WO2001018036A2

WO2001018036A2 - Methods and reagents for regulating gene expression

Info

Publication number: WO2001018036A2
Application number: PCT/US2000/024010
Authority: WO
Inventors: John V. Frangioni; Lewis C. Cantley; Marc R. Montminy
Original assignee: Beth Israel Deaconess Medical Center; Joslin Diabetes Center, Inc.
Priority date: 1999-09-03
Filing date: 2000-08-31
Publication date: 2001-03-15
Also published as: AU7099400A; WO2001018036A3

Abstract

The present invention relates to a generalized approach for identifying minimal domain-specific transcriptional activation sequences. In particular, applicants have discovered that molecular diversity approaches can be used to discover minimal, co-activator domain specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment.

Description

METHODS AND RE A GENTS FOR REGULA TING GENE EXPRESSION

Background of the Invention To respond to changes in its environment, the cell utilizes mechanisms that integrate extracellular signals with specific changes in gene expression. Although they have remained elusive for researchers for many years, the mechanisms underlying transcriptional activation and repression have become clearer in recent years. For example, it has become apparent that non-DNA binding transcriptional co-activators, such as p300 and CREB binding protein (CBP), previously thought to function primarily as "bridging" proteins between DNA-bound transcription factors and the transcription complex, play a critical regulatory role as integrators of diverse signaling pathways with the selective induction of gene expression. Upon interaction with CREB-binding protein, (CBP, which bridges a CRE (cAMP response element)/CREB complex to components of the basal transcriptional apparatus), active Serl33-phosphorylated CREB effects transcription of CRE-dependent genes. The mechanism of CREB activation is effected by diverse signals, including those regulating the intracellular levels of cAMP and Ca+2, growth factors, and cellular stress. Accordingly, CREB-mediated transcription regulates diverse cellular responses, including intermediary metabolism, neuronal signaling, cell proliferation, and apoptosis.

Because transcriptional co-activators, such as p300 and CBP, are believed to play a critical regulatory role in cellular signaling, there has been interest in elucidating the structures and interactions involved in specific binding domains. It has been reported that multiple signal transduction pathways are coupled to gene activation via a KIX domain found in co-activators p300 and CBP (p300/CBP). The KIX domain of the co- activators p300/CBP integrates signals from a variety of molecules including CREB, c- Myb, c-Jun, Statla, SREBP, and the HTLV-1 Tax protein. A solution structure of the KIX domain reveals that it is composed of three -helices, two of which are parallel and form a hydrophobic groove (Radhakrishnan, 1997). Clearly, because of the importance of the co-activators p300/CBP, and more specifically the KIX domain, in cellular signaling, it would be desirable to discover moieties capable of binding to the KLX domain, other than the natural ligands.

There remains a need, however, to discover small molecules such as short peptides (e.g., less than 20 amino acids in length), that are capable of binding to the KIX domain. These compositions would be valuable, in one aspect, as a tool to study domain-specific co-activator recruitment in isolation from the multiple and complex interactions of p300/CBP with natural binding proteins. Additionally, these compositions would also be useful either as specific transactivators or as specific inhibitors of cellular signaling, thus regulating many of the activities and disease pathways (e.g., cancer induced by excessive cell proliferation) in cells.

Summary of the Invention

One aspect of the present invention relates to a generalized approach for identifying minimal domain-specific transcriptional activation sequences. The results of the present studies with KIX binding domains demonstrate that molecular diversity approaches can be used to discover minimal, co-activator domain specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment. In general, the present invention provides a method for identify optimal activator sequences by employing a partially or completely degenerate peptide display library with peptides of, e.g., 3-20 residues in length, more preferably 4- 10, and even more preferably 4-8. The peptides can be displayed in virtually any format, e.g., as encoded combinatorial libraries or as part of genetic packages. In certain embodiments, the peptides are provided as part of a fusion protein.

To further illustrate, the subject invention provides a method for identifying co- activator domain specific synthetic activators comprising:

(i) contacting a target domain of a selected transcription factor with a peptide display library comprising a variegated population of test peptide sequences of 3-20 amino residues in length;

(ii) identifying peptide sequences from the peptide display library which bind to the target domain; and

(iii) identifying peptide sequences from step (ii) which have the further characteristic(s):

(a) inhibit transcriptional activation by the selected transcription factor, and/or

(b) activates transcription of a target gene when provided as a fusion protein with a DNA binding sequence or other domain which promotes localization of the peptide to the target gene.

The peptide library can be provided as a soluble library or a library of small peptides, but is most preferably provided as part of a fusion protein, e.g., as part of a phage display library. By permitting the identification of small peptides which maintain the ability to bind to transcription factors, the present invention also contemplates compositions including transcriptional inhibitors comprising a peptide sequence identified according to the subject method, or a peptidomimetic thereof, wherein the inhibitor reduces the rate of transcription of a gene in a manner dependent on the selected transcription factor.

Likewise, the invention also contemplates chimeric transcriptional activators comprising a peptide sequence identified according to the subject method, and a DNA binding sequence or other domain which promotes localization of the activator to a target gene. In still other aspects, the present invention provides a method for identifying a compound which inhibits interaction of the selected transcription factor with one or more of its natural ligands, comprising: a. generating a reaction mixture comprising a polypeptide including the target domain of the selected transcription factor and a peptide or polypeptide including an active peptide sequence identified according to the methods described herein; b. contacting the reaction mixture with one or more test compounds; c. ascertaining the ability of the test compound to inhibit the interaction of the target domain and the active peptide sequence. Another aspects of the present invention relates to a peptide sequence termed

"KIX binding sequence", e.g., a peptide sequence, preferably a helical peptide sequence, that binds to a KIX domain, e.g., which preferably binds in the hydrophobic groove of a KIX domain.

Preferred KTX binding sequences are represented in the general formula B1-B2-B3-B4-B5-B6 wherein,

Bl represents an amino acid residue with a large hydrophobic sidechain, preferably I, L, M, F, P, W or Y, more preferably I, Y, L or W, and even more preferably; L or W; B2 represents a dipeptide linker, and preferably represents X2-X3 as described below, or it can be a dipeptide linker of two amino acid residues having polar sidechains, e.g., R, N, D, C, E, Q, H, K, S or T;

B3 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably an amino acid residue with a large hydrophobic sidechain, such as I, L, M, F, W or Y, and even more preferably Y, I or L, though B3 can be H in certain embodiments;

B4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably an acidic sidechain, such as D or E, or an amino acid residue with a large hydrophobic sidechain, preferably L;

B5 represents a dipeptide linker, and preferably represents X4-X5 as described below, or it can be a dipeptide linker of two amino acid residues having polar sidechains, e.g., R, N, D, C, E, Q, H, K, S or T; or hydrophobic sidechains, e,g, A, G, I, L, M, F, P, W, Y or V B6 represents an amino acid residue with a hydrophobic sidechain, preferably A,

G, I, L, M, F, P, W, Y or V, and more preferably L, V or F;

In certain embodiments, the KD binding sequence will be represented in the general formula:

Xl-X2-X3-X'-X4-X5-L-X" (I) wherein

XI represents an amino acid residue with a large hydrophobic sidechain, preferably I, L, M, F, P, W or Y, and more preferably L or W;

X2 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably A or W; X3 represents an amino acid residue with a small hydrophobic sidechain, preferably A, G, or V, and more preferably A or V;

X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably an acidic sidechain, such as D or E;

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V;

X' represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably an amino acid residue with a large hydrophobic sidechain, such as I, L, M, F, W or Y, and even more preferably Y, I or L.;

X" represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L, V or F; wherein the polypeptide binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence.

In certain embodiments, X' will be a histidine In certain embodiments, B3 represent phosphorylated tyrosine (pT) or an analog thereof. The phosphotyrosine moiety, pT, can be represented by the general formula

where Y is selected from a group consisting of

, — (CH₂)_m-BeF₃ , and — (CH₂)_m-AlF₃ wherein m is zero or an integer in the range of 1 to 6; X is absent or represents O, S, or N; Dj represents O or S; D2 represents N3, SH2, NH₂, or NO2; and R15 and Rjg each independently represent hydrogen, a lower alkyl, or a pharmaceutically acceptable salt, or R₁₅ and R₁₆ taken together with the O-P-O, O-B-O, O-V-O or O-As-O atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the. ring structure. In a preferred embodiment, pTyr is a non-hydrolyzable phosphotyrosine analog. The »αra-substituted phenylalanine is also the most preferred.

A KLX peptide (or peptidomimetic) can be linear, cyclic or heterocyclic (e.g., includes two or more intrachain binds to form 2 or more loops).

Preferably, the KLX binding sequence is 3-20 residues in length, more preferably 4-10, and even more preferably 4-6. In general, the KLX binding sequence competitively inhibits binding of a transcription factor, especially an inducible transcription factor, to a KLX domain. For example, the KIX sequence competes with binding of a KLX domain with CREB or Myb. In certain preferred embodiments, the KIX binding sequence competitively inhibits binding of pCREB (CREB phosphorylated on Ser-133) to a KLX domain. Preferred KIX domains are those from CBP and p300. In certain embodiments, the BOX binding sequence, depending on whether it is being used as an antagonist (e.g., as an oligopeptide) or an agonist (e.g., as an activation domain of a chimeric transcription factor), preferably is able to modulate "KLX- dependent" transcription of a gene, e.g., transcription that is dependent on a transcription factor having a KIX domain. In certain preferred embodiments, the KIX binding sequence modulates transcription dependent on certain preferred embodiments, the KIX binding sequence modulates transcription dependent on such factors as CREB, Myb, p53, nuclear hormone receptors (such as PPARγ and TH receptor), HIV Tat protein, HTLV-I oncoprotein Tax, Epstein-Barr virus (EBV) immediate-early protein BZLF1, Stats (such as Statό), CCAAT/enhancer binding proteins related activating transcription factor (C/ATF), or ATF4.

