PT97388A - METHOD FOR THE IDENTIFICATION OF A PROTEIN PARTNER OR A PROTEIN INHIBITOR COMPOUND - Google Patents

Description

* *

Further overexpression of c-myc and normal mouse fiberbladders, coupled with the expression of a ras-activated mouse product, transforms fibroblasts and gives them the ability to form tumors in live animals (Land, H '). (1983), Ruley, H "E", Nature 304: 602-602 (1983)). The function of c-myc protein remains unknown despite. Evidence suggests possible roles in regulating transcription, RNA processing and replication. Myc is currently one. the protein family (Derin et al., et al., 5, et al., R, A, et al., Battisti, Dell 34, 8, 1311-1326 (1987), L-myc 13131 -1326 (987) and -CH2 -CH3 -CH2 -CH2 -CH2 -CH2 -CH2 -CH2 -CH2CH2CH (1986), Kelly, K. et al., Annu Rev. (1986)) Recent studies suggest that ss. such as c-myc alter gene expression and

The cells are immortalized through regulation of activating activity of or promoter of the specific target genes and thereby repressing the staining of target genes, for example. Varmus, HE, Science 238 21337-1339 (1987)) 2 Kingston, R. et al., LS, 41, 3-5 (1985) Bishop, ?

Weinberg, R.A >, Science 230: 770-776 (eds.), ≪ / RTI >

Many regulatory proteins are heterodimeric, or. are composed of two different peptide chains that inter-act to generate the native protein. Among such regulatory proteins are DMA-binding proteins which are capable of binding to specific DMA sequences and thus regulating the transcription of DMA into RNA. The dimerization of such proteins is necessary for these proteins to have such binding specificity . A large number of Myc, Fos5 Jun5 Ebp, Fra-i, Jun-B, Spl, H2TF-1 / NF-kB, PRDI, TDF, GLI, Evi-I, transcriptional regulatory proteins were identified

ptor glucocorticoid 9 i receptor < of progesterone thyroid hormones C-erbA) and ZIF / 268j, 2 (0CT2> and PIT-I5 the yeast putative yeast cells QTF-1 (QTC1) SWISj AR8RII and LACv, conjugation factors MATal, MATa2 and MATalp jJ & gt Neuromorphs with the 25SBs protein, "Kruppel &quot;, and suppressors of &quot; hairy wing% &quot; paireti &quot;, &quot; f ushi tarazu &quot;, &quot; cut,! antennapeoxa- k &quot;, &quot; seren αip11y: pc-l; and a!

Hul trabi thoraKSl

Transcriptional regulatory proteins in eukaryotes and methods used to characterize such proteins have been recently reviewed (Johnson et al., Ann. Rev. Bioch. 58, 799-839 (1989)).

Members of the Jun / Fos and ATF / CREB mammalian transcriptional regulatory protein families only bind to DMA as dimers. The proteins in these families are proteins with a &quot; leucine zipper region &quot;). C-olectively. the combination of a basic region and a leucine zyper region is designated as the bZIP domain. i gc.

in general, it is predominant '

the basic region, which is involved in contact i

observed in the DMA dimerization, are possible while the siper region mediates many dimeric combinations, however, the particular nature of the matching siper specificities are permissive (Abel, T. et al., Nature 341: 24-25 (1989) &gt; .

Another large family of proteins contains the DNA / dimerization motif known as the basic helix-loop-helix (bHLH) motif (Jones, N, Cell 61: 9-11 (1990)). A bHLH protein generally contains a basic N-terminus followed by a helix-loop-helix structure, two unsympathetic short helices containing hydrophobic residues in all third or fourth positions. The sequence of the basic region does not necessarily show any indication of an amphipathic helix. The loop region intervening agent generally contains one or more propeller breaking residues.

The bHLH motif was first detected in two proteins, E12 and E47, which bind to a specific DMA sequence of the E &quot; box enhancer &quot; found in the immunoglobulin enhancers (Murrs C, et al., Cell 5, &quot; 77-7S3 (1989)). The E motifs are generally double-stranded variants of the consensus sequence 5R-CAGBTBSC-35. For example, the reason μΕΙ è BTCAABATBBC, the motif μΕ2 crx! is A8CA8CTG8C-, μΕ3 and 8TCATST88C »μΕ is T8CA88T8T (Murre, C. et al. Cell 56: 777-783 (1989)). Like many transcription factors, peptides containing the bHLH motif often dimer with the others either as a homodimer containing two identical peptides or as a heterodimer containing two different peptides. Examples of heterodimeric complexes of two bHLH proteins that bind to DMA are known to be more efficiently than the homodimeric complexes of both polypeptides in the heterodimer (Murre C. et al., Cell 5Ã © s777-783 (1989); Murre, C et al., Cell 58: 537-544 (1989)).

