PT97387A - Test for detection of c-myc protein - Google Patents

Abstract

The present invention relates to a method for detecting and classifying compounds as inhibitors of the action of c-myc protein, which detects the ability of a compound to inhibit or in some other way interfere with the formation of c-myc heterodimers. Said method involves the evaluation of the ability of said compound to alter the expression of a reporter gene in a host cell, in which the expression of said reporter gene is operationally bound to the DNA bond by said heterodimer to the sequence μE2.

Description

1 ì

FIELD OF THE INVENTION

This invention is directed to methods for a. identification of compounds that inhibit c-myc transcriptional activity and specifically compounds that inhibit the binding of complete c-myc heterodimeric to specific DNA sequences.

BACKGROUND OF THE INVENTION

The induction of many types of cancer is thought to be caused by the activation of cellular oncogenes (Bishop, J., Science 235, 305-310 (1987)). Barbacid, M., Ann. Rev. Biochem. 56, 779-827 (1987)) Cole, H.D., Ann. Rev. Benet. 20, 361-384 (1986), and Neinberg, R.A., Science 23, 770-776 (1985)). Such oncogenes will express oncoproteins residing in the cell, often located at a specific site such as nucleus, cytoplasm or cell membrane. (1985) s

For example, the c-myc cell gene encodes the protein and X-pressure of large amounts of c-myc in a cell variety allows the expression of c-myc. (Reviewed in Bishop, JM, Ce11 42s23-38 s " 3rd of Weinberg, RA, Science 230s / 70-776 (1985)). Express-myc has been implicated as a factor in at least 10% of all human cancers. The title compound was prepared according to the procedure described in Examples 1 and 2,

Furthermore, c-myc overexpression in rat normal, together with expression of an activated oncogenesis, transforms fibroblasts and the ability to form tumors in live animals (Land Nature, 1984, 506-60i) N-ature

II < 1983)) r

The function of c-myc protein remains unknown despite evidence suggesting possible roles in the regulation of mycoplasma. transcription, RNA processing and replication. Recent studies suggest that oncoproteins such as c-myc alter the expression of genes and immortalize cells by regulating the activity of specific target gene promoters and thus activating or repressing the transcription of those target genes (see, for example, Varmus HE, Science 238: 1337-1339 (1987)); Kingston. R.E. et al., Cell 41: 3-5 (1985); Bishop, J. M., et al., 42: 23-38 (1985); Weinberg, R.A., Science 230: 770-776 (19S5)).

It has been proposed that c-myc proteins are sequence-specific DNA binding proteins. However, only nonspecific intersections with c-myc and par-N-myc DNA were reported. (Donner, P. et al., Nature 296: 262-265 (1982); Persson, H. et al., Science 225: 718-721 (1984); Ramsay, 6. et al., Proc Natl Acad , Sei, USA 81: 7742-7746 (1984); Ikegaki, N. et al., Proc., Natl Acad, Sci, USA 83: 5929-5933 (1986)).

Immunoglobulin heavy chains contain specific binding sites known as E motifs. E motifs are generally variants of the consensus sequence 5J-CASSTBBC-35. For example, the motif μΕΙ is STCAA0ATGGC, the motif μΕ2 is AGCAGCT8SC, the μβ motif is STCATGTGB, μΕ5 is TBBCABGTGT (Murre, C. et al., Cell 56: 777-783 (1989)). W-myc binding to any of the E-box sequences (Murre, C, et al., Cell 56: 777-783 (1989)), to be

Thus, while it is desirable to identify compounds that inhibit c-myc oncoprotein activity, this has not been possible so far. By inhibiting c-myc activity, inhibition and / or control of c-myc-induced cell growth can be achieved. Administration of such inhibitors will provide therapeutic benefits in the treatment of diseases in which e -pression and c-myc activity is a factor in promoting cell growth or maintaining the cells in a transformed state.

