CA2525255A1 - Minimalist bzip proteins and uses thereof - Google Patents

Abstract

The present invention provides minimalist bZIP proteins having a basic region derived from bHLH proteins fused to a leucine zipper dimerization domain derived from bZIP proteins and methods and uses thereof in the treatment of cancer. The present invention also provides pharmaceutical compositions for treating cancer.

Description

TITLE: MINIMALIST BZIP PROTEINS AND USES THEREOF.
FIELD OF THE INVENTION

The invention relates to minimalist bZIP proteins and uses thereof.
Specifically, the minimalist bZIP proteins are made from the fusion of the basic region of a bHLH protein to the leucine zipper dimerization domain of a bZIP protein. These proteins are particularly useful in treating cancer.

BACKGROUND OF THE INVENTION

Nature's use of the protein a-helix for specific DNA recognition is ubiquitous and maximally utilized by the basic region/leucine zipper motif (bZIP), which comprises a pair of short a-helices that recognize the DNA major groove with sequence-specificity and high affinity (Struhl, K., Trends Biochem. Sci. 1989, 14, 137-140; Landschulz, W. H. et al., Science 1988, 240, 1759-1764).
Crystal structures of the bZIP domain of GCN4 bound to two different DNA
sites (Konig, P. and Richmond, T. J., J. Mol. Biol. 1993, 233, 139-154;
Ellenberger, T. E. et al., Cell 1992, 71, 1223-1237; Keller, W. et al., J.
Mol.
Biol. 1995, 254, 657-667) and the Jun-Fos heterodimer bZIP-DNA crystal (Glover, J. N. M. and Harrison, S. C., Nature 1995, 373, 257-261) show that a continuous a-helix of -60 amino acids provides the basic-region interface for binding to specific DNA sites, as well as the leucine zipper coiled-coil dimerization structure. Remarkably, these crystal structures also demonstrate astonishing conservation of protein backbone structure across species between the two yeast GCN4 and avian Jun-Fos structures.

Myc, Max, and Mad Proteins The basic-region/helix-loop-helix (bHLH) motif, including the subvariant basic-region/helix-loop-helix/leucine-zipper (bHLHZ) motif, is very similar to the bZIP
in that a dimer of a-helices binds specific sites in the DNA major groove;
protein dimerization is effected by the helix-loop-helix, a tetramer of a-helices in the bHLH, or by the helix-loop-helix/leucine-zipper in the bHLHZ, in which dimerization is mediated by both the tetrameric HLH and adjacent leucine zipper (compare structures in Figure 1) (Murre, C. Cell, 1989, 56, 777-783).
The bHLH comprises bHLH proteins as well as subfamily variants: the bHLHZ (such as Max and USF), and the bHLH/PAS (such as AhR and Arnt), where the PAS domain assists in efficient protein dimerization. Unlike the leucine zipper, the PAS structure is unknown. The PAS has been found in the Per, Arnt, and Sim proteins-hence, "PAS"-as well as AhR and HIF-1a (Gradin, K., et al., Mol. Cell. Biol. 1996, 16, 5221-5231). The PAS domain comprises 200-300 amino acids and contains characteristic repeats termed the "A" and "B" domains.

Like bZIP proteins, the bHLH protein family also regulates transcription. In particular, the Myc, Max, and Mad transcription factor network comprises widely expressed bHLHZ proteins critical for control of normal cell proliferation and differentiation (Amati, B. and Land, H., Curr. Opin. Gene. Dev. 1994, 4, 102-108; Orian, A. et al., Genes Dev., 2003, 17, 1101-1114). Myc is proto-oncogenic; deregulated overexpression of myc genes leads to malignant transformation, and myc genes are suspected of being among the most frequently affected in human tumors and disease (Nesbit, C. D. et al., Oncogene 1999, 18, 3004-3016) including Burkitt's lymphoma (Taub, R. et al., Proc. Natl. Acad. Sci. USA 1982, 79, 7837-7841; Dalla-Favera, R. et al.. M., Proc. Natl. Acad. Sci. USA 1982, 79, 7824-7827), neuroblastomas (Schwab, M. et al., Nature 1984, 308, 288-291), and small cell lung cancers (Nau, M. M.
et al., Nature 1985, 318, 69-73).

In contrast, Max is a stable, constitutively expressed dimerization partner that heterodimerizes with Myc, Mad, and Mxi, thereby controlling their DNA-binding and gene-regulatory activities (Amati, B. and Land, H., Curr. Opin.
Gene. Dev. 1994, 4, 102-108; Orian, A. et al., Genes Dev., 2003, 17, 1101-1114). Myc-Max is a transcriptional activator that binds the Enhancer box (E-box) sequence 5'-CACGTG (Blackwood, E. M. et al., Science 1991, 251, 1211-1217; Blackwell, T. K. et al., Mol. Cell. Biol. 1993, 13, 5216-5224). Myc does not homodimerize in vivo or at physiological concentrations, so its activity is mediated by heterodimerization with Max. In contrast Max can homodimerize, although it preferentially heterodimerizes; Max homodimers can bind the E-box, albeit with lower affinities than that of the heterodimers (Blackwood, E. M. et al., Science 1991, 251, 1211-1217). Several promoters contain the E-box sequence 5'-CACGTG, including that for p53 tumor suppressor (Reisman, D. et al., Cell Growth Differ. 1993, 4, 57-65). Mad-Max (Amati, B. et al., Cell 1993, 72, 233-245) and the related Mxi-Max (Zervos, A.
S. et al., Cell 1993, 72, 223-232) are transcriptional repressors that antagonize Myc-Max by competing for the same E-box sequence.

The Max network is highly conserved in vertebrates and mammals and ubiquitous; in Drosophila, for instance, a conservative estimate is that Max network proteins interact with approximately 2000 genes (Orian, A. et al., Genes Dev., 2003, 17, 1101-1114). The transactivation domain mediating the gene-regulatory activities of the Myc-Max heterodimer lies in the amino-terminal region of Myc; Max's role is to allow Myc to bind DNA, thereby mediating its cellular activities (Amati, B. and Land, H., Curr. Opin. Gene.
Dev. 1994, 4, 102-108). Therefore, mutant proteins that interfere with Myc-Max recognition of the E-box site may also interfere with Myc's disease-promoting activities.

AhR and Arnt Proteins Not only interesting from a protein-design perspective, the AhR-Arnt system is notable for its possible role in disease pathways. The AhR, also known as the dioxin receptor, mediates signal transduction (Fisher, J. M., et al., Mol.
Carcinogen. 1989, 1, 216-221) by dioxins and related polycyclic aromatic hydrocarbons, including benzo[a]yrenes found in cigarette smoke and smog, heterocyclic amines in cooked meat, and polychlorinated biphenyls (PCBs).
In analogy to the glucocorticoid receptor, the latent AhR is found associated with heat-shock protein hsp90 in the cytosol (Cadepond, F. et al., J. Biol.
Chem. 1991, 266, 5834-5841.). Ligand binding induces nuclear translocation of the AhR (Pollenz, R. S. et al., Mol. Pharmacol. 1995, 45, 428-438), release of hsp90, and dimerization with the nuclear protein Arnt (Reyes, H. et al., Science 1992, 256, 1193-1195); this activated complex (Whitelaw, M. et al., Mol. Cell. Biol. 1993, 13, 2504-2514; Cuthill, S., et al., Mol. Cell. Biol.
1991, 11, 401-411) then binds specific xenobiotic response elements (XRE sites) and activates gene transcription (Wu, L. and Whitlock, J. P. Nucl. Acid. Res.
1993, 21, 119-125; Fujisawa-Sehara, A. et al., Nucl. Acid. Res. 1987, 15, 4179-4191). The endogenous ligand, if any, for the dioxin receptor has yet to be discovered. During evolution, plant flavones and later, certain combustion products like dioxin, appear to have appropriated the AhR for stimulating their own metabolism.
AhR and Arnt are bHLH/PAS proteins; they differ from most other bHLH
transcription factors in that AhR-Arnt dimerization occurs only in the presence of ligand. The PAS domain is remote from the basic region, and importantly, it does not affect DNA binding, as it is purely necessary for dimerization and ligand binding; Poellinger and coworkers found that the minimal bHLH
domains of AhR and Arnt are solely capable of recognition of XRE sites and dimerization (Pongratz, I., et al., Mol. Cell. Biol. 1998, 18, 4079-4088).
Previous work has shown that within the bZIP family, basic regions and leucine zippers from different proteins can be exchanged with no resulting change in a-helical structure or DNA-binding function (Agre, P. et al., Science 1989, 246, 922-926; Lajmi, A. R. et al., J. Am. Chem. Soc. 2000, 122, 5638-5639; Sellers, J. W. et al., Nature 1989, 341, 74-76). Likewise, the bHLH/bHLHZ is well conserved structurally and essentially identical among bHLH/bHLHZ family members (Nair, S. K. and Burley, S. K., Cel12003, 112, 193-205). Protein-DNA crystal structures for bHLH proteins MyoD (Ma, P. C
et al., Cell 1994, 77, 451-459) and E47 (Ellenberger, T. et al., Genes Dev.
1994, 8, 970-980), and bHLHZ proteins Max (Ferre-D'Amare, A. R. et al., Nature 1993, 363, 38-45; Brownlie, P. et al., Structure 1997, 5, 509-520) and USF (Ferre-D'Amare, A. R. et al., EMBO J. 1994, 13, 180-189) show closely related structures and DNA-binding functions. Exchange of basic regions and dimerization elements in the bHLHZ family also yields native-like proteins:
Prochownik and coworkers showed that the Max basic region could be fused to the USF HLHZ domain to generate hybrids that could homodimerize and bind the E-box (Yin, X. et al., Oncogene 1998, 16, 2629-2637).

The crystal structures of bZIP and bHLH demonstrate that although they are distinct protein structural families, they share the most similarity in comparison to other families of DNA-binding proteins: in particular, the a-helix DNA
recognition element is highly conserved in the two families (Konig, P. and Richmond, T. J., J. Mol. Biol. 1993, 233, 139-154; Ellenberger, T. E. et al., Cell 1992, 71, 1223-1237; Keller, W. et al., J. Mol. Biol. 1995, 254, 657-667;
Glover, J. N. M. and Harrison, S. C., Nature 1995, 373, 257-26; Ma, P. C et al., Cell 1994, 77, 451-459; Ellenberger, T. et al., Genes Dev. 1994, 8, 970-980; Ferre-D'Amare, A. R. et al., Nature 1993, 363, 38-45; Brownlie, P. et al., Structure 1997, 5, 509-520; Ferre-D'Amare, A. R. et al., EMBO J. 1994, 13, 180-189). In contrast, there are differences in the hinge angles which govern positioning of the basic regions in the major grooves between bZIP and bHLH.
Additionally, the dimerization element in the bHLH is more complicated than the smaller, simpler leucine zipper.

No simple code exists for protein-DNA recognition, and this fact has made design of sequence-specific DNA-binding proteins a major challenge.

SUMMARY OF THE INVENTION

The invention relates to novel minimalist bZIP proteins that are small, simplified molecular recognition scaffolds. These proteins are useful for design of helical proteins that can target specific DNA ligands.

Accordingly, the invention provides a minimalist bZIP protein comprising:
a) a basic region of a basic helix-loop-helix protein (bHLH);
b) a hinge region; and c) a leucine zipper domain of a bZIP protein, wherein the minimalist bZIP protein binds a target DNA sequence.

The bHLH proteins include bHLH subvariants, bHLHZ subvariants and bHLH/PAS subvariants. In one embodiment, the target DNA sequence is an E-box or XRE1 site.

In a particular embodiment, the invention provides a minimalist bZIP protein comprising:
a) a basic region from Max;
b) a hinge region; and c) a leucine zipper region from C/EBP, wherein the minimalist bZIP protein binds an E-box target DNA sequence.

In another particular embodiment, the invention provides a minimalist bZIP
protein comprising:
a) a basic region from Arnt;
b) a hinge region; and c) a leucine zipper region from C/EBP, wherein the minimalist bZIP protein binds an XRE1 target DNA sequence or an E-box target DNA sequence. .

