# Patent

## Title
Method for identifying compounds that inhibit the activity of Myc

## Abstract

Method for identifying compounds useful for the therapy of cancers in which the activity of Myc is causally involved. The method is based on measuring the activity of the E3 ligase HectH9, which is required for ubiquitination and activation of Myc.

## Background
RELATED APPLICATIONS
This application claims priority to European Patent Application EP 04011505.7, filed May 14, 2004. The contents of which are incorporated herein.
DISCLOSURE OF THE INVENTION
The present invention relates to the therapy of cancer. In particular, the present invention relates to methods for identifying compounds that have the potential to inhibit the activity of the proto-oncogene Myc and may thus be useful for the therapy of cancer.
The proto-oncogene c-myc encodes a transcription factor (Myc) that can both activate and repress transcription. Myc activates transcription as part of a binary complex with its partner protein, Max. The complex binds to specific DNA sequences, termed E-boxes, and recruits a number of co-activators to DNA; indeed, four distinct classes of co-activators have been described that may mediate transcriptional activation by Myc.
First, a short conserved domain in the amino-terminus of Myc (“MycboxII”) binds to the TRRAP protein and Myc recruits the histone acetyl-transferases, Tip60 and GCN5 via binding to TRAPP. Similarly, Myc recruits the p300 and CBP acetyl-transferases though a direct interaction of the terminus-terminus of Myc with each protein. Second, Myc interacts with mediator complexes in vitro and recruits mediator to E-boxes in vivo independently of histone acetylation, suggesting that interactions with mediator complexes contribute to transcriptional activation by Myc. Third, Myc recruits the E3-ligase Skp2 to DNA and recruitment of Skp2 contributes to transcriptional activation by Myc. Fourth, Myc binds to BAF53, a subunit of the chromatin-remodeling BAF complex, implying chromatin-remodeling as a mechanism that may contribute to Myc-dependent gene activation. The precise contribution of each co-factor to the activation of individual genes by Myc is largely unknown.
Myc represses transcription at least in part through the formation of a ternary complex that contains Max and the transcription factor, Miz1. Miz1 binds to the start site of several Myc-repressed genes and activates these genes. Myc does not bind to the start sites of Myc-repressed genes directly but is recruited to these sites through interaction with Miz1. In contrast to free Miz1, the Myc/Miz1 complex acts as a transcriptional repressor complex.
Free Miz1 requires p300 as co-activator and binding of Myc to Miz1 displaces p300 from Miz1, explaining in part how Myc inhibits transactivation by Miz1. However, transcriptional repression by Myc requires domains in the amino-terminus that are not required for displacement of p300 from Miz1, suggesting that additional co-factors contribute to transcriptional repression by Myc.
The (c-) MYC gene and two of its relatives, MYCN or MYCL, contribute to the genesis of a wide variety of human tumors. In these tumors, the expression of MYC genes is enhanced relative to the surrounding or normal tissue, arguing that there is a selective pressure for high expression of Myc proteins during tumor development. For example, the MYCN gene is amplified in a subset of childhood neuroblastoma, correlating with extremely poor prognosis of the affected children (Brodeur et al., 1984; Schwab et al., 1983). In other tumors, expression of a MYC family gene is increased because mutations occur in the signaling pathway that control their expression: one example are the mutations in the APC pathway that affect cMYC expression in colorectal carcinomas (He et al., 1998; van de Wetering et al., 2002).
Besides colon cancer, elevated or deregulated expression of c-Myc has been detected in a wide range of human cancers and is often associated with aggressive, poorly differentiated tumors. Such cancers include breast, cervical, small cell lung carcinomas, osteosarcomas, glioblastomas, melanoma and myeloid leukemias (reviewed in Pelengaris et al., 2002b).
Examples of Myc-activated target genes, whose activation results in stimulation of tumor growth, proliferation and survival are cyclin D2 and CDK4, which are important for cell cycle progression, the translation initiation factors eIF4 and eIF2 that are important for cell growth, as well as ornithine decarboxylase and carbamyl-phosphate synthase, enzymes required for polyamine and pyrimidine biosynthesis, respectively (reviewed in Grandori and Eisenman, 1997; Pelengaris et al., 2002b). Examples of Myc-repressed genes, whose repression results in cell proliferation, are the cell cycle inhibitors p15ink4b and p21Cip1.
Inhibition of Myc activity is a highly attractive approach for drug discovery in oncology, since recent experimental data suggest that even a brief inhibition of Myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Jain et al. (2002) engineered a conditional transgenic mouse to overexpress Myc, which induced formation of highly malignant osteogenic sarcoma. A brief loss of Myc overexpression caused the tumor cells to differentiate into mature osteocytes that formed histologically normal bone. Felsher and Bishop (1999) showed that transgenic mice expressing the myc oncogene in hematopoietic cells developed malignant T cell leukemias and acute myeloid leukemias. However, when this gene was switched off the leukemic cells underwent proliferative arrest, differentiation, and apoptosis. Pelengaris et al. (1999) targeted expression of an inducible form of the c-Myc protein to the epidermis of mice and observed formation of angiogenic premalignant skin lesions, which regressed when the c-Myc protein was deactivated.
In general, specific pharmacological interference with the function of transcription factors has been difficult to achieve. This is particularly true for Myc: despite its obvious value as a potential target for tumor therapy, no drugs have emerged that specifically interfere with its function. For example, screens aimed at disrupting the Myc/Max interface have only yielded compounds with extremely low potency (Berg et al., 2002).
It was an object of the invention to elucidate the mechanisms that regulate the activation and thus the transcriptional activity of Myc. The knowledge about such mechanisms is the key for providing screening assay methods for identifying compounds that have the potential to interfere with the activation of Myc and thus to be useful in the therapy of cancer.
As mentioned above, it has been suggested that Myc may exist in an activating and in a repressive state, potentially depending on the site its binds to DNA and/or its site-specific interaction partners.
In the experiments of the invention, it has surprisingly been found that the E3-ligase HectH9 ubiquitinates Myc on lysine residues close to the carboxyl-terminus of Myc. It could be shown that ubiquitination does not target Myc for degradation, but is required for the recruitment of the co-activator p300. It was shown that ubiquitination of Myc by HectH9 is inhibited by Miz1. Since Miz1 binds to the start site of many Myc-repressed genes but is absent from E-box elements in vivo, the findings of the invention show a mechanism how the transcriptional properties of Myc are regulated by the local chromatin environment. In particular, the findings of the present invention show for the first time the critical role of the E3-ligase HectH9 for Myc activation.
Miz1 is a zinc finger transcription factor that carries at its amino-terminus a POZ-domain. POZ domains are protein-protein interaction modules; in Miz1, the POZ-domain is required for transcriptional activation by Miz1.