However to date no c-myc inhibitors have been identified. The identification of such inhibitors was due to the absence of identification of a specific DNA binding sequence to which c-myc binds and the absence of an inexpensive and reproducible detection assay that could readily identify potential inhibitors and their activated derivatives.

There remains a need for rapid and inexpensive detection assays that identify specific inhibitors of c-myc activity.

SUMMARY OF THE INVENTION Recognizing the potential importance of inhibitors of c-myc anoprotein activity in the therapeutic treatment of many forms of cancer and knowing of the absence of a simple assay system in which such inhibitors can be identified, the present inventors have investigated the binding of c-myc to DNA. These studies have led to the discovery that c-myc, when in the form of a heterodimer, binds especially to the sequence μΕ2 A8CABCT68C and not to any other motif E 'The c-myc homodimers do not bind to the motif μΕ2.

These efforts have made it possible to identify a simple, rapid and inexpensive assay for the detection of compounds that interfere with the ability of the c-myc heterodimers to bind to specific DMA sequences and thus inhibit the transcriptional activity of the c-myc oncoprotein. The invention provides a reproducible and precise method for objectively classifying compounds, including human pharmaceutical compounds, as an inhibitor of c-rayc activity. The invention further provides a method for identifying and classifying the mechanism of action of a bio-active c-myc inhibitor compound. The invention further provides an assay for the control of the isolation and / or purification of a c-myc inhibitor compound or mixtures of such compounds from a crude preparation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, a number of terms used in recombinant DNA technology are extensively used. To provide a clearer and more consistent understanding of the specification and claims, including the scope of such terms, the following definitions are provided,

As used herein, two macromolecular elements are operatively linked when the two macromolecular elements are physically arranged such that the features influencing the activity of the first element cause the first element to induce an effect on the second element. For example, a. transcription of a coding sequence which is operably linked to a promoter element is induced by facets which " the activity of the promoter? transcription of a coding sequence that is operably linked to a promoter element is inhibited by

LuTiS acts that " repress " promoter activity. Thus, the promoter region will be operably linked to a coding sequence of a protein if the transcription of the activity of the coding sequence is influenced by the activity of the promoter.

Answer. As used herein, the term " response " it is intended to refer to a change in any parameter that can be used to measure, indicate or otherwise describe the binding of the c-myc heterodimer to the DMA sequence μΕ2. The response may be elucidated as a physical change (such as a change in phenotype) or may be revealed as a molecular change (such as a change in a reaction rate or affinity constant). Detection of the response can be performed by suitable means.

Compound. The term " compound " is intended to refer to a chemical entity 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.

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

PromoterA " promoter " is a DNA sequence located near the start of the S-terminal transcript " of the transcribed sequence. The promoter can count on multiple regulatory elements that interact in the modulation of transcription of the operably linked gene. s r

Empression A Expression is a process by which information encoded within a gene is revealed. If the gene encodes a protein? the expression involves transcription of the mRNA into DNA, processing of the 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 referred to as " expressing layers " a polypeptide if the DNA has the polypeptide coding sequences and expression control sequences which? in the environment of the appropriate host provides 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 linked to the nucleotide sequence encoding the peptide.

Cloning vehicle, A " cloning vehicle " is any molecular entity that is capable of releasing a nucleic acid sequence in a host cell for cloning purposes. Examples of cloning vehicles include plasmids or phage genomes. A plasmid that can be replicated autonomously in the host cell is especially desired mint. Alternatively, a nucleic acid molecule that can be inserted 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 in which such DNA sequences can be cut in a certain way without loss of an essential biological function of the carrier and in which the DNA can be inserted to be replicated and cloned. The carrier vehicle may further contain a label suitable for use in identifying cells transformed with the tetracycline carrier. The tags are, for example, or è. ampicillin. The word " vector " is resistance sometimes used for " cloning vehicle "

Expression vehicle. An " expression vehicle " is a vehicle or vector similar to a clone vector but is intended to provide sequences capable of expressing the cloned qene after transformation into a host.