In certain preferred embodiments, the KIX binding sequence modulates CBP- and/or p300-dependent transcription.

In certain preferred embodiments, the KIX binding sequence modulates Ca2+ and/or cAMP-dependent transcription.

Where provided in the form of an oligopeptide, e.g., of 3-20 amino acid residues in length, or in the context of a larger polypeptide, the subject KIX binding sequence can be selected by criteria which include its binding constant to a KIX domain. In such a manner, the Ki of an agonist, or the ability of an agonist to activate transcription, can be fine tuned. For example, in the case of the KLX binding sequence being provided as an activation domain of a chimeric transcription, the actual peptide sequence can be selected to provide a desired level transcriptional activation, e.g., ranging from 2 fold to orders of magnitude.

Moreover, as we demonstrate herein, the KIX binding sequence is a modular component, and can be added at various positions to a chimeric protein with no more than routine experimentation.

One aspect of the invention relates to a nucleic acid encoding a polypeptide which includes a KLX binding sequence, such as a KIK binding sequence set forth above, or represented in the general formula:

X1-X2-X3-Y-X4-X5-L-F (I) wherein

XI represents an amino acid residue with a large hydrophobic sidechain, preferably I, L, M, F, P, W or Y, and more preferably L or W; X2 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably A or W;

X3 represents an amino acid residue with a small hydrophobic sidechain, preferably A, G, or V, and more preferably A or V; X4 represents an amino acid residue with a charged sidechain, preferably R, D,

E, H or K, and more preferably and acidic sidechain, such as D or E;

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the polypeptide binds to a KIX domain in a manner dependent upon the presence of the KLX binding sequence.

In certain preferred embodiments, KIX binding sequence is represented in the general formula (W/L)(W/A)(V/A)Y(D/E)(L/V)LF.

In certain preferred embodiments, the KLX binding sequence mediates binding of the polypeptide to the CREB-binding protein (CBP) or p300 transcriptional coactivators. In certain preferred embodiments, the KLX binding sequence mediates binding of the polypeptide to the KIX domain with a Kd of 10^~5M or less, and more preferably 10^-6, 10^-7 or even 10"⁸M or less.

In certain preferred embodiments, the polypeptide, upon association with a transcriptional regulatory sequence of a target gene, upregulates expression of the target gene in a manner dependent upon the presence of the KLX binding sequence, e.g., the polypeptide upregulates expression of the target gene by at least two-fold relative to the absence of the polypeptide, and more preferably at least two or three orders of magnitude.

In certain preferred embodiments, the polypeptide further includes a DNA binding domain.

In certain preferred embodiments, the polypeptide further includes one or more additional activation domains.

In certain preferred embodiments, the polypeptide further includes at least one additional KIX binding sequence, and preferably 3-10. Another aspect of the invention provides a recombinant polypeptide which includes a KLX binding sequence represented in the general formula:

X1-X2-X3-Y-X4-X5-L-F (I) wherein XI represents an amino acid residue with a large hydrophobic sidechain, preferably I, L, M, F, P, W or Y, and more preferably L or W;

X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably and acidic sidechain, such as D or E;

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the polypeptide binds to a KLX domain in a manner dependent upon the presence of the KIX binding sequence.

Yet another embodiment of the subject invention relates to a peptide or peptidomimetic of 6-20 amino acid residues in length, which includes a KLX binding sequence represented in the general formula:

X1-X2-X3-Y-X4-X5-L-F (I) wherein

XI represents an amino acid residue with a large hydrophobic sidechain, preferably I, L, M, F, P, W or Y, and more preferably L or W; X2 represents an amino acid residue with a hydrophobic sidechain, preferably A,

G, I, L, M, F, P, W, Y or V, and more preferably A or W;

X3 represents an amino acid residue with a small hydrophobic sidechain, preferably A, G, or V, and more preferably A or V;

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the peptide binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence. In still other embodiments, the subject peptide (or peptidomimetic analogs thereof) are cyclic, e.g., and have an amino acid sequence represented by formula VI:

S-c-Xj -X₂-X₃-X₄-X₅-X₆-X₇-X₈-c-G-S (IV) Xi represents any amino acid residue, more preferably an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V), polar (e.g., R, N, D, C, E, Q, H, K, S or T), acidic (D or E) or basic (R, H or K) side chain, and more preferably is P, W, E, S, L, N or R; X₂ represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L,

M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, L V, K H, T or G;

X represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is W, T, D or V;

X4 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, R, M, G, T, A, Y, L;

X5 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is V, Y, L, N, W, E, F, G;

X6 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is D, S, L, P, G, R, K, V; X7 represents an amino acid residue with an aromatic (F, Y, W) or acidic (D or

E) sidechain, and more preferably is F, Y, W or D;

X8 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably, preferably an aromatic side chain (F, Y, W), and more preferably S, E, V, Y, R, W, T or P; and c is a molecule capable of forming ringed structures with a covalent bond, e.g., c=Cysteine for a peptide composed of all natural amino acids and oxidized such that a disulfide bond between the two cysteines is formed. In general, the peptide or peptidomimetic of the present invention can be selected for its ability to inhibit binding of cyclic AMP-responsive factor (CREB) or c-Myb to the CBP or p300 transcriptional coactivators, preferably with a Ki of lOOμM or less, and more preferably with a Ki less than lOμM, lμM, lOOnM, lOnM or even InM. Preferably, the peptide or peptidomimetic inhibits expression of CREB or c-Myb dependent gene transcription.

In certain embodiments, the peptide or peptidomimetic is formulated in a pharmaceutically acceptable excipient. Yet another aspect of the present invention provides a method for stimulating transcription of a target gene, comprising

(i) transfecting the cell with an expression construct encoding a polypeptide which includes a KIX binding sequence represented in the general formula: X1-X2-X3-Y-X4-X5-L-F (I) wherein

X2 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably A or W;

X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably and acidic sidechain, such as D or E; X5 represents an amino acid residue with a hydrophobic sidechain, preferably A,

G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the polypeptide binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence.

(ii) causing the cell to express the polypeptide; (iii) causing the polypeptide to become associated with a transcriptional regulatory sequence of the target gene.

The present invention describes, for the first time, the ability of small molecules (e.g., having a molecular weight less than 5000 amu, more preferably less than 2500 amu, and most preferably less than 1000 amu) to interact with the KLX domain of polypeptides (e.g., p300/CBP) and thus act as transactivators. In general, the present invention provides compositions capable of binding to the KLX domain of a polypeptide comprising nucleic acids encoding polypeptides which include a KLX binding sequence, recombinant polypeptides which include a KLX binding sequence, and peptides, peptidomimetics, and libraries thereof of 6-20 amino acid residues in length which include a KLX binding sequence.

In one particularly preferred embodiment, each of these compositions includes a KIX binding sequence represented in the general formula (I): X1-X2-X3-Y-X4-X5-L-F (I) wherein

X3 represents an amino acid residue with a small hydrophobic sidechain, preferably A, G, or V, and more preferably A or V; X4 represents an amino acid residue with a charged sidechain, preferably

R, D, E, H or K, and more preferably and acidic sidechain, such as D or E;

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V.

In another preferred embodiment, each of these compositions includes a KLX binding sequence represented in the general formula (II)

X1-X2-X3-A1-X4-A2-A3-A4 (II) wherein

XI, X2 and X3 each independently comprise a natural or non-natural amino acid or a peptidomimetic thereof; Al, A2, A3 and A4 each independently comprise an aromatic or aliphatic natural or non-natural amino acid or peptidomimetic thereof; and

X4 comprises a charged natural or non-natural amino acid or peptidomimetic thereof.

In certain preferred embodiments, X4 is selected from the group consisting of aspartate, glutamate and a peptidomimetic thereof. In certain other preferred embodiments, Al, A2, A3 and A4 each independently comprise an aromatic or aliphatic moiety selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, methionine, proline, and a peptidomimetic thereof. In particularly preferred embodiments, X4 comprises an acidic moiety.

The present invention also provides compositions capable of interacting with the KLX domain of p300/CBP having a formula (HI) comprising:

A1-A2-A3-A4-X1-A5-A6-A7 (IH) wherein Al, A2, A3, A4, A5, A6 and A7 each independently comprise an aromatic or aliphatic natural or non-natural amino acid or peptidomimetic thereof; and

XI comprises a charged natural or non-natural amino acid or peptidomimetic thereof.

In preferred embodiments, Al, A2, A3, A4, A5, A6 and A7 each independently comprise an aromatic or aliphatic moiety selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, methionine, proline, and a peptidomimetic thereof; and XI is selected from the group consisting of aspartate, glutamate and a peptidomimetic thereof. In certain other preferred embodiments, A4 comprises tyrosine or a peptidomimetic thereof; XI comprises aspartate, glutamate or a peptidomimetic thereof; and A6 comprises leucine or a peptidomimetic thereof; and wherein A7 comprises phenylalanine or a peptidomimetic thereof. In still other preferred embodiments, Al comprises tryptophan, leucine or a peptidomimetc thereof; A2 comprises tryptophan, alanine, or a peptidomimetic thereof; A3 comprises valine, alanine, or a peptidomimetic thereof; A4 comprises tyrosine or a peptidomimetic thereof; XI comprises aspartate, glutamate, or a peptidomimetic thereof; A5 comprises leucine, valine or a peptidomimetic thereof; A6 comprises leucine or a peptidomimetic thereof; and A7 comprises phenylalanine or a peptidomimetic thereof. In particularly preferred embodiments, XI comprises an acidic moiety.