In order to understand the function of this domain in more detail, the inventor performed a two-hybrid screen looking for novel interaction partners. In this screen, the inventor obtained multiple clones that are derived from a single gene, which encodes a HECT-domain E3-ligase alternatively named “HectH9”, LASU1 (GenBank Accession Nos. AB071605 and BAC06833) or KIAA0312 (GenBank Accession No. AB002310) (FIG. 1a). A protein corresponding to the carboxyl-terminus of HectH9 has also been described as a DNA binding protein (“UREB 1; Upstream-element-binding protein 1” SwissProt Accession No. Q7Z6Z7; Gu et al. 1994). The full-length HectH9 protein is available as GenBank Accession No. NP—113584 (SEQ ID NO:2; DNA sequence SEQ ID NO:1). Although UREBI has been described as a DNA binding protein, the inventor has been unable to confirm a significant DNA binding activity of HectH9.
HectH9 belongs to the HECT-domain family of ubiquitin ligases (“homologous to E6AP carboxy-terminus”), which are characterized by a conserved carboxy-terminal catalytic domain. Among the conserved amino acids is a cysteine residue, which is required for thioester formation with ubiquitin. The clones recovered in the two-hybrid screen conducted in the experiments of the invention define a small region sufficient for interaction with Miz1. The amino-terminus of HectH9 contains two UBA (ubiquitin associated motif) domains, that are known to bind ubiquitin in several ubiquitin-binding proteins, a WWE protein-interaction domain and two putative nuclear localization signals (NLS). Consistent with the presence of two NLS, two different antibodies directed against HectH9 detected the endogenous protein in the cell nucleus of Hela cells (FIG. 1b and data not shown). Controls using cells that were stably transfected with a pSUPER vector, which directs the synthesis of a siRNA against HectH9, showed that the signal detected in the immune-fluorescence is specific for HectH9 (FIG. 1b, middle panel). Immunoblots confirmed that cells transfected with pSUPER-HectH9 expressed lower levels of protein than cells transfected with an empty pSUPER vector (FIG. 1b, lower panel).
To test whether HectH9 binds to Miz1 in vitro, Miz1 and HectH9 were synthesized by coupled transcription-translation in a reticulocyte lysate in the presence of 35S-methionine (FIG. 1c). After synthesis, lysates were mixed and immuno-precipitated either with antibodies directed against HectH9 or a control serum. Miz1 was clearly detectable in anti-HectH9 immunoprecipitates, whereas control precipitates contained significantly lower levels of Miz1. To test whether the POZ domain of Miz1 is required for binding, the experiment was repeated with ΔPOZ 1. No ΔPOZ-Miz1 was recovered in anti-HectH9 precipitates, demonstrating that the POZ-domain of Miz1 mediates binding to HectH9 in vitro. The inventor also tested whether HectH9 binds to Myc in vitro and did not detect an interaction between HectH9 and Myc in these experiments.
To test whether Miz1 binds to HectH9 in vivo, the inventor transfected HeLa cells with expression vectors encoding Miz1, Miz1ΔPOZ and HectH9. Miz1 was recovered in anti-HectH9 immunoprecipitates, whereas control immunoprecipitates contained significantly less Miz1. No specific binding of Miz1 ΔPOZ to HectH9 could be detected in vivo, consistent with the in vitro and two-hybrid data. α-HectH9 immunoprecipitates contained significant amounts of Myc, demonstrating that Myc binds to HectH9 in vivo. Most likely, therefore, binding of Myc to HectH9 is stabilized by additional proteins in vivo.
Hect-domain proteins act as E3-ligases that ubiquitinate proteins to which they bind. To test whether HectH9 ubiquitinates either Miz1 or Myc, the inventor transfected cells with expression plasmids encoding Myc, Miz1, HectH9 and a histidine-tagged ubiquitin. Transfected cells were lysed and ubiquitinated proteins were isolated by binding to Ni-agarose. Immunoblots of the eluates were probed with antibodies directed against Miz1 and Myc (FIG. 2a and data not shown). In these experiments, the inventor did not detect any activity of HectH9 against Miz1. In contrast, HectH9 efficiently ubiquitinated Myc when both protein were co-expressed in the absence of Miz1. In these experiments, the inventor noted ubiquitinated forms of Myc up to a molecular weight of 250 kd, strongly suggesting that HectH9 catalyzes the transfer of multiple ubiquitin moieties on Myc. Co-expression of Miz1 essentially abolished ubiquitination of Myc by Miz1. Progressive amino-terminal deletions in HectH9 revealed that the Miz1 binding domain on HectH9 is required for ubiquitination of Myc, suggesting that Myc and Miz1 compete for binding to HectH9 (data not shown).
HectH9 might catalyze ubiquitination of Myc in two different manners: it might either catalyze the complex formation of Myc with an unknown E3-ligase or itself be the catalytically active E3-ligase. To distinguish between the two possibilities, the inventor introduced a single point mutation into the carboxyl-terminus of HectH9 that replaces the catalytic cysteine with a serine. HectH9CS, in contrast to wild type HectH9, did not ubiquitinate Myc in vivo, demonstrating that the catalytic activity of HectH9 is required for ubiquitination of Myc in vivo (FIG. 2b).
Surprisingly, neither overexpression nor depletion of HectH9 had any detectable effect of Myc stability in several assays (data not shown). In order to be able to assess the functional consequences of HectH9-mediated ubiquitination of Myc, the inventor therefore decided to map the lysine residues in Myc that are targeted by HectH9 (FIG. 3). To do so, the inventor measured in parallel both binding to HectH9 and ubiquitination by HectH9 of a series of both N-terminal and C-terminal deletion mutants described by Tansey and colleagues (FIG. 3a). In these experiments, full-length Myc bound efficiently to HectH9 and was efficiently ubiquitinated by HectH9, as described above (FIG. 3b). Deletion from the amino-terminus led to a progressive loss of both binding and ubiquitination: for both ΔN1Myc and ΔN2Myc, residual binding could be detected and there was some residual ubiquitination. In contrast, ΔN3Myc neither bound HectH9 nor was ubiquitinated by HectH9. Most likely, therefore, the amino-terminus of Myc is required for binding to HectH9 and the loss of ubiquitination of amino-terminally deleted mutants of Myc by HectH9 is an indirect consequence of the reduction in binding of such proteins.
Both ΔC1Myc and ΔC2Myc bound efficiently to Myc in vivo (FIG. 3c). ΔC1Myc was ubiquitinated by HectH9 to the same extent as wild type Myc. In contrast, ΔC2Myc was not detectably ubiquitinated by HectH9. Since ΔC2Myc bound to HectH9 but was not ubiquitinated, the inventor speculated that the region between AC1 and AC2 might contain the target lysines for HectH9. To test this idea, the inventor replaced the six lysines in this region by arginine in full-length Myc, generating MycKR6.
Immunofluorescence experiments revealed that K6R was localized in the cell nucleus, demonstrating that replacement of the lysines in the nuclear localisation signal by arginine did not induce gross alterations in the subcellular localisation. The inventor next repeated the ubiquitination assays using wild type Myc and MycKR6 in parallel. Ubiquitination of MycKR6 by HectH9 was strongly reduced relative to wild type Myc (FIG. 3d), although it was not completely abolished. Strikingly, also the basal ubiquitination that occurs in the absence of co-expressed HectH9 was strongly reduced relative to wild-type Myc, demonstrating that a significant percentage of ubiquitination of Myc occurs on one or more of the six lysine residues the inventor has identified (FIG. 3d, lower panel).