In yet another particular embodiment, the invention provides for a first and second minimalist bZIP protein comprising a leucine zipper region in the first minimalist bZIP protein and a leucine zipper region in the second minimalist bZIP protein capable of forming a heterodimer. In a specific embodiment, the leucine zipper region in the first minimalist bZIP protein is from Jun and the leucine zipper region in the second minimalist bZIP protein is from Fos.

The invention also provides for the use of the minimalist bZIP proteins of the invention for repressing myc-related transcriptional activation. The invention further provides the use of the minimalist bZIP proteins able to bind to an E-box target DNA sequence for treating cancer. The invention also provides for a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein able to bind to an E-box target DNA sequence to a mammal in need thereof. In one embodiment, the cancer is selected from the group consisting of breast cancer, colon cancer, gynecological cancer, hepatocellular carcinomas, hematological tumors, Burkitt lymphoma, neuroblastoma and small cell lung cancer In another embodiment, the invention provides for the use of the minimalist bZIP proteins able to bind to an XRE1 target DNA sequence for treating cancer. The invention also provides for a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein able to bind to an XRE1 target DNA sequence to a mammal in need thereof.
In one embodiment, the cancer is a soft tissue carcinoma or respiratory cancer.

The invention also provides minimalist bZIP proteins further fused to an activation domain, a repressor domain or a drug and uses thereof.

The invention also provides pharmaceutical compositions comprising the minimalist bZIP proteins of the invention and a pharmaceutically acceptable carrier, diluent or excipient, and uses thereof.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:
Figure 1 shows the protein-DNA crystal structure of protein bound to DNA
Left. GCN4 bZIP in complex with the AP-1 DNA site, 5'-TGACTCA
(Ellenberger, T. E., et al. Cell, 1992, 71, 1223-1237) DNA is the dark double helix at the bottom of the figure, and the bZIP is the a-helical dimer above (left). The leucine zipper dimerizes into the coiled-coil, which then smoothly forks to either side of the DNA, allowing the basic region dimer to bind opposite sides of the DNA major groove. Right. Max bHLH/ZIP in complex with the E-box DNA site, 5'-CACGTG (Ferre-D'Amare, A. R., et al. Nature, 1993, 363, 38-45) Note that the basic region and helix 1 are contiguous, and that helix 2 and the leucine zipper are contiguous.

Figure 2 shows Arntl-C/EBP binding E-box. Plates were grown at 30 deg C
for 6 days. (A) Arntl-C-EBP inserted into vector pGAD424 and grown on SD-His/-Leu plates with 10 mM 3-AT. (B) Control of vector alone also grown on SD-His/-Leu plates with 10 mM 3-AT.

Figure 3 shows a schematic diagram of the Yeast One Hybrid system used to evolve the minimalist bZIP proteins of the invention.

Figure 4 shows binding between MM3 and E-box. Plates were incubated four days, 30 C. a. Max/C-EBP plated on SD/-His/-Leu plus 10 mM 3-AT.
Binding undetectable (some bubbles arise from sorbitol in plate medium). b.
MM3 plated on SD/-His/-Leu plus 10 mM 3-AT. c. MM3 plated on SD/-His/-Leu plus 20 mM 3-AT. d. MM3 plated on SD/-His/-Leu plus 60 mM 3-AT.

DETAILED DESCRIPTION OF THE INVENTION

The a-helical bZIP motif serves as a manipulable scaffold for protein design.
The small, simple bZIP can be a very tractable scaffold for design of new proteins with new DNA recognition properties. The present inventors have examined heterodimeric and homodimeric bZIP motifs in which portions of bHLH and bZIP proteins are fused to create novel hybrid proteins, thus enlarging their binding capabilities. These small proteins still retain the structure and function of native proteins, thus condensing these proteins to minimal units of functionality.

(a) Novel Minimalist bZIP Proteins:

The invention relates to the creation of novel minimalist bZIP proteins based on fusion of the DNA-binding domain from a bHLH protein and the leucine zipper dimerization domain originating from a bZIP protein.

Accordingly, in one embodiment, the invention provides a minimalist bZIP
protein comprising a) a basic region of a basic helix-loop-helix protein (bHLH);
b) a hinge region; and c) a leucine zipper domain of a bZIP protein, wherein the minimalist bZIP protein binds a target DNA sequence.

In one embodiment, the minimalist bZIP protein of the invention is 30-100 amino acids in length. In a preferred embodiment, the minimalist bZIP protein is 40-60 amino acids in length.

The hinge offers a large amount of flexibility in length and identity.
Technically, a hinge could have zero amino acids, as long as the lengths of basic region and zipper compensate for the flexibility. Accordingly, in one embodiment, the hinge region has 0-20 amino acids. In another embodiment the hinge region has 3 amino acids. In a specific embodiment, the hinge region comprises the amino acid sequence RIR or GIR. As the orientation of the minimalist bZIP protein is significant for binding to DNA, each desired minimalist bZIP protein is constructed in triplicate using an additional 1, 2 or 3 amino acids from the C-terminal end of helix 1 in order to select for the best orientation for binding to DNA. One of skill in the art could readily determine which of the 3 hinge regions best orients the protein to the DNA. For example, if the structure is known, this can be analysed by the crystal structure or NMR
structure. Alternatively the modeling can be based on the high-resolution structure of a protein known to be similar. Accordingly, in an embodiment of the invention, the invention provides the minimalist bZIP proteins of the invention having an additional 1, 2 or 3 amino acids from the C-terminal end of helix I of the bHLH protein between the basic region and the hinge region.
A minimalist bZIP protein that does not provide an ideal orientation for DNA
binding may be used as a negative control.

The leucine zipper domain is derived from bZIP proteins selected from the group consisting of C/EBP, Jun, Fos, GCN4 and CREB. Where it is desired to construct minimalist bZIP proteins that homodimerize, the preferred leucine zipper domain is derived from C/EBP. Where it is desired to construct minimalist bZIP proteins that heterodimerize, use of the leucine zipper domains of Jun or Fos is preferred.

In one embodiment, the basic region is derived from a bHLH protein selected from the group consisting of a bHLH subvariant, bHLHZ subvariant or bHLH/PAS subvariant. The bHLH subvariant may be selected from the group consisting of MyoD, Myc, E2A, E47, E12, TALL, Id proteins, GL3 and EGL3, TFEB, PIF1, PIL6, ATH, NGN and HAND1. The bHLHZ subvariant may be selected from the group consisting of Mad, Mxi, Max, Myc, Spzl, USF, Mash, BMP, TFE3 and AP4. The bHLH/PAS subvariant may be selected from the group consisting of AhR, Arnt (also known as HIF-1(3), HIF-1a, HIF-2a, HIF-3a, Per and Sim.

In a particular embodiment, the bHLHZ subvariant is Max. In another particular embodiment, the bHLH/PAS subvariant is Arnt.
In one embodiment, the minimalist bZIP protein comprises a) a basic region from Max; b) a hinge region; and c) a leucine zipper region from C/EBP, wherein the minimalist bZIP protein binds an E-box target DNA sequence.

In a particular embodiment, the minimalist bZIP protein comprises the amino acid sequence as shown in Table 1(SEQ ID NOs 1-6), differing only in the number of amino acids in the hinge region.

In another embodiment, the invention provides a minimalist bZIP protein comprising a) a basic region from Arnt; b) a hinge region; and c) a leucine zipper region from C/EBP, wherein the minimalist bZIP protein binds an XRE1 target DNA sequence or an E-box target DNA sequence.

In a particular embodiment, the minimalist bZIP protein comprises the amino acid sequence as shown in Table 2 (SEQ ID NOs 7-12), differing only in the number of amino acids in the hinge region.

In another particular embodiment, the invention provides a minimalist bZIP
protein comprising the amino acid sequence as shown in SEQ ID NOs 14-22, 52, 53 or 54.

In yet another particular embodiment, the invention provides for a first and second minimalist bZIP protein comprising a leucine zipper region in the first minimalist bZIP protein and a leucine zipper region in the second minimalist bZIP protein capable of forming a heterodimer. In a specific embodiment, the leucine zipper region in the first minimalist bZIP protein is from Jun and the leucine zipper region in the second minimalist bZIP protein is from Fos.
The minimalist bZIP proteins of the invention are able to bind target DNA
sequences. In one embodiment, the minimalist bZIP proteins bind to the homodimeric E-box DNA sequence 5'-CAG-CTG-3' (Class A proteins) and 5'-CAC-GTG-3' (Class B proteins). In another embodiment, the minimalist bZIP
proteins of the invention bind to the heterodimeric XRE1 site 5'-TTGC-GTG-3' (Class C proteins). Class A proteins include MyoD, E47, AP4, E12, Tall and have the consensus as shown in Table 6. Class B proteins include Max, Myc, USF, TFE3, TFEB, Arnt and have the consensus as shown in Table 6 of grant. Class C proteins include AhR and Sim and recognize half sites 5'-T(CfT)GC-3' or 5'-GT(A/G)C-3'. Finally, the minimalist proteins may be generated to heterodimerize with a protein fused to a basic region of a bZIP
protein, such as GCN4 which binds to the half site: 5'-TGAC.

The different combinations of basic regions and leucine zipper regions generates a large variety of binding repertoires. The ability to bind to a particular target DNA sequence may depend on the spacing of the target DNA
sequences and the flanking sequences found in an endogenous DNA
promoter. A person skilled in the art would readily be able to test a minimalist bZIP protein to determine its ability to bind to particular target DNA
sequences.

For example, the structure and function of the proteins of the invention can be quantitatively characterized using techniques known in the art, such as by DNase I footprinting; chemical footprinting; circular dichroism;
thermodynamics by fluorescence anisotropy and calorimetry; and high-resolution x-ray crystallography and molecular modeling. DNAse I footprinting is used to demonstrate the binding of a protein to a specific DNA sequence.
When a protein binds the specific DNA sequence, clear footprints can be seen. A wild type bHLH protein that is expected to bind the sequence can be used as a positive control. Electrophoretic mobility shift assay (EMSA) is a gel assay in which one can detect whether the DNA sequence is bound by the particular protein. If protein binds to the DNA, the DNA's mobility through the gel is retarded, and therefore, the band corresponding to DNA shifts. One can also measure free energies of protein-DNA binding by titrating different concentrations of protein with DNA and measuring the shift. Capillary electrophoresis and other chromatography variants are similar to EMSA gel assay in that free DNA would move through the column matrix differently from DNA bound by protein. Therefore, these assays can measure where free DNA elutes from the matrix; if protein binds DNA, there is a change in mobility of DNA. Normally, protein-bound DNA mobility would be slower. Alternatively, the Yeast two-hybrid or Yeast one-hybrid system can be used to detect DNA-protein interactions as described in the examples section below.

Circular Dichroism is an excellent method for characterizing the structure of bZIP proteins, as the a-helix displays distinctive minima at 208 nm and 222 nm. Fluorescence anisotropy measures the tumbling motion of molecules containing a fluorophore; nanosecond lifetimes of most fluorophores are comparable to the time necessary for rotation of a molecule or complex of molecular weight less than 105 D, the range of many protein-DNA complexes (the excited state fluorescence lifetime for fluorescein is -4 ns) (Lakowicz, J.R. Principles of Fluorescence Spectroscopy; 2nd Edition ed.; Plenum Press:
New York, 1999). Large complexes tumble slowly relative to the lifetime of fluorophore and exhibit only slight depolarization of emission with respect to polarized excitation, and a higher anisotropy than small complexes that tumble rapidiy (Hill, J.J. and Royer, C.A. "Fluorescence Approaches to Study of Protein-Nucleic Acid Complexation." Meth. Enzymol. 1997, 278, 390-416).

Thus, the anisotropy of free, short DNA duplexes should be significantly less than that for the same DNA bound by a protein such as a minimalist bZIP
protein of the invention. High-resolution NMR and/or X-ray crystallography for structural characterization would also be useful for characterizing the novel bZIP proteins of the present invention. One can obtain detailed pictures of molecules showing positions of atoms, bond lengths, and distances, producing high-resolution detail of atomic interactions. Calorimetry can also be used for characterization of thermodynamics of complexation. One can measure enthalpies and free energies (and can then calculate entropies) as well as heat capacities. These thermodynamic parameters provide information about how strong the complexation is, how stable it is, how factors like temperature or a mutation or salts can affect the binding strength and stability. Other fluorescence experiments besides anisotropy, including fluorescence resonance energy transfer (FRET) and fluorescence homoquenching can also be used. With FRET and homoquenching, one can label the protein and DNA with fluorophores whose properties are distance-dependent. Therefore, if the fluorophores are close to each other, which would occur when the protein and DNA bind, one would see fluorescence in FRET or would see quenching of fluorescence in homoquenching. Thus, this is useful for detecting binding of the minimalist bZIP protein of the invention with specific target DNA sequences. Mass spectrometry (MS) can be used to determine the molecular weight of the molecule and can even detect molecular complexes, like a protein-DNA complex.