One aspect of the present invention provides a method of treating, e.g., lessening the severity or preventing the occurrence of a condition induced by either (1) constitutive interaction of the KLX domain of p300/CBP with a natural KLX domain ligand or (2) a lack of interaction of the KLX domain of p300/CBP with a natural KLX domain ligand. In general, the subject method comprises administering to an animal, e.g. a human, infected with specific condition a pharmaceutical preparation comprising a therapeutically effective amount of a small organic molecule which can inhibit the interaction between the KLX domain and a natural KLX domain ligand. In preferred embodiments, the inhibitor has a molecular weight of less than 10,000 amu, more preferably less than 7500 amu, 5000 amu, and even more preferably less than 3000 amu. For instance, the inhibitor can be either (i) KIX peptide or peptidomimetic, preferably corresponding in length to an 8-mer, e.g., in certain embodiments, or (ii) a gene construct for expressing the KLX peptide. The KLX peptide, peptidomimetic or gene construct is formulated in the pharmaceutical preparation for delivery into infected cells of the animal. The subject method can be used to inhibit pathological progression of diseases and disorders involving aberrant KlX-dependent gene transcription. Yet another aspect of the invention relates to a pharmaceutical preparation comprising a therapeutically effective amount of a KIX peptide or peptidomimetic, formulated in the pharmaceutical preparation for delivery into infected cells of an animal. In preferred embodiments, the polypeptide is formulated as a liposome. As will be appreciated by one of ordinary skill in the art, the compositions and preparations described herein can also be utilized serially or in combination with conventional therapeutic agents or regimens including, but not limited to, salicylic acid, podophyllotoxin, retinoic acid, surgery, laser therapy, radiation, and cryotherapy.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and JJ (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- TV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Brief Description of the Drawings

Figure 1 - Alignment and Helicity of Natural and Synthetic p300/CBP Binding Sequences:

A. Alignment of the phosphorylation-induced helical segment of CREB, the constitutively helical segment of c-Myb, and p300/CBP-binding sequences of

SREBPla, SREBP2, Cubitus interruptus (Ci) and the optimal first (KBP 1.66) and second (KBP 2.20) round phage display-derived peptides.

Aliphatic/Aromatic residues comprising the putative hydrophobic ridge are shown in boldface. Note a conserved acidic residue at position 5 of most sequences.

B. Helical wheel representation of synthetic KBP 2.20 (left) and the homologous sequences from natural KIX domain ligands c-Myb (amino acids 295-302); middle) and the KID domain of phospho-CREB (amino acids 134-141; right).

Aliphatic/Aromatic residues comprising the putative hydrophobic ridge are starred.

C. Molecular modeling of KBP 2.20 showing the lowest energy state (-24.4 kcal/mole), as calculated by Quanta (Molecular Simulations). A continuous dielectric model, assuming a dielectric constant of 10, was employed. The only assumption was a helical conformation. Energies ranged from -16.1 to -24.4 kcal/mol (Mean -20.5 ± 2.6 kcal/mol). Van der Waals surfaces are shown for the starred residues from Fig. IB.

Figure 2 - Intermolecular Contacts of KBP 2.20 with the Hydrophobic Groove of the KIX Domain of p300/CBP:

A. GST alone, the wild type KLX domain (GST/KLX 10-672; see Experimental Protocol), or KLX domain point mutants were incubated with KBP-alkaline phosphatase fusion protein pAPi/KBP 2.20 and assayed for binding as detailed in Experimental Protocol. Total activity bound (mean ± S.E.M.), expressed in nmols p-nitrophenol liberated, is displayed on the ordinate. The wild type KIX domain bound 31% of the total supernatant activity (data not shown). Point mutations are labeled as residue number flanked by the single letter code for wild type (prefix) and mutant (suffix) amino acid. Below each lane is shown the corresponding GST protein used for each condition after separation on a 12%

SDS-polyacrylamide gel and staining with Coumassie Brilliant Blue. Note that the R600Q mutant is comprised of amino acids 553-679 of CBP¹¹, whereas all other mutants are comprised of 586-672 . The R600Q mutant was included as a negative control since previously published NMR data¹⁰ suggests that the KLX domain does not properly fold in the presence of this mutation, and therefore should not be capable of binding to KBP 2.20.

B. Intermolecular contacts of KBP 2.20 with the wild type KLX domain (derived from Fig. 2A) are superimposed on the previously published NMR structure¹⁰ of the KLX domain. In gray is shown the ribbon diagram of the three alpha helices comprising the wild type KLX domain¹⁰. In black are the residues whose mutation resulted in greater than 50% inhibition of KBP 2.20 binding.

Figure 3 - Confirmation of Binding Surface Overlap with Phospho-CREB and c-Myb, and Precipitation of p300/CBP from Whole Cell Lysates using KBPs:

A. Overlap of KLX domain contacts between KBP 2.20 and phospho-CREB was confirmed by a competition experiment. Alkaline phosphatase-KIX 10-672 (a fusion of the wild type KLX domain with C terminus of sPLAP; see Experimental

Protocol) was incubated with GST/KBP 2.20 (a fusion of KBP 2.20 with the C terminus of GST) in the absence of peptide, or with 3.75 μM KID-29 or 3.75 μM phospho-KTD-29 peptides as described in Experimental Protocol. The Kj of phospho-KID-29 for the KIX domain is approximately 750 nM (data not shown). Total activity bound (mean ± S.E.M.), expressed in nmols p-nitrophenol liberated, is displayed on the ordinate. In the absence of peptide, approximately 28% of the total supernatant activity was bound by GST/KBP 2.20 (data not shown).

B. Repression of c-Myb-dependent gene activation was tested by co-transfection of c-Myb, a Myb-Luciferase reporter plasmid, and a single copy of KBP 1.66 or

KBP 2.20 fused to the C terminus of GAL4 DBD (using vector pAL^ see Experimental Protocol). Cells were transfected without (-) or with (+) 100 ng of pCMV/c-Myb¹⁵ as indicated. Cells were transfected with 100 ng of pALi alone (1,2), pALi/ B? 1.66 (3) or pAL,/KBP 2.20 (4). Fold activation (mean ± S.E.M.), relative to -c-Myb, of a Myb-Luciferase reporter plasmid (see

Experimental Protocol) is shown on the ordinate. Below the plot is a Western blot of the corresponding cell lysates after separation on a 16% SDS- polyacrylamide gel and detection with an anti-GAL4 DBD monoclonal antibody.

C. Western blot of p300/CBP (solid arrow) in 293 whole cell lysates following pulldown assay using synthetic peptides covalently coupled to an affinity resin

(see Experimental Protocol). Conditions included: 10 μg (1), 20 μg (2), and 40 μg (3) of total cell lysate, and 160 μg of total cell lysate precipitated with either Affι-Gel-10/KBP 1.66 (4), Affi-Gel-10/KBP 2.20 (5), or Affi-Gel- 10/Hydrophobic Control Peptide (6; sequence of control peptide = FRYFSYWY). Proteins were separated on a 6% polyacrylamide gel. Figure 4 - KBPs Derived from Phage Display Screening Comprise a Family of Modular Transactivators Whose Binding Affinity Correlates with the Level of Transcriptional Activation:

A. A single copy of the optimal first round (KBP 1.66) or optimal second round (KBP 2.20) KLX domain binding peptide was fused to the C terminus of the GAL4 DNA binding domain (DBD) and compared directly to natural activators. Cells were transfected with 5 ng of each construct, and assayed for GAL4 activation as described in Experimental Protocol. Conditions tested were: GAL4

DBD alone (1), or the GAL4 DBD fused to KBP 1.66 (2), KBP 2.20 (3), the minimal activation domain of c-Myb (4; amino acids 290-315; Ref. 15), full- length Spl (5), and full-length c-Fos (6; Ref 31). Fold activation (mean ± S.E.M.), relative to the GAL4 DBD alone is shown on the ordinate. Shown below the figure is a Western blot for the GAL4 DBD fusion protein after separation, on an 18% (1-4) or 12% (5,6) SDS-polyacrylamide gel, and detection with an anti-GAL4 DBD monoclonal antibody. Protein products (arrowheads) have an apparent molecular weight of 18 kDa (GAL4 DBD with or without KBPs), 21 kDa (c-Myb), 99 kDa (Spl), and 59 kDa (c-Fos). Data shown is representative of three independent experiments.

B. Modulation of KBP 2.20 transcriptional activation by wild type and mutant El A. GAL4 transcriptional activation was assayed after transfection of 5 ng of: GAL4 DBD alone (1) or GAL4/KBP 2.20 (2-8). Percent activation (mean ± S.E.M.), shown on the ordinate, is relative to pAL_t/KBP 2.20 (2). Increasing amounts of wild type El A, or a mutant El A protein incapable of binding to p300/CBP due to deletion of amino acids 15-35³² (El A ), was co-transfected with pALi/KBP 2.20 at a ratio of 1 :1 (3,6), 2:1 (4,7) or 5:1 (5,8). Like pALi, E1A proteins were driven by a CMV promoter. GAL4 DBD expression, as described in (A), is shown below each condition. Data shown is representative of four independent experiments.