Myc is a bHLH protein and the c-myc bHLH domain is encoded at amino acids 255-410 of c-myc. The homology of sequences between the proteins expressed by the three human myc CN-myc genes 393-437 "human c-myc 346-4 and L-myc human 289-338) and other genes containing a bHLH domain were compared (Murra C. et al., Cell 56: 777-783 (1989)).

In the absence of partner proteins with which it can dimerize, a homodimer of two myc chains does not bind to the DMA of μΕ2. Thus, it is desirable to identify the partners that direct the binding of myc to DNA and compounds that inhibit myc activity by inhibiting such intersection of myc with the partner. Proteins such as myc that contain the bHLH motif also have the ability to hybridize with other proteins of the bHLH motif so it is highly probable that the myc partners also contain the bHLH motif. By inhibiting such intersections, inhibition and / or control of myc-induced cell growth can be achieved. Administration of inhibitors of the formation of myc partners provides therapeutic benefits in the treatment of diseases in which myc expression and activity is a factor in myc. promotion of cell growth or maintenance of cells in a transformed state '

However, to date, no myc inhibitors have been identified. The identification of such inhibitors has suffered from the absence of a simple, inexpensive and reproducible assay assay which can readily identify potential inhibitors and their active derivatives. Thus there is still a need for rapid and inexpensive detection assays that identify specific inhibitors of oncogene activity. -X.

SUMMARY OF THE INVENTION

Recognizing the potential importance of oncoprotein inhibitors in the therapeutic treatment of many forms of cancer and knowing of the lack of a simple assay system in which such inhibitors can be identified, the inventors have investigated the use of chimeric oncogene constructs in in vitro experiments in hosts prokaryotic as a model system in which to identify agents that alter the expression of oncogenes.

These efforts culminated in the development of a simple and inexpensive assay that can be used to identify partners of proteins in general and partners of transcriptional regulatory proteins in particular

The methods of the invention are especially useful for the preparation of partners which influence regulatory regulation and especially oncoprotein activity. The method of the invention further provides a method of stacking, isolating and characterizing inhibitors from such partners and especially inhibitors of rapid oncoprotein activity, including activity. The invention further provides a reproducible method for objectively classifying human pharmaceutical compounds as an inhibitor of oncogenes. The method of the invention provides for the protection of protein partners by means of their ability to destroy host-mediated repression in host hosts by counting the dimerization domain B of the partner. are in a framework characterized by lambda growth promoters expressing DNA binding proteins cl and the domain of the partners which are thus identified and can be easily cloned

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the sequence of the &quot; 5 dt-1 ny-one year DS sites used to synthesize the HLH / LZ agonists and c-myc HLH. s

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Various terms of recombinant DNA technology have been extensively used in the description which is used. In order to provide a clear economic understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided '

As used herein, two macromolecular elements are operably linked when the two macromolecular elements are physically arranged in such a way that the motifs which influence the activity of the first element cause the first element to have an effect on the next element

Fusion protein. Such a term is used herein, &quot; fusion protein &quot; is a hybrid protein that has been constructed to contain domains of two different proteins. The term &quot; protein gene, &quot; is intended to refer to a DMA sequence encoding a fusion protein, including where appropriate its transcriptional and translational regulatory elements.

Variant »A" variant &quot; of one. fusion protein is a protein which contains a sequence of amino acids which is substantially similar but not identical to the amino acid sequence of a fusion protein constructed from the natural domains, ie, domains containing the native aromatic amino acid sequence

By a substantially similar "amino acid sequence" is meant an amino acid sequence which is highly homologous but not identical to the amino acid sequence found in a fusion protein. Highly homologous amino acid sequences include sequences with 80% or of horology, and possibly less homology, especially if homology is concentrated in the domains of interest "

A "functional derivative" of a fusion protein is a protein that has the ability to dimerize with a fusion protein or ability to bind to the target DMA target, which is substantially similar to the ability of the fusion protein constructs By substantially similar is meant that the biological activities described above are qualitatively similar to the fusion proteins of the invention but differently different. For example, a functional derivative of a fusion protein may recognize the same target of the fusion protein or form heterodimers with the same partner protein but not a. same affinity, a peptide is

As used herein, I have been referred to as a &quot; functional derivative &quot; when it has the amino acid skeleton c 1 - ¾ D Γ O t i i. Fraction plus additional chemical fractions that are not generated. 1 part of the fusion protein. Such fractions may improve the solubility of the derivatives, absorption, biological half-life, etc. Fractions may alternatively decrease the tonicity of the derivative or eliminate or attenuate any undesirable side effects of the derivative , etc. Fractions capable of mediating such strokes are described in