Insertion of alanine residues into the basic region of bZIP has been shown to retain the helical structure and DNA binding of the native protein (Lajmi et al.
JACS, 2000, 122, 5638-5639). Accordingly, the minimalist bZIP proteins may be further refined and/or simplified by generating Ala-rich basic regions. The minimalist bZIP proteins may also be strung together such that the helical units can generate multimeric proteins capable of binding longer sequences.

In a further embodiment, the sequences encoding the minimalist bZIP
proteins are mutagenized and then the protein products are selected for better binding to the DNA. Accordingly, in one embodiment, the minimalist bZIP
proteins are minimalist bZIP proteins of the invention that have been further evolved by mutagenesis and selection. In a particular embodiment, the mutagenesis and the selection occurs in a yeast one-hybrid or yeast two-hybrid system.

In another embodiment, the minimalist bZIP proteins of the invention are fused to an activation domain or a repressor domain. The activation domain may be derived from the proteins Ga14, Myc, Mad (Mxi), and VP16, and HIF-3a. An actual transcriptional repressor is not always necessary for repression of gene expression because if the minimalist bZIP protein sits on a gene or promoter, it may be enough to block or repress the transcription of the gene.
However, a repressor may be fused to the minimalist bZIP proteins of the invention. A repressor domain may be derived from Id, Mad (Mxi), and HIF-3a.

The minimalist bZIP proteins of the invention may also contain or be used to obtain or design "peptide mimetics". "Peptide mimetics" are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review).
Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of the proteins of the invention, including biological activity and a reduced propensity to activate human T cells. Peptide mimetics also include peptoids and oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA
89:9367).

Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements.
Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of the secondary structures of the proteins of the invention. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.

The molecules of this invention can be prepared in any of several ways but it is most preferably conducted exploiting routine recombinant methods. It is a relatively straightforward procedure to use the protein sequences and information provided herein to deduce a polynucleotide (DNA) encoding any of the preferred protein sequences. This can be achieved for example using computer software tools such as the DNSstar software suite [DNAstar Inc, Madison, WI, USA] or similar. Any such DNA sequence with the capability of encoding the preferred polypeptides of the present or significant homologues thereof, should be considered as embodiments of this invention.

As a general scheme, genes encoding any of the preferred minimalist bZIP
protein sequences can be made using gene synthesis and cloned into a suitable expression vector. In turn the expression vector is introduced into a host cell and cells selected and cultured. The proteins of the invention are purified from the culture medium and formulated into a preparation for therapeutic administration.

Methods for purifying and manipulating recombinant proteins including fusion proteins are well known in the art. Necessary techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991).

The proteins of the invention can be prepared using recombinant DNA
methods. The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

The present invention also provides a purified and isolated nucleic acid molecule comprising a sequence encoding the minimalist bZIP proteins of the invention, preferably a sequence encoding the protein described herein as shown in SEQ ID NOs. 1-12, 14-22, 52, 53 or 54.

The term "isolated and purified" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An "isolated and purified" nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e.
sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

The term "nucleic acid" as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly. The nucleic acid sequences of the present invention may be ribonucleic (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

In one embodiment, the purified and isolated nucleic acid molecule comprises:

(a) a nucleic acid sequence encoding the amino acid sequences of the proteins of the invention, preferably as shown in SEQ ID NOs. 1-12, 14-22, 52, 53 or 54;

(b) nucleic acid sequences complementary to (a);

(c) nucleic acid sequences which are homologous to (a) or (b);

(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions; or (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in codon sequences due to the degeneracy of the genetic code.

Further, it will be appreciated that the invention includes nucleic acid molecules comprising nucleic acid sequences having substantial sequence homology with the nucleic acid sequences encoding the proteins and peptides of the invention, and fragments thereof. The term "sequences having substantial sequence homology" means those nucleic acid sequences which have slight or inconsequential sequence variations from these sequences, i.e., the sequences function in substantially the same manner to produce functionally equivalent proteins. The variations may be attributable to local mutations or structural modifications.

Nucleic acid sequences having substantial homology include nucleic acid sequences having at least 80%, preferably 90% identity with the nucleic acid sequence encoding the proteins of the invention.

Another aspect of the invention provides a nucleic acid molecule, and fragments thereof having at least 15 bases, which hybridize to nucleic acid molecules of the invention under hybridization conditions, preferably stringent hybridization conditions. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed: 6.0 x sodium chloride/sodium citrate (SSC) at about 45 C, followed by a wash of 2.0 x SSC
at 50 C. The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50 C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65 C.

Accordingly, nucleic acid molecules of the present invention having a sequence which encodes a protein of the invention may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno associated viruses), so long as the vector is compatible with the host cell used. The expression "vectors suitable for transformation of a host cell", means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule.
"Operatively linked" is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal.
Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.

The recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, 11-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as 13-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of a target recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The term "transformed host cell" is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms "transformed with", "transfected with", "transformation" and "transfection"
are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation. Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other such laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).

The nucleic acid molecules of the invention may also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No.
4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S.
Patent Nos. 4,401,796 and 4,373,071).

The invention also provides nucleic acids encoding fusion proteins comprising a novel protein of the invention and a selected protein, or a selectable marker protein.

Another aspect of the present invention is a cultured cell comprising at least one of the above-mentioned vectors.

A further aspect of the present invention is a method for preparing a minimalist bZIP protein comprising culturing the above mentioned cell under conditions permitting expression of the minimalist bZIP protein from the expression vector and purifying the minimalist bZIP protein from the cell.

Uses of the Minimalist bZIP proteins of the Invention:

Mutant proteins that interfere with Myc-Max recognition of the E-box may interfere with Myc's disease promoting activities. Accordingly, the invention provides a use of the minimalist bZIP proteins of the invention able to bind to an E-box target DNA sequence for repressing myc-related transcriptional activation.

Myc is an oncoprotein known to be overexpressed in a wide variety of human diseases, including 80% breast, 70% colon, and 90% gynecological cancers, 50% hepatocellular carcinomas and a variety of hematological tumors (Gardner, L. et al., Encyclopedia of Cancer, Bertino, J. R. Ed., 2002, Academic Press).

Accordingly, in one embodiment, the invention provides the use of the minimalist bZIP proteins able to bind to an E-box target DNA sequence for treating cancer. In antoher embodiment, the invention provides a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein able to bind to an E-box target DNA sequence to a mammal in need thereof. In one embodiment, the cancer is selected from the group consisting of breast cancer, colon cancer, gynecological cancer, hepatocellular carcinomas, hematological tumors, Burkitt lymphoma, neuroblastoma and small cell lung cancer. In another embodiment, the mammal is human.

As discussed above, the minimalist bZIP protein may also be fused to a repressor of transcription. Accordingly, the invention provides the use of a minimalist bZIP protein able to bind to an E-box target DNA sequence fused to a repressor for repressing myc-related transcriptional activation and/or for treating cancer. In another embodiment, the invention provides a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein able to bind to an E-box target DNA sequence fused to a repressor to a mammal in need thereof.

The Myc, Max and Mad transcription factor network are critical for control of normal cell proliferation and differentiation. Accordingly, the invention also provides the use of the minimalist bZIP proteins able to bind to an E-box target DNA sequence for controlling cell proliferation and/or differentiation.
In another embodiment, the invention provides the use of a minimalist bZIP
protein able to bind to an E-box target DNA sequence fused to an activation domain for activating cell proliferation and/or differentiation. In yet another embodiment, the invention provides the use of a minimalist bZIP protein able to target an E-box target DNA sequence fused to a repressor for repressing cell proliferation and/or differentiation.

In another embodiment, the invention provides for a method of regulating a desired gene by inserting at least one E-box sequence upstream of the desired gene and introducing a minimalist bZIP protein capable of recognizing the inserted E-box sequence, wherein the minimalist bZIP protein then acts to regulate the expression of the gene. The minimalist bZIP protein used in the method can be further fused to an activation or repressor domain.

The Ahr/Arnt system is notable for its possible role in disease pathways given its role in mediating signal transduction by dioxins and related polycyclic aromatic hydrocarbons. Given that the endogenous ligand for the dioxin receptor is not yet known, the minimalist bZIP proteins of the invention that target the XRE1 site are useful for regulating the dioxin pathway.
2,3,7,8-tetrachlorodibenzo-p-dioxin, commonly referred to as TCDD or dioxin, produces a variety of highly toxic effects, including chloracne, teratogenesis, tumor promotion, and immunotoxicity (Whitelaw, M. et al., Mol. Cell Biol.
1993, 13, 2504-2514; Poland, A. and Knutson, J. C., Ann. Rev. Pharmacol.
Toxicol. 1982, 22, 517). Dioxin is an industrial byproduct produced during herbicide manufacture, the bleaching of paper pulp, and combustion of chlorinated organic materials. Dioxin can accumulate in the environment;
although it decomposes rapidly in organic solution under artificial or natural light, no photodecomposition occurs in aqueous environments or on wet or dry soil (Crosby, D. G., et al., Science, 1971, 173, 748-749). The resistance of dioxin to metabolic degradation and the stability of the dioxin-receptor complex may account for its persistence and toxicity (Johnson, E. F., Science, 1991, 252, 924).

Animal studies have proven this ubiquitous pollutant to be extremely lethal, perhaps the most powerful carcinogen tested (Roberts, L., Science, 1991, 251, 624-626; Gray, L. E. Jr. and Ostby, J. S., Toxicol. Appl. Pharmacol., 1995, 133, 285-294]. Human effects, however, have been subject to wide controversy, especially in studies concerning dioxin-tainted Agent Orange used by the United States during the Vietnam War as a defoliant.

In 1991, the National Institute of Occupational Safety and Health published an exhaustive examination of 5172 male chemical workers exposed to dioxin on the job from 1942 to 1982 (Fingerhut, M. A., et al., N. Engi. J. Med., 1991, 324, 212-218). The low exposure cohort worked for less than one year in a dioxin-tainted occupation, the high exposure cohort for at least a year; the latency period for cancer occurrence was at least twenty years (Roberts, L., Science, 1991, 251, 624-626). The low-exposure group showed no increased risk in cancer, despite exposure to dioxin at levels 90 times higher than that for the general population. The high exposure group, however, was estimated to be exposed to dioxin levels 500 times higher than that for the general population and had nearly a 50% increase in cancer mortality, mostly in soft tissue sarcomas and, unexpectedly, respiratory cancer.

Dioxin's disease/cancerous effects can be mediated by aryl hydrocarbon receptor. This receptor must first bind the ligand to initiate its detrimental effects. A receptor-binding model for dioxin may explain the result that the low-exposure cohort in the NIOSH study discussed above exhibited no increase in cancer risk. Response to dioxin increases slowly at low dioxin concentrations but elevates rapidly after reaching a critical concentration (dissociation constant, Kd). Thus, instead of a linear model for dioxin toxicity, the binding curve would be sigmoidal, and there may exist a practical threshold below which dioxin concentrations may be deemed to be "safe."
This receptor-mediated mechanism for dioxin action provides a target for monitoring levels of ligand.

The AhR mediates signal transduction by dioxins and related polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrenes found in cigarette smoke and smog, heterocyclic amines found in cooked meat, and polychlorinated biphenyis (PCBs) (Fisher, J. M. et al., Mol. Carcinogen., 1989, 1, 216-221). In analogy to the glucocorticoid receptor, the latent dioxin receptor is found associated with heat-shock protein hsp90 in the cytosol (Cadepond, F., et al., J. Biol. Chem. 1991, 266, 5834-5831). Ligand binding induces release of hsp90, nuclear translocation of the AhR (Pollenz, R. S., et al., Mol. Pharmacol., 1995, 45, 428-438), and dimerization with the nuclear protein Arnt (Reyes, H., et al., Science, 1992, 256, 1193-1195); this activated complex then binds specific DNA sites (xenobiotic response elements or XREs) and activates gene transcription (Wu, L. and Whitlock, J. P. Nucl.