To test whether ubiquitination of Myc at these sites triggers degradation of Myc, the inventor infected NIH3T3 cells with recombinant retroviruses that express Myc or MycKR6. Pools of infected cells were treated with cycloheximide for different lengths of time (FIG. 4a). Cell lysates were prepared and immunoblots were probed with the monoclonal antibody 9E10, which specifically recognizes human Myc. In these experiments, the inventor observed that the rate of degradation of wild type Myc and MycKR6 was identical. Quantitation of the results (FIG. 4b) showed that the half-time of MycKR6 was unaltered relative to wild type Myc. From these results and those described above, the inventor concluded that ubiquitination by HectH9 does not regulate the stability of the Myc protein.
Several types of poly-ubiquitin chains can be attached to target proteins and only those, in which ubiquitin is linked through lysine-48 target proteins for proteasomal degradation. In contrast, poly-ubiquitin chains with a lysine-63 linkage do not target proteins to the proteasome. In order to determine the type of linkage, which HectH9 transfers to Myc, the inventor performed ubiquitination assays using specific point mutants of his-tagged ubiquitin, in which either lysine 48 or lysine 63 is replaced by an arginine (FIG. 4c). Mutation of lysine 48 did not affect the ability of HectH9 to transfer ubiquitin on Myc; in contrast, mutation of lysine 63 abolished the ability of HectH9 to transfer ubiquitin on Myc. The inventor concluded that HectH9 catalyzes the assembly of a lysine-63 linked poly-ubiquitin chain on Myc.
Polyubiquitination can affect the function of the protein in different ways. In order to test how ubiquitination affects the function of Myc, the inventor initially performed transient reporter assays. No difference was seen in the ability of MycKR6 to inhibit Miz1 dependent transactivation in these assays (data not shown). Also, no difference was seen in the ability of Myc and MycKR6 to activate an E-box dependent reporter derived from the prothymosin-α gene, in transient transfection assays.
In order to test whether these effects extend to the regulation of endogenous target genes by Myc, the inventor infected NIH3T3 cells with retroviruses expressing either Myc, MycKR6 or an empty control vector (pBabepuro). Cells were then serum-starved for 48 hours to deplete endogenous Myc and then RNA was extracted and subjected to an array analysis using an 11.5 k cDNA array (FIG. 5b). This analysis revealed a class of genes that was consistently downregulated in cells expressing KR6 relative to cells expressing wildtype Myc. Comparison with a database of Myc target genes revealed that 80% of these genes were known target for activation of Myc. RT-PCR analysis confirmed the differences in expression of these genes between cells expressing Myc and MycKR6 (FIG. 5c). However, not all targets of Myc were differentially expressed between cells expressing Myc and MycKR6: for example, nucleolin and Rbb2 were induced equally well by both Myc and MycKR6. Taken together, the data show that MycKR6 is deficient in activation of a subset of Myc target genes in vivo.
The six lysines that are replaced by arginines in KR6 are located in a domain that has recently been shown to be involved in binding of the p300 and CBP co-activator. The inventor therefore speculated that ubiquitination of Myc might be required for efficient recruitment of p300. To test this idea, the inventor co-transfected cells with HA-tagged p300 and either Myc or MyckKR6, either by itself or in the presence of HectH9. Lysates were then immunoprecipitated with antibodies against the HA-tag of p300 and the precipitates probed with antibodies directed against Myc (FIG. 5c). In these experiments, efficient complex formation between Myc and p300 was observed in the presence of HectH9, but not in its absence. Importantly, the efficiency of complex formation was reduced for MycKR6, suggesting that the lysine residues need to be ubiquitinated for complex formation to occur. The data strongly suggest that MycKR6 is deficient in transactivation of some target genes becuase binding of Myc to p300 requires ubiquitination at one or more of the six lysines targeted by HectH9.
One prediction from this model is that Myc should recruit p300 sleectively to E-box elements, but not to Miz1 sites, since Miz1 inhibits ubiquitination by HectH9 (see FIG. 2a). The inventor tested this model using a cell line that carries a tetracyclin-inducible Myc. Chromatin-immunoprecipitation was performed both before and after induction of Myc; as targets, the inventor chose nucleolin (E-box dependent activation by Myc) and c/EBPα (a Miz1-dependent target of repression), since initial experiments had shown that the amount of Myc bound to both loci is comparable to each other and higher than for several other genes tested (FIG. 5d, and data not shown). Induction of Myc led to a similar increase of Myc binding at both loci. At the nucleolin locus, there was a clear increase in binding of p300; in contrast, the amount of p300 at the c/EBPα locus remained unaltered. Similar experiments using antibodies directed against TRRAP, p400, Tip60 and Tip48/49 did not reveal a similar difference (data not shown), arguing that MycboxII-dependent interactions occur at both sites of activation and repression.
In summary, the experiments of the present invention have shown that the HECT-domain E3-Ligase HectH9 ubiquitinates Myc; Miz1 inhibits ubiquitination of Myc. Ubiquitination of Myc by HectH9 does not regulate Myc stability, since the polyubiquitin chain is linked via lysine-63 of ubiquitin. Rather, ubiquitination is required for transactivation of specific Myc target genes and for recruitment of the co-activator, p300. From this it is concluded that the transcriptional properties of Myc are regulated by site-specific ubiquitination. These findings make, for the first time, the cancer target Myc accessible for screening assays that allow for identifying Myc inhibitors useful in cancer therapy. (In the context of Myc inhibition, “activation of Myc” and “activity of Myc” are used interchangeably).
WO 01/75145 describes a method for assaying ubiquitin ligase, in which the amount of a tagged ubiquitin that is bound to an E3 is measured, without using a specific target. While HECT domain E3-ligases are mentioned and exemplified by the mammalian E6AP-E6 complex, which functions as a ubiquitin ligase for the tumor suppressor p53, WO 01/75145 does not mention HectH9, neither as such nor in the context with myc.
U.S. Pat. No. 5,968,761 describes an assay for identifying an inhibitor of ubiquitin-mediated proteolysis of a cell-cycle regulatory protein, inter alia myc, in which the level of ubiquitination of the regulatory protein is measured. Again, apart from the fact that this assay aims at identifying inhibitors of proteolysis of a cell-cycle regulatory protein and not of its activation, HectH9 is not among the E3-ligases that are mentioned. The same is true for the review article by Sun, 2003, which suggests targeting E3 ubiquitin ligases for cancer therapy.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1a-1d shows the interaction of Myc and Miz1 with HectH9
FIG. 2a-b shows the ubiquitination of Myc by HectH9
FIG. 3a, 3b1, 3b2, 3c and 3d show HectH9 targets lysines in the carboxyl-terminus of Myc
FIG. 4a-c shows that HectH9 mediates lysine-63 linked poly-ubiquitination of Myc
FIG. 5a-d shows transcriptional properties of Myc and MycKR
FIG. 6 shows a model of Myc activation and Myc repression.