In an expression vehicle, the gene to be cloned is generally operably linked to certain control sequences such as promoter sequences. Expression control sequences will vary depending on whether the vector is intended to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcription elements such as potentiating elements, termination sequences, tissue specific elements and / or initiation sites and termination of translation »

Host. By " host " is meant any organism which is the recipient of a cloning or expression vehicle.

In the hosts of the invention, at least two genetic constructs have been used. First is a recombinant construct) capable of expressing c-myc, and according to a reporter whose expression is operably linked to the c-myc heterodimer that binds to the sequence μΕ2. If desired, a recombinant construct capable of expressing a c-myc partner protein may also be used. 9

Constructs that are capable of expressing c-myc protein can be constructed using the guidelines described below or commercially acquired. The pE2 motif sequence may be operably linked to any gene which confers a yeast selectable marker. In a preferred embodiment a labeling is used which allows for phenotypic selection in Baccharomvces cerevisiae.

Yeast that can be co-transfected with a c-myc gene and the binding sequence at μΕΞ can be used to (1) identify the presence or absence of host endogenous proteins that interact with c-myc in a manner that allows their connection è sequence μΕ2? (2) classify a protein as a c-myc partner protein? and < 3 > identify ε classify compounds as agents that destroy the formation of heterodimers between c-myc and its protein partners. >

All three applications are based on the same principles in the presence of a c-myc partner protein, a c-myc complex and the partner protein are formed. The complement of c-myc with its partner binds to its motif μΕ2 and the expression of the brand gene will be changed. In the absence of the c-myc partner protein, the formation of the ε-heterodimer complex does not occur, the expression of the tag protein will not be altered. The partner protein can bind endogenously to the μ.Ε2 sequence even in the absence of c-myc. However, when c-myc is present in the cell, the amount and strength of binding of the c-myc heterodimer partner protein is increased and the binding strength at μΕ2 is greater. 10 10

Hosts who have AIDS transfused with an expressible c-myc gene and a binding sequence to μΕ2 can be used to identify the presence or absence of host endogenous proteins that interact with c-myc in order to allow c-myc to bind to the sequence μΕ2 if the expression of c-myc in such hosts is sufficiently low so that the moles of c-myc that are expressed do not overlap the moles of the partner protein that is endogenous in the cell. If such analyzes reveal that the host contains proteins c-myc binding sites that induce binding of the c-myc heterodimer to the μΕ2 sequence, such a partner protein may be isolated using techniques known as gel mobility alteration analysis, cloacal vectors and cDNA expression such as for example, lambda gil'3 and lambda gtil, or other full-length cDNA cloning and expression systems in yeast such as, for example, pBi and pTRP Only, all of them can be purchased commercially (Clontech, Pala Alta, California). It is not necessary for the host to be completely deficient in the c-myc partner proteins to be useful in the method of the invention. As described below, if c-myc is expressed at levels much higher than those found in the host, transcription of the reporter gene from endogenous partner proteins may be negligible or in such low amounts as not to render the utility of the methods of the invention unfeasible.

If c-myc expression is transcribed from a strong promoter, and / or if the c-myc expression cassette is provided in a high copy number vector, the c-myc levels are high enough to exceed one low background level and such c-osyc constructs can be used to analyze the ability of cloned c-myc partners to influence the binding of DMA to c-myc. Someone familiar with it

The material can adapt the expression system to the desired level by using known methods.

The sequence of c-myc is known (Battey, J. et al., C-3447779-787 and 1983)) and probes that are able to identify a c-myc clone can be purchased commercially from New England Nuclear / Dupont Biotechnology Boston, MA).