Acid. Res. 1993, 21, 119-125; Fujisawa-Sehara, A. et al., Nucl. Acid. Res.
1987, 95, 4179-4191). Dioxins are very potent inducers of transcription target genes, including cytochrome P4501A1, which codes for aryl hydrocarbon hydroxylase, a catalyst for oxygenation of polycyclic hydrocarbons to phenois and epoxides, some of which are mutagenic and carcinogenic (Whitelaw, M.
et al., Mol. Ce118io1. 1993, 13, 2504-2514).

Accordingly, the invention also provides the use of a minimalist bZIP protein able to bind to an XRE1 target DNA sequence for treating cancer. In another embodiment, the invention provides a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein able to bind to an XRE1 target DNA sequence to a mammal in need thereof. In another embodiment, the invention provides the use of a minimalist bZIP protein able to bind to an XRE1 target DNA sequence fused to a repressor domain for treating cancer. In yet another embodiment, the invention provides a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein able to bind to an XRE1 target DNA sequence fused to a repressor domain to a mammal in need thereof. In one embodiment, the cancer is a soft tissue carcinoma or respiratory cancer. In another embodiment, the mammal is human.

Since the minimalist bZIP proteins of the invention bind to specific DNA
sequences, the proteins may be used as targeting agents. In another embodiment, the minimalist bZIP proteins are fused to a drug. For example, the drug may be an anti-cancer agent. In another embodiment, the invention provides a method of treating cancer comprising administering an effective amount of a minimalist bZIP protein fused to a drug to a mammal in need thereof.

Additionally, these a-helical, bZIP/bHLH hybrids have agricultural and biological (nonhuman) applications. For example, the plant G-box is 5'-CACGTG, the same sequence as the mammalian E-box; the G-box refers specifically to plants. The G-box is bound by bZIP proteins; however, the bZIP/bHLH hybrids of the present invention also bind the G-box, as it is identical to the E-box. Plants use bZIP proteins ubiquitously; Arabidopsis thaliana has four times as many bZIP proteins as do humans and yeast (Jakoby, M. et al., Trends Plant Sci., 2002, 7, 106-111). In Arabidopsis, many G-box regulated genes are linked to ultraviolet and blue light signal transduction and regulation of light-sensitive promoters, and there is evidence that some GBF-like proteins (G-box binding factor), including ROM1 and ROM1, may regulate storage protein expression, and therefore, play a role in seed maturation. Control of storage protein expression may achieve healthier, more vigorous, larger plants and crops. In addition, the E-box/G-box can be cloned upstream of a gene that one wishes to control: hence, a genetically modified plant. By extension, the genetically modified organism does not have to be a plant, but it could be an animal; these proteins can also have veterinary applications in cases of genes that fortuitously possess an E-box (or E-box-related) or XRE1 (or XRE1-related) sequence in the promoter.
This plant could then be engineered to express a bHLH/bZIP hybrid that would then target the cloned E-box/G-box, thereby, regulating the desired gene. Numerous other plant applications are contemplated by the use of minimalist bZIP proteins including controlled regulation of proteins to either enhance or decrease the growth of specific plant organs and tissues by reducing the expression or effectiveness of endogenous growth-associated proteins. The regulation of gene expression by the use of minimalist bZIP
proteins and insertions of E-box sequences can result in the modification of various traits, such as durability, size, succulence, texture, and longevity.
It is envisioned that both monocotyledonous and dicotyledonous plants can be used, as well as stem and leaf vegetables (e.g., broccoli, lettuce, spinach, cabbage), fruit and seed vegetables (e.g., tomato), fiber crops and cereals (e.g., corn, oats, wheat), and forest and ornamental crops (e.g., cotton). For example, regulation of grape growth in the wine industry in order to provide the ability to fight common grape afflictions, such as phylloxera, or to regulate leaf, root, stem or petiole growth for improved cabbage, spinach, celery, beets, soybeans, sugarcane, flower stalks can be envisioned.

The invention also provides a pharmaceutical composition for treating a mammal with cancer comprising a minimalist bZIP protein of the invention and a pharmaceutically acceptable carrier, diluent or excipient. In a preferred embodiment, the mammal is human. In one embodiment, the minimalist bZIP
protein recognizes an E-box/G-box DNA sequence. In a specific embodiment, the cancer is selected from the group consisting of breast cancer, colon cancer, gynecological cancer, hepatocellular carcinomas, hematological tumors, Burkitt lymphoma, neuroblastoma and small cell lung cancer. In another embodiment, the minimalist bZIP protein recognizes an XRE site. In a specific embodiment, the cancer is a soft tissue carcinoma or respiratory cancer. In a further embodiment, the minimalist bZIP protein of the pharmaceutical composition is fused to a repressor.

The proteins of the invention may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans and animals. Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of protein to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, intramuscular, etc.), oral administration, inhalation, transdermal administration (such as topical cream or ointment, etc.), or suppository applications. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.

The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences (2000 - 20th edition) Mack Publishing Company). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

Methods of Evolving Better DNA Binding Minimalist bZIP Proteins In order to examine the binding activities of the minimalist bZIP proteins in vivo, a useful strategy based on the yeast one-hybrid system was used.
Examination of in vivo binding was chosen, rather than in vitro, in order to mimic the native cellular environment. Additionally, the system allows for in vivo directed evolution of proteins targeting specific DNA sites:
particularly, bZIP-like homodimers that bind the E-box.

An excellent assay for monitoring in vivo interactions between proteins and DNA is provided by the yeast one-hybrid system (Kumar, R. et al., J. Biol.
Chem., 1996, 271, 29612-29618; Chen, X. et al., Nature, 1996, 383, 691-696). The basis for the yeast one-hybrid assay is the useful fact that eukaryotic transcriptional activators comprise physically and functionally independent DNA-binding domains and activation domains. Therefore, a hybrid protein can be constructed that comprises a DNA-binding domain fused to a suitable transcriptional activator domain. This hybrid can then be assayed for binding to a specific DNA target, because successful protein-DNA
complexation results in transcription of one or more reporter genes. Thus, the yeast one-hybrid assay is particularly useful for isolating proteins that bind a specific DNA target; for instance, this system can be used to map the DNA-binding domains of previously known proteins as well as discovery of new proteins capable of recognizing a desired target site.

Because the yeast one-hybrid assay is an in vivo system, it offers the advantage of examination of protein-DNA recognition in the native eukaryotic environment, in contrast to other in vitro surface display technologies (Benhar, I., Biotech. Adv., 2001, 19, 1-33). Both in vivo and in vitro binding assays, such as the yeast one- and two-hybrid assays and phage display, enable monitoring of specific protein-DNA and protein-protein interactions. The main application of such systems is their use for selection of individual targets from large libraries of different clones. Diversity in these libraries can be generated by various means, such as using degenerate oligonucleotides for randomization of large sections of a gene's coding sequence (Wang, B. S.
and Pabo, C. 0., Proc. Natl. Acad. Sci. USA, 1999, 96, 9568-9573) or mutagenic PCR protocols to generate small numbers of random mutations in genes (Cherry, J. R. et al., Nat. Biotech, 1999, 17, 379-384). Similarly, a directed evolution process, in which molecules with desired traits can be evolved and isolated, can be achieved by starting with large, diverse libraries of mutants followed by an appropriate selection procedure. Multiple rounds of directed evolution can be performed to give improvement in the desired molecular function.

Creating large libraries of mutant clones can be a tedious and difficult task.
This is partially due to the low efficiency of transformation obtained with ligated plasmid vectors-typically one to three orders of magnitude lower transformation efficiency than that obtained with supercoiled plasmids (Tobias, A. V. in Directed Evolution Library Creation, Arnold, F. H. and Georgiou, G., Eds, 2003, Humana Press, Totowa, NJ). An excellent alternative to classical cloning methods involving ligated vectors is cloning by use of homologous recombination (Aylon, Y. and Kupiec, M., Mut. Res., 2004, 566, 231-248). Whereas this method is not applicable in bacteria, due to low frequency of recombination, it is very useful in yeast. In yeast, homologous recombination can substitute for ligation in order to give very high transformation frequencies (Butler, T. and Alcalde, M., in Directed Evolution Library Creation, Arnold, F. H. and Georgiou, G., Eds, 2003, Humana Press, Totowa, NJ). In homologous recombination, mutant gene inserts are cotransformed with linearized plasmid; the end sequences on the inserts and linearized plasmid are homologous. Yeast can then perform homologous recombination on the inserts and plasmids to form closed circular plasmids.
Another advantage is that the homologous recombination procedure also allows the mutated linear PCR products to recombine amongst themselves prior to creation of circular plasmid (Swers, J. S., et al., Nucl. Acids.
Res., 2004, 32, 36-44). This process drives shuffling of acquired mutations within the PCR products. Thus, successive rounds of directed evolution using homologous recombination and appropriate selection steps leads to loss of unwanted mutations and accumulation of positive ones-this procedure resembles the natural evolution process.

Accordingly, the minimalist bZIP proteins of the invention can be evolved by:
(a) linearizing DNA duplexes carrying a minimalist bZIP protein sequence;

(b) subjecting the DNA in step (a) to mutagenic PCR to create a mutated minimalist bZIP library;

(c) linearizing an appropriate yeast vector that has anchors homologous with the genes encoded in the minimalize bZIP library in (b);

(d) cotransforming the products of step (c) with the linearized vector into yeast with a genome integrated with target DNA sites; and (e) plating on selective medium;

wherein the colonies that grow in the selective medium have evolved minimalist bZIP proteins that bind to the target DNA site.

The above method can further comprise repeating steps (a) to (e) with the sequence encoding the evolved minimalist bZIP protein.

The following non-limiting examples are illustrative of the present invention:
EXAMPLES

Generation of Max-C/EBP bZIP minimalist proteins:

The basic region of Max was fused to the leucine zipper of C/EBP. Three different fusion proteins were constructed Max-1, -2, -3 which differ by one amino acid in order to leave some flexibility in the hinge region-where the zipper is fused to the basic region, which splays out to straddle DNA. Table I
shows the sequences of Max-1, -2 and -3-C/EBP with two different hinge regions, RIR and GIR, which are expected to have different helical capabilities. A BamH I site was used for joining the basic regions to the zipper regions. Because there are -3.5 amino acids per turn in an a-helix, Max-1, -2, and -3 should be sufficient for generating a protein capable of targeting the E-box site. Indeed, the Max-1-C/EBP has been evolved into MM3 which binds to the E-box.

Generation of Arnt-C/EBP bZIP minimalist proteins:

Arnt is a bHLH protein (no leucine zipper). Arnt basic region binds 5'-GTG, half of the canonical E-box. Native Arnt preferentially heterodimerizes, but does homodimerize. Three different fusion proteins were constructed Arnt-1, -2, -3 which differ by one amino acid in order to leave some flexibility in the hinge region-where the zipper is fused to the basic region, which splays out to straddle DNA. Table 2 shows the sequences of Arnt-1, -2 and -3-C/EBP
with two different hinge regions, RIR and GIR, which are expected to have different helical capabilities.

Desi_gn of Protein Heterodimers that Target Asymmetric DNA Site.

The xenobiotic response element 1(XRE1) resides in the 5' flanking region of the CYP9A1 (cytochrome P450) gene. The XRE1 sequence is 5'-TTGC=GTG. Arnt binds to 5'-GTG, which is half of the E-box sequence 5'-CAC=GTG. AhR binds to 5'-TTGC. Fortuitously, well-characterized bHLH
and bZIP proteins bind to these same half sites. The bHLH protein Max binds to 5'-CAC=GTG; the bZIP protein C/EBP binds 5'-TTGC=GCAA (Agre, P. et al., Science 1989, 246, 922-926; Landschulz, W. H. et al., Science 1988, 240, 1759-1764). A heterodimer comprising the Max and C/EBP basic regions may therefore recognize 5'-TTGC=GTG, same as the AhR-Arnt heterodimer.
Because of the wealth of information on C/EBP and Max, including Max bHLH-DNA crystal structures (Ferre-D'Amare, A. R. et al., Nature 1993, 363, 38-45; Ellenberger, T. et al., Genes Dev. 1994, 8, 970-980), these proteins were a sound first-generation choice.

