC 50 measurement of 2.99xlO "5 M.

Claims: CLAIMS 1. An assay for specifically identifying inhibitors of muscle protein degradation, comprising (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin- carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and means for determining the extent of inhibition of ubiquitin transfer to a target protein. 2. An assay for specifically identifying inhibitors of muscle protein degradation, comprising, in the absence of an E3 protein, (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and means for determining the extent of inhibition of ubiquitin transfer to a target protein. 3. An assay for specifically identifying inhibitors of muscle protein degradation, according to claims 1 and 2. wherein the human ubiquitin-carrier E2 protein is E2i7k . 4. An assay, according to claims 1 to 3, wherein the means for determining the extent of inhibition of ubiquitin transfer to a target protein is by a scintillation proximity assay (SPA), by an enzyme linked immuno-sorbent assay (ELISA) or by a radio-immuno assay (RIA). 5. An assay, according to claim 4, wherein the means for determining the extent of inhibition of ubiquitin transfer to a target protein is by a scintillation proximity assay (SPA). 6. An assay, according to claims 1 to 5, wherein the target protein used is histone 2A, biotinylated histone 2A, troponin T or α actin. 7. An assay, according to claim 6, wherein the target protein used is biotinylated histone 2A. 8. A gel-based version of the assay described in claims 1 to 7 for identifying the stage in the ubiquitinylation reaction at which the inhibitor is acting. 9. A method for specifically identifying inhibitors of muscle protein degradation, comprising contacting the inhibitor with the components of an assay as described in claims 1 to 7.

Inventors: HOLLOWAY BRIAN ROY, WADDELL IAN DAVID, HOLLOWAY, BRIAN, ROY, WADDELL, IAN, DAVID
Applicants: HOLLOWAY BRIAN ROY, WADDELL IAN DAVID, ZENECA LTD, HOLLOWAY, BRIAN, ROY, WADDELL, IAN, DAVID, ZENECA LIMITED
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1, G01N33