The processes for

Rsminqton3s Pharmaceutical Sciences coupling such fractions to a molecule are well known '

A functional derivative of a fusion protein may or may not contain post-translational modifications such as covalently bound sugars, depending on the need for such modifications for carrying out the methods of the invention. The term &quot; functional derivative &quot; is intended to encompass &quot; frag &quot; variants &quot;, &quot; analogs &quot; or &quot; d &gt;: &quot; functional

H molecules of a molecule,

Promoter »A &quot; promoter &quot; is a DMA sequence located at the beginning of the art in Fig. tremo 5? of the transcribed sequence to which RN-polymerase is bound or initiates transcription. The promoter may contain multiple regulatory elements that interact in the modulation of transcription of the operably linked gene,

Expression. Expression is the process by which the information encoded within a penile is transcribed and translated into a protein.

A nucleic acid molecule, such as a DMA or. gene is referred to as &quot; capable of expressing a polypeptide if the molecule has the sequences encoding the polypeptide and the expression control sequences which, within the environment of the appropriate host, provide the ability to transcribe, process and translate the genetic information contained in DNA in a protein product and if such expression control sequences are operably linked to the nucleotide sequence encoding the polypeptide.

Cloning vehicle. A &quot; clonagsm vehicle &quot; is any molecular entity that is capable of providing a nucleic acid sequence to a host cell for cloning purposes. Examples of cloning vehicles include plasmid or phage genomes. Especially preferred is a peptide that can replicate autonomously in the host cell. Alternatively, a nucleic acid molecule that can be inserted into the host cell's chromosomal DNA is especially useful.

The screening vehicles are often characterized by one or a small number of endonuclease recognition sites in which such DNA sequences can be cut in a determined manner without loss of one. essential biological function of the carrier and in which the DNA can be cut in order to perform its replication and cloning. A cloning vehicle still contains a label suitable for use in identifying cells transformed with the cloning vehicle. The marks are for example of strength 4 &gt;

tetracycline or. ampicillin "The word" vector &quot; is by vessels used for &quot; cloning vehicle &quot;

Expression vehicle An "expression vehicle" is a vehicle or vehicle-like cloning vector but is especially intended to provide sequences capable of expressing the cloned gene upon transformation into a host.

In an expression vehicle, the gene to be cloned is operably linked to certain control sequences such as promoter sequences. The expression control sequences will vary depending on whether the vector is intended for. expressing the operably linked gene in a prokaryotic or eukaryotic host and may further contain host-specific transcriptional elements such as operator elements. upstream stowaway regions, enhancing elements. terminology, specific tissue elements and / or translation initiation and termination sites.

Host. By "host &quot; is meant to be the organism which is the recipient of a cloning or expression vehicle.

Answer. The term &quot; response &quot; is intended to refer to a change in any parameter that can be used to measure and describe the effect of a compound on the activity of a protein. The response may be revealed 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). The detection of the response can be performed by any suitable means. - 13

an

Compound, the term &quot; compound &quot; is intended to refer to a chemical entity, liquid or gaseous solid phase, the term shall be read as including synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides or lipids and also small entities such as neurotransmitters, ligands, hormones or sclonal compounds "any

Bioactive Compound. It is intended to refer to compound which induces a measurable response in the assays of the invention.

Heterodimeric proteins are proteins which contain two different polypeptide chains which associate with one another due to hydrogen bonds, ionic interactions, hydrophobic interactions, disulfide bridges and the like, but which are not linked to one another by a bond The two polypeptide chains and one heterodimeric protein are hereinafter referred to as "partners" of one another. The heterodimeric transcriptional regulatory proteins have a discrete domain that is required for dimerization to occur. A second discrete domain is required for e. the DMA.

These observations have been made by the inventors to enable the identification of a partner of a heterodimeric protein. Such protein partners may be identified by constructing a chimeric peptide which retains its own DNA binding domain capable of conferring phenotypic expression in a host cell (preferably a bacterial host cell, such as E coli). By control of such phenotype, the formation and activity of a heterodimer can be controlled. The most preferred chimeric peptides contain a dimerization domain of a putative heterodimeric protein partner and the DNA binding domain of a bacteriophage repressor, such as the bacteriophage lambda repressor. The bacteriophage lambda possesses the ability of a bacterial host and (a) replicate and grow from infectious lymphocytes (1). (b) integrating into the host's sound in a non-infectious lysogenic lysogenic form. "When integrated as part of the chromosome, inert phage DNA is referred to as a" prophage. "Due to having prophylactic lysogenic bacteria are immune to the super- in-fection by other phage particles of the same type. The bacteriofa-go lambda is a member of a lambdae bacteriophage family (such as 434, 21 and PSD5

These are all valid values for the nodes of the present invention.