We use the leucine zippers from bZIP proteins Jun and Fos to ensure heterodimerization. In the crystal structure (Glover, J. N. M. and Harrison, S.
C., Nature 1995, 373, 257-261), the Fos-Jun heterodimer is oriented on the AP-1 DNA site (5'-TGACTCA) such that the Fos basic region binds to 5'-TGAC and Jun binds 5'-CTCA. Therefore, the C/EBP basic region is fused to the Fos zipper, whereas the Max basic is fused to the Jun zipper (Table 3).
These fusions should maintain proper orientation for heterodimerization such that binding will occur at 5'-TTGC=GTG, rather than 5'-GTG=TTGC.
Incidentally, although Jun and Fos preferentially heterodimerize, Jun can homodimerize, whereas Fos does not. Both Max and Arnt can homodimerize, so the Max-Jun and Arnt-Jun homodimers that may occur will be reflective of the natural system.

Second Generation Hybrids: AhR or Amt basic region, Fos or Jun leucine zipper The AhR bHLH is promiscuous in dimerization partners; the AhR bHLH/PAS
construct, however, specifically heterodimerizes with only the Arnt bHLH/PAS, and therefore, the PAS domain confers dimerization specificity. The proteins of the invention do not contain the PAS domain, but do use the Jun and Fos leucine zippers to promote heterodimerization.

Hybrids are constructed comprising the basic regions of human AhR
(Dolwick, K. M. et al., Mol. Pharm., 1993, 44, 911-917) and human Arnt (Hoffman, E. C., et al., Science, 1991, 252, 954) with the leucine zippers of Fos and Jun, respectively (Table 4). The same flexibility in the fusion junction is explored in these hybrids as well. Thus, portions of Helix 1 in the AhR and Arnt sequences are used to lengthen the hinge between the basic region and zipper. This junction may not be as straightforward as for the well-characterized bHLH proteins used in the first-generation studies, as AhR and Arnt are bHLH/PAS proteins (Hogenesch, J. B. et al., J. Biol. Chem., 1997, 272, 8581-8593; Lindebro, M. C. et al., EMBO J., 1995, 14, 3528-3539), which are closely related, but not identical, to the bHLH and bHLHZ motifs.
The reference sequences are shown in Table 5.

Comparison of protein-DNA interactions: bHLH bZIP and the unknown AhR

Arnt is believed to form a complex with the 5'-GTG site resembling that of Max, E47, USF, and MyoD-these are all bHLH or bHLHZ proteins that use the same basic-region residues to contact the E-box (Swanson, H. I. and Yang, J.-H., J. Biol. Chem., 1996, 271, 31657-35661). The AhR complex with 5'-TTGC likely represents a unique variant of the bHLH motif; AhR's basic region is unlikely to be strongly helical, as it contains four prolines and one glycine, residues known to be helix breakers (Luque, I. et al., Biochemistry, 1996, 35, 13681-13688). The AhR basic may also not display the characteristic transition from disordered to stable a-helix upon binding to specific DNA, as do other known bHLH proteins (Ferre-D'Amare, A. R. et al., EMBO J. 1994, 13, 180-189).

That the AhR basic may employ a different mechanism from known bHLH
proteins for DNA recognition expands the present invention's ability to recognize diverse sequences. Most bZIP and bHLH heterodimers bind half sites that are palindromic or pseudopalindromic. The AhR-Arnt heterodimer binds two distinct, unrelated half sites (Rowlands, J. C. and Gustafsson, J.
A., Crit. Rev. Toxicol., 1997, 27, 109-134; Bacsi, S. C. et al., Mol. Pharmacol., 1995, 47, 432-438). Thus, a pair of basic a-helices (or new motifs that AhR
represents) is capable of tremendous molecular diversity in ligand-binding.

= The Max bHLHZ Structure. The structures of bHLH and bHLHZ proteins in complex with E-box sites show a high level of conservation of specific protein-DNA interactions. The Max bHLH/ZIP complex with the Class B E-box shows three highly conserved specific contacts (Table III): His2$ makes a hydrogen bond to N7 of G3', GIu32 accepts hydrogen bonds from N4 of C3 and N6 of A2, and Arg36 makes a hydrogen bond to N7 of G1' at the dyad axis. The intact Max structure also shows the same specific contacts (Brownlie, P. et al., Structure 1997, 5, 509-520), and the USF bHLH/ZIP
complex displays these specific contacts and makes equivalent backbone contacts to phosphodiester groups with Asn29, Arg33, and Arg35 as does Max (Ferre-D'Amare, A. R. et al., EMBO J. 1994, 13, 180-189). Both MyoD and E47 are bHLH proteins that bind the Class A E-box. For MyoD, the contacts to the outer base pairs are similar to those of Max: GIu32 accepts hydrogen bonds from N4 of C3 and N6 of A2 (weak, 3.5-3.8 A distance) (Ma, P. C et al., Cell 1994, 77, 451-459). This essential glutamic acid is absolutely conserved in both Class A and B proteins, and the bifurcated interaction with the CA
step is conserved in all structures discussed. E47 makes this same contact with GIu32, plus its side chain methylenes make van der Waals contact with the C5 methyl group on T2' (Ellenberger, T. et al., Genes Dev. 1994, 8, 970-980).

= The MyoD bHLH Structure. The other specific contacts that MyoD bHLH
makes are not identical to Max bHLH/ZIP: Arg25 makes a hydrogen bond to N7 of G10' and a water-mediated contact to 06 of G10'; Thr29 makes a van der Waals interaction with T2'-the corresponding amino acids in Max and USF do not make specific DNA contacts. Notably, Arg25 is buried in the major groove in MyoD, but in Max, it swings away from the major groove and makes a phosphodiester contact; in its stead, the highly conserved His28 of Max (one helical turn from Arg25) contacts N7 of G3' and displaces the Arg25 side chain. Arg3l of MyoD makes electrostatic contacts with phosphodiesters flanking the E-box and maybe a weak interaction with a flanking adenine; in Class B proteins Max and USF, the corresponding amino acid is hydrophobic and makes no contact at all to DNA. Finally, position 36 is critical for discrimination between the Class A and B E-box sequences. In Class B
proteins Max and USF, Arg36 specifically contacts N7 of G1' at the dyad axis.
In Class A proteins MyoD and E47, position 36 is occupied by Leu and Val, respectively. Curiously, the MyoD cocrystal structure, shows no contact of Leu36 whatsover with DNA. When Leu36 is mutated to Arg, MyoD now binds to the Class B site. So this position is critical for discrimination of the central base pairs (Blackwell, T. K. et al., Mol. Cell. Biol. 1993, 13, 5216-5224).

= Comparison of AhR and Arnt with Known Structures: Swanson and Yang performed extensive mutation and deletion analyses on both the AhR
and Arnt basic regions; they show that Arnt behaves similarly to Class B
proteins Max and USF (Swanson, H. I. and Yang, J.-H., J. Biol. Chem., 1996, 271, 31657-35661). For Arnt, they found that GIu32, Arg35, and Arg36 were critical for recognition of 5'-GTG, same as Max. They do not discuss His28.
When the Glu32 side chain is shortened by mutation to Asp, or when Arg35 and Arg36 are mutated to Gln, DNA binding is abolished, but heterodimerization to AhR is unaffected. When Arg33 and Arg34 are mutated to Gin, DNA-binding is reduced somewhat, so it is likely that these residues are involved in nonspecific phosphodiester interactions, same as for the corresponding amino acids in Max, MyoD, and USF.

Unlike Arnt, AhR does not display classic bHLH behavior. When AhR Pro3l and Ser32 are substituted with Leu and Glu to give a Max-like sequence, binding is abolished. Replacements at Arg34 and Arg36 also virtually abolished DNA-binding. When the two prolines in the AhR sequence KPIPAE
(see Table 6) are replaced with alanines, -70% binding is retained, so these Pro's are not involved in critical interactions. Critical residues for specific DNA
binding are Pro3l, Ser32, Lys33, Arg34, His35, and Arg36 (Swanson, H. I.

and Yang, J.-H., J. Biol. Chem., 1996, 271, 31657-35661). Although AhR is similar to other bHLH and bHLHZ proteins in that it uses the same stretch of amino acids to contact DNA (Table 6), given the four prolines and glycine in its basic region, AhR must be using a nonhelical DNA-binding structure.

= How do AhR and Arnt Interact with DNA? The absolutely conserved Asn235 of GCN4 spans the major groove to accept a hydrogen bond from N4 of C3' and donate a hydrogen bond to 04 of T4 (Table 6; Asn235 of GCN4 is aligned with His28 of Max) (Ellenberger, T. E. et al., Cell 1992, 71, 1223-1237). This crucial interaction requires that the basic region lie deep in the major groove and specifies two of four base pairs in each half site. GCN4 Asn235 corresponds to Max His28, as far as comparisons between different protein families can be made. Asn is capable of a bifurcated hydrogen bond and can therefore dictate the identities of two base pairs, and this may explain how bZIP proteins bind a four bp half site, whereas bHLH proteins bind only three bp. In GCN4, the methyl side chains of Ala238 and Ala239 make van der Waals contacts with the C5 methyl groups on T4 and T2'. The highly conserved GIu32 of Max corresponds in position with Ala239; Glu32 dictates the outer CA step in Max, much as GCN4 Asn235 dictates the outer TG step.
Noteworthy is the van der Waals contact that the USF GIu32 side chain makes to thymine, akin to that of Ala238 and Ala239 in GCN4. GCN4 Arg243 makes bidentate contact with N7 and 06 of G1' in the AP-1 complex (Ellenberger, T.
E. et al., Cell 1992, 71, 1223-1237), and N7 of G1' and the phosphodiester group of Cl with ATF/CREB (Konig, P. and Richmond, T. J., J. Mol. Biol.
1993). In bHLHZ proteins, the corresponding Arg36 makes a single contact with N7 of the central G, and Leu36 in the MyoD bHLH makes no DNA
contact. Although the bHLH and bZIP are distinct protein families, their DNA-binding domains display significant similarity in structure and alignment.
Because the bZIP, bHLH, and bHLHZ all use basic a-helical structures to bind the major groove, the corresponding amino acids are similarly placed for DNA-binding function (see alignment in Table 6). Therefore, as long as the basic regions are properly situated in the major groove, a-helical structure and DNA-binding function will be retained. The caveat is to maintain proper orientation for binding; the hinge region is responsible for orienting the basic regions, and therefore flexibility is designed into the hinge by generating variants of each bHLH/bZIP hybrid differing by a single amino acid in the hinge. Although the AhR basic region is unlikely to have substantial helical structure, it is possible that the conserved Asn3l in AhR and Sim may also make a bidentate interaction with DNA, thereby specifying the outer two base pairs. In order to recognize the XRE1 5'-TTGC sequence, Asn3l needs to span the major groove to contact 04 of T4 and N6 of A3' to specify the outer TT step. The inner GC base pairs are recognized by the conserved Lys/Arg34 and Arg37. Although the Class C protein AhR is unlikely to utilize a helical structure to bind the major groove, its basic region contains several conserved amino acids capable of making DNA contacts similar to those discussed for the bHLH and bZIP.

Further Simplification of the Protein Scaffold with Alanine Mutagenesis Alanine is substituted in the basic region of the minimalist bZIP proteins of the invention (Table 7). Because Arnt is similar to Max and USF, Ala substitutions for Arnt is more straightforward than for AhR, which is not helical, but Ala replacements in AhR should become so.

The Ala-mutants Max-13A, C/EBP-18A, Arnt-16A, and AhR-17A are designed to maintain both specific and nonspecific protein-DNA interactions, akin to the GCN4 mutant 1 1A; even more heavily mutated proteins are designed wherein only specific contacts are conserved, similar to 18A. In Table 7, specific interactions are boldfaced, nonspecific phosphodiester contacts are underlined, and proposed alanine substitutions are italicized; the Max structure and mutagenesis work on AhR and Arnt are the basis for Table 7 (Ferre-D'Amare, A. R., et al. Nature 1993, 363, 38-45. Swanson, H. I. and Yang, J.-H. J. Biol. Chem. 1996, 271, 31657-31665.) At the bottom of Table 7, the GCN4 sequence is shown as a reference; note that in C/EBP, the highly conserved position 239 is occupied by Val rather than Ala; Va1239 is conserved in C/EBP-18A. Most bZIP proteins have A1a239. Johnson has shown that Va1239 is an important determinant for C/EBP binding to the half site 5'-TTGC (Johnson, P. F., Mol. Cell. Biol. 1993, 13, 6919-6930). Johnson generated numerous GCN4-C/EBP hybrids wherein the fusion junctions of the two proteins varied; Va1239 was critical for discrimination between GCN4 vs.
C/EBP sites. Therefore, another mutant would be C/EBP-19A, in which Va1239 4 Ala.