C. KBP transcriptional activation is potentiated by p300 co-expression. 5 ng of PAL, (1,2), pAL]/KBP 1.66 (3,4), or pALj/KBP 2.20 (5,6) was transfected as described for (A). Full length p300 driven by a CMV promoter (2,4,6), or the empty vector (1,3,5) was co-transfected at a 10:1 ratio. Fold activation (mean ± S.E.M.), relative to pAL] alone is shown on the ordinate. GAL4 DBD expression is shown below each condition as described for (A). Data shown is representative of four independent experiments.

Detailed Description of the Invention

I. Overview

Protein-encoding genes in eukaryotes are transcribed by RNA polymerase π (pol H), a multisubunit enzyme that is brought to an appropriate gene promoter (pol π promoter) through the assembly of a pre-initiation complex comprising a number of general transcription factors. The multisubunit protein complex TFHD is required for transcription by most, if not all, promoters targeted by pol II. Whereas the TATA-box binding protein (TBP) of TFHD is sufficient for basal transcription, pol π transcription is also regulated by gene-specific activator proteins. Activator-dependent transcription requires, inter alia, TBP-associated proteins (TAFs) and other transcriptional cofactors. One of the important concepts to emerge from studies of eukaryotic gene expression is that activators of pol II-dependent transcription are composed of functional modules whose abilities to bind to subunits of the ultimate pol π complexes regulates transcriptional activity of a nearby gene. The histone acetyltransferases (HATs) p300 and CBP are potent transcriptional co-activators. Many extremely important and diverse, intracellular signals are transduced by p300/CBP, including cAMP- and c-Myb-dependent gene activation. P300/CBP share an important domain termed "KLX" through which these signals are transduced. We have discovered that short peptides are sufficient to direct binding to KLX domains and, in certain embodiments, are sufficient to function as transcriptional activators when associated with DNA binding domains

The present invention pertains to nucleic acid molecules and proteins which can be used to regulate the expression of genes in eukaryotic cells.

One aspect of the present invention relates to transcriptional activators ("chimeric activators") which are derived to include a KIX binding sequence and optionally additional transcriptional activation sequences, also referred to herein as "activation tags" (further defined infra). The transcriptional activators of the invention are capable of affecting transcriptional activation, as for example, affecting the assembly or stability of an active polymerase complex. It has been discovered that a polypeptide comprising a KLX binding sequence is capable of binding to, and recruiting p300/CBP to the polypeptide, thereby increasing the transcriptional activity of the polypeptide. Thus, e.g., the addition of a KLX binding sequence to a transcriptional activator having one or more activation tags, increases its transcriptional activity. The transcriptional activators of the invention preferably further comprise a DNA binding domain; a domain mediating interaction with one or more other polypeptides, e.g., a multimerizing domain; or a ligand-binding domain.

Another aspect of the present invention relates to agents for inhibiting transcriptional activation. Preferred agents comprise a peptide comprising a KIX binding sequence or peptidomimetic or analog thereof. Without wanting to be limiting in the mechanism of action, such agents may inhibit transcriptional activation by occupying the KIX domain of p300/CBP and thereby competitively inhibiting its access by transcription factors, e.g., CREB, c-Myb, c-Iun, and Stats.

It is recognized herein the importance of learning more about the structural, size and sequence requirements for binding to the KIX domain. In realization of this goal, the present invention describes short peptide sequences capable of binding to the KLX domain of a polypeptide. Prior to this demonstration, it was believed that mammalian activators were generally large (40-80 amino acids) featureless peptide tracts with a high content of acidic residues (Ptashne, 1997). Thus, it is demonstrated for the first time herein, the ability of a small molecule (preferably less than 5000 amu, more preferably less than 2500 amu, and most preferably less than 1000 amu) to bind to the KIX domain of a polypeptide. It will be appreciated that the present invention encompasses not only the ability of short peptidic sequences, as disclosed herein, to bind to the KLX domain, but also encompasses the ability of small peptidomimetic and small non-peptidic compounds, as described herein, to bind to the KLX domain of a polypeptide.

II. Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence. The term "intron" refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product, e.g., as may be encoded by a coding sequence. "Transcriptional regulatory sequence", also termed herein "regulatory element",

"regulatory sequence" or "regulatory element", are generic terms used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. The term "enhancer", also referred to herein as "enhancer element", is intended to include regulatory elements capable of increasing, stimulating, or enhancing transcription from a basic promoter. The term "silencer", also referred to herein as "silencer element" is intended to include regulatory elements capable of decreasing, inhibiting, or repressing transcription from a basic promoter. Regulatory elements can also be present in genes other than in 5' flanking sequences. Thus, it is possible that regulatory elements of a gene are located in introns, exons, coding regions, and 3' flanking sequences.

The terms "basic promoter" or "minimal promoter", as used herein, are intended to refer to the minimal transcriptional regulatory sequence that is capable of initiating transcription of a selected DNA sequence to which it is operably linked. This term is intended to represent a promoter element providing basal transcription. A basic promoter frequently consists of a TATA box or TATA-like box and is bound by an RNA polymerase and by numerous transcription factors, such as GTFs and TATA box Binding Proteins (TBPs).

The terms "basic promoter" and "regulatory element" further encompass "tissue specific" promoters and regulatory elements, i.e., promoters and regulatory elements which effect expression of the selected DNA sequence preferentially in specific cells

(e.g., cells of a specific tissue). Gene expression occurs preferentially in a specific cell if expression in this cell type is significantly higher than expression in other cell types.

The terms "promoter" and "regulatory element" also encompass so-called "leaky" promoters and "regulatory elements", which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well. The terms

"promoter" and "regulatory element" also encompass non-tissue specific promoters and regulatory elements, i.e., promoters and regulatory elements which are active in most cell types. Furthermore, a promoter or regulatory element can be a constitutive promoter or regulatory element, i.e., a promoter or regulatory element which constitutively regulates transcription, as opposed to a promoter or regulatory element which is inducible, i.e., a promoter or regulatory element which is active primarily in response to a stimulus. A stimulus can be, e.g., a molecule, such as a hormone, a cytokine, a heavy metal, phorbol esters, cyclic AMP (cAMP), or retinoic acid.

The term "core promoter element" is intended to include the TATA box and the initiator element. "DNA recognition sequence" or "DNA recognition element", as those phrases are used herein, mean a DNA sequence which is capable of binding to one or more DNA-binding domains, e.g., of a transcription factor.

The term "initiator" refers to a short, weakly conserved element that encompasses the transcription start site and which is important for directing the synthesis of properly initiated transcripts.

The term "transcription factor" refers to any protein or modified form thereof that is involved in the initiation of transcription but which is not itself a part of the polymerase. Transcription factors are proteins or modified forms thereof, which interact preferentially with specific nucleic acid sequences, i.e., regulatory elements, and which in appropriate conditions stimulate transcription ("transcriptional activators") or repress transcription ("transcriptional repressors"). Some transcription factors are active when they are in the form of a monomer. Alternatively, other transcription factors are active in the form of ohgomers consisting of two or more identical proteins or different proteins (heterodimer). The factors have different actions during the transcription initiation: they may interact with other factors, with the RNA polymerase, with the entire complex, with activators, or with DNA. The factors are generally classifiable into two groups: (i) the general transcription factors, and (ii) the transcription activators. Transcription factors usually contain one or more regulatory domains.

The term "regulatory domain" refers to any domain which regulates transcription, and includes both activation and repression domains. The term "activation domain" denotes a domain in a transcription factor which positively regulates (increases) the rate of gene transcription. The term "repression domain" denotes a domain in a transcription factor which negatively regulates (inhibits or decreases) the rate of gene transcription.

The term "general transcription factor" used interchangeably herein with the term "GTF" and with "basic transcription factor" refers to proteins or protein complexes which work in concert with RNA Polymerase π to bring about promoter recognition and accurate transcription initiation. These proteins constitute, together with the RNA polymerase π, the Transcription Initiation Complex. GTFs include TFILA, TFEDB, TFHD, TFΠE, TFHF, and TFHH. These GTFs are usually sufficient to direct basal levels of transcription in vitro from strong promoters (i.e., those containing TATA boxes). Several GTF interact with one another and/or with RNA Polymerase II. For example, TFHE interacts with TFHH and RNA Polymerase π, TFH F interacts with RNA Polymerase π and with TFHB, and TFII B interacts with TBP from TFHD and RNA Polymerase II.

The term "transcriptional activator" as used herein refers to a protein or protein complex which is capable of enhancing the efficiency with which the basal transcription complex performs, i.e., activating transcription. Thus, as used herein, a transcriptional activator can be a single protein or alternatively it can be composed of several units at least some of which are not covalently linked to each other. A transcriptional activator typically has a modular structure, i.e., comprises various domains, such as a DNA binding domain, and one or more transcriptional activation tags. Some transcriptional activators may engage a subset of GTFs. For example, some transcriptional activators may include activation tags which collectively contact both TFHD and TFHB. Other transcriptional activators may contact a single GTF.

The term "cofactor" which is used interchangeably herein with the terms "co- activator", "adaptor" and "mediator" refers to proteins which either enhance or repress transcription in a non-gene specific manner, e.g., which lack intrinsic DNA binding specificity. Thus, cofactors are general effectors. Positively acting cofactors do not stimulate basal transcription, but enhance the response to an activator. Positively acting cofactors include the histone acetyltransferases (HAT), such as p300 and CBP. Other positively acting cofactors include PCI, PC2, PC3, PC4, and ACF. TAFs which interact directly with transcriptional activators are also referred to as cofactors.