When lambda is grown in a bacterial form in infected bacteria, the bacteria are finally lysed. This lysis results in a clarification of the turbides or opaque appearance associated with the presence of a bacterial culture. The growth and replicative activity of a phage that smooths bacterial cells in their lytic cycle can be tested by examining their ability to lyse bacteria, thereby forming plaques (clear areas in which the bacteria have been lysed) in a bacterial culture grown in water; (The Baeteriophaqe Lambda &quot;, Hershev, AD

Harbor Laboratory, New York (197); &quot; Lambda II &quot;, Hendrix, R.W. et al., eds., Cold Spring Harbor Laboratory, New York, (1983), (Maniatis, T. et al., Molecular Cioning (A Laboratory Manual), 21st edition, Cold Spring Harbor Laboratory, New York (1988) ). The lambda cl gene encodes a repressor protein, cl, which is required to maintain lysogeny. In its native state, cl, which is a homodimer (a dimer of two identical strands) and the close linking of the repensor to lambda DNA requires the state of

The leader binds to two operators that control the transcription of adjacent genes whose products are required for the expression of the genes responsible for lytic development. By binding to these operators, the repressor prevents transcription of all lambda genes except itself. The complete amino acid and nucleotide sequences of the cl gene (&quot; Lambda 11% Hendrix, R.W. et al., Eds., Cold Spring Harbor Abortion, New York, (1983)) are known.

In the constructs of the invention, a fusion protein is created which contains the DNA-binding domain of cl (the N-terminal 112 amino acids of lambda cl) and the dimerization domain of a putative heterodimeric protein partner. In a highly preferred embodiment the dimerization domain, the faHLH region, of c-myc (c-myc amino acids 255-41®) is used. If desired, other dimerization domains may be used.

Dimerization domains can be predicted by analyzing the three-dimensional structure of a protein using the amino acid sequence and computer-known techniques of analysis in the field, for example the Chou-Fasman algorithm. Such techniques enable the identification of helical domains and other areas of interest, for example hydrophobic or hydrophilic domains in the peptide structure. The HLH dimerization domain in a protein can be defined by comparing an amino acid sequence with that of ten known HLH dimerization domains (amino acids 336-393 at E12, 336-393 at E47, 554-61.3 at &quot; dauqhterless &quot;, 357-4Θ7 in twist, 393-437 at N-myc and UI-Sn-OOD. human Î ± -cyl, 346-4 β1 in human c-myc, Î »8-164 in MyoD, and locus genes &quot; achaete-scute &quot;, 1.167 of Ϊ4, -95 of T5 (Murre, C , et al., Gel 56, 777-783 (1989).) The domain of HLH deletion contains

two unsympathetic propellers separated by a intervening loop. The first helix sings 12 amino acids and the second helix contains 13 amino acids. Do certain amino acids appear to be conserved in the HLH format? especially the hydrophobic residues which are present in the helices. Comparisons of the two sequences designated above show that there are five virtually identical hydrophilic residues within the 55th end of the homologous region and a series of essentially hydrophobic residues in two short segments that are separated from one another by a which sequence generally contains proline or glycine grouped '

A leucine siper domain is generally about 35 amino acids in length and contains an arrangement of seven repeated leucine residues and a very high density of amino acids with opposing charges (acidic and basic) suitably juxtaposed to anneal intra-helical ions.

Leucines extending from the helix of a polypeptide are believed to interlace with those of the analogous helix of a second peptide (the partner) and form the lock designated leucine sipere. The fusion protein of the invention was constructed as molecule of recombinant DNA that is capable of expressing that of fusion in a bacterial host. The transformation of ros ". coli with a recombinant lambda construct expressing such a fusion protein results in immunity of the bacterial yeast due to dimerization as a host host protein of the fusion protein and especially the clone domain in a homodimer form which is binding to the appropriate lambda DNA operators' after transformation and expression of the fusion protein is therefore avoided by lithic lambda growth or subsequent lambda infection.