BRIEF SUMMARY OF THE INVENTION
The present invention relates to a method for determining whether a compound has the potential to inhibit the activation of Myc, wherein a test compound's ability to interfere with the activity of the E3-ligase HectH9, which has the amino acid sequence shown in SEQ ID NO:2, or of a fragment or variant thereof that has E3-ligase activity, is determined, said HectH9 activity being selected from

    a) the transfer of ubiquitin from a ubiquitin conjugating enzyme E2 to HectH9, which has the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof that has E3-ligase activity; or
    b) the transfer of ubiquitin from HectH9 to Myc,

and wherein a decrease in the level of HectH9 activity in the presence of the test compound, as compared to the level of HectH9 activity in the absence of the test compound, is indicative of the compound's potential to inhibit the activation of Myc.



DETAILED DESCRIPTION AND EMBODIMENTS OF THE INVENTION
In the following, the term “HectH9” or “HectH9 activity”, respectively, encompasses HectH9 and variants and fragments thereof that have an E3-ligase activity similar to that of the naturally occurring HectH9 (SEQ ID NO:2), whereby “similar” means that such E3-ligase activity may be enhanced or decreased as compared to the naturally occurring HectH9. Since E3-ligase activity is determined by the HECT domain, it is essential that this domain, which contains the active site, is present.
For the present invention, the term “Myc” encompasses the HectH9 substrate c-Myc or any of its relatives that are activated in vivo by a ubiquitination reaction mediated by HectH9 (n-Myc or 1-Myc). With regard to the method of the invention, “Myc” refers to a protein corresponding to the naturally occurring Myc protein or a fragment of variant thereof to which ubiquitin can be transferred in a quantifiable way.
In the embodiment a), the present invention relates to a method for identifying compounds that inhibit transfer of ubiquitin from a ubiquitin-conjugating enzyme E2 to HectH9, i.e. ubiquitination of HectH9. In this method, HectH9 is incubated, together with a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), ubiquitin and ATP for a period of time sufficient to obtain a measurable level of ubiquitin associated with HectH9, and the level of ubiquitination of HectH9 in the presence or absence of a test compound is compared, and wherein a decrease in the level of HectH9 ubiquitination in the presence of the test compound, as compared to the level of HectH9 ubiquitination in the absence of the test compound, is indicative of the compound's ability to inhibit the activation of Myc.
The reaction that this embodiment of the invention is based on is a thioester formation between the active site cysteine of HectH9 and the terminal glycine of ubiquitin.
The components for this type of assay can be obtained as follows:
The proteins used in the method of the invention are preferably recombinant proteins, which can be obtained according to conventional methods by transforming a suitable host with a plasmid carrying the sequence encoding the protein. Alternatively, the recombinant proteins may be obtained by in vitro translation or coupled in vitro transcription/translations systems, which are commercially available, e.g. from Roche. The proteins obtained by such systems, optionally in already radioactively labeled form, may be used without further purification. In an embodiment of the invention, the substrate protein Myc is obtained by that method.
If the assay components, in particular HectH9, E1, E2 and/or ubiquitin, are produced recombinantly by expression in host cells, the host cells may, inter alia, be insect cells that are combined with the well-established baculovirus expression system, or bacterial cells, in particular E. coli cells. The cDNA sequences encoding the protein components are available from the literature and from databases:
The assay components HectH9, E2 and ubiquitin are usually produced and purified as fusion proteins. The proteins may be fused to an affinity tag, which is a protein suitable for affinity purification, such as gluthathion S-transferase (GST, Amersham Pharmacia), maltose binding protein (MBP, New England Biolabs), chitin binding domain (New England Biolabs), the myc-epitope (EQKLISEEDL) or the His(6) tag (Qiagen, Novagene). The fusion protein can be expressed, e.g. in E. coli, and purified according to standard protocols.
E1 may be produced and purified as the other assay components; advantageously it is purified due to its reversible interaction with ubiquitin according to known methods (e.g. Hatfield and Vierstra, 1990 and 1992). In the case of using untagged ubiquitin, a commercially available product (e.g. from Sigma, Fluka) may be used as this assay component.
In the method of the invention, preferably human HectH9 is used, whose DNA and protein sequences are readily available, as described above: GenBank Accession No. BAC06833 (“Lasul”); GenBank Accession No. NP—113584 (full-length HectH9 protein, SEQ ID NO:2), GenBank Accession No. NM—031407 (DNA sequence encoding full-length HectH9, corresponding to SEQ ID NO:1).
The ubiquitin activating enzyme (E1) can be selected from a variety of E1 molecules; an example for a suitable E1 is the wheat UBAI E1 (GenBank Accession No. M55604), however, UBAI E1 from other species, e.g. from Xenopus laevis or human (GenBank Accession No. M58028) or from mouse (SwissProt P30138) may also be used. Other E1 molecules have been described in WO 01/75145; their suitability in the method of the invention can be easily tested in preliminary experiments by combining E1 candidates with the other assay components and testing the functionality of the E1 candidates in the desired assay format. E1 can be purified on a ubiquitin affinity matrix according to published procedures (e.g. Hatfield and Vierstra, 1990 and 1992).
Also, the ubiquitin conjugating enzyme (E2) can be selected from a variety of E2 molecules; an example is the human variant UBCH5b (GenBank Accession No. U39317), which has proven useful in the present invention, although, also in this case, UBCH5b homologues from other species, e.g. Xenopus laevis, may be employed. Alternatively, UBCH5a (GenBank Accession No. AAH05980) or UBCH5c (GenBank Accession No. AAH66917) can be used. Alternatively, ubiquitin conjugating enzymes different from UBCH5a, b or c can be used, as long as these enzymes support the transfer of ubiquitin to the HectH9 protein. Other E2 molecules have been described in WO 01/75145 the contents of which are incorporated herein; their suitability in the method of the invention can be easily tested in preliminary experiments, as described above for E1. Preferably, the ubiquitin conjugating enzyme E2 is fused to an affinity tag which is selected from the ones listed above as suitable for HectH9, but different from the tag chosen for HectH9. For example, in the case that GST-HectH9 is used, His(6) or another tag different from GST is used for tagging E2.
Preferably, the naturally occurring proteins are used as assay components; however, the proteins may contain deviations from the natural amino acid sequence as long as these deviations do not impair their functional activity.
Thus, the components used in the method of the invention may also be variants or fragments of the above-mentioned protein components, e.g. ubiquitin, E1, E2 and/or HectH9 variants or fragments. The variants may be substitutional, insertional or deletional variants that can be obtained by site specific mutagenesis of nucleotides in the DNA encoding the respective protein, e.g. using PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant or fragment, and thereafter expressing it, As long as such variants or fragments exhibit the same biological activity as the naturally occurring protein, they are useful in the method of the invention. Variants or fragments that have modified characteristics, e.g. a stronger enzymatic activity, can also be used. In order to improve the performance of a protein with a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed variants screened for the optimal desired activity. Techniques for making substitution mutations at predetermined sites in well known, e.g. PCR mutagenesis.
Since the full-length HectH9 protein is 4374 amino acids long, it is advantageous to use a HectH9 fragment, which can be more readily produced in recombinant form. Essential for the suitability of a HectH9 fragment for use in the present invention is that it contains the HECT domain, i.e. the C-terminal region comprising a stretch of ca. C-terminal 350 amino acids. To verify that a pre-selected HectH9 variant, in particular a fragment, is useful for the method of the present invention, it can be tested in preliminary tests by measuring whether it associates with ubiquitin at the active cysteine site (in its non-mutated form) within the HECT domain, which results, in the absence of a substrate, in formation of a polyubiquitin chain, which, in certain embodiments of the invention, is measured. By way of example, a HectH9 fragment that has been shown to have HectH9 activity for the purpose of the present invention, comprises the C-terminal 638 amino acids of HectH9 (or LASU1, respectively).