The term "transcriptional activation tag", also referred to herein as "activation tag", "transcriptional activation unit" and "activation unit", refers to a peptide sequence which is capable of inducing or otherwise potentiating activator-dependent transcription, either on its own or when linked covalently or non-covalently to another transcriptional activation unit. As opposed to a transcriptional activator generally, an activation tag corresponds to a minimal polypeptide sequence that retains the ability to interact directly or indirectly with a transcription factor. Of course, unless otherwise clear from the context, where a chimeric protein is referred to as "including" or "comprising" an activation tag, particularly one including a KLX binding sequence, it will be understood that portions of other proteins can be included.

In addition to subject KLX binding sequence, the subject chimeric proteins can include other activation tags. For example, a transcriptional activation unit can be a peptide rich in acidic residues, glutamine, proline, or serine and threonine residues. Yet other transcriptional activators can be rich in isoleucine or basic amino acid residues (see, e.g., Triezenberg (1995) Cur. Opin. Gen. Develop. 5:190, and references therein). For instance, an activation tag can be a peptide motif of at least about 6 amino acid residues associated with a transcription activation domain, including the well-known "acidic", "glutamine-rich" and "proline-rich" motifs such as the K13 motif from p65, the OCT2 Q domain and the OCT2 P domain, respectively.

A "dimerization domain" is defined as a domain that induces formation of dimers between two proteins having that domain, while a "tetramerization domain" is defined as a domain that induces formation of tetramers amongst proteins containing the tetramerization domain. An "oligomerization domain", generic for both dimerization and tetramerization domains, facilitates formation of ohgomers, which can be of any subunit stoiechiometry (of course greater than one). The term "interact" as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a yeast two hybrid assay or by immunoprecipitation. The term interact is also meant to include "binding" interactions between molecules. Interactions may be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature. Preferred binding affinities have a Kd of 10^"6 or less, preferably 10^"8 or less, 10^"9 or less, 10^"10 or less, 10^"11 or less, or most preferably 10^"12 or less.

The term "holoenzyme complex" refers to RNA Polymerase H-containing complexes.

The term "squelching" which is used interchangeably herein with the term "activator interference" refers to the inhibition of transcription observed when an activator is present at artificially high concentrations (Ptashne and Gann (1990) Nature 346:329). While not bound by any particular theory, this inhibition is understood to result from the sequestration by the activator (unbound to DNA) of either (i) an adaptor component necessary to bridge the activator with the basal machinery, and/or (ii) a basal component.

The term "subunit", when referring to the subunit of a transcriptional activator, refers to any unit of the transcriptional activator, e.g., a transcriptional activation unit, a DNA binding domain, or a ligand binding domain.

The term "unit", when referring to a unit of a transcription factor, refers generally to a minimum portion of a transcription factor having a specific activity, e.g., transcriptional activation, transcriptional repression, DNA binding, or ligand binding.

As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.

The term "transduction" is generally used herein when the transfection with a nucleic acid is by viral delivery of the nucleic acid. "Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the recombinant protein is disrupted. As used herein, the term "transgene" refers to a nucleic acid sequence which has been introduced into a cell. Daughter cells deriving from a cell in which a transgene has been introduced are also said to contain the transgene (unless it has been deleted). A transgene can encode, e.g., a polypeptide, partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene). Alternatively, a transgene can also be present in an episome. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, (e.g. intron), that may be necessary for optimal expression of a selected coding sequence.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. "Derived from" as that phrase is used herein indicates a peptide or nucleotide sequence selected from within a given sequence. A peptide or nucleotide sequence derived from a named sequence may contain a small number of modifications relative to the parent sequence, in most cases representing deletion, replacement or insertion of less than about 15%, preferably less than about 10%, and in many cases less than about 5%, of amino acid residues or base pairs present in the parent sequence. In the case of DNAs, one DNA molecule is also considered to be derived from another if the two are capable of selectively hybridizing to one another. The terms "chimeric", "fusion" and "composite" are used to denote a protein, peptide domain or nucleotide sequence or molecule containing at least two component portions which are mutually heterologous in the sense that they are not, otherwise, found directly (covalently) linked in nature. More specifically, the component portions are not found in the same continuous polypeptide or gene in nature, at least not in the same order or orientation or with the same spacing present in the chimeric protein or composite domain. Such materials contain components derived from at least two different proteins or genes or from at least two non-adjacent portions of the same protein or gene. Composite proteins, and DNA sequences which encode them, are recombinant in the sense that they contain at least two constituent portions which are not otherwise found directly linked (covalently) together in nature.

The term "CBP" refers to CREB binding protein.

The term "p300/CBP" refers to the histone acetyltransferase p300 or to the histone acetyltransferase CBP. The term "KLX domain" refers to the domain of a histone acetyltransferase, e.g., p300 or CBP, that interacts with a transcription factor, such as CREB, c-Myb, c-Iun, Stat lot, SREBP, and the HTLV-1 Tax protein. A KLX domain is composed of three α- helices, two of which are parallel and form a hydrophobic groove Exemplary guidance for identifying KLX domains is provided by Radhakrishnan et al. (1999) I Mol Biol 1999 Apr 16;287(5):859. An exemplary KIX domain is amino acid residues 586-672 of the mouse CBP. Also see Brookhaven database PDB Id: 1KDX.

The term "KIX binding agent," which is used interchangeably herein with "KLX domain binding agent," "KLX binding ligand," and with "KIX domain binding ligand," refers to a KLX binding peptide or peptidomimetic or analog thereof that binds specifically to the KIX domain of a histone acetyltransferase (HAT), e.g., p300 and CBP, with an affinity constant (Kd) of 10^"5 or less, and more preferably 10^_6M, 10^"7M,10^-8M, 10^"9M, 10^"10M, 10^"nM or less, or most preferably 10^"12 or less.

A "KLX binding peptide," which is used interchangeably herein with "KLX domain binding peptide," refers to a peptide that binds specifically to the KIX domain of a histone acetyltransferase (HAT), e.g., p300 and CBP, with an affinity constant (Kd) of 1 of 10^"5 or less, and more preferably 10"⁶M, 10"⁷M,10-⁸M, 10^"9M, 10^"10M, 10^"nM or less, or most preferably 10^"12 or less. KIX binding peptides can be naturally-occurring of synthetic. Naturally occurring KIX binding peptides include the transcription factors CREB, c-Myb, c-Jun, Stat l , SREBP, and the HTLV-1 Tax protein, either phosphorylated or non-phosphorylated, depending on the particular transcription factor. Thus, for example, binding of CREB to a KLX domain is significantly enhanced by phosphorylation of CREB in the portion that interacts with a KIX domain, i.e., at serine 133. On the contrary, c-Myb interacts with a KLX domain with a higher affinity in a non-phosphorylated state. As further described herein, KIX binding peptides preferably have an alpha-helical structure, which is either constitutively present in a peptide, or induced by phosphorylation of the peptide. Examples of KIX binding peptides are provided in the Examples, and include, e.g., KBP2.20. In a preferred embodiment, a KLX binding peptide has an amino acid sequence represented by the general formula I. In an even more preferred embodiment, a KLX binding peptide has the amino acid sequence represented in the general formula (W/L)(W/A)(V/A)Y(D/E)(L/V)LF. The term "KIX Therapeutic" as used herein is intended to generically encompass, unless otherwise obvious from its context, such molecules as polypeptides or peptides including a KLX binding sequence, peptidomitnetics and other small molecule mimics thereof, as well as expressions constructs of such peptides and polypeptides. The term covers both agonists and antagonists of KLX-dependent transcription. The term "KIX binding sequence," which is used interchangeably herein with

"KIX domain binding sequence," refers to the amino acid sequence of a KIX binding peptide. When referring to a KLX binding agent which is not a peptide, but, e.g., a peptidomimetic or other chemical derivative or analog of a KIX binding peptide, the term "KLX binding sequence" refers to the sequence of a peptide from which the KLX binding agent was derived or to which it is analogous. Preferred KLX binding sequences are set forth under the definition of "KLX binding peptide."

The term "ED50" means the dose of a drug, e.g. a KLX therapeutic, which produces 50% of its maximum response or effect.

An "effective amount" of, e.g., a KIX Therapeutic with respect to the subject methods of treatment, refers to an amount of the antagonist in a preparation which, when applied as part of a desired dosage regimen brings about, e.g., a change in the rate of cell proliferation and/or the state of differentiation of a cell and/or rate of survival of a cell according to clinically acceptable standards for the disorder to be treated or the cosmetic purpose. The "growth state" of a cell refers to the rate of proliferation of the cell and/or the state of differentiation of the cell. An "altered growth state" is a growth state characterized by an abnormal rate of proliferation, e.g., a cell exhibiting an increased or decreased rate of proliferation relative to a normal cell.

As used herein, "immortalized cells" refers to cells which have been altered via chemical and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture. The term "LD50" means the dose of a drug which is lethal in 50% of test subjects.

The term "therapeutic index" refers to the therapeutic index of a drug defined as LD50/ED50 A "patient" or "subject" to be treated by the subject method can mean either a human or non-human animal.

The term "prodrug" is intended to encompass compounds which, under physiological conditions, are converted into the therapeutically active agents of the present invention. A common method for making a prodrug is to include selected moieties which are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal.