The partner, host protein is provided as a recombinant construct if fDr supplied as a recombinant construct to the host cell should be expressed at levels comparable to those of c-myc protein. C-myc partner proteins can be identified through the use of an iest plaque assay, as described in U.S. patent application Ser. The invention relates to a method for the preparation of a pharmaceutical composition comprising the preparation of a pharmaceutical composition comprising a pharmaceutical composition comprising a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier. The proteins intended by the screening assay may be subcloned into known expression vectors to provide a recombinant source of the expression of the partner protein gene.

The genetic constructs of the invention may be placed on different plasmids or combined into a plasmid. A construct may also be inserted into the genome of a host cell. Preferably, the c-myc protein coding construct and the partner protein coding construct are provided to the host on two different plasmids. is due to an effect on the effect it may be important to establish that the effect of the compound is effect on the formation of the c-myc heterodimer and not the activity of the reporter gene product per se. Such is established by comparing the results found for c-myc or in hosts which do not have the expression vector the c-myc partner expression vector or both motif μΕ2 can be located anywhere on the transcription cassette - which can allow the transcription of that gene to be operably linked to the c-myc-partner binding complex. Thus, such a motif may be located at the 5 'end relative to the transcription initiation site. 35 relative to the transcription initiation site, for example in an intron, similar to its location relative to the promoter region in the immunoglobulin genes. The reporter gene whose expression is operably linked to the c-myc heterodimer that binds to the sequence μΕ2 can be any gene whose expression can be controlled. Any detectable phenotypic change may serve as a basis for the methods of the invention. In a preferred embodiment, the reporter gene is a gene which is not normally expressed by the host or a gene that replaces the endogenous host gene Any reporter gene that is capable of being operably linked to a promoter capable of responding to the binding of the c-myc-partner partner heterodimers to μΕ2 can be used. For example, in yeast the use of the yeast LEU2 gene as a reporter gene in strains which do not normally have LEU2 allows such a yeast to grow in leucine as a single carbon source, the expression of the reporter gene is controlled by the mere observation that the host cells retain the ability to grow in leucine. Similarly, use of the suc2 gene as a reporter gene will allow the growth of a sucrose yeast host in sucrose to be used as a detection method. In both examples, growth on the indicated substrate is indicative of binding to the Î ± 2 DNA of the heterodimer and the absence of such growth indicates the absence of or binding. absence of the formation of the heterodimer.

In another example, a host construct which is galactose will respond to galactose in the media, a host construct which is lac2 gali will be sensitive to lactose. Other reporter genes include hista3, ura3 and trp5. One skilled in the art can envisage many other suitable reporter systems which will reveal the presence or inhibition of the biological activity of the c-myc heterodimers.

Reporter constructs in which the motif μ.Ε2 and the lacZ reporter gene are operably linked will express β-galactosidase in response to binding of a partner c-myc-protein complex. Such expression can be easily assessed by controlling the capacity of the host to produce β-galactosidase (Maniatis et al., Molecular Cloninq (A Laboratorv Manual), 2nd edition, Cold Spring Harbor Laboratory, 1989). The production of β-galactosidase can be monitored visually by detecting its activity to reduce the chromophore dye, X-gal (available from International Biotechnologists, Inc., New Haven, CD, β-galactosidase reduces X -gal to a form having a blue color. In another embodiment, the chloramphenicol acetyltransferase (CAT) coding sequence was used as the reporter gene.

Any detection method that can identify expression of the reporter gene may be used. For example, reporter gene product levels can be tested directly with an immunoassay. Such immunoassays include those in which the antibody is in a liquid phase or bound to a solid phase carrier. In addition, the reporter gene may be detectably labeled in various ways for use in immunoassays. Preferred immunoassays for the detection of a reporter protein include radioimmunoassays, enzyme linked immunosorbent assays (ELISA's, or other known assays, such as immunoassay immunoassays, chemiluminescence assays or hioluminescence assays'

In an assay for screening for the ability of a compound to alter the c-myc heterodimer's activity, yeast strains expressing such heterodimers and which contain the .beta.2 binding site, can be seeded and cultured and the compound to be isolated. can be applied to plates on a filter paper disc which can be impregnated with such a compound. Alternatively, the compound may be incorporated into the medium in which the host cells are growing.