Preferably, a low copy number plasmid is used together with a weak promoter (for example the 17β-

and the lactose operator) for transcription of the fusion gene so as not to fill the bacterial host with a large excess of protein of the invention. If a large excess of fusion protein is synthesized, the ratio of the homodimer moles of the fusion protein heterodimer to the fusion protein (as described below) may be so high that the homodimer effectively maintains phage growth repression and avoids detection of the heterodimer '

Any one of the foregoing or a protein having a thermodymer binding domain with the inhibitory fusion protein will prevent the formation of fusion protein homodimers. Such proteins may therefore be identified by their ability to interfere with mediated phage growth repression by the fusion protein.

In one embodiment, the bacterial host expressing the fusion protein described above was transformed with a lambda expression library expressing cloned eukaryotic genes. For example lambda gtll packaging systems for the generation of expression libraries from mRNA that are useful in the methods of the invention are known and can be obtained commercially (for example from Promega Corporation Hadison, Wisconsin). Further, the libraries can be generated with the use of poly (A) -containing cytoplasmic mRNA derived from any source animal. To induce expression of the cloned proteins contained therein may be added 1βmM IPTS (isopropylthiogalactoside) "

Since all host cells produce the lambda repressor of the fusion protein, the lytic propagation of the expressing library. in infected lambda will be repressed. Any infectious member of the lambda expression library that does not express a protein having a dimerization domain that is capable of binding to the dimerization domain of the fusion protein will not confer mediated phage repression, by the fusion protein homodimer. If any member of the lambda expression library expresses a protein capable of interfering with the homodimerization of the fusion protein (such as through the formation of a heteradimer with, for example, the faHLH domain of a fusion peptide, &gt; the repressor function will be lost and lithic growth of lambda will occur. Thus, the identification of heterodimer-forming partners can be readily accomplished by screening the plaque-forming phages. The method of the invention is generally applicable for the identification of The advantage of the method of the invention for identifying partners of the protein is that the partner that is identified is one that has a higher affinity for the fusion protein than the partner that is identified as having a higher affinity for the fusion protein than the affinity of the homodimer and therefore is a protein that has a high probability of being an important regulator tarsteda act In addition, the partner who is identified already is. in a cloned expression form which can be used to obtain larger amounts of the protein for its isolation and still -characterization by known molecular protein and molecular biology techniques. Identification of protein partners in expression libraries allows the identification of compounds that inhibit the ability of such partners to form heterodimers through

! i

the ability of the compound to inhibit plaque formation.

For example, once a partner is identified by the appearance of clear or cloudy plaques as described above, the identification of compounds that prevent or otherwise interfere with the formation of heterodimers of the proteinaceous partners can be identified by screening the ability of such compounds to inhibit the formation of plaques. A compound which exhibits inhibition of plaque formation in this example will be a compound which (a) avoids the association of the fusion protein with the partner peptide which is also being expressed in the host and (b) does not prevent the formation of homodimers, i.e. the dimerization of the cl domains of the fusion protein and (c) does not inhibit cell growth under the plate assay conditions.

To ensure that the partner selected by the above process is specific to the dimerization regulation of the fusion protein and does not inhibit transcription in general, lambda expressing such a partner can be used to infect a host bacterial strain expressing fusion proteins constructed with dimerization domain of other proteins and the ability of the partner to induce lytic growth in such examined hosts. For example, where it is of interest to identify a compound which inhibits the dimerization of bHLH but not the dimerization of bZIP, a fusion protein containing cl as above and the appropriate dimer-forming domain is constructed from a bZIP protein.

Partners and compounds that inhibit the association of such partners with any type of transcription regulatory protein that is associated with the bacterial methods in cells may be 1. Identified

Using the above techniques, the inventors also include: specific partner proteins that associate with c-mvc in vivo and which help the binding of c-asyc to DNA. These partners strengthen the degree and duration of binding of c-myc to its binding partner, the DMA sequence of μΕ2. One of these partner proteins has a molecular weight of 46,000 daltons and binds to the mE2 sequence in the absence of c- myc. The identification of an E * NA-binding domain in a protein can be accomplished by a variety of techniques known and previously used to identify such domains (see Johnson, EP et al., Ann. Rev. Biochem., 58, 799-839 1989) for a review of such areas).

H

DMA-binding proteins and DMA-binding domains in such proteins are identified and purified by their DNA affinity. For example, binding to DMA can be revealed in filter hybridization experiments wherein the protein (generally labeled to facilitate detention) is bound to the DMA immobilized on a filter or, vice versa, wherein the DMA binding site ( generally labeled) binds to a filter on which the immobilized protein is present. The sequence and affinity specificity of such binding is revealed with DNA profiling assays and gel retardation assays. The purification of such proteins can be performed using techniques of affinity chromatography for specific DNA sequences, i.e. column chromatography with a resin, derivatized with the DNA to which the domains bind. The proteolytic degradation of the. DMA may be used to reveal the domain that retains the ability to bind to DMA, -. The dimerization domain in a protein is recognized by its homology with known dimerization domains and can be predicted from the amino acid sequence of the protein using computer aided structural analysis as described above. The binding domain and the dimerization domain are arranged in the fusion protein so as not to destroy the function of the two domains ie the DMA binding domain, when suitably diroerized it could recognize the DMA element to which it binds naturally and the dimerization domain retain the ability to dimerize with their partners. Those familiar with the subject carrying out the control assays will be able to establish that the fusion protein functions properly.