As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis. As used herein, "transformed cells" refers to cells which have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and or hyperplastic, with respect to their loss of growth control. "Basal cell carcinomas" exist in a variety of clinical and histological forms such as nodular-ulcerative, superficial, pigmented, morphealike, fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the most common cutaneous neoplasms found in humans. The majority of new cases of nonmelanoma skin cancers fall into this category.

The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate surrounding tissues and to give rise to metastases. Exemplary carcinomas include: "basal cell carcinoma", which is an epithelial tumor of the skin that, while seldom metastasizing, has potentialities for local invasion and destruction;

"squamous cell carcinoma", which refers to carcinomas arising from squamous epithelium and having cuboid cells; "carcinosarcoma", which include malignant tumors composed of carcinomatous and sarcomatous tissues; "adenocystic carcinoma", carcinoma marked by cylinders or bands of hyaline or mucinous stroma separated or surrounded by nests or cords of small epithelial cells, occurring in the mammary and salivary glands, and mucous glands of the respiratory tract; "epidermoid carcinoma", which refers to cancerous cells which tend to differentiate in the same way as those of the epidermis; i.e., they tend to form prickle cells and undergo comification;

"nasopharyngeal carcinoma", which refers to a malignant tumor arising in the epithelial lining of the space behind the nose; and "renal cell carcinoma", which pertains to carcinoma of the renal parenchyma composed of tubular cells in varying arrangements. Other carcinomatous epithelial growths are "papillomas", which refers to benign tumors derived from epithelium and having a papillomavirus as a causative agent; and "epidermoidomas", which refers to a cerebral or meningeal tumor formed by inclusion of ectodermal elements at the time of closure of the neural groove.

Throughout this application, the term "proliferative skin disorder" refers to any disease/disorder of the skin marked by unwanted or aberrant proliferation of cutaneous tissue. These conditions are typically characterized by epidermal cell proliferation or incomplete cell differentiation, and include, for example, X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, and seborrheic dermatitis. For example, epidermodysplasia is a form of faulty development of the epidermis. Another example is "epidermolysis", which refers to a loosened state of the epidermis with formation of blebs and bullae either spontaneously or at the site of trauma.

The term "small molecule" as used herein refers to a compound either synthesized in the laboratory or found in nature. Typically, a small molecule refers to an organic, i.e., carbon-containing compound, characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 5000 amu, more preferably less than 2500 amu and most preferably less than 1500 amu. As used herein, the term "small molecule" may refer to short (i.e., preferably less than 20 amino acid sequences) peptides, non-peptide natural products and non-peptide compounds synthesized in the laboratory. Preferably, non-peptide compounds synthesized in the laboratory are "natural product-like" , that is, possess stereochemical and functional group diversity as well as diversity of spatial orientation.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1 -C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl

(including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.

Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like. The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.

The term "aryl" as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon. As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -

Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-. The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:

wherein R9, RI Q and R'10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)_m-Rg, or R9 and R\Q taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rg represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In preferred embodiments, only one of R9 or Rio ^{can De a} carbonyl, e.g., R9, R Q and the nitrogen together do not form an imide. In even more preferred embodiments, R9 and RJQ (and optionally R'10) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)_m-Rg. Thus, the term "alkylamine" as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and R Q is an alkyl group.

The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R is as defined above, and R'n represents a hydrogen, an alkyl, an alkenyl or -(CH2)_m-R8, where m and Rg are as defined above.

The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

wherein R9, R Q are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.

The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2)_m-Rg, wherein m and Rg are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.

The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R\ \ represents a hydrogen, an alkyl, an alkenyl, -(CH2)_m-Rg or a pharmaceutically acceptable salt, R' represents a hydrogen, an alkyl, an alkenyl or -(CH2)_m-Rg, where m and Rg are as defined above. Where X is an oxygen and R\ or R'n is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R\ \ is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R\ \ is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'n is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and Rn or R'n is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and Rn is hydrogen, the formula represents a "thiolcarboxylic acid." Where

X is a sulfur and R\ \' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and Rn is not hydrogen, the above formula represents a

"ketone" group. Where X is a bond, and Rn is hydrogen, the above formula represents an "aldehyde" group.

The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2)_m-Rg, where m and Rg are described above.

The term "sulfonate" is art recognized and includes a moiety that can be represented by the general formula:

in which R4 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl. The term "sulfate" is art recognized and includes a moiety that can be represented by the general formula:

0

II O— S-OR₄₁

II o in which R4 is as defined above. The term "sulfonamido" is art recognized and includes a moiety that can be represented by the general formula:

in which R9 and R' are as defined above.

The term "sulfamoyl" is art-recognized and includes a moiety that can be represented by the general formula:

⁰ S-N M

II \_R

in which R9 and R Q are as defined above.

The term "sulfonyl", as used herein, refers to a moiety that can be represented by the general formula:

O

II

O in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term "sulfoxido" as used herein, refers to a moiety that can be represented by the general formula:

O

II S-R 44 in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A "phosphoryl" can in general be represented by the formula:

wherein Q represented S or O, and R46 represents hydrogen, a lower alkyl or an aryl.

When used to substitute, e.g., an alkyl, the phosphoryl group of the phosphorylalkyl can be represented by the general formula:

wherein Q represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N. When Q is an S, the phosphoryl moiety is a "phosphorothioate".

A "phosphoramidite" can be represented in the general formula:

O 0

— Qr-P— O — — Q— P — OR₄₆

I , or I N (R₉ ) R₁₀ N (R₉ ) R₁₀ wherein R9 and R Q are as defined above, and Q2 represents O, S or N. A "phosphonamidite" can be represented in the general formula:

wherein R9 and R Q are as defined above, Q2 represents O, S or N, and R4g represents a lower alkyl or an aryl, Q2 represents O, S or N.

A "selenoalkyl" refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -Se-(CH2)_m-R.7_> m and R7 being defined above. Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls. As used herein, the definition of each expression, e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

The phrase "protecting group" as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2^nd ed.; Wiley: New York, 1991). The term "amino acid residue" is known in the art. In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-iUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11 :1726-1732). In certain embodiments, the amino acids used in the application of this invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan. The term "amino acid residue" further includes analogs, derivatives and congeners of any specific amino acid referred to herein, as well as C-terminal or N- terminal protected amino acid derivatives (e.g. modified with an N-terminal or C- terminal protecting group). For example, the present invention contemplates the use of amino acid analogs wherein a side chain is lengthened or shortened while still providing a carboxyl, amino or other reactive precursor functional group for cyclization, as well as amino acid analogs having variant side chains with appropriate functional groups). For instance, the subject compound can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains which are suitable herein will be recognized by those skilled in the art and are included in the scope of the present invention. Also included are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms. The configuration of the amino acids and amino acid residues herein are designated by the appropriate symbols (D), (L) or (DL), furthermore when the configuration is not designated the amino acid or residue can have the configuration (D), (L) or (DL). It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly noted to the contrary, a named amino acid shall be construed to include both the (D) or (L) stereoisomers. D- and L-α-Amino acids are represented by the following Fischer projections and wedge-and-dash drawings. In the majority of cases, D- and L-amino acids have R- and S-absolute configurations, respectively.

Ek-arrimaicfc a-βm>aid>

A "reversed" or "retro" peptide sequence as disclosed herein refers to that part of an overall sequence of covalently-bonded amino acid residues (or analogs or mimetics thereof) wherein the normal carboxyl-to amino direction of peptide bond formation in the amino acid backbone has been reversed such that, reading in the conventional left-to- right direction, the amino portion of the peptide bond precedes (rather than follows) the carbonyl portion. See, generally, Goodman, M. and Chorev, M. Accounts of Chem. Res. 1979, 12, 423.

The reversed orientation peptides described herein include (a) those wherein one or more amino-terminal residues are converted to a reversed ("rev") orientation (thus yielding a second "carboxyl terminus" at the left-most portion of the molecule), and (b) those wherein one or more carboxyl-terminal residues are converted to a reversed ("rev") orientation (yielding a second "amino terminus" at the right-most portion of the molecule). A peptide (amide) bond cannot be formed at the interface between a normal orientation residue and a reverse orientation residue.

Therefore, certain reversed peptide compounds of the invention can be formed by utilizing an appropriate amino acid mimetic moiety to link the two adjacent portions of the sequences depicted above utilizing a reversed peptide (reversed amide) bond. In case (a) above, a central residue of a diketo compound may conveniently be utilized to link structures with two amide bonds to achieve a peptidomimetic structure. In case (b) above, a central residue of a diamino compound will likewise be useful to link structures with two amide bonds to form a peptidomimetic structure.

The reversed direction of bonding in such compounds will generally, in addition, require inversion of the enantiomeric configuration of the reversed amino acid residues in order to maintain a spatial orientation of side chains that is similar to that of the non- reversed peptide. The configuration of amino acids in the reversed portion of the peptides is preferably (D), and the configuration of the non-reversed portion is preferably (L). Opposite or mixed configurations are acceptable when appropriate to optimize a binding activity.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trα/w-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g. the ability to bind to the KLX binding domain), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in binding to the KLX binding domain. In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. Thus, the contemplated equivalents include peptidomimetic or non-peptide small molecule binders of the KLX domain. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Also for purposes of this invention, the term "hydrocarbon" is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted.

As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. "Transcriptional regulatory sequence" is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.

Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the subject peptide. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

The term "gene construct" refers to a vector, plasmid, viral genome or the like which includes a coding sequence, can transfect cells, preferably mammalian cells, and can cause expression of the KIX peptide or peptidomimetic of the cells transfected with the construct. As used herein, the term "pharmaceutically acceptable" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not excessively toxic to the hosts of the concentrations of which it is administered. The administration(s) may take place by any suitable technique, including subcutaneous and parenteral administration, preferably parenteral. Examples of parenteral administration include intravenous, intraarterial, intramuscular, and intraperitoneal, with intravenous being preferred.

As used herein, the term "prophylactic or therapeutic" treatment refers to administration to the host of the medical condition. If it is administered prior to exposure to the condition, the treatment is prophylactic (i.e., it protects the host against infection), whereas if administered after infection or initiation of the disease, the treatment is therapeutic (i.e., it combats the existing infection or cancer).

The term "cell-proliferative disorder" denotes malignant as well as nonmalignant cell populations which morphologically often appear to differ from the surrounding tissue. III. Description of Preferred Embodiments

A. Exemplary Chimeric Activators

In one aspect, the invention provides chimeric transcriptional activators comprising at least one KIX binding peptide fused to one or more additional protein domains, e.g., a DNA binding domain, an activation tag, a ligand binding domain and an oligomerizing domain.

In one embodiment, the chimeric activator comprises one KIX binding peptide. In other embodiments, the chimeric activator comprises two or more KIX binding peptides, three or more, five or more, or ten or more KIX binding peptides that are covalently linked. When referring to a polypeptide comprising a peptide, e.g., a KIX binding peptide, herein, it is meant that the polypeptide comprises the amino acid sequence of a KLX peptide covalently linked to other amino acids or peptides to form one polypeptide. The chimeric activator of the invention can further comprise one or more transcriptional activation domains, i.e., transcriptional activation tags. The activation tag(s) can be from the same protein as the KLX binding peptide. Alternatively, the activation tag(s) can be from a different protein, or can be synthetic activation tags. In the latter case, they are said to be heterologous to the KLX binding peptide. Activation tags and KLX binding peptides are also said to be heterologous with respect to each other if they do not occur in the nature in the same order or orientation as in the chimeric activator or they do not occur in nature with the same spacing between them as they have in the chimeric activator. The order of the KLX binding peptide(s) relative to each other and relative to the one or more activation tags can be as desired. Although certain arrangements may provide stronger transactivators, the arrangement necessary to obtain the desired transcriptional activity can easily be determined by a person of skill in the art, and as further described herein. Thus, a chimeric transcription activation region consists of a continuous polypeptide region containing two or more reiterated or otherwise heterologous activation tags, such as KLX binding peptides.

For obtaining strong transcriptional activity, it may be desirable to select activation tags that contact different components of the basic transcriptional machinery, so as to increase the chances of obtaining synergy between the different activation tags.

Where multiple copies of a particular activation tag are included in the same contiguous polypeptide, the composite activator preferably includes at least 3 copies of the activation tag, but more preferably at least 5, 10, 15, or even at least 20 copies of the tag. In a preferred embodiment, the chimeric transcriptional activator includes at least two different activation tags from the group of acidic activation tags, proline-rich transcription activation tags, serine/threonine-rich activation tags, glutamine-rich activation tags, and even more preferably, at least two of those activation tags are selected from disparate proteins (i.e., that do not naturally occur together in the same protein). As desired, the composite activation sequence can be provided as part of a fusion protein including a DNA binding domain.

In other embodiments, the composite activation sequence can be fused with a ligand binding domain which, in the presence of a multivalent ligand, can facilitate recruitment of the composite activator to a DNA-bound complex. The complex can be loaded with multiple activators, in a ligand-dependent manner, by inclusion of multiple ligand binding domains. In yet another embodiment, the subject chimeric proteins include one or more oligomerization sequences which permits non-covalent oligomerization of multiple activators, or of an activator and a DNA binding protein. For instance, a transcriptional activator can include a tetramerization domain. In the instance where the composite activator contains one or more oligomerization domains and/or ligand binding domains, but is not contiguous with a DNA binding domain, the composite activator can be coexpressed in cells with a second protein including a DNA binding domain and appropriate oligomerization or ligand binding domains to form complexes with the composite activator proteins. Thus, composite activator proteins can be recruited to a site of transcriptional regulation by interaction with a DNA binding protein by oligomerization, which may be constitutive or inducible.

Techniques for making the subject fusion proteins are adapted from well-known procedures. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. Alternatively, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. In another method, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments. Amplification products can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel et al. ohn Wiley & Sons: 1992).

(i) KIX Binding Peptides In a preferred embodiment, a KIX binding peptide has an amino acid sequence represented by one or more of the general formulas (I), (II), (III) or (IV), supra, and more preferably (W/L)(W/A)(V/A)Y(D/E)(L/V)LF. Certain preferred KIX binding peptides are shown in Table 1 in the Examples. A KIX binding peptide preferably forms an alpha-helical structure, either constitutively, or after posttranslational modification of the peptide, such as by phosphorylation. Thus, for example a KIX binding peptide derived from CREB is preferably phosphorylated at serine 133 for interacting with a KIX domain. Formation of an alpha helical structure by a peptide can be determined, e.g., by crystallography. A KLX binding peptide is also preferably capable of precipitating p300/CBP from a cell lysate, as further described herein.

(ii) Other Activation Tags

The activation tags useful in the composite activators of the present invention can be derived from one or more transcription factors. Polypeptides which can function to activate transcription in eukaryotic cells are well known in the art. In particular, transcriptional activation domains which contain suitable activation tags have been described for many DNA binding proteins and have been shown to retain their activation function when the transcriptional activation domain, or a suitable fragment thereof, is transferred to a heterologous protein.

Any particular activation tag is preferably at least 6 amino acids in length, and preferably contains no more than about 300 amino acid residues, though even more preferably, less than 200 or even less than 100 residues.

Activation tags can be naturally occurring activation units, e.g., portions of transcription factors, or they can be synthetic.

Types of transcriptional activation domains that can be used in the invention include, in addition to the acidic transcription activation domains, proline-rich transcription activation domains, serine/threonine-rich transcription activation domains and glutamine-rich transcription activation domains. Examples of proline-rich activation domains include amino acid residues 399-499 of CTF/NFl and amino acid residues 31- 76 of AP2. Examples of serine/threonine-rich transcription activation domains include amino acid residues 1-427 of ITFl and amino acid residues 2-451 of ITF2. Examples of glutamine-rich activation domains include amino acid residues 175-269 of Octl and amino acid residues 132-243 of Spl. The amino acid sequences of each of the above described regions, and of other useful transcriptional activation domains, are disclosed in Seipel, K. et al. (EMBO I. (1992) 13:4961-4968).

Preferred peptides for use in the fusion protein of the invention is the herpes simplex virus virion protein 16 (refeπed to herein as VP16, the amino acid sequence of which is disclosed in Triezenberg, S.J. et al. (1988) Genes Dev. 2:718-729). In one embodiment, an activation tag coπesponding to about 127 of the C-terminal amino acids of VP16 is used. For example, a polypeptide having an amino acid sequence coπesponding to positions 208-335 of VP16 can be used as the second polypeptide in the fusion protein. Suitable C-terminal peptide portions of VP16 are described in Seipel, K. et al. (EMBO I. (1992) 13:4961-4968). One particularly important source of transcription activation tags which are featured in a number of embodiments of the invention is the (human) NF-kB subunit p65. In one embodiment the chimeric activator contains one or more copies of a peptide sequence comprising all or part of the p65 sequence spanning residues 450-550, or a peptide sequence derived therefrom. Another prefeπed activation tag is provided in residues 753-881 of GAL4. Still other illustrative activation domains and motifs of human origin include the activation domain of human CTF, the 18 amino acid (NFLQLPQQTQGALLTSQP) glutamine rich region of Oct-2, the N-terminal 72 amino acids of p53, the SYGQQS repeat in Ewing sarcoma gene and an 11 amino acid (535-545) acidic rich region of Rel A protein. In addition to previously described transcriptional activation domains, novel transcriptional activation tags, which can be identified by standard techniques, are within the scope of the invention. The transcriptional activation ability of a polypeptide can be assayed by linking the polypeptide to another polypeptide having DNA binding activity and determining the amount of transcription of a target sequence that is stimulated by the fusion protein. For example, a standard assay used in the art utilizes a fusion protein of a putative activation tag and a GAL4 DNA binding domain (e.g., amino acid residues 1- 93). This fusion protein is then used to stimulate expression of a reporter gene linked to GAL4 binding sites (see e.g., Seipel, K. et al. (1992) EMBO I. 11:4961-4968 and references cited therein). It is well known in the art that certain transcription factors are active only in specific cell types, i.e., that transcription factors can act in a tissue specific manner. Without wanting to be limited to a specific mechanism of action, it is possible that this tissue specificity results from the fact that the transcription factor interacts with specific factors, e.g, cofactors, which are present only in certain cell types. This tissue specificity can be localized to a specific portion of the transcription factor. In certain transcription factors, this portion is located outside activation domains, whereas in other transcription factors, this portion may be localized within an activation domain. Thus, by using activation tags which are functional essentially in specific cells, it is possible to design a transcriptional activator of the invention having a certain tissue specificity.

The multiple activation units and other domains of the transcriptional of the invention can be from any eukaryotic species, and it is not necessary that every unit or domain be from the same species. Prefeπed species include vertebrates, such as mammals. Even more prefeπed units or domains are from humans. For use of the transcriptional activators of the invention in gene therapy in a subject of a specific species, e.g., human, it is preferable to use units and domains from the same species to avoid immune reactions against the transcriptional activator or complex.