Eniarging the DNA-Binding Repertoire: Determinants of Specificity in bHLH and bHLH/ZIP Proteins Class A proteins target 5'-CAG=CTG, whereas Class B proteins target 5'-CAC=GTG. bHLH proteins MyoD and E47 have highly conserved Arg31 and Leu/VaI36, in contrast to Class B bHLH/ZIP proteins, which contain the absolutely conserved Arg36 and a hydrophobic amino acid at position 31 (Leu, Ile, Val, Met-no contact with DNA). MyoD can be changed from Class A to B
specificity by mutating Leu36 to Arg ( Blackwell, T. K. et al., Mol. Cell.
Biol.
1993, 13, 5216-5224). If Max-13A is a functional Class B protein, then Max-13A-RL may have Class A binding specificity. In this case, Arg31 from MyoD
is also retained, because it is highly conserved, and the MyoD crystal structure shows that it makes nonspecific interactions with the DNA backbone plus a weak specific contact in the major groove.

These Ala-based minimalist bZIP proteins and the mutants that switch binding between C/EBP and GCN4 or Max and MyoD are tests of the protein-design capabilities of the present invention (Table 8).

Yeast One-Hybrid Assay for Monitoring the In Vivo interactions between Proteins and DNA

Construction of Reporter Strain A reporter strain was constructed such that the E-box target, 5'-CACGTG, resides upstream of the HIS3 reporter gene. The bHLH/bZIP hybrid was fused to the GAL4 transcriptional activation domain so that if a hybrid binds the E-box, the HIS3 protein will be expressed, thus allowing yeast to survive under conditions of histidine auxotrophy. Four tandem copies of the E-box (as shown in Table 9) were cloned into the pHISi-1 integrating reporter vector (Matchmaker One-Hybrid System, Clontech). After insertion of the E-box insert, the pHISi-1 vector was linearized and incorporated into the yeast genome by homologous recombination to generate Saccharomyces cerevisiae YM4271[pHlSi-1/E-box]. To assess background due to the reporter, 3-aminotriazole (3-AT) was used as a competitive inhibitor of the HIS3 protein. Because it is possible that the reporter may be activated by endogenous factors, YM4271 [pHISi-1/E-box] was subjected to titration with varying amounts of 3-AT to measure the concentration of 3-AT sufficient for suppression of background growth. Results demonstrate that 10 mM 3-AT
suppresses background expression.

The recombinant plasmid pGAD424 having the Arntl-C/EBP (SEQ ID NO. 7) insertion was transformed into the reporter strain and plated on SD -His -Leu with 10mM3-AT to test for binding activity (Figure 2A). The vector alone was transformed as a negative control (Figure 2B). The results show that the Arntl-C/EBP minimalist bZIP protein was able to bind to the E-box target DNA
sequence.

Evolution of the Minimalist bZIP proteins The minimalist bZIP protein carrying the basic region of Max, a hinge region and the leucine zipper domain of C/EBP was evolved into better binders of the E-box DNA sequence by use of a modified yeast one-hybrid assay, in which mutated PCR fragments were cloned via homologous recombination. A
schematic of the process is shown in Figure 3.

These resultant new protein constructs may compete efficiently with the Myc-Max heterodimer for binding the E-box site and would be therefore able to repress myc transcriptional activity and control the aberrant activity of myc upon oncogenic transformation. Because these fusion proteins do not contain the native Max HLHZ dimerization domain, as it has been replaced by the C/EBP leucine zipper, they are unable to heterodimerize with Myc.

MM3 Protein Construction of Protein Library The gene for Max-C/EBP served as the template for mutagenic PCR for generation of the protein library. PCR reaction conditions were adjusted to minimize mutational bias and to yield 1-3 mutations/gene. The mutated Max-C/EBP genes were inserted into vector pGAD424, which carries a GAL4 activation domain and LEU2 selection marker (Matchmaker One-Hybrid System, Clontech).

The original Max-C/EBP hybrid, as shown in Table 10, was tested in the standard yeast one-hybrid assay using the reporter strain described above, and the binding was undetectable. The binding was undetectable likely due to the fact that the E-box used lacks flanking regions and also based on the spacing between the tandem repeats. For example, two other E-box variants, the Max E-box (favored target site for Max) and Arnt E-box (favored target site for Arnt) are also shown in Table 9. Although Max and Arnt both target the core E-box (5'-CACGTG), they have flanking sequence preferences.
Additionally, the spacing between the four E-boxes as denoted by N(o, 2, 4, 8) may play a role.

Therefore, the Matchmaker One-Hybrid System from Clontech was modified in order to perform directed evolution. Vector pGAD424 was linearized and cotransformed with an excess of mutagenized Max-C/EBP genes. These mutant PCR products share a 48 base-pair homology with both 5' and 3' ends of the linearized pGAD424 vector. Approximately 106 independent clones were generated during one round of selection.

Library Screening Transformation of the yeast cells was performed by electroporation.
Following electroporation, cells were plated on minimal selective medium lacking leucine and histidine with the appropriate amount of 3-AT to suppress background. Plasmid pGAD424 was also transformed as a negative control;
the activation domain alone did not activate the reporter system.

Selection and Validation of Positive Clones In order to confirm positive clones, that is, those clones expressing protein that bind the DNA target site, a number of validation experiments were performed. Positive colonies indicating potential protein-DNA recognition at the E-box appeared after 4-6 days incubation. Colonies were considered positive if their diameters exceeded 2 mm. Only one grew after replating.
Plasmid DNA from this positive clone was transformed into the control yeast strain containing pHISi-1 plasmid YM4271[pHISi-1], with no integrated target DNA, to test specific binding to E-box. This serves as a negative control to confirm that the selected protein is unable to activate the HIS3 reporter in the absence of target DNA. After successfully passing these two validations, plasmid DNA was sequenced. The sequence is shown in Table 10 as MM3 (mutant Max 3).

The sequence demonstrates that there has been a frameshift resulting in 8 different amino acids in the C-terminal end of the basic region compared to the original Max1-C/EBP minimalist protein sequence. This change would affect spacing and orientation. The changes make sense because they are good alpha-helix formers, one basic residue (R) which is good for making an electrostatic interaction with DNA (maybe far from DNA, but reasonable), and two hydrophilic residues (S and T) that help solubility in water. The sequence also optionally has a mutation in the leucine zipper domain as shown in Table 10.

To reconfirm that the plasmid is truly the source of protein that binds E-box and activates reporter, the sequenced plasmid was again transformed into YM4271[pHISi-1/E-box] and assayed for growth under library screening conditions. Plasmid was extracted from colonies and sequenced a second timee The second sequence matched the first sequence for MM3, thereby confirming the result.

A plasmid containing just the C/EBP leucine zipper was constructed to test the indispensability of the basic region for DNA binding. Although the C/EBP
zipper is expected to play some role in aiding DNA binding, for the well-structured, a-helical zipper helps to stabilize the more disordered basic region, the zipper itself is not part of the DNA-binding domain of the bZIP.
This control containing just the leucine zipper showed no colony growth as expected.

The MM3 is a stronger binder of the E-box than Max-C/EBP (Figure 4) in this assay; because the Yl H assay is not quantitative, it is not possible to say how strongly MM3 binds E-box. yeast cells expressing MM3 were plated on plates containing 0-60 mM 3-AT to test the strength of binding between MM3 and E-box. Since 3-AT inhibits HIS3 protein necessary for cell survival under histidine auxotrophy, cell growth on higher concentrations of 3-AT
demonstrate strong binding of MM3 to E-box. Even at 60 mM 3-AT, significant colony growth occurs after four days.

MM3 was discovered after only one round of evolution; further evolution may uncover better E-box binding mutants.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Table 1: Max basic repion, C/EBP leucine zipper (with RIRIGIR linker) Maxl- ADKRAHHNALERKRRDHIKDSFHS-RIR-LEQKVLELTSDNDRLRKRVEQLSRELDTL SEQ ID No. 1 C/EBP R
Max2- ADKRAHHNALERKRRDHIKDSFHSL-RIR-LEQKVLELTSDNDRLRKRVEQLSRELDTL SEQ ID No. 2 C/EBP(R) Max3- ADKRAHHNALERKRRDHIKDSFHSLR-RIR-LEQKVLELTSDNDRLRKRVEQLSRELDTL SEQ ID No.

C/EBP(R) Maxl- ADKRAHHNALERKRRDHIKDSFHS-GIR-LEQKVLELTSDNDRLRKRVEQLSRELDTL SEQ ID No. 4 C/EBP G
cn cn Max2- ADKRAHHNALERKRRDHIKDSFHSL-GIR-LEQKVLELTSDNDRLRKRVEQLSRELDTL SEQ ID No. 5 N
N
C/EBP(G U'I
L"
Max3- ADKRAHHNALERKRRDHIKDSFHSLR-GIR-LEQKVLELTSDNDRLRKRVEQLSRELDTL SEQ ID No.

C/EBP(G) cn ~
Note: RIR/GIR spacer added for facile cloning (BamH / site for joining basics and zippers).
w Table 2: Arnt basic region, C/EBP leucine zipper (with R/R/GIR linker) Arntl- KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITE-RIR- SEQ ID No. 7 C/EBP(R) LEQKVLELTSDNDRLRKRVEQLSRELDTL
Arnt2- KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITEL-RIR- SEQ ID No. 8 C/EBP R) LEQKVLELTSDNDRLRKRVEQLSRELDTL
Arnt3- KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITELS-RIR- SEQ ID No. 9 C/EBP(R) LEQKVLELTSDNDRLRKRVEQLSRELDTL
Arntl- KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITE-GIR- SEQ ID No. 10 C/EBP(G) LEQKVLELTSDNDRLRKRVEQLSRELDTL
Arnt2- KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITEL-GIR- SEQ ID No. 11 C/EBP G LEQKVLELTSDNDRLRKRVEQLSRELDTL N
cn N
Arnt3- KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITELS-GIR- SEQ ID No. 12 C/EBP(G LEQKVLELTSDNDRLRKRVEQLSRELDTL o cn r r i w Table 3: First Generation Fusion Proteins: C/EBP or Max basic region. Fos or Jun leucine zipper.Dashes within protein sequences are place holders for sequence alignment purposes. Highly conserved amino acids are in bold. The putative basic helix-loop-helix is marked. The basic regions are aligned at highly conserved positions 32 and 36 using numbering for Max.

basic 32 36 ~ ZIP SEQ ID
No.
*C/EBP-Fos SNEYRVRRERNNIAVRKSRDKAKQRNVE---Maxl-Jun ADKRAHHNALERKRRDHIKDSFHS---LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVN 14 Max2-Jun ADKRAHHNALERKRRDHIKDSFHSL-LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVN 15 Max3-Jun ADKRAHHNALERKRRDHIKDSFHSLR-LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVN 16 *replace Q with E in the ZIP for cloning with Xho I enzyme.
O

O
O

F' F' I
O
W

Table 4: Second Generation Fusion Proteins: AhR or Arnt basic region, Fos or Jun leucine zipper.
Dashes within protein sequences are place holders for sequence alignment purposes. Highly conserved amino acids are in bold. The putative basic helix-loop-helix is marked. The basic regions are aligned at highly conserved positions 32 and 36 using numbering for Max.

basic 32 36 ~ ZIP SEQ
ID No.
AhR1- ASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDR---Fos AhR2- ASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDRL--Fos AhR3- ASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDRLA-Fos *replace Q with E in the ZIP for cloning with Xho I enzyme. N
cn N

basic 32 36 / ZIP SEQ
ID No.
cn Arntl KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITE----Jun Arnt2 KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITEL--LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVN 21 ow -Jun Arnt3 KFLRCDDDQMSNDKERFARENHSEIERRRRNKMTAYITELS--Jun Table 5 Reference Sequences: Dashes within protein sequences are place holders for sequence alignment purposes. Highly conserved amino acids are in bold. The putative basic helix-loop-helix is marked.

basic ~ Helix I ~ loop ~ Helix II SEQ IDNO.
AhR ASRKRRKPVQKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLPF-PQDVINKLDKL-Arnt KFLRCDDDQMSNDKERFARENHSEIERRRR-NKMTAYITELSDMVPT-CSALARKPDKL-basic ~ ZIP SEQ ID NO.
cn N

Fos KRRIRRERNKMAAAKCRNRRRELTDT-LQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRP 25 Jun KAERKRMRNRIAASKCRKRKLERIAR-LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVN 26 "' F-' F-' Table 6: Sequence alignment of basic regions of bHLH, bHLHZ, and bZIP
proteins. Numbering is same as that for Max (Ferre-D'Amare, A. R. et al., Nature 1993, 363, 38-45). Highly conserved amino acids are in bold. Adapted from (Swanson, H. I., et al., J. Biol. Chem., 1995, 270, 26292-26302).