To sustain a sufficient ATP level during the entire ubiquitination reaction, a so-called “ATP regenerating system” (e.g. comprising 0.5 mM ATP, 60 μg/ml creatine phosphokinase, 6.6 mM phosphocreatine, 10 mM Tris-HCl, 0.5 mM MgCl2, 1 mM KCl, 0.05 mM DTT) may be advantageously employed (Murray, 1991), in particular in assays that employ in vitro-translated proteins. When the assay employs purified proteins, ATP may be present by itself.
By “ubiquitin” is meant a polypeptide which is ligated to another polypeptide by ubiquitin ligase enzymes. Ubiquitin is a highly conserved 76 amino acid protein expressed in all eukaryotic cells. The ubiquitin used in the method of the present invention can be from any species of organism, preferably a eukaryotic species. Preferably, the ubiquitin is mammalian, in particular human (SwissProt Accession No. PO2248). Ubiquitin is commercially available (Sigma), it may also be recombinantly produced; in this case it may be fused to various tags for purification, i.e. His(6), GST or for detection, i.e. myc-epitope, HA-epitope. In both cases, ubiquitin comprises the 76 amino acids that are required for its function. Preferably, a tagged ubiquitin is employed in the assay. The ubiquitin used in the assay may also carry a non-proteinacious tag, e.g. biotin. Other suitable ubiquitin tags have been described in WO 01/75145 the contents of which are incorporated herein.
The above-described assay essentially comprises the steps of the ubiquitination reaction itself and the step of measuring the extent of ubiquitin transfer to HectH9.
The first step comprises reacting the assay compounds listed above for a period of time sufficient to allow for the ubiquitination reaction, e.g. for 30 min.
The reaction may either be conducted in solution by simply mixing the assay components, or alternatively, the reaction may be carried out by using immobilized HectH9. In this case, HectH9 carries an affinity tag (GST or one of the alternative tags mentioned above) that is used for its binding to a solid phase carrying the ligand for the respective affinity moiety, e.g. glutathione agarose or sepharose beads or microtiter plates coated with antibodies against the affinity moiety, e.g. commercially available anti-GST antibodies
After the reaction has been completed, the extent of ubiquitin associated with HectH9 can be measured in different ways:
In case the ubiquitination reaction has been carried out in solution, the affinity-tagged, e.g. GST-tagged, HectH9 is captured on microtiter plates that are coated with an antibody against GST (this step can be omitted in case the reaction has been carried out with HectH9 bound to a solid phase). The unbound GST-HectH9, the unincorporated ubiquitin and the other reaction partners are then washed off. Subsequently, the immobilized ubiquitin can be visualized by using an antibody that is directed against a tag epitope, e.g. the myc-epitope present in the recombinant tagged ubiquitin, which antibody carries a detectable label. Suitable labels are well known in the art, they have, inter alia, been described in WO 01/75145 the contents of which are incorporated herein. Examples are radioactive labels, e.g. 125I, enzymatic labels, e.g. horseradish peroxidase or alkaline phosphatase, or fluorometric labels. In a preferred embodiment, quenched fluorophors, e.g. Europium (Delphia System by Perkin Elmer/Wallac) that will be dequenched upon incubation with an enhancer solution (Perkin Elmer/Wallac), are used.
The obtained values are compared to values obtained from reactions without HectH9 (negative control, background) and to values obtained from a reaction mixture incubated in the presence of the solvent (usually DMSO) only (positive control).
Alternatively to using the ELISA-type assay described above to detect the amount of bound ubiquitin, the physical proximity of ubiquitin molecules associated with HectH9 (in the case of measuring the ubiquitin transfer to HectH9) or to the HectH9 substrate (in the case of determining ubiquitination of a HectH9 substrate; see below) upon incubation at 37° C. can be used to measure the extent of ubiquitin association with HectH9 or its substrate by fluorescence resonance energy transfer (FRET, as described by Gershkovich et al., 1996, or by Matayoshi et al., 1990, or recently reviewed by Selvin, 2000). FRET can only be achieved if certain conditions are fulfilled, i.e. fluorophor pairs with overlapping emission and excitation wavelengths, like europiurn/allophycocyanin, europium/Cy5, europium/PE (all commercially available from Perkin Elmer/Wallac) and an minimal proximity of these fluorophors below 5-10 nM. These fluorophors can be added either bound to antibodies directed against the affinity label, e.g. GST, or the epitope, e.g. the myc epitope, or can be directly coupled to HectH9, HectH9 substrate or ubiquitin (custom service of Wallac). When coupled to antibodies, the fluorophors are added to the reaction after its completion. No further washing steps are necessary and signals (excitation at 340 nm and emission measurement at 665 nm in the case of the FRET pair allophycocyanin and europium) are measured after incubation at 4° C. for 30 min, allowing the binding of the antibodies and the subsequent energy transfer between the fluorophors. In case of direct labeling of reaction components, i.e. ubiquitin or HectH9, real time measurements can be performed allowing the detection of kinetic differences in the reaction.
In the alternative embodiment b), the invention relates to a method for determining whether a compound has the ability to inhibit the transfer of ubiquitin from HectH9 to Myc, i.e. ubiquitination mediated by HectH9. In this embodiment HectH9 is incubated, together with a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), ubiquitin, ATP and a HectH9 substrate protein (Myc) for a period of time sufficient to obtain a measurable level of ubiquitination of the substrate protein Myc and comparing the level of ubiquitination of Myc in the presence or absence of a test compound, wherein a decrease in the level of Myc ubiquitination in the presence of the test compound, as compared to the level of Myc ubiquitination in the absence of the test compound, is indicative of the compound's ability to inhibit the activation of Myc.
This embodiment is based on the observation obtained in the experiments of the invention that HectH9 is able to ubiquitinate substrate proteins, but not substrate proteins that have mutations at positions necessary for the attachment of ubiquitin.
In a preferred embodiment, Myc is the c-Myc onco-protein.
With regard to the assay components and the reaction conditions, the features described for embodiment a), i.e. the assay based on transfer of ubiquitin to HectH9, also apply to embodiment b), i.e. the assay based on substrate (Myc) ubiquitination. These features refer both to assay components and their production as well as assays and the reaction conditions.
The substrate protein and DNA sequences are known from the literature and from data bases, e.g. c-Myc (Watt et al., 1983, GenBank Accession No. P01106), 1-myc (GenBank Accession No. AA038672) or n-myc (Kohl et al., 1986, GenBank Accession No. PO4198). Instead of using the full-length Myc protein, a C-terminal fragment that contains, as a minimum requirement, a relevant site for ubiquitination by HectH9 can be used. With respect to c-Myc, it has been shown in the experiments of the invention that the region from lysine 298 to lysine 355 is critical for ubiquitination. This means that at least one of the lysine residues in this region needs to be present, e.g. on a polypeptide encompassing approximately the region from lysine 298 to lysine 355. The size of the fragment may vary and can be easily determined in preliminary experiments by employing different sized Myc peptide fragments in the intended assay format and determining and selecting suitable (poly)peptides.
In the embodiment b) that uses the HectH9 substrate protein Myc, the substrate protein is captured by virtue of a tag, e.g. MBP, and the amount of associated ubiquitin is quantified in the presence or absence of test compounds.
Preferably, the method of the invention is in the form of a screening method in the high throughput format. By way of example, such a screening assay is performed in 96 or 384 well plates in a suitable reaction volume, e.g. 50 μl, in the presence or absence of the test compounds. The test compounds are usually dissolved in DMSO. In such a screening method, a test compound from a compound pool, e.g. from a chemical library, including combinatorial libraries, is applied to each well of an assay plate. Compounds that exhibit the desired properties, i.e. inhibit HectH9 activity, are hits that will be further evaluated.
Compounds identified as positive hits in the above-described primary screening assays according to embodiment a) and/or b) are next confirmed to be specific inhibitors of HectH9 activity and not to be inhibitors of any of the other enzymes present in the reaction mixture.