One may be able to detect the ability of a compound to alter the activation activity of the compound. transcription of the c-myc heterodimer through the appearance of a zone, which often resembles a halo, around the disc impregnated with the compound. If for example, the compound is toxic to host survival per se, the host does not grow on the host. zone containing the compound.

The methods of the invention may also be used to screen for the presence of such inhibitors in crude extracts and to follow the methods of the invention. the purification of the useful ones as inhibitors from them. The methods of the invention are also in the evaluation of the stability of the inhibitors identified above, to evaluate the effectiveness of various preparations. S. The permeability of the cells to various compounds may be increased if necessary through the use of a mutant cell line having increased permeability or using compounds known to increase permeability. For example, in yeast compounds such as α-nonapeptide polym in B cells can be used to increase yeast permeability to small organic compounds. In higher eukaryote cells, dimethylsulfoxide can be used to increase permeability. Analogs of such compounds which are more permeable through the yeast membranes may also be used. For example, dibutyryl derivatives often have a higher permeability.

In a preferred embodiment, the genetic constructs and methods of using them are used in eukaryotic hosts and especially in yeast, insect and mammalian cells. The introduced sequence is incorporated into a plasmid or vector capable of replicating autonomously or having integrative activity . The DMA sequence of the fusion protein and / or target gene may be chemically constructed if a genomic or mRNA clone is not to be used as a source of genetic information. The methods of DMA synthesis are well known (Oligonucleotide Synthesis, A Practical Approach, M.J. Sail, ed., IRL Press, Washington, DC, 1094; Synthesis and Applications of DNA and RNA, S. Marang, ed. Academic Press, San Diego, CA, 1937). Because the genetic code is degenerate, more than one codon can be used to construct the DMA sequence encoding a particular amino acid CWatson, J, D, Em; Molecular Biology of the Sene, 3rd edition, Vol. Benjamin, Inc, Menlo Park, CA, 1977, pp. 356-357)

16 - J

In order to express the constructs of recombinant fusions of the invention, transcription and translation signals recognizable by the host are required. A cloned protein encoding the DNA sequence, obtained by the methods described above, preferably in a double-stranded form), may be operably linked to sequences that control trans-expression in an expression vector and introduced, for example by transformation , in a host cell to produce recombinant proteins useful in the methods of the invention or functional derivatives thereof, such techniques are well known (Recommant DNA Methodology, Wu, R, et al., Academic Press, (1990), 5 Maniatis, T. et al., Molecular Cloninq < A Laboratorv Manual), Second Edition, Cold Spring Barbar Laboratorv, 1989).

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

Where the signals of the native expression control sequences do not function satisfactorily then the functional sequences in the host cell may be substituted. Expression of the constructs of the invention in different hosts may result in different paddle-translation 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 biological function is not inhibited. Expression of proteins in yeast hosts is preferably achieved using yeast regulatory signals. Vectors of the invention may contain operably linked regulatory elements such as upstream activating sequences in yeast or DNA elements that confer species, tissue or cell specific expression in an operably linked gene.

In general, expression vectors containing transcriptional regulatory sequences, such as promoter sequences and transcription termination sequences, are used in conjunction with a host. These sequences facilitate efficient transcription of gene fragments which are useful for them linked. In addition, expression vectors also typically contain discrete DNA elements such as, for example. Ca) an origin of replication that allows autonomous replication of the vector or elements that promote vector insertion into the host chromosome in a stable manner and (c) specific genes that are capable of providing phenotypic selection in transformed cells. Eukaryotic expression vectors may also contain elements that allow their maintenance in prokaryotic hosts; such vectors are known as shuttle vectors. The precise nature of the regulatory regions necessary for the expression of genes will vary between species or cell types and there are many suitable expression vector systems. which can be purchased commercially.