Basic Regioas(PartiaZ) DNA binding sites Class B 25 30 321 SEQ ID No.
Max ADKRAHHNALERKRR 5'-CAC=GTG 27 Myc NVKRRTHNVLERQRR 28 Arnt RFARENHSEIERRRR 32 CONSENSUS --BB--HN--ERRRR N
cn Class A 321 SEQ ID No. Ln MyoD ADRRKAATMRERRRL 5'-CAG=CTG 33 'n E12 KERRVANNARERLRV 36 I Tall VVRRIFTNSRERWRQ 37 CONSENSUS --RR---N-RER-R-w Class C 4 3 21 SEQ ID No.
AhR KPIPAEGIKSNPSKRHRD 5'-T(C/T)GC half site 38 Sim MKEKSKNAARTRRE 5'-GT(A/G)C half site 39 CONSENSUS ------N--B--R-bZZP 235 4321 SEQ ID NO.
GCN4 DPAALKRARNTEAARRSR 5'-TGAC half site 40 Table 7: Alanine-based mutants. Numbering is same as that for Max (Ferre-D'Amare, A. R. et al., Nature 1993, 363, 38-45).

Basic Helix I

Max ADKRAHHNALERKRRDHIKDSFHS SEQ ID NO. 41 Max-13A AAKRAAHNAAERARRAAAAAAAAA SEQ ID NO. 42 Arnt KFLRCDDDQMSNDKERLARENHSEIERRRRNKMTAYITE SEQ ID NO. 43 Arnt-16A AAARAAHSAAERARRAAAAAAAAA SEQ ID NO. 44 AhR ASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDR SEQ ID NO. 45 AhR-17A AAAAAAAAAPSKRHRAAAAAAAAA SEQ ID NO. 46 bzlP Sequences 235 Ln C/EBP SNEYRVRRERNNIAVRKSRDKAKQRNVE SEQ ID NO. 47 L' C/EBP-18A AAAAAAARARNNAAVRKSRAAAAAAAAA SEQ ID NO. 48 - - - Ln GCN4 DPAALKRARNTEAARRSRARKLQRMKQ SEQ ID NO. 49 - - ~

w Table 8: Alanine-based mutants designed to switch between Class A and Class B
binding sites. Numbering is same as that for Max Basic Helix I

Max ADKRAHHNALERKRRDHIKDSFHS SEQ ID NO. 41 MyoD ADRRKAATMRERRRLSKVNEAFET SEQ ID NO. 50 Max-13A AAKRAAHNAAERARRAAAAAAAAA binds 5'-CAC=GTG SEQ ID NO. 42 Max-13A-RL AAKRAAHNARERARLAAAAAAAAA binds 5'-CAG=CTG SEQ ID NO. 51 ~
O

N

N

O
O

F' F' I
O
W

Table 9: E-box Inserts Four Tandem Copies of E-box Insert 5'- CACGTG 4-3' Max E-box Insert 5'-[CCACGTGGN o 2 6]4-3' Arnt E-box Insert 5'-[TCACGTGAN(o, 2, 6)]4-3' Table 10: Evolution of Minimalist bZIP Proteins: Sequence of original Max-C/EBP comprising the basic region of Max, residues 22-47, an RIR linker providing a BamH I site that facilitates cloning, and the C/EBP leucine zipper, residues 310-338. Sequence of MM3 that was evolved after one round of directed evolution in the modified yeast one-hybrid system. Mutations are in bold underline. In the Max basic region, a Leu is mutated to Ser; a frameshift mutation occurs at the C-terminal end of the basic region and in the C/EBP
zipper, there is optionally an Asn mutated to Ser.

Max- ADKRAHHNALERKRRDHIKDSFHS-RIR- SEQ ID NO. 52 C/EBP LEQKVLELTSDNDRLRKRVEQLSRELDTL
MM3a ADKRAHHNASERKRRDTSRTLSTL-RIR- SEQ ID NO. 53 LEQKVLELTSDSDRLRKRVEQLSRELDTL
MM3b ADKRAHHNASERKRRDTSRTLSTL-RIR- SEQ ID NO. 54 LEQKVLELTSDNDRLRKRVEQLSRELDTL

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE
SPECIFICATION

Agre P, Johnson PF, McKnight SL (1989) Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and C/EBP.
Science 246: 922-926.
Amati B, Land H (1994) Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death. Curr Opin Gene Dev 4(102-108).
Amati B, Brooks MW, Levy N, Littlewood TD, Evan GI et al. (1993) Oncogenic Activity of the c-Myc Protein Requires Dimerization with Max. Cell 72:
233-245.
Aylon Y, Kupiec M (2004) New insights into the mechanism of homologous recombination in yeast. Mut Res 566(231-248).
Bacsi SG, Reisz-Porszasz S, Hankinson 0 (1995) Orientation of the Heterodimeric Aryl Hydrocarbon (Dioxin) Receptor Complex on Its Asymmetric DNA Recognition Sequence. Mol Pharmacol 47: 432-438.
Benhar I(2001) Biotechnological applications of phage and cell display.
Biotech Adv 19: 1-33.
Blackwell TK, Huang J, Ma A, Kretzner L, Alt FW et al. (1993) Binding of myc proteins to canonical and noncanonical DNA sequences. Mol Cell Biol 13: 5216-5224.
Blackwood EM, Eisenman RN (1991) Max: A Helix-Loop-Helix Zipper Protein That Forms a Sequence-Specific DNA-Binding Complex with Myc.
Science 251: 1211-1217.
Brownlie P, Ceska TA, Lamers M, Romier C, Stier G et al. (1997) The crystal structure of an intact human Max-DNA complex: new insights into mechanisms of transcriptional control. Structure 5: 509-520.
Butler T, Alcalde M (2003) Preparing libraries in Saccharomyces cerevisiae.
In: Arnold FH, Georgiou G, editors. Directed Evolution Library Creation.
Totowa, New Jersey: Humana Press.
Cadepond F, Schweizer-Groyer G, Segard-Maurel I, Jibard N, Hollenberg SM
et al. (1991) Heat Shock Protein 90 as a Critical Factor in Maintaining Glucocorticosteroid Receptor in a Nonfunctional State. J Biol Chem 266: 5834-5841.
Casimiro DR, Wright PE, Dyson HJ (1997) PCR-based gene synthesis and protein NMR spectroscopy. Structure 5: 1407-1412.
Casimiro DR, Toy-Palmer A, Blake II RC, Dyson HJ (1995) Gene synthesis, high-level expression, and mutagenesis of Thiobacillus ferrooxidans rusticyanin: His 85 is a ligand to the blue copper center. Biochemistry 34: 6640-6648.
Chen X, Rubock MJ, Whitman M (1996) A transcriptional partner for MAD
proteins in TGF-(3 signalling. Nature 383: 691-696.
Cherry JR, Lamsa MH, Scneider P, Vind J, Svendsen A et al. (1999) Directed evolution of a fungal peroxidase. Nat Biotech 17: 379-384.

Crosby DG, Wong AS, Plimmer JR, Woolson EA (1971) Photodecomposition of Chlorinated Dibenzo-p-Dioxins. Science 173: 748-749.
Cuthill S, Wilhelmsson A, Poellinger L (1991) Role of the Ligand in Intracellular Receptor Function: Receptor Affinity Determines Activation In Vitro of the Latent Dioxin Receptor to a DNA-Binding Form. Mol Cell Biol 11: 401-411.
Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC et al. (1982) Human c-myc oncogene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 79:
7824-7827.
Dolwick KM, Schmidt JV, Carver LA, Swanson HI, Bradfield CA (1993) Cloning and Expression of a Human Ah Receptor cDNA. Mol Pharm 44: 911-917.
Ellenberger T, Fass D, Arnaud M, Harrison SC (1994) Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer. Genes Dev 8: 970-980.
Ellenberger TE, Brandl CJ, Struhl K, Harrison SC (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted a helices:
Crystal stucture of the protein-DNA complex. Cell 71: 1223-1237.
Ferre-D'Amare AR, Prendergast GC, Ziff EB, Burley SK (1993) Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature 363: 38-45.
Ferre-D'Amare AR, Pogonec P, Roeder RG, Burley SK (1994) Structure and function of the b/HLH/Z domain of USF. EMBO J 13: 180-189.
Fingerhut MA, Halperin WE, Marlow DA, Piagitelli LA, Honchar PA et al.
(1991) Cancer Mortality in Workers Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin. N Engl J Med 324: 212-218.
Fisher JM, Jones KW, Whitlock JP (1989) Activation of Transcription as a General Mechanism of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Action. Mol Carcinogen 1: 216-221.
Fujisawa-Sehara A, Sogawa K, Yamane M, Fujii-Kuriyama Y (1987) Characterization of xenobiotic responsive elements upstream from the drug-metabolizing cytochrome P-450c gene: a similarity to glucocorticoid regulatory elements. Nucl Acids Res 15: 4179-4191.
Gardner L, Lee L, Dang C (2002) The c-Myc Oncogenic Transcription Factor.
In: Bertino JR, editor. Encyclopedia of Cancer. San Diego, CA:
Academic Press.
Glover JNM, Harrison SC (1995) Crystal structure of the heterodimeric bZIP
transcription factor c-Fos-c-Jun bound to DNA. Nature 373: 257-261.
Gradin K, McGuire J, Wenger RH, Kvietikova I, Whitelaw ML et al. (1996) Functional Interference between Hypoxia and Dioxin Signal Transduction Pathways: Competition for Recruitment of the Arnt Transcription Factor. Mol Cell Biol 16: 5221-5231.
Gray LEJ, Ostby JS (1995) In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicol Appl Pharmacol 133: 285-294.
Hoffman EC, Reyes H, Chu F, Sander F, Conley LH et al. (1991) Cloning of a Factor Required for Activity of the Ah (Dioxin) Receptor. Science 252:
954.