Compounds possibly obtained as unspecific (“false”) hits are inhibitors of ubiquitin activating enzyme (E1) and ubiquitin conjugating enzyme (E2). E1 is necessary to activate ubiquitin for the subsequent reaction, as it is the only enzyme that is able to recognize free ubiquitin in solution. E1 forms a thioester with ubiquitin in an ATP-dependent manner and transfers this now activated ubiquitin molecule to an E2 enzyme, where another thioester between E2 and ubiquitin is formed. The thioester formation of E2 with ubiquitin is strictly dependent on the presence of E1, which means that E2 alone is unable to form a thioester with free ubiquitin. Compounds that inhibit any step in this cascade, i.e. compounds that interfere with the ATP binding to E1 or that prevent the formation of the thioesters between ubiquitin and E1 or ubiquitin and E2 like any reducing agent will subsequently inhibit the formation of multiubiquitin chains and appear as positive hits. The ATP-dependent thioester formation can be used to identify such false hits, i.e. compounds that inhibit this first step in the formation of multiubiquitin chains. Assays that are able to measure thioester formation and can thus be used in a secondary assay to exclude false hits, are known in the literature (Yu, et al., 1996). In brief, they are based on using the nature of a thioester with regard to reducibility by agents like dithiothreitol or β-mercapto ethanol. A mixture of E1, E2, ATP and ubiquitin is incubated at 37° C. in the presence of the compounds identified to inhibit the transfer of ubiquitin to HectH9 or DMSO as control. These samples are subsequently subjected to SDS-PAGE under non-reducing conditions; i.e. thioesters that have formed will not be broken up and transferred to PVDF or nitrocellulose membranes. Ubiquitin can be detected either by using monoclonal antibodies against ubiquitin (commercially available, e.g. from Santa Cruz) or by using and detecting tagged versions of ubiquitin (GST-ubiquitin, anti-GST antibodies; 3-9×myc-ubiquitin, 9E10 antibodies). In control reactions, free ubiquitin can be detected as well as ubiquitin associated with an approx. 20 kDa protein (E2) and an approximately 120 kDa protein (E1). Reduction or disappearance of these thioesters upon incubation with inhibitory compounds can be taken as indication for inactivation of either E1 or E2 or both and considered as unspecific.
Advantageously, a further secondary assay may be performed to confirm that the compounds identified in the assays of the invention are inhibitors specific for a HectH9-mediated ubiquitination reaction and not inhibitors of reactions mediated by other HECT domain containing proteins. Such (one or more) assay(s) use one or more other HECT domain containing protein(s), e.g. NEDD4 (GenBank Accession No. P46934), SMURFI (GenBank Accession No. Q9HCE7) or ITCHY (GenBank Accession No. Q96J02).
Alternatively, the activity of Myc can be inhibited by preventing the interaction of the transcriptional co-activator p300 and the ubiquitinated Myc or a variant or fragment thereof. A screening assay based on this principle is a protein-protein interaction assay, in which one of the interaction partners is usually bound to a solid support and binding of the other partner, which may carry a detectable epitope tag, is quantified. A compound that interferes with binding of the two proteins is a candidate for reduction of Myc activation.
Compounds identified in the screening methods of the invention, which function as inhibitors of HectH9 activity, are expected to completely or partially inhibit proliferation of tumor cells; this effect is expected to subsequently induce apoptotic cell death. Due to this ability, such compounds are drug candidates for the therapy of cancer.
To test the ability of a candidate compound to inhibit tumor cell proliferation, primary human tumor cells or various different human cell lines (e.g. the colon cancer cell line HCT116 or the breast cancer cell line MCF7) are incubated with the candidate compound identified by the method of the invention and the inhibition of tumor cell proliferation is tested by conventional methods, e.g. bromo-desoxyuridine or 3H incorporation, Alamar BLUE. Compounds that exhibit an anti-proliferative effect in these assays may be further tested in tumor animal models and used for the therapy of tumors.
Animal models useful for testing these compounds include those described above (Felsher and Bishop, 1999; Jain et al., 2002; Pelengaris et al., 1999; Pelengaris et al., 2002a) that exhibit Myc-dependent tumorigenesis. Moreover, xenograft models of human cancer cell lines containing elevated and/or deregulated Myc, such as breast, colon, cervical, small cell lung carcinomas, osteosarcomas, glioblastomas, melanoma and myeloid leukemias, can be used to test the efficacy of such compounds.
Toxicity and therapeutic efficacy of the compounds identified as drug candidates can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD50, ED50. The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal). A pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using one or more physiologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. “Remington Pharmaceutical Sciences”.
The following Examples are provided to show how the invention can be practices they are not intended to limit the invention in any way. In the following Examples, if not otherwise stated, the following materials and methods were used:
Protein Interaction
To generate the bait plasmid, the POZ domain of Miz-1 (amino acids 1-117) was amplified by PCR and inserted into pGBT9 (Clontech). A total of 1×106 independent transformants of a human HeLa cDNA library fused to the GAL4 activation domain (Matchmaker, Clontech) were screened. In vitro transcription/translation was carried out in reticulocyte lysate (Promega) in the presence of [S35]-methionine. The translated product were mixed and incubated for 90 min at 4° C. Immunoprecipitation was carried out using the indicated antibodies immobilized on protein G-sepharose beads. The beads were washed four times in 20 mM HEPES pH 7.8, 100 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 0.5% Igepal CA-630. Bound proteins were analyzed by SDS-gel electrophoresis and fluorography.
Cell Culture
HeLa cells were grown in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Recombinant retroviruses were generated and used as described (Peukert et al., 1997). Infected cells were selected and analyzed within 2 passages after selection.
Transfections and Expression Analysis
All transient transfection experiments were carried out using a standard CaPO4-protocol. The prothyinosina-reporter plasmids have been described in (Desbarats et al., 1996). CMV-based expression plasmids encoding Myc, Miz1 and ΔPOZMiz1 have been described (Herold et al., 2002). Point mutants were introduced into Myc using the Quick-exchange kit from Stratagene. Expression plasmids encoding his-tagged ubiquitin and ubiquitin-mutants were a kind gift of Martin Scheffner (University of Cologne). A full-length cDNA encoding HectH9 was inserted into pcDNA3 (Invitrogen) to generate CMV-HectH9 (kind gift of Kristian Helin). pSUPER plasmids were a kind gift of Rene Bemards (Brummelkamp et al., 2002).
Antibodies
Immunofluorescence and immunoprecipitation was carried out as described previously (Staller et al., 2001). The following antibodies were used: Miz1: Monoclonal antibody 10E2; Myc: 9E10 (Evan et al., 1985) and N262 (Santa Cruz); HectH9: affinity-purified polyclonal antiserum against the carboxyl-terminus and monoclonal antibody Ax8D 1 (Kind gift of Kristian Helin).
Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed as described previously (Bouchard et al., 2001) using the following antibodies: anti-Myc (N-262; sc-764), anti-p300 and control antibody. Immunoprecipitated DNA samples were amplified by real-time PCR (ABI-Prism) using a Eurogentech Cybr-Green Core kit and primers specific for the E-boxes of the indicated genes. Primer sequences are available upon request.
EXAMPLE 1
Two-Hybrid Screen for Identifying Novel Interaction Partners of Miz1.
Interaction of Myc and Miz 1 with HectH9
The results of the experiments are shown in FIG. 1:
Panel a: The schematic diagram shows the carboxyl-terminal catalytic domain (“HECT”), two putative nuclear localization signals (“NLS”), a leucine zipper (“LZ”) and an UBA and a WWE domain. The sequence corresponding to the clones recovered in the two hybrid screens is underlined.
Panel b shows the in vitro interaction of HectH9 and Miz1. Miz1, ΔPOZMiz1 and Myc were translated in a reticulocyte lysate in the presence of 35S-methionine; after synthesis, the lysates containing the proteins indicated at the top were mixed and precipitated with the indicated antibodies. Shown is an autoradiogram of the precipitates; the input corresponds to 20% of the synthesized proteins.
Panel c shows that HectH9 is a nuclear protein. Shown are immunofluorescence pictures of HeLa cells stained with a polyclonal antiserum against HectH9. The top panel shows HeLa cells stably transfected with empty pSUPER; the bottom panel shows HeLa cell transfected with pSUPER-HectH9.
Panel d shows the in vivo interaction of HectH9 and Myc. HeLa cells were transiently transfected with expression plasmids encoding the indicated proteins. The lanes at the left show the input controls. The lanes on the right show the immunoprecipitates using the indicated antibodies. The top panel was probed with antibodies against HectH9, the bottom panel probed with antibodies against Myc.