The activation units of a transcriptional coactivator can be covalently linked to each other in a linear aπay, i.e., the NH2 -terminus of one activation unit is linked to the COOH-terminus of another activation unit. The activation units can be aπanged in any order. However, as described herein, it has been observed, that certain arrangements of activation units results in higher levels of transcriptional activation than other aπangements. The order in which the activation units should be arranged will depend on the result desired, i.e., the degree of transactivation that one desires to achieve, and can be determined, e.g., by performing cotransfection experiments, as described in the Examples. Briefly, expression vector encoding the activation units in various aπangements linked to a DNA binding domain are cotransfected together with a reporter construct containing a reporter gene operably linked to a promoter containing a DNA site recognized by the DNA binding domain, and expression of the reporter gene is measured. For such assays, it is preferable to use a cell line in which the activation units are known to be active. In another embodiment, combinatorial intron splicing can be used to generate a diverse library of composite activation sequences. U.S. Patent 5,498,531 describes a means for carrying out the equivalent of "exon shuffling" by intron-mediated trans- splicing.

(iii) DNA binding domain

In certain embodiments of the invention, the KIX binding peptide is provided as part of a chimeric protein which further comprises a DNA-binding domain. In other embodiments, the transcriptional activator of the invention is localized to a DNA sequence by virtue of constitutive or inducible oligomerization with a DNA binding polypeptide comprising a DNA binding domain. In such instances, the DNA binding domain can be provided in a fusion protein which one or more oligomerization domains or ligand binding domains. The choice of component DNA-binding domains may be influenced by a number of considerations, including the species, system and cell type to which is targeted; the feasibility of its incorporation into a chimeric protein, as may be shown by modeling; and the desired application or utility. The DNA binding domain can be a naturally occurring DNA-binding domain from a transcription factor. Alternatively, the DNA binding domain can be an artificial (or partially artificial) polypeptide sequence having DNA binding activity. For example, the DNA-binding domain can be a naturally occurring DNA binding domain that has been modified to recognize a different DNA binding site. The particular DNA-binding domain chosen will depend on the target promoter. For example, if the gene to be transcriptionally activated by the subject method is an endogenous gene, the DNA- binding domain must be able to interact with the promoter of the endogenous gene (endogenous promoter). Alternatively, as described in greater detail below, the endogenous promoter could be replaced, e.g., by homologous recombination, with a heterologous promoter for which the DNA binding domain is selected. Such a substitution may be necessary if no transcription factor is known to bind the endogenous promoter of interest. Alternatively, in such a situation, it is also possible to clone a DNA-binding domain interacting specifically with a sequence in the promoter of interest. This can be done, e.g., by phage display screening with a DNA molecule comprising at least a portion of the promoter of interest.

Desirable properties of DNA binding domains include high affinity for specific nucleotide sequences, termed herein "target sequences", low affinity for most other sequences in a complex genome (such as a mammalian genome), low dissociation rates from specific DNA sites, and novel DNA recognition specificities distinct from those of known natural DNA-binding proteins. Preferably, binding of a DNA-binding domain to a specific target sequence is at least two, more preferably three and even more preferably more than four orders of magnitude greater than binding to any one alternative DNA sequence, as may be measured by relative Kd values or by relative rates or levels of transcription of genes associated with the selected and any alternative DNA sequences. It is also prefeπed that the selected DNA sequence be recognized to a substantially greater degree by the DNA binding domain of the trancriptional activator of the invention than by an endogenous protein. Thus, for example, target gene expression in a cell is preferably two, more preferably three, and even more preferably more than four orders of magnitude greater in the presence of the transcriptional activator of the invention containing a DNA-binding region than in its absence.

Prefeπed DNA binding domains have a dissociation constant for a target sequence below 10^-8 M, preferably 10"⁹ M, more preferably below 10"¹⁰ M, even more preferably below 10^-11 M. For gene therapy applications, they are preferably derived from human proteins.

From a structural perspective, DNA-binding that can be used in the invention may be classified as DNA-binding proteins with a helix-turn-helix structural design, such as, but not limited to, Myb, Ultrabithorax, Engrailed, Paired, Fushi tarazu, HOX, Unc86, the Ets and homeobox families of transcription factors, and the previously noted Octl, Oct2 and Pit; zinc finger proteins, such as Zif268, SWI5, Krueppel and Hunchback; steroid receptors; DNA-binding proteins with the helix-loop-helix structural design, such as Daughterless, Achaete-scute (T3), MyoD, E12 and E47; and other helical motifs like the leucine-zipper, which includes GCN4, C/EBP, c-Fos/c-Jun and JunB. The amino acid sequences of the component DNA-binding domains may be naturally-occurring or non-naturally-occurring (or modified).

One strategy for obtaining component DNA-binding domains with properties suitable for this invention is to modify an existing DNA-binding domain to reduce its affinity for DNA into the appropriate range. For example, a homeodomain such as that derived from the human transcription factor Phoxl, may be modified by substitution of the glutamine residue at position 50 of the homeodomain. Substitutions at this position remove or change an important point of contact between the protein and one or two base pairs of the 6-bp DNA sequence recognized by the protein. Thus, such substitutions reduce the free energy of binding and the affinity of the interaction with this sequence and may or may not simultaneously increase the affinity for other sequences. Such a reduction in affinity is sufficient to effectively eliminate occupancy of the natural target site by this protein when produced at typical levels in mammalian cells. But it would allow this domain to contribute binding energy to and therefore cooperate with a second linked DNA-binding domain. Other domains that amenable to this type of manipulation include the paired box, the zinc-finger class represented by steroid hormone receptors, the myb domain, and the ets domain.

In another embodiment, the DNA binding domain is created from the assembly of DNA binding domains from various transcription factors, resulting in a DNA binding domain having a novel DNA binding specificity. Such DNA binding domains, referred to herein as "composite DNA binding domains" can be designed to specifically recognize unique binding sites. For example, a DNA binding domain can be constructed that comprises DNA binding regions from a zinc finger protein and a homeobox protein. One such DNA binding domain is ZFHD1, a composite DNA binding domain comprising an Oct-1 homeodomain and zinc fingers 1 and 2 of Zif268, which is further described in PCT Application WO 96/20951 by Pomerantz et al.

The DNA sequences recognized by a chimeric protein containing a composite DNA-binding domain can be determined experimentally, as described below, or the proteins can be manipulated to direct their specificity toward a desired sequence. A desirable nucleic acid recognition sequence consists of a nucleotide sequence spanning at least ten, preferably eleven, and more preferably twelve or more bases. The component binding portions (putative or demonstrated) within the nucleotide sequence need not be fully contiguous; they may be interspersed with "spacer" base pairs that need not be directly contacted by the chimeric protein but rather impose proper spacing between the nucleic acid subsites recognized by each module. These sequences should not impart expression to linked genes when introduced into cells in the absence of the engineered DNA-binding protein.

To identify a nucleotide sequence that is recognized by a transcriptional activator protein containing the composite DNA-binding region, preferably recognized with high affinity (dissociation constant 10^"11 M or lower are especially prefeπed), several methods can be used. If high-affinity binding sites for individual subdomains of the composite DNA-binding region are already known, then these sequences can be joined with various spacing and orientation and the optimum configuration determined experimentally (see below for methods for determining affinities). Alternatively, high- affinity binding sites for the protein or protein complex can be selected from a large pool of random DNA sequences by adaptation of published methods (Pollock, R. and Treisman, R., 1990, A sensitive method for the determination of protein-DNA binding specificities. Nucl. Acids Res. 18, 6197-6204). Bound sequences are cloned into a plasmid and their precise sequence and affinity for the proteins are determined. From this collection of sequences, individual sequences with desirable characteristics (i.e., maximal affinity for composite protein, minimal affinity for individual subdomains) are selected for use. Alternatively, the collection of sequences is used to derive a consensus sequence that carries the favored base pairs at each position. Such a consensus sequence is synthesized and tested (see below) to confirm that it has an appropriate level of affinity and specificity.

A number of well-characterized assays are available for determining the binding affinity, usually expressed as dissociation constant, for DNA-binding proteins and the cognate DNA sequences to which they bind. These assays usually require the preparation of purified protein and binding site (usually a synthetic oligonucleotide) of known concentration and specific activity. Examples include electrophoretic mobility- shift assays, DNasel protection or "footprinting", and filter-binding. These assays can also be used to get rough estimates of association and dissociation rate constants. These values may be determined with greater precision using a BIAcore instrument. In this assay, the synthetic oligonucleotide is bound to the assay "chip," and purified DNA- binding protein is passed through the flow-cell. Binding of the protein to the DNA immobilized on the chip is measured as an increase in refractive index. Once protein is bound at equilibrium, buffer without protein is passed over the chip, and the dissociation of the protein results in a return of the refractive index to baseline value. The rates of association and dissociation are calculated from these curves, and the affinity or dissociation constant is calculated from these rates. Binding rates and affinities for the high affinity composite site may be compared with the values obtained for subsites recognized by each subdomain of the protein. As noted above, the difference in these dissociation constants should be at least two orders of magnitude and preferably three or greater.

The invention further provides chimeric activators of the present invention provided as a fusion protein with an inducible DNA binding domain(s). In one embodiment, the inducible DNA binding domain is the E. coli tet repressor (TetR), which binds to tet operator (tetO) sequences upstream of target genes. In the presence of tetracycline, or an analog, which bind to tetR, DNA binding is abolished and thus transactivation is abolished. This system, in which the TetR had previously been linked to transcription activation domains, e.g, from VP16, is