Hogenesch JB, Chan WK, Jackiw VH, Brown RC, Gu Y-Z et al. (1997) Characterization of a Subset of the Basic-Helix-Loop-Helix-PAS
Superfamily That Interacts with Components of the Dioxin Signaling Pathway. J Biol Chem 272: 8581-8593.
Jakoby M, Weisshaar B, Droge-Laser w, Vicente-Carbajosa J, Tiedemann J
et al. (2002) bZIP transcription factors in Arabidopsis. Trends Plant Sci 7: 106-111.
Johnson EF (1991) A Partnership between the Dioxin Receptor and a Basic Helix-Loop-Helix Protein. Science 252: 924.
Keller W, Konig P, Richmond TJ (1995) Crystal structure of a bZIP/DNA
Complex at 2.2 A: Determinants of DNA specific recognition. J Mol Biol 254: 657-667.
Kumar R, Chen S, Scheurer D, Wang Q-L, Duh E et al. (1996) The bZIP
Transcription Factor Nrl Stimulates Rhodopsin Promoter Activity in Primary Retinal Cell Cultures. J Biol Chem 271: 29612-29618.
Konig P, Richmond TJ (1993) The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA
flexibility. J Mol Biol 233: 139-154.
Lajmi AR, Wallace TR, Shin JA (2000a) Short, Hydrophobic, Alanine-based Proteins Based on the bZIP Motif: Overcoming Inclusion Body Formation and Protein Aggregation During Overexpression, Purification, and Renaturation. Prot Exp Purif 18: 394-403.
Lajmi AR, Lovrencic ME, Wallace TR, Thomlinson RR, Shin JA (2000b) Minimalist, Alanine-based, Helical Protein Dimers Bind to Specific DNA
Sites. J Am Chem Soc 122: 5638-5639.
Landschulz WH, Johnson PF, McKnight SL (1988) The Leucine Zipper: A
Hypothetical Structure Common to a New Class of DNA Binding Proteins. Science 240: 1759-1764.
Lindebro MC, Poellinger L, Whitelaw ML (1995) Protein-protein interaction via PAS domains: role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt transcription factor complex. EMBO J 14: 3528-3539.
Luque I, Mayorga OL, Freire E (1996) Structure-based thermodynamic scale of a-helix propensities in amino acids. Biochemistry 35: 13681-13688.
Ma PCM, Rould MA, Weintraub H, Pabo CO (1994) Crystal Structure of MyoD
bHLH Domain-DNA Complex: Perspectives on DNA Recognition and Implications for Transcriptional Activation. Cell 77: 451-459.
Murre C, McCaw PS, Baltimore D (1989) A New DNA-Binding and Dimerization Motif in Immunoglobulin Enhancer Binding, daughteriess, MyoD, and myc Proteins. Cell 56: 777-783.
Nair SK, Burley SK (2003) X-Ray Structure of Myc-Max and Mad-Max Recognizing DNA: Molecular Bases of Regulation by Proto-Oncogenic Transcription Factors. Cell 112: 193-205.
Nau MM, Brooks BM, Battey J, Sausville E, Gazdar AF et al. (1985) L-Myc, a new Myc-related gene amplified and expressed in human small cell lung cancer. Nature 318: 69-73.
Nesbit CD, Tersak JM, Prochownik EV (1999) MYC oncogenes and human neoplastic disease. Oncogene 18: 3004-3016.

Oliphant AR, Struhl K (1987) The use of random-sequence oligonucleotides for determining consensus sequences. Methods Enzymol 155: 568-582.
Oliphant AR, Nussbaum AL, Struhl K (1986) Cloning of random-sequence oligodeoxynucleotides. Gene 44: 177-183.
Orian A, van Steensel B, Deirow J, Bussemaker HJ, Li L et al. (2003) Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev 17: 1101-1114.
Poland A, Knutson JC (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Ann Rev Pharmacol Toxicol 22: 517.
Pollenz RS, Sattler CA, Poland A (1995) The Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Receptor Nuclear Translocator Protein Show Distinct Subcellular Localization in Hepa 1 c1 c7 Cells by Immunofluorescence Microscopy. Mol Pharmacol 45: 428-438.
Pongratz I, Antonsson C, Whitelaw ML, Poellinger L (1998) Role of the PAS
Domain in Regulation of Dimerization and DNA Binding Specificity of the Dioxin Receptor. Mol Cell Biol 18: 4079-4088.
Prytulla S, Dyson HJ, Wright PE (1996) Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin. FEBS Left 399: 283-289.
Reisman D, Elkind NB, Roy B, Beamon J, Rotter V (1993) c-Myc Transactivates the p53 Promoter through a Required Downstream CACGTG Motif. Cell Growth Differ 4: 57-65.
Reyes H, Reisz-Porszasz S, Hankinson 0 (1992) Identification of the Ah Receptor Nuclear Translocator Protein (Arnt) as a Component of the DNA Binding Form of the Ah Receptor. Science 256: 1193-1195.
Roberts L (1991) Research News: Dioxin Risks Revisited. Science 251: 624-626.
Rowlands JC, Gustafsson J-A (1997) Aryl Hydrocarbon Receptor-Mediated Signal Transduction. Crit Rev Toxicol 27: 109-134.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition. New York: Cold Spring Harbor Press.
Schwab M, Varmus HE, Bishop JM, Grzeschik KH, Naylor SL et al. (1984) Chromosome localization in normal human cells and neuroblastomas of a gene related to c-Myc. Nature 308: 288-291.
Sellers JW, Struhl K (1989) Changing Fos oncoprotein to a Jun-independent DNA-binding protein with GCN4 dimerization specificity by swapping 'leucine zippers'. Nature 341: 74-76.
Struhl K (1989) Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eucaryotic transcriptional regulatory proteins. Trends Biochem Sci 14:
137-140.
Suga M, Hatakeyama T (2003) High efficiency electroporation by freezing intact cells with addition of calcium. Curr Genet 43(206-211).
Swanson HI, Yang J-H (1996) Mapping the Protein/DNA Contact Sites of the Ah Receptor and Ah Receptor Nuclear Translocator. J Biol Chem 271:
31657-31665.

Swanson HI, Chan WK, Bradfield CA (1995) DNA Binding Specificities and Pairing Rules of the Ah Receptor, ARNT, and SIM Proteins. J Biol Chem 270: 26292-26302.
Swers JS, Kellogg BA, Wittrup KD (2004) Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display. Nuci Acid Res 32: 36-44.
Taub R, Kirsch I, Morton C, Lenoir G, Swan D et al. (1982) Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc Natl Acad Sci USA 79: 7837-7841.
Tobias AV (2003) Preparing libraries in Escherichia coli. In: Arnold FH, Georgiou G, editors. Directed Evolution Library Creation. Totowa, New Jersey: Humana Press.
Wang BS, Pabo CO (1999) Dimerization of zinc fingers mediated by peptides evolved in vitro from random sequences. Proc Natl Acad Sci USA 96:
9568-9573.
Whitelaw M, Pongratz I, Wilhelmsson A, Gustafsson J-A, Poellinger L (1993) Ligand-Dependent Recruitment of the Arnt Coregulator Determines DNA Recognition of the Dioxin Receptor. Mol Cell Biol 13: 2504-2514.
Wu L, Whitlock JP (1993) Mechanism of dioxin action: receptor-enhancer interactions in intact cells. Nuci Acid Res 21: 119-125.
Yin X, Grove L, Prochownik EV (1998) Lack of transcriptional repression by max homodimers. Oncogene 16: 2629-2637.
Zervos AS, Gyuris J, Brent R (1993) Mxi1, a Protein hat Specifically Interacts with Max to Bind Myc-Max Recognition Sites. Cell 72: 223-232.

Claims (41)

1. A minimalist bZIP protein comprising:

a) a basic region of a basic helix-loop-helix protein (bHLH);
b) a hinge region; and c) a leucine zipper domain of a bZIP protein, wherein the minimalist bZIP protein binds a target DNA sequence.

2. The minimalist bZIP protein of claim 1, wherein the hinge region comprises 0-20 amino acids.

3. The minimalist bZIP protein of claim 2, wherein the hinge region comprises 3 amino acids.

4. The minimalist bZIP protein of claim 3, wherein the hinge region comprises the sequence RIR or GIR.

5. The minimalist bZIP protein of any one of claims 1-4, wherein the hinge region further comprises an additional 1, 2 or 3 amino acids derived from the C-terminal end of helix 1 of the bHLH protein between the basic region and the hinge region.

6. The minimalist bZIP protein of any one of claims 1-5, comprising 30 to 100 amino acids.

7. The minimalist bZIP protein of claim 6, comprising 40 to 60 amino acids.

8. The minimalist bZIP protein of any one of claims 1-7, wherein the bZIP
protein is selected from the group consisting of C/EBP, Jun, Fos, GCN4 and CREB.

9. The minimalist bZIP protein of any one of claims 1-8, wherein the bHLH protein is selected from the group consisting of a bHLH
subvariant, a bHLHZ subvariant and a bHLH/PAS subvariant.

10. The minimalist bZIP protein of claim 9, wherein the bHLH subvariant is selected from the group consisting of MyoD, Myc, E2A, E47, E12, TALL, Id proteins, GL3, EGL3, TFEB, PIF1, PIL6, ATH, NGN and HAND1.

11. The minimalist bZIP protein of claim 9, wherein the bHLHZ subvariant is selected from the group consisting of Mad, Mxi, Max, Myc, Spz1, USF, Mash, BMP, TFE3 and AP4.

12. The minimalist bZIP protein of claim 9, wherein the bHLH/PAS
subvariant is selected from the group consisting of AhR, ARNT, HIF1.alpha., HIF-2.alpha., HIF-3.alpha., Per and Sim.

13. The minimalist bZIP protein of any one of claims 1-12, wherein the target DNA sequence is an E-box sequence selected from 5'-CAG
CTG and 5'-CAC-GTG.

14. The minimalist bZIP protein of any one of claims 1-12, wherein the target DNA sequence is an XRE1 sequence 5'-TTGC-GTG.

15. The minimalist bZIP protein of any one of claims 1-12, wherein the target DNA sequence is a half-site sequence 5'-T(C/T)GC or 5'-GT(A/G)C.

16. The minimalist bZIP proteins of any one of claims 1-15, wherein the basic region is mutated to generate an alanine rich sequence.

17. The minimalist bZIP protein of any one of claims 1-16 further evolved into a stronger DNA binding protein by mutagenesis and selection.

18. A minimalist bZIP protein comprising the amino acid sequence as shown in SEQ ID NO. 53.

19. A minimalist bZIP protein comprising the amino acid sequence as shown in SEQ ID NO. 54.

20. A minimalist bZIP protein comprising:
a) a basic region from Max;

b) a hinge region; and c) a leucine zipper region from C/EBP, wherein the minimalist bZIP protein binds an E-box target DNA
sequence.

21. The minimalist bZIP protein of claim 20, comprising an amino acid sequence selected from the sequences as shown in SEQ ID NOs. 1-6.

22. A minimalist bZIP protein comprising:

a) a basic region from Arnt;
b) a hinge region; and c) a leucine zipper region from C/EBP, wherein the minimalist bZIP protein binds an XRE1 target DNA
sequence or an E-box target DNA sequence.

23. The minimalist bZIP protein of claim 22, comprising an amino acid sequence selected from the sequences as shown in SEQ ID NOs. 7-12.

24. The minimalist bZIP protein of any one of claims 1-23, wherein the protein is further fused to an activation domain.

25. The minimalist bZIP protein of claim 24, wherein the activation domain is derived from Ga14, Mad, Myc, and VP16.

26. The minimalist bZIP protein of any one of claims 1-23, wherein the protein is further fused to a drug for drug delivery.

27. The minimalist bZIP protein of any one of claims 1-23, wherein the protein is further fused to a repressor domain.

28. The minimalist bZIP protein of claim 27, wherein the repressor domain is Mxi, Id and HIF-3.alpha..

29. A minimalist bZIP protein comprising the amino acid sequence as shown in SEQ ID NOs. 14-22 or 52.

30. A minimalist bZIP protein heterodimer comprising a first and second minimalist bZIP protein comprising a leucine zipper region in the first minimalist bZIP protein and a leucine zipper region in the second minimalist bZIP protein capable of forming a heterodimer.

31. The minimalist bZIP protein heterodimer of claim 30, wherein the leucine zipper region in the first minimalist bZIP protein is from Jun and the leucine zipper in the second minimalist bZIP protein is from Fos.

32. A use of a minimalist bZIP protein according to any one of claims 1-13, 16-23, and 26-29 for repressing myc-related transcriptional activation.

33. A use of a minimalist bZIP protein according to any one of claims 1-23 and 26-31 for treating cancer.

34. The use of claim 33, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, gynecological cancer, hepatocellular carcinomas, hematological tumors, Burkitt lymphoma, neuroblastoma and small cell lung cancer.

35. The use of claim 33, wherein the cancer is a soft tissue carcinoma or respiratory cancer.

36. A use of a minimalist bZIP protein according to any one of claims 1-29 for modulating cell proliferation or differentiation.

37. A pharmaceutical composition comprising the minimalist bZIP protein according to any one of claims 1-23 and 26-31 and a pharmaceutically acceptable carrier, diluent or excipient.

38. A use of the pharmaceutical composition of claim 37 for treating cancer.

39. The use of claim 38, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, gynecological cancer, hepatocellular carcinomas, hematological tumors, Burkitt lymphoma, neuroblastoma and small cell lung cancer.

40. The use of claim 38, wherein the cancer is a soft tissue carcinoma or respiratory cancer.

41. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a minimalist bZIP protein having an amino acid sequence as shown in SEQ ID NOs. 1-12, 14-22, 52, 53 or 54.