The fusion protein constructs of the invention may be kept off the chromosome as plasmids or inserted into a host cell.

The methods of the invention may be used to screen for compounds in their pure form in a variety of concentrations and also in their impure form. The methods of the invention may also be used to identify the presence of such inhibitors in crude and to follow the purification of the inhibitors from them. The methods of the invention are also useful in evaluating the stability of the inhibitors identified above to evaluate the effectiveness of various preparations.

Analogues of such compounds may also be used which are more permeable through the membranes of bacterial host cells. For example the dibutyryl derivatives often have a higher permeability.

The methods may also be used to identify partners and compounds that interfere with the partner of membrane-laden proteins and / or with cytoplasmic proteins if such proteins are capable of associating with the domain of the fusion protein rimerization. The DMA sequence of the fusion protein and / or the target gene may be chemically constructed or constructed by known recombinant means. Methods for the chemical synthesis of DMA are well known (Scheme 1, Synthesis, A Practical Approach &quot;, SJ1, ed., 3RL Press, Washington, D.C., 1994, "Synthesis and Applications of DMA and RNA", S. Narang, ed. Academic Press, San Diego, CA, 1987). coding sequence for a particular amino acid (Watson, JD, Em 5 &quot; Molecular Biology of the 8th ed., 3rd ed., W. Benjamin, Inc. Menlo Park, CA, 1977, pp. 356-357),

Expression of the recombinant fusion constructs of the invention requires transcription and translation signals recognizable by the host. A cloned fusion protein, obtained by the methods described above and in double stranded form, may be operably linked to sequences which control the transcriptional expression in an expression vector and introduced, for example by transformation, into a host cell to produce the recombinant fusion proteins or their functional derivatives, for use in the methods of the invention, V:

Regulatory signals may be selected from the initiation of transcription which enable repression or activation of the expression ε -pressure of the fusion construct can be modulated if desired. Regulatory signals are sensitive to temperature so that by varying the temperature the expression can be repressed either. started or. which are chemically upregulated, for example by a metabolite or a substrate added to the growth medium. Alternatively, the fusion construct may be constitutively expressed in the host cell. It is necessary to express the proteins in a host wherein the ability of the protein to maintain its biological function is not prevented. Expression of proteins in bacterial hosts is preferably achieved using prokaryotic regulatory signals.

The splicing vectors typically contain such discrete DMA elements as for example. a &gt; an origin of rheology that prompts autonomous replication of the vector or elements that promote the insertion of the vector on the host chromosome in a stable manner and &lt; h) specific genes that are capable of providing phenotypic selection in transformed cells. Many suitable expression vector systems useful in the methods of the invention can be commercially purchased. Once the DMA vector or sequence containing such constructs is generated, (s) are introduced into a suitable host cell by any of a variety of suitable methods, for example by transformation. After introduction of the vector, the recipient cells are cultured in a selective medium, which selects the growth of vector-containing cells. Cloned sequence expression (s) results in the production of. fusion protein

If the DNA sequence encoding the fusion protein and an operably linked promoter were introduced into a recipient host cell as a non-replicative DNA or RNA molecule, which may be a linear molecule or preferably a molecule covalently closed circular cell that is incapable of autonomous replication, the expression of the fusion protein can occur through transient expression of the introduced sequence.

Genetically stable transformants can be constructed with vector systems or transformation systems by which the DNA of the fusion protein is integrated into the host chromosome. Such integration may occur again within the cell or be assisted by transfection with a vector that functions in a non- inserts itself into the host chromosome, for example with bacteriophages, transposons or other DNA elements that promote the integration of DNA sequences in the chromosome '

Cells that have been transformed with the DNA vectors of the fusion protein of the invention are also selected by the introduction of one or more tags allowing the selection of host cells containing the vector, for example, the tag can provide resistance to biocides, e.g., resistance to antibiotics or the like. The transformed host cell may be fermented according to known means to achieve optimum cell growth and also to achieve optimum expression of the damaged fragments of the fusion protein sequence. Optimal expression of the fusion protein is the expression that provides no

more than the same moles of fusion protein subunit as the partner protein moieties to be expressed. However, variations in this amount are acceptable so long as they do not interfere with the partner's ability, when in heterodimer form, to overcome the repressor activity

One may wish to further characterize the c-myc partner proteins which are identified by the methods of the invention in a eukaryotic expression system. Such characterization can be performed according to the methods described in co-inventor U.S. patent application entitled &quot; C-MYC SCREEMIMS ASSAYS &quot;, Serial MS &quot; 7/21253 &quot; delivered on the same day of this application, 19 April, 199Θ and included here as a reference '

The examples are not intended to limit the invention in any way. The examples are not intended to limit the invention in any way.