Panel e shows that in vivo interaction of HectH9 and Miz1 depends on the POZ domain. The experiment was carried out as in panel d, except that Miz1 or ΔPOZMiz1 were co-expressed with HectH9. The blot was probed with antibodies against Miz1.
EXAMPLE 2
Analysis of whether HectH9 Ubiquitinates Miz 1 or Myc
HeLa cells were transfected with expression plasmids encoding the indicated proteins. Lysates were precipitated with Ni-agarose and the precipitates probed with antibodies against Myc.
FIG. 2 shows that it is Myc that is ubiquitinated by HectH9.
EXAMPLE 3
Analysis of the Functional Consequences of HectH9-Mediated Ubiquitination of Myc
To test the functional consequences of HectH9-mediated ubiquitination of Myc, the lysine residues in Myc that are targeted by HectH9 were mapped. It was found that HectH9 targets lysines in the carboxyl-terminus of Myc. The findings of these experiments are shown in detail in FIG. 3.
Panel a is a schematic drawing of the Myc mutants used in these experiments. Two conserved “Mycboxes” and the carboxyl-terminal BR/HLH/LZ-domain are shown. NLS indicates the nuclear localization signal.
Panel b shows the analysis of N-terminally truncated mutants of Myc. Top: HeLa cells were transfected with expression plasmids encoding HectH9 and the indicated mutants of Myc. Lysates were immunoprecipitated with the indicated antibodies and the precipitates probed with an α-Myc antibody (9E10, which recognizes an epitope in the carboxyl-terminus of Myc). Bottom: HeLa cells were transfected with expression plasmids encoding His-tagged ubiquitin, HectH9 (where indicated) and the indicated mutants of Myc. Lysates were precipitated with Ni-agarose and the precipitates probed with anti-Myc antibodies.
Panel c shows the binding and ubiquitination of carboxyl-terminal mutants of Myc by HectH9. The experiment was carried out as in “b”, except that a different antibody (N262 raised against the amino-terminal 262 amino acids of Myc) was used to detect Myc proteins.
Panel d shows that a mutant of Myc that has six lysines replaced by arginine (MycKR6) has reduced basal and HectH9-mediated ubiquitination.
The experiment was carried out as in panels b and c. The top panel shows Ni-agarose precipitates of lysates of transfected cells, which were probed with an antibody directed against Myc. The lower panels show immunoblots of aliquots of the transfected cells with α-Myc and α-Cdk2 antibodies showing equal expression of Myc and MycKR6 after transfection.
EXAMPLE 4
Testing whether ubiquitination triggers degradation of Myc
The results of the experiments, which show that HectH9 mediates lysine-63 linked poly-ubiquitination of Myc, is depicted in FIG. 4:
Panel a shows unaltered stability of Myc and MycKR6. NIH3T3 cells were infected with retroviruses expressing either Myc or MycKR6 Cells were treated with cycloheximide for the indicated times and lysates were probed with antibody 9E 10, which is specific for human Myc.
Panel b presents the quantitation of the results show in panel a.
Panel c: Ubiquitination of Myc by HectH9 was carried out as in FIG. 2 in the presence of wither wild-type ubiquitin or the ubiquitin mutants in which the indicated residue is replaced by an arginine.
EXAMPLE 5
Testing how ubiquitination affects the function of Myc
The transcriptional properties of Myc and MycKR6, obtained as a result of the transient reporter assays, are shown in FIG. 5:
Panel a: Transient transfection assays with the indicated amounts of expression plasmids for either Myc or MycKR6 in HeLa cells using an E-box reporter plasmid derived from the prothymosin-a gene, a target of Myc.
Panel b: Relative expression levels of endogenous Myc target genes. NIH3T3 cells were infected with retroviruses expressing either Myc or MycKR6 and serum-starved for 48 hours to deplete the endogenous Myc proteins. RNA was isolated and subjected to microarray analysis using an 11.5 kcDNA array. Shown are relative expression levels of selected direct target genes of Myc. The values show the fold expression in cells expressing wild type Myc relative to cells expressing MycKR6.
Panel c: HectH9 stimulates binding of Myc to p300. HeLa cells were transfected with expression plasmids encoding HA-tagged p300, Myc or MycKR6 and HectH9 where indicated. Lysates were precipitated with a-HA antibody and probed with antibodies directed against Myc. The left panel shows an input control.
Panel d: Myc recruits p300 selectively to E-Boxes, not to Miz1 binding sites in vivo. Shown are chromatin-immunoprecipitation (Chip) experiments with the indicated antibodies from a B-cell line harboring a tetracyclin-regulatable Myc gene. Chips were carried out either in the presence or absence of Myc with the indicated antibodies. The percentage DNA bound was calculated from real-time PCR amplifying the precipitates with primers for the indicated genes.
EXAMPLE 6
Screening assay in high throughput format for determining the transfer of ubiquitin to HectH9
The proteins used in the following example are obtained by recombinant expression in suitable host cells, i.e. E. coli JM 109, and purified by virtue of their fusion to an affinity tag.
In a 384 well amylase-coated microtiter plate (Pierce), an enzyme mixture containing 1 ng his-tagged UBAI (E1), 30 ng his-tagged UBCH5 (E2), 50 ng MBP-HectH9, consisting of the C-terminal 638 amino acids of HectH9, and 100 ng GST-ubiquitin are mixed in reaction buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 5% gylcerol, 0.5% DTT and 0.5% BSA) in a volume of 20 μl. This solution is mixed with 5 μl of the test compound (100 μM in 20% DMSO), being dispensed by aspirating from a compound source plate. The enzymatic reaction is started by the addition of 5 μl 4 mM ATP. Plates are incubated for 60 min at room temperature and washed 3 times in reaction buffer. Subsequently, Europium-labeled antibodies recognizing the GST protein (Wallac/Perkin Elmer) are added (30 μl of a solution containing 3 μg/ml labeled antibody in PBS containing 0.05% Tween 20). After incubation for 60 min, the plates are washed with PBST and fluorescence is enhanced by addition of DELFIA enhancement solution (Wallac/PerkinElmer). Europium fluorescence is measured in a fluorescence plate reader (i.e. Victor2, Wallac/PerkinElmer) at an excitation wavelength of 340 nm and an emission wavelength of 615 nM. For normalization, each plate contains a column of negative controls with 5 μl 20% DMSO solution instead of test compound. The values of the negative control show the assays maximum signal intensity. Inhibition of GST-ubiquitin transfer to HectH9 leads to a reduced signal. Results are calculated using the following procedure: value of test compound divided by the average of the negative controls on the same plate, multiplied by 100 (=% control). Compounds that give a reduced signal are further evaluated whether they are specific inhibitors of HectH9 activity. Compounds confirmed to be specific are further tested by applying them to a panel of tumor cells and testing them for their anti-proliferative effect.
EXAMPLE 7
Screening Assay in High Throughput Format for Determining the Transfer of Ubiquitin from HectH9 to Myc
In a 384 well amylase-coated microtiter plate (Pierce), an enzyme mixture containing 1 ng his-tagged UBAI (E1), 30 ng his-tagged UBCH5 (E2), 50 ng his-tagged HectH9, (all three assay components were obtained as described in Example 6) 50 ng of MBP-tagged c-Myc (the full-length protein obtained by in vitro translation using a kit from Roche) and 100 ng GST-ubiquitin (see Example 6) are mixed in reaction buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 5% glycerol, 0.5% DTT and 0.5% BSA) in a volume of 20 μl. This solution is mixed with 5 μl of the test compound (100 μM in 20% DMSO), being dispensed by aspirating from a compound source plate. The enzymatic reaction is started by the addition of an ATP regenerating system (1 mm ATP, 1 μg/μL creatine phosphokinase, 10 mm phosphocreatine). Plates are incubated for 60 min at room temperature and washed 3 times in reaction buffer. Subsequently, Europium-labeled antibodies recognizing the GST protein (Wallac/Perkin Elmer) are added (30 μl of a solution containing 3 μg/ml labeled antibody in PBS containing 0.05% Tween 20). After incubation for 60 min plates are washed with PBST and fluorescence is enhanced by addition of DELFIA enhancement solution (Wallac/PerkinElmer). Europium fluorescence is measured in a fluorescence plate reader (i.e. Victor2, Wallac/PerkinElmer) at an excitation wavelength of 340 nm and an emission wavelength of 615 nM. For normalization, each plate contains a column of negative controls with 5 μl 20% DMSO solution instead of test compound. The values of the negative control show the assays maximum signal intensity. Inhibition of GST-ubiquitin transfer to Myc leads to a reduced signal. Results are calculated using the following procedure: value of test compound divided by the average of the negative controls on the same plate, multiplied by 100 (=% control). Compounds that give a reduced signal are further evaluated whether they are specific inhibitors of HectH9 activity. Compounds confirmed to be specific are further tested by applying them to a panel of tumor cells and testing them for their anti-proliferative effect.
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## Claims