In a particularly preferred embodiment yeasts are used as host cells. The elements necessary for the transcriptional expression of a gene in yeast were

In the yeast most of the promoters contain three basic DMA elements; in the yeast, most of the promoters contain three basic DMA elements; (1) an upstream activator sequence (UAS) j (2) a TATAj element and (3) an initiation element (I). Some promoters also contain functional elements. Methods in yeast genetics are well known " Stru.hl, 1 " Nature 385; 391-397 (1983); Sherman, et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1983)).

In another embodiment mammalian cells have been used as host cells. A variety of transcriptional and translational regulatory signals may be derived for expression of protein in mammalian cells and especially from genomic sequences of viruses infecting eukaryotic cells.

Once the vector or DMA sequence containing < s) construction < Ses), a < s) construct (Ses) is introduced into a suitable host cell through any one of a variety of suitable means, for example by transformation. After introduction of the vector, the recipient cells are cultured in a suitable host cell. selective medium, which selects the growth of vector-containing cells. The expression of the clones (s) of cloned genes results in the production of the protein. This expression may take place continuously in the transformed cells or in a controlled manner. sx

Genetically stable transformants can be constructed with episomal vector systems or the integrated vector systems by which the DMA of the fusion protein is integrated into the host chromosome. Such integration may occur again within the cell or may be aided by transformation with a vector which functionally inserts into the host's own host vectors, for example, with transcripts, transposons or other DNA elements, which integration of sequences from DNA on chromosomes.

Cells that have been transformed with the inventive DNA vectors have been screened by introducing one or more tags that allow the selection of host cells that contain the vector, for example, the label may provide resistance to biocides, e.g. resistance to antibiotics or. to heavy metals such as copper or the like. The transformed host cell may be fermented or cultured according to means known in the art to achieve optimum cell growth and also to achieve optimum expression of the cloned fragments of the fusion protein sequence. As described below, a high level of fusion protein expression for the damaging sequences encoding the fusion proteins according to a preferred process of this invention.

The following examples further describe the methods and methods used in the practice of the invention. The examples are not intended to limit the invention in any way.

EXAMPLES

Example I

Screening of a cDNft expression library for helix-helix-domain proteins of the host cell

To do " the deletion of proteins with the helix-ansa-helix CHLH) of c-DNA from amino acids 255-410 CSattey, J. st capable of human mycobacteria codified, Ce11 34s779-787

(1983)), which contains the basic β-HLH repand of the c-myc protein was ligated, in the same reading frame with a fragment encoding the N-terminal 112 amino acids of the lambda (cl) repressor protein. Expression of this chimeric protein was placed under the control of the very weak β-lactamase promoter and the lactose operator. This unit of expression was. subcloned into pACYC 177, a low copy number plasmid (10-15 copies / cell) with a commercially available kanamycin gene,

Cells transformed with this construct were found to be resistant to phage lambda infection by a " dots " of plates. The above construct, p ¥ C192cIHLH, made the cells resistant to phage infection by more than 10 "pfu, while cells expressing the N-terminal region of the monkey were infected at < 1® * " pfu. The Y strain was used to make E. coli transformed with E. coli transformed with pYC192CIHLH and screened a human amygdala / B cell expression library in lambda gtil. 5 x 105 pfu were transformed above, Y190 :: 1bda. Purification was performed on isolated plates and resuspended in purified sera were pooled in duplicate in the strain as well as a lysogenic strain where there were no plaque plates and a suspension medium plate (SM) was peeled off. The 16 plate of the agreement has the size of the small, 70 medium, 70 large placings. (Four from the " small group " did not form plaques in the plaque purification step). Each of these was then screened by an assay of " two " of plaques in a strain of E. coli, 3M109, transformed with plasmid pJH370, which expresses a chimeric protein consisting of the N-terminus of the fused G-terminal domain of GGN4. Each of the phages was also tested in the above-mentioned strain used in primary screening, as well as in the lysogenic strain for lambda and the parental strain ¥ 1090.