EXAMPLES

Example i

Construction of c-myc fusion proteins following operator

The pre-operative / operative regions all these constructs themselves consist of the {5-lactamase and lac promoter and Sbine Delgarno sequence (S "D =). The sequence is 26 GGA TCC TCT AAA TAC ATT CAA ATA AGT ATC CGC TCA TGA Bam Hl

GAC AAT AAC GGT AAC CAG AAT TGT GAG CGC TCA CAA TTT TG -10 BstEII ATC GAT AGG AAA CTC GAG ATG ...

Ciai S.D. Xhol +1 cl cl in each of these constructs consists of the first 336 bp (112 amino acids) corresponding to the N-terminal polypeptide generated by recA cleavage. Polymerase chain reaction (PCR) primers having Xhol and XbaI sites at ends 51 and 3 ', respectively, were synthesized. The promoter / operator and cl DNAs were cloned into BamHI and XbaI digested pUC18. The sequence around the Xbal site is as follows: 5 'CAG GCA GGG TCT AGA ...

Gin Ala Gli Xbal seq. encoder of cl.

The HLH / LZ (helix-helix / leucine zipper) and c-myc HLH fragments were generated by PCR using a c-myc cDNA as template. HLH / LZ is a 258 bp fragment synthesized with primers starting at sites # 2 and # 9 (Figure 1) with 5 'and 3' XbaI and Sall sites, respectively. HLH is a 165 bp fragment with Xbal and PstI sites at the 511 and 3 'ends, respectively; the boundaries of HLH are at the sites marked # 2 and # 10. The primer used in site # 10 included a stop codon 27

(I.e.

as used in site # 9. The insertion of c-myc sequences into pU33cI was at the restriction sites corresponding to those indicated in the PCR primers.

Each of the constructs in pUC18 was subcloned into pYC177 as follows. pYC177 was digested with BglII (filled with Klenow) and BamHI and each of the pUC18-based constructs was digested with HindIII (filled with Klenow) and BamHI. The resulting pYC constructs provide resistance to canamicillin.

Example 2

Screening of a cDNA expression library for protein partners capable of interacting with the c-myc helix-loop-helix domain

To detect proteins capable of interacting with human c-myc helix-loop-helix (HLH), DNA encoding amino acids 355-409 of c-Myc, which contain the base region and HLH of the c-myc peptide was linked as above in the same reading frame to a DNA fragment encoding the N-terminal 112 amino acids of the lambda repressor protein (cl). Expression of this chimeric protein was placed under the control of the weak B-lactamase promoter and the lactose operator. This expression unit was subcloned into pACYC177, a low copy plasmid (10-15 copies / cell) with a canami-kin gene. Cells transformed with this construct were found to be resistant to phage lambda infection by a &quot; dots &quot; of plates. The above construction, pYC192cIHLH, became the 28

cells resistant to phage infection at &gt; 1β pfu, while cells expressing the N-terminal region of cl alone were. 2 infected mice at &lt; 10 pfu. E. coli strain Y1090 transformed with pYC192clHLH was used to screen a human amygdala / B cell library in lambda gtll, constructed according to the manufacturer's recommendations (Promega). 5 x 10 pfu were tested in duplicate in the above transformed strain, as well as a lysogenic Y1090 :: lambda strain. 800 plates were formed on each of the test plates. In the control plates (treated with lysogenic strain Y1090 :: lambda) no plaques appeared. From the test plates, a total of 160 plates were plaque-purified once and an isolated plaque was picked and resuspended in suspension medium (SM). The 160 purified plates were grouped according to the size of plates: small, medium, large 70. (Four from the &quot; small group &quot; did not form plaques in the plaque purification step.) Each of these was then tested by a &quot; dots &quot; of plaques in a E. coli strain JM109, transformed with the plasmid pJH370, which expresses a chimeric protein consisting of the N-terminus of the fused domain with the leucine zipper domain of GCN4. Each of the phages was also tested again in the above strain used in the initial screening, as well as in the lambda lysogenic strain and in the Y1090 parent strain.