1. A method for identifying a Myc activation inhibitor compound comprising the steps of measuring the ability of a test compound to interfere with the activity of the E3-ligase HectH9, which has the amino acid sequence of SEQ ID NO: 2, wherein HectH9 activity is measured by:
a) the transfer of ubiquitin from a ubiquitin conjugating enzyme E2 to HectH9, or to a fragment thereof that has E3-ligase activity, or
b) the transfer of ubiquitin from HectH9 to Myc or a C-terminal fragment thereof that contains a critical site for ubiquitination by HectH9,
wherein said measurement is conducted after a period of time sufficient to allow for the ubiquitination reaction to occur; and wherein a decrease in the level of said HectH9 activity in the presence of a test compound, as compared to the level of said HectH9 activity in the absence of said test compound, is indicative of said compound's potential to inhibit the activation of Myc.

2. The method of claim 1, wherein Myc is c-Myc.

3. The method of claim 1, for determining whether a test compound has the ability to inhibit transfer of ubiquitin to HectH9, wherein HectH9 or a fragment thereof is incubated together with a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), ubiquitin and ATP for a period of time sufficient to obtain a measurable level of ubiquitin associated with HectH9, and wherein the levels of ubiquitination, of HectH9 in the presence or absence of said test compound are compared, and wherein a decrease in the level ofHectH9 ubiquitination in the presence of said test compound, as compared to the level of HectH9 ubiquitination in the absence of said test compound, is indicative of said compound's potential to inhibit the activation of Myc.

4. The method of claim 1, wherein said period of time is about 30 minutes.

5. The method of claim 1 for determining whether a compound has the ability to inhibit the transfer of ubiquitin from HectH9 to its substrate protein Myc, wherein HectH9 or a fragment thereof is incubated together together with a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), ubiquitin, ATP and Myc or a fragment thereof for a period of time sufficient to obtain a measurable level of ubiquitination of Myc, wherein the levels of ubiquitination of Myc in the presence or absence of a test compound are compared and wherein a decrease in the level of Myc ubiquitination in the presence of said test compound, as compared to the level of Myc ubiquitination in the absence of said test compound, is indicative of said compound's potential to inhibit the activation of Myc.

6. The method of of claim 1, wherein the HectH9 fragment used is comprised of a HECT domain.

7. The method of claim 1, wherein said method is performed in a high throughput format.

8. The method of claim 7, wherein said high throughput format is comprised of a step that uses a 96 to 384 well format.

9. The method of claim 1, wherein the HectH9 activity is measured by the transfer of ubiquitin from HectH9 to a C-terminal fragment thereof that is comprised o lysine 298 and/or lysine 355.