Phages that formed plaques in parent strain V109Θ and in the strain it expresses. d-Myc but not in the lysogenic strain or in the cI-SCM4-expressing strain were defined as " positives ", ", " positives " were screened twice in these four strains. In the " dots " of plaques 5-20 μΐ of " positive " typically give 50-100 plaques. Even if a very small plaque in cI-6CN4 can be seen then the phage is defined as " negative ". Through this assay, a total of 10 " positives " of the 156 phages tested. Of these, 3 were from the " small " and 7 were from the " medium " group. Such positives can be synthesized using known methods for expression in eukaryotic hosts useful in the -invent 'methods.

Example 2

Identification of an inhibitor of the formation of c-Myc heterodimers in yeast cells Yeast host cells were transformed with plasmids bearing c-royc (host " a "); or c-myc and a 46,000 dal tone partner protein identified as above (host " b " >). In addition all yeast strains were cotransformed with a plasmid containing the sequence 1 * s

encoding the β-galactosidase operably linked to the sequence μΕ2 as described above.

A carpet of each of the yeast strains transformed into agar plates containing β-β in the medium and small circular filters containing the compound W, X, Y or. Z were placed on the mats. The yeast grew and the plaques were controlled for colony and colon growth by visual observation. Typical results of such experiment are shown in Table 1.

Identification of inhibitors of c-myc protein Compound Yeast Colony growth Color derived from the β-gal assay with X-gal None 4- White b + Blue W a + White b + White Λ a - - b - Y 5 4 * White b + Blue zs + Blue b + Blue

The results from the above table indicate that α-compound W avoids the induction of β-aalactosidase in the host cells " b ". Thus, compound W is an inhibitor of the formation of heterodimers and an inhibitor of the biological activity of c-myc. Compound X inhibits yeast growth per se and therefore will not be a compound of interest. 24

Campaste Υ does not prevent the induction of β-galactosidase activity in host cells " b **. Thus, compound Y is not an inhibitor of the formation of heterodimers. Compound Z has an interesting effect of inducing the activity " a " which do not contain β-galactosidase β-host hosts in the host cells used c-myc partner protein in addition to preventing the formation of heterodimers.

This suggests that the partner protein hosts host protein. compound Z may induce the synthesis that otherwise will not be present in yeast or which may be (or will be) one in the same. From these results compound W will be identified as an inhibitor of the formation of c-myc heterodimers and therefore the transcriptional activity of c.-myc in vivo.

I

All references cited herein are hereby incorporated by reference. Having fully described the invention, it will be understood by those skilled in the art that the scope of the invention is within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any of the embodiments thereof.

Claims (1)

* 75 - REHABILITATION. A method for the identification and classification of a compound as a DMA-binding inhibitor of C-10 heterodimers, characterized in that it comprises evaluating the ability of said compound to alter the expression of a reporter gene in a host cell - wherein the expression of said reporter gene is operably linked to the binding to DMA by said heterodimer to the sequence μΕ2. The method of claim 1, wherein the expression of said reporter gene induces a phenotypic change in a cell hostess. 3â. 5. A method according to claim 1, wherein said reporter gene is lacZ. 4ã. A method according to claim 1, wherein said reporter gene is CAT2. A method according to claim 1, wherein said reporter gene is U2. A method according to claim 8, characterized in that said phenotype is detected by visual observation of the host cell. The method of claim 1 wherein said host is a mammalian cell wherein said host is a mammalian cell, wherein said mammalian cell is a mammalian cell. ro, Lisboaj April 17, 1991 J. PEREIRA DA CRUZ Official Agent of Industrial Property RUA VICTOR CORDON, 10- A 3 * 1200 USBOA