Phages that formed plaques in the Y1090 parent strain and the cI-Myc-expressing strain, but not in the lysogenic strain or in the CI-GCN4 expressing strain were defined as &quot; positives &quot;. &Quot; positives &quot; were tested twice in these four strains. In the &quot; dots &quot; in plaques 5-20 μΐ of the &quot; positive &quot; typically gave 50-100 plaques. If a very small plaque was seen on CI-GCN4, then the phage was defined as &quot; negative &quot;. By this assay, a total of 10 d &quot; positives &quot; of the 156 phages tested. Of these, 3 were from group 29

&quot; small &quot; and 7 were from the &quot; medium &quot; group. These positives represent proteins that associate c-myc sufficiently to inhibit the formation of homodimers.

Example 3

Identification of a compound and avoids heterodimerization of the c-mcc partner. E. coli host cells expressing a protein identified as a c-myc partner protein &quot; medium &quot; as described in Example 2 were further exposed to compounds W, X / Y and Z, and the effect of such compounds on the ability of phage lambda to grow in a lytic manner was determined by looking at the ability of the compound to inhibit plaque formation in plaques solid agar.

Typical results of such an experiment are presented in Table 1. * Jt.

Table ls Identification of partners of the c-myc protein Compound partner Formation of gtu plates none - no 4 yes W - no 4 yes X - yes + yes Y ~ no 4 · no

The results from the above table indicate that in the absence of the partner protein, compound W had no effect on the ability of the lambda cl protein to form dimers and suppress lytic growth. Still, the 16-valent had no effect on the ability of the partner to form heterodimers with the fusion protein. Therefore, the compound U will not be a compound of interest. Compound X interfered with the formation of homodimers and not with the combination of heterodimers. Therefore compound X is not an inhibitor of c-mye function. Compound Y is an inhibitor of the formation of heterodimers. Compound Y is not an integer with the formation of homodimers but does not interfere with the formation of heterodimers. Therefore, X Λ>

compound Υ is a compound which can inhibit the action of c-myc in vivo, includes-invention, invention equiva-scope

All references cited herein are fully as references. Having now been fully described, it will be understood by those skilled in the art that the pruning can be carried out within a wide range of conditions, lenses, parameters and the like, without affecting the spirit or the invention or any accomplishment thereof.

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Claims (1)

The method for the identification and classification of a protein partner is characterized by comprising: (a) transforming a bacterial host cell capable of expressing the fusion protein by inserting a protein of item a fusion protein domain forms a homodimer that represses the growth of a lytic bacteriophage (b) transforming said host cell from part &lt; a) to a genetic construct capable of expressing said protein partner; &lt; c &gt; the culture of said host cell of part &lt; b &gt; under conditions allowing expression of said fusion protein and said protein partner; (d) determining the ability of said lytic bacteriophage to induce lysis of said host cell; and &lt; e) the classification of said protein partner based on the presence or absence of said lysine. A method of identifying and classifying a compound as a dimerization inhibitor of a protein partner, characterized in that it comprises s (a) transforming a bacterial host cell with a genetic construct capable of expressing a V ' .λ binding to DMA and a dimerization domain and wherein said fusion protein forms a homodimer that represses the growth of a lithium bacteriophage? (a) a. transforming said host cell of part (a) with a genetic construct capable of expressing said protein partner; (c) culturing said host cell (b) in the presence of said compound and under conditions that the expression of said fusion protein is said protein; (d) determining the ability of said compound to prevent growth induced by the protein partner of said bacteriophage and lysing said host cell and Ce &gt; the classification of said compound as an inhibitor of the formation of the protein partner based on the presence or presence of said lysis. The method of any one of claims 1 or 2, wherein said binding domain is DMA is the DNA binding domain of the repressor protein of the bacteriophage bacterium. The method of claim 1 wherein the DMA binding domain of said repressor protein cI is the W-terminal amino acids of said repressor protein. 34. 2. A method according to any of claims 1 to 2, characterized in that the method of claim 1, wherein said domain is a domain. with any one of the aforementioned dimerization domain 7 '. Method according to claim 6, characterized by wherein said heterocyclic heterocyclic group is selected from the group consisting of: The method according to any one of claims 1 to 5, wherein said BHLH domain is amino acids 10 '. A method according to any one of claims 1 or 2, characterized in that said claimed- The method of any one of claims 1 to 2, wherein said domain of claim 1 is a zinc finger domain. Official Agent of Industrial Property RUA V1CT0R CORDON, 10-A 3. "1200 LISBOA