NZ237608A - Identifying compounds which inhibit c-myc transcriptional activity - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £37608 237608 No.: Date: f 0":.A *' 3 .A^.r.Lv'^O-v i - cp „ . 1 ... ... : ; f ... , Q \is)..33.(-5.O.,• ..c i . . . ./.3.^G --- — ^ IMEW 'iEALfcihS '•' 3 PATENTS ACT, 1953 COMPLETE SPECIFICATION "C-MYC SCREENING ASSAYS' We, THE GENERAL HOSPITAL CORPORATION, a Corporation of the State of Massachusetts, United States of America, of Fruit Street, Boston, Massachusetts 02114, United States of America, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- (followed by page la) n 237608 A - la- TITLE OF THE INVENTION C-MYC SCREENING ASSAYS Field of the Invention This invention is directed to methods for identifying compounds which inhibit c-myc transcriptional activity, and specifically compounds which inhibit c-myc heterodimer complex binding to specific DNA sequences.

BACKGROUND OF THE INVENTION The induction of many types of cancer is thought to be ultimately caused by activation of cellular oncogenes (Bishop, J.M., Science 235:305-310 (1987); Barbacid, M., Ann. Rev. Biochem. 56:779-827 (1987); Cole, M.D., Ann. Rev. Genet. 20:361-384 (1986); and Weinberg, R.A., Science 230:770-776 (1985)). Such oncogenes express oncoproteins that reside in the cell, often localized to a specific site such as the nucleus, cytoplasm or the cell membrane.

For example, the cellular c-myc gene encodes the c-myc protein. Expression of large amounts of c-myc in a variety of cell types allows the cells to grow indefinitely in cell culture (reviewed in Bishop, J.M., Cell 42:23-38 (1985); and Weinberg, R.A., Science 230:770-776 (1985)). C-myc expression has been implicated as a factor in at least 10% of all human cancers.

Further, overexpression of c-myc in normal rat fibroblasts, together with expression of an activated ras oncogene product, transforms the fibroblasts and endows them with the ability to form tumors in living animals (Land, H. et al., Nature 304:596-601 (1983); Ruley, H.E., Nature 304:602-606 (1983)).

The function of the c-myc protein remains unknown despite evidence suggesting possible roles in transcriptional regulation, RNA processing, and replication. Recent studies suggest that oncoproteins such as c-myc alter gene expression and immortalize cells by regulating the promoter activity of specific target genes and thus activating or repressing transcription of those target genes (see, for example, Varmus, H.E., Science £38: 1337-1339 (1987)); Kingston, R.E. et al.} Cell 41:3-5 (1985); Bishop, J.M., Cell 42: 23-38 (1985); Weinberg, R.A., Science 230:170-776 (1985)).

It has been proposed that the myc proteins are sequence specific DNA binding proteins. However, only nonspecific interactions with DNA have been reported for c-myc and for N-myc. (Donner, P. et al., Nature 296:262-265 (1982); Persson, H. et al., Science 225:718-721 (1984); Ramsay, G. et al., Proc. Natl. Acad. Sci. USA 81:7742-7746 (1984); Ikegaki, N. et al., Proc. Natl. Acad. Sci. USA 53:5929-5933 (1986)).

Immunoglobin heavy chains contain specific protein-binding enhancer sites known as E motifs. E motifs generally are variants of the 5'-CAGGTGGC-3' consensus sequence. For example, the /iEl motif is GTCAAGATGGC, /iE2 motif is AGCAGCTGGC, /iE3 is GTCATGTGG, fiES is TGGCAGGTGT (Murre, C. et al., Cell 56:777-783 (1989). One report found no binding of N-myc to any the E box sequences (Murre, C. et al., Cell 56:777-783 (1989)).

Thus, although it is desirable to identify compounds that inhibit c-myc oncoprotein activity, such has not previously been possible. By inhibiting c-myc activity, inhibition 237 6 0 8 V and/or control of c-myc-induced cell growth may be achieved. Administration of such inhibitors would provide therapeutic benefits in the treatment of diseases in which expression and activity of c-myc is a factor in promoting cell growth or in maintaining the cell in a transformed state.

However, to date, no inhibitors of c-myc have been identified. The identification of such inhibitors has suffered for lack of identification of a specific DNA binding sequence to which c-myc binds, and for lack of a simple, inexpensive and reliable screening assay which could rapidly identify potential inhibitors and active derivatives thereof.

Thus a need still exists for rapid, economical screening assays which identify specific inhibitors of c-myc activity.

SUMMARY OF THE INVENTION Recognizing the potential importance of inhibitors of c-myc oncoprotein activity in the therapeutic treatment of many forms of cancer, and cognizant of the lack of a simple assay system in which such inhibitors might be identified, the inventors have investigated c-myc DNA binding. These studies have resulted in the discovery that c-myc, when in a heterodimer form, specifically binds to the /iE2 sequence AGCAGCTGGC, and not to any other E motif. Homodimers of c-myc will not bind to the fiE2 motif.

These efforts have made possible the identification of a simple, rapid and inexpensive assay for the detection of compounds which interfere with the ability of c-myc heterodimers to bind to specific DNA sequences and thus inhibit the transcriptional activity of the c-myc oncoprotein.

The invention provides a reliable and accurate method for objectively classifying compounds, including human pharmaceuticals, as an inhibitor of c-myc activity.

The invention further provides a method for identifying and classifying the mechanism of action of a bioactive c-myc-inhibiting compound.

The invention further provides an assay for the monitoring of the isolation and/or purification of an c-myc-inhibiting compound or mixture of such compounds from a crude preparation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the description that follows, a number of terms used in recombinant DNA technology are extensively utilized. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Qperablv-linked. As used herein, two macromolecular elements are operably-1inked when the two macromolecular elements are physically arranged such that factors which influence the activity of the first element cause the first element to induce an effect on the second element. For example, the transcription of a codi'ng sequence which is operably-1inked to a promoter element is induced by factors which "activate" the promoter's activity; transcription of a coding sequence which is operably-1inked to a promoter element is inhibited by factors which "repress" the promoter's activity. Thus, a promoter region would be operably-1inked to the coding sequence of a protein if transcription of the coding sequence activity was influenced by the activity of the promoter.

Response. As used herein, the term "response" is intended to refer to a change in any parameter which can be used to measure, indicate or otherwise describe c-myc heterodimer binding to the /iE2 DNA sequence. The response may be revealed as a physical change (such as a change in phenotype) or, it may be revealed as a molecular change (such 237 as a change in a reaction rate or affinity constant). Detection of the response may be performed by any means appropriate.

Compound. The term "compound" is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase. The term should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides, or lipids, and also small entities such as neurotransmitters, ligands, hormones or elemental compounds.

Bioactive Compound. The term "bioactive compound" is intended to refer to any compound which induces a detectable or measurable response in the methods of the invention.

Promoter. A "promoter" is a DNA sequence located proximal to the start of transcription at the 5' end of the transcribed sequence. The promoter may contain multiple regulatory elements which interact in modulating transcription of the operably-1inked gene.

Expression. Expression is the process by which the information encoded within a gene is revealed. If the gene encodes a protein, expression involves transcription of the DNA into mRNA, the processing of mRNA (if necessary) into a mature mRNA product, and translation of the mature mRNA into protein.

A nucleic acid molecule, such as a DNA or gene is said to be "capable of expressing" a polypeptide if the DNA contains the coding sequences for the polypeptide and expression control sequences which, in the appropriate host environment, provide the ability to transcribe, process and translate the genetic information contained in the DNA into a protein product, and if such expression control sequences are operably-1inked to the nucleotide sequence which encodes the polypeptide.

Cloning vehicle. A "cloning vehicle" is any molecular In 2 entity which is capable of delivering a nucleic acid sequence into a host cell for cloning purposes. Examples of cloning vehicles include plasmids or phage genomes. A plasmid which can replicate autonomously in the host cell is especially desired. Alternatively, a nucleic acid molecule which can insert into the host cell's chromosomal DNA is especially useful.

Cloning vehicles are often characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning.

The cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance. The word "vector" is sometimes used for "cloning vehicle." Expression vehicle. An "expression vehicle" is a vehicle or vector similar to a cloning vehicle but is especially designed to provide sequences capable of expressing the cloned gene after transformation into a host.

In an expression vehicle, the gene to be cloned is usually operably-1 inked to certain control sequences such as promoter sequences. Expression control sequences will vary depending on whether the vector is designed to express the operably-1inked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.

Host. By "host" is meant any organism that is the recipient of a cloning or expression vehicle. 237608 In the hosts of the invention, at least two genetic constructs are utilized. First, a recombinant construct capable of expressing c-myc; and second, a reporter gene whose expression is operably linked to c-myc heterodimer binding to 5 the /iE2 sequence. If desired, a recombinant construct capable of expressing a c-myc partner protein may also be used.

Constructs which are capable of expressing c-myc protein may be constructed utilizing the guidelines as described below or purchased commercially.

The ntZ motif sequence may be operably linked to any gene which confers a selectable marker in yeast. In a perferred embodiment, a marker gene which allows phenotypic selection in Saccharomyces cerevisiae is used.

Yeast which have been co-transformed with both an 15 expressible c-myc gene and with the ^E2 binding sequence may be used to (1) identify the presence or absence of endogenous host proteins which interact with c-myc in a manner which permits it to bind to the ^E2 sequence; (2) classify a protein as a c-myc partner protein; and (3) identify and classify 20 compounds as agents which disrupt heterodimer formation between c-myc and its partner proteins.

All three applications are based on the same principle: in the presence of a c-myc partner protein, a complex of c-myc and the partner protein will be formed. The complex of c-25 myc and its partner will bind to the fj.E2 motif, and expression of the marker gene will be altered. In the absence of the c-myc partner protein, heterodimer complex formation will not occur and expression of the marker protein will not be altered.

The partner protein may endogenously bind to the /iE2 sequence even in the absence of c-myc. However, when c-myc is present in the cell, the amount and strength of the c-myc-partner protein heterodimer binding is increased and the strength of the /*E2 binding is greater.

Hosts which have been co-transformed with both an expressible c-myc gene and with the pE2 binding sequence may be used to identify the presence or absence of endogenous host proteins which interact with c-myc in a manner which permits c-myc to bind to the /iE2 sequence if c-myc expression in such host is sufficiently low that the moles of c-myc which are expressed do not overwhelm the moles of the partner protein which is endogenously in the cell. If such analyses reveal that the host contains c-myc binding proteins which induce c-myc heterodimer binding to the /xE2 sequence, such partner protein may be isolated using techniques known in the art such as gel mobility shift analysis, cDNA expresison cloning vectors such as, for example, AgtlO and Xgtll, or other cloning systems specifically designed for high-efficiency cloning and expression of full-length cDNA in yeast such as, for example, pGl and pTRP56, all of which are commerically available (Clontech, Palo Alto, California).

It is not necessary that the host be completely deficient in c-myc partner proteins to be useful in the method of the invention. As described below, if c-myc is expressed at levels much greater than those found in the host, reporter gene transcription from endogenous partner proteins may be negligible, or of such low amount that it does not otherwise prevent the utility of the methods of the invention.

If the c-myc expression is transcribed with a strong promoter, and/or if the c-myc expression cassette is supplied on a high copy number vector, the levels of c-myc will be high enough to overcome a low level background and such c-myc constructs may be used to analyze the ability of cloned c-myc partners to influence c-myc DNA binding. One of ordinary skill in the art can adapt the expression system to the level of expression desired using methods known in the art.

The sequence of c-myc is known (Battey, J. et al., Cell 34:779-787 (1983)) and probes which are capable of identifying a c-myc clone are commercially available (New England Nuclear/DuPont Biotechnology Boston, MA).

The partner protein, if supplied as a recombinant construct to the host cell, should be capable of expressing at levels comparable to that of the c-myc protein. C-myc partner proteins can be identified by utilizing a phage plaque accau ac rlacr* vi hoH *5 n wn Q1 /I fiAOQ ' reference. Proteins identified by the screening assay can be subcloned into eukaryotic expression vectors known in the art and commerically available so as to provide a recombinant source of partner protein gene expression.

The genetic constructs of the invention may be placed on different plasmids, or combined on one plasmid.

A construct may also be inserted into the genome of a host cell. Preferably, the construct coding for the c-myc protein and the construct coding for the partner protein are provided to the host on two different plasmids.

It is important to establish that the effect of the compound is due to an effect on c-myc heterodimer formation and not an effect on the activity of the reporter product per se. Such effect can be established by comparing the results found in hosts which lack either the c-myc expression vector or the c-myc partner expression vector or both.

The [iE2 motif may be located at any site in the transcription cassette of the reporter gene which allows for the transcription of that gene to be operably-1 inked to c-myc-partner complex binding. Thus, such motif may be located 5' to the transcriptional start site or 3' to the transcriptional start site, for example, in an intron, similar to its location relative to the promoter region in the immunoglobulin genes. and incorporated herein by -.., c r>,/' The reporter gene whose expression is operably linked to c-myc heterodimer binding to the ill2 sequence may be any gene whose expression can be monitored. Any detectable phenotype change may serve as the basis for the methods of the invention. In a preferred embodiment, the reporter gene is a gene not normally expressed by the host, or a gene which replaces the host's endogenous gene. Any reporter gene which is capable of being operably-1inked to a promoter capable of responding to the binding of the the c-myc-partner protein heterodimers to /iE2 may be used.

Especially, for example, genes which endow the host with an ability to grow on a selective medium are useful. For example, in yeast, use of the yeast LEU2 gene as a reporter gene in strains which normally lack LEU2 allows such yeast to grow on leucine as a sole carbon source. Expression the reporter gene is monitored by merely observing whether the host retains the ability to grow on leucine. In a similar manner, use of the suc2 gene as a reporter gene would allow growth of the a suc2" yeast host on sucrose to be used as the detection method. In both examples, growth on the indicated substrate indicates /zE2 DNA binding of the c-myc-partner protein heterodimer and lack of such growth indicates lack of binding or lack of heterodimer formation.

In another example, a construct (and host) which is gall*gallO~ would respond to galactose in the medium; a construct (and host) which is lac2+gall+ would be lactose sensitive. Other reporter genes include his3, ura2 and trp5. One of ordinary skill in the art can imagine many other appropriate reporter systems which would reveal the presence or inhibition of biological activity of the c-myc heterodimers.

Reporter constructs in which the /iE2a motif and the lacZ reporter gene are operably linked will express 0-galactosidase in response to binding of a c-myc-partner 237 protein complex. Such expression can be easily scored by monitoring the ability of the host to produce /?-galactosidase (Maniatis, T. et al.. Molecular Cloning (A Laboratory Manual). 2nd edition, Cold Spring Harbor Laboratory, 1989). The production of ^-galactosidase may be visually monitored by detecting its activity to reduce the chromophoric dye, X-gal (commercially available from International Biotechnologiers, Inc., New Haven, CT). /?-galactosidase reduces X-gal to a form which possesses a blue color. In another embodiment, the coding sequence of chloramphenicol acetyl transferase (CAT) is used as the reporter gene.

Any detection method which can identify expression of the reporter gene may be used. For example, levels of the product of the reporter gene may be directly assayed with an immunoassay. Such immunoassays include those wherein the antibody is in a liquid phase or bound to a solid phase carrier. In addition, the reporter gene can be detectably labeled in various ways for use in immunoassays. The preferred immunoassays for detecting a reporter protein using the include radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), or other assays known in the art, such as immunofluorescent assays, chemiluminescent assays, or bioluminescent assays.

In an assay to screen for the ability of a compound to alter activity of the c-myc heterodimer, yeast strains which express such heterodimers and which contain the /iE2 binding site, may be plated and grown as lawns and the compound to be tested may be applied to the plates on a filter paper disk that is impregnated with such compound. Alternatively, the compound may be incorporated into the media within which the host cells are growing.

One may be able to detect the ability of a compound to alter c-myc heterodimer transcription activation activity by the appearance of a zone, which often resembles a halo, around J 15 20 ; ; > 0 KTTf '"'*■ <7 the compound-impregnated disk. If for example, the compound is toxic to the host's survival per se. the host will not grow in the zone containing the compound.

The methods of the invention can be used to screen compounds in their pure form, at a variety of concentrations, and also in their impure form. The methods of the invention can also be used to identify the presence of such inhibitors in crude extracts, and to follow the purification of the inhibitors therefrom. The methods of the invention are also useful in the evaluation of the stability of the inhibitors identified as above, to evaluate the efficacy of various preparations.

The permeability of cells to various compounds can be enhanced, if necessary, by use of a mutant cell strain which possess an enhanced permeability or by using compounds which are known to increase permeability. For example, in yeast compounds such as polymyxin B nonapeptide may be used to increase the yeast's permeability to small organic compounds. In cells from the higher eukaryotes, dimethyl sulfoxide (DMSO) may be used to increase permeability. Analogs of such compounds which are more permeable across yeast membranes may also be used. For example, dibutyryl derivatives often display an enhanced permeability.

In a preferred embodiment, the genetic constructs and the methods for using them are utilized in eukaryotic hosts, and especially in yeast, insect and mammalian cells. The introduced sequence is incorporated into a plasmid or vector capable of either autonomous replication or integrative activity.

The DNA sequence of the fusion protein and/or target gene may be chemically constructed if it is not desired to utilize a clone of the genome or mRNA as the source of the genetic information. Methods of chemically synthesizing DNA are well known in the art {Oligonucleotide Synthesis, A Practical 237608 Approach, M.J. Gail, ed., IRL Press, Washington, D.C., 1094; Synthesis and Applications of DNA and RNA, S.A. Narang, ed., Academic Press, San Diego, CA, 1987). Because the genetic code is degenerate, more than one codon may be used to 5 construct the DNA sequence encoding a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd edition, W.A. Benjamin, Inc., Menlo Park, CA, 1977, pp. 356-357).

To express the recombinant fusion constructs of the invention, transcriptional and translational signals recog-10 nizable by the host are necessary. A cloned protein encoding DNA sequence, obtained through the methods described above, (preferably in a double-stranded form), may be operably-1 inked to sequences controlling transcriptional expression in an expression vector, and introduced, for example by 15 transformation, into a host cell to produce recombinant proteins useful in the methods of the invention, or functional derivatives thereof. Such techniques are well known in the art (Recombinant DNA Methodology, Wu, R. et al., eds., Academic Press, (1989); Maniatis, T. et al., Molecul ar 20 Cloning (A Laboratory Manual), second edition, Cold Spring Harbor Laboratory, 1989).

Transcriptional initiation regulatory signals can be selected which allow for repression or activation of the expression of the c-myc construct or the partner protein 25 construct or both, so that expression of such constructs can be modulated, if desired. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, for example, by a metabolite, 30 salt, or substrate added to the growth medium.

Where the native expression control sequences signals do not function satisfactorily in the host cell, then sequences functional in the host cell may be substituted.

Expression of the constructs of the invention in * i different hosts may result in different post-translational modifications which may alter the properties of the proteins expressed by these constructs. It is necessary to express the proteins in a host wherein the ability of the protein to retain its biological function is not hindered. Expression of proteins in yeast hosts is preferably achieved using yeast regulatory signals. The vectors of the invention may contain operably-1inked regulatory elements such as upstream activator sequences in yeast, or DNA elements which confer species, tissue or cell-type specific expression on an operably-1inked gene.

In general, expression vectors containing transcriptional regulatory sequences, such as promoter sequences and transcription termination sequences, are used in connection with a host. These sequences facilitate the efficient transcription of the gene fragment operably-1inked to them. In addition, expression vectors also typically contain discrete DNA elements such as, for example, (a) an origin of replication which allows for autonomous replication of the vector, or, elements which promote insertion of the vector into the host's chromosome in a stable manner, and (b) specific genes which are capable of providing phenotypic selection in transformed cells. Eukaryotic expression vectors may also contain elements which allow it to be maintained in prokaryo-tic hosts; such vector are known as shuttle vectors.

The precise nature of the regulatory regions needed for gene expression will vary between species or cell types and there are many appropriate expression vector systems that are commercially available.

In a highly preferred embodiment, yeast are used as the host cells. The elements necessary for transcriptional expression of a gene in yeast have been recently reviewed (Struhl, K. Ann. Rev. Biochem. 58:1051-1077 (1989)). In yeast, most promoters contain three basic DNA elements: (1) an m 237 6 0 8 upstream activator sequence (UAS); (2) a TATA element; and, (3) an initiation (I) element. Some promoters also contain operator elements. Methods in yeast genetics are well known (Struhl, K. Nature 305:391-397 (1983); Sherman, et al., 5 Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1983)).

In another embodiment, mammalian cells are used as the host cells. A wide variety of transcriptional and translational regulatory signals can be derived for expression 10 of proteins in mammalian cells and especially from the genomic sequences of viruses which infect eukaryotic cells.

Once the vector or DNA sequence containing the construct^) is prepared for expression, the DNA construct(s) is introduced into an appropriate host cell by any of a variety 15 of suitable means, for example by transformation. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the protein. This expression can 20 take place in a continuous manner in the transformed cells, or in a controlled manner.

Genetically stable transformants may be constructed with episomal vector systems, or with integrated vector systems whereby the fusion protein DNA is integrated into the host 25 chromosome. Such integration may occur de novo within the cell or be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in 30 chromosomes.

Cells which have been transformed with the DNA vectors of the invention are selected by also introducing one or more markers which allow for selection of host cells which contain the vector, for example, the marker may provide biocide resis- 237608 •16- tance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.

The transformed host cell can be fermented or cultured according to means known in the art to achieve optimal cell 5 growth, and also to achieve optimal expression of the cloned fusion protein sequence fragments. As described hereinbelow, a high level of fusion protein expression for the cloned sequences coding for the proteins can be achieved according to a preferred procedure of this invention.

The following examples further describe the materials and methods used in carrying out the invention. The examples are not intended to limit the invention in any manner.

EXAMPLES Example 1 Screening a cDNA expression library for proteins able to interact with the helix-loop-helix domain of c-Myc.

To screen for proteins able to interact with the helix-loop-helix (HLH) of human c-Myc DNA encoding amino acids 255-410 (Battey, J. et al., Cell 34:779-787 (1983)), which contains the basic region and HLH of the c-myc protein was 25 ligated, in frame, to a DNA fragment encoding the N-terminal 112 amino acids of the lambda repressor (cl) protein. The expression of this chimeric protein was placed under the control of the very weak ^-lactamase promoter and the lactose operator. This expression unit was subcloned into pACYC177, a 30 low copy number plasmid (10-15 copies/cell) with a kanamycin^ gene which is commercially available. Cells transformed with this construct were shown to be resistant to lambda phage infection by a dot plaque assay. The above construct, pYC192cIHLH, made cells resistant- to phage infection by >108 ■US'? r pfu, whereas cells expressing the N-terminal region of cl alone were infected at <10^ pfu.

The JL. coli strain Y1090 transformed with pYC192cIHLH was used to screen a human tonsil/B cell Agtll expression library. 5 x 106 pfu were sceened in duplicate on the above transformed strain, as well as a Y1090::X lysogen strain. On each of test plates 800 plaques formed. On the control plates there were no plaques were plaque-purified once and a single plaque was picked and suspended in suspension medium (SM). The 160 purified plaques were grouped according to plaque size: 20 small, 70 medium, 70 large. (Four of the "small" group did not form plaques in the plaque purification step.) Each of these was then screened by a dot plaque assay on an JL. coli. strain, JM109, transformed with the plasmid pJH370, which expresses a chimeric protein consisting of the N-terminus of cl fused to the leucine zipper domain of GCN4. Each phage was also retested on the above mentioned strain used in the primary screen, as well as the X lysogenic strain and the parental Y1090 strain.

Phage which formed plaques on the parental Y1090 strain and the cI-Myc-expressing strain, but not on either the lysogenic strain or the cI-GCN4-expressing strain were defined as "positive". "Positives" were screened a total of twice on these four strains. In the dot plaque assay 5-20/il of the "positive" phage typically yielded 50-100 plaques. If a single, tiny plaque could be seen on cI-GCN4, then the phage was defined as "negative". By this assay, a total of 10 "positives" of the 156 phage tested were found. Of these, 3 were of the "small" group and 7 were of the "medium" group. Such positives may be subcloned using methods known in the art for expression in eukaryotic hosts useful in the methods of the invention. 237 6 -18-Example 2 Identification of an Inhibitor of c-Mvc Heterodimer Formation in Yeast Cells Yeast host cells are transformed with plasmids carrying c-myc (host 'a'); or c-myc and a 46,000 dalton partner protein identified as above (host 'b'). In addition all yeast strains are cotransformed with a plasmid which contains the coding sequence for ^-galactosidase operably-1 inked to the /iE2 sequence as described above.

A lawn of each of the transformed yeast strains is spread on agar plates containing X-gal in the medium and small filter disks containing compound W, X, Y, or Z are placed on the lawns. The yeast are allowed to grow and the plates are monitored for colony growth and colony color by visual observation. Typical results from such an experiment are shown in Table 1. 237 6 Table 1: Identification of C-myc-protein Inhibitors Compound Yeast Colony Color from Growth j9-gal Assay with X-gal none a + White b + Blue W a + White b + White X a _ b - - Y a + White b + Blue Z a + Blue b + Blue The results of the above table indicate that compound W prevents the induction of £-galactosidease in the 'b' host cells. Therefore, compound W is an inhibitor of heterodimer formation and an inhibitor of c-myc biological activity.

Compound X inhibits the growth of yeast per se and thus would not be a compound of interest.

Compound Y does not prevent induction of ^-galactosidase activity in the 'b' host cells. Therefore, compound Y is not an inhibitor of heterodimer formation.

Compound Z shows an interesting effect of inducing P-galactosidase activity in the 'a' host cells which does not contain the c-myc partner protein used in the 'b' hosts, rather than preventing heterodimer formation. This suggests that compound Z may induce synthesis of a partner protein which is not otherwise present in the yeast host cells or that it may be (or mimic) such a protein.

From these results, compound W would be identified as an

Claims (9)

£ -20- inhibitor of c-myc heterodimer fomration and thus of c-myc transcriptional activity in vivo. All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof. 10 wsv"45-v: : *v i • . •21- o t 7 / o o ■' j / o u o

1. A method for identifying and classifying a compound as an inhibitor of c-myc heterodimer DNA binding wherein said method comprises evaluating the ability of said compound to alter expression of a reporter gene in a host cell, wherein expression of said reporter gene is operably-1inked to DNA binding by said heterodimer to the /jE2 sequence.

2. The method of claim 1, wherein expression of said reporter gene induces a phenotypic change in a host cell.

3. The method of claim 1, wherein said reporter gene is 15 lacZ.

4. The method of claim 1, wherein said reporter gene is CAT. 20

5. The method of claim 1, wherein said reporter gene is LEU2.

6. The method of claim 2, wherein said phenotypic change is detected by visual inspection of the host cell. 25

7. The method of claim 1, wherein said host is 5. cerevisiae.

8. The method of claim 1, wherein said host is a 30 ■ mammalian cell.

9. A method for identifying and classifying a compound as an inhibitor of c-myc heterodimer DNA binding as defined in claim 1 substantially as herein described with reference to any example thereof. ; £ i\i 'f- o\ H * „ . ' o: By Jii«rrhcSf authorised Agent -'1P<?J992*f AJ.fAf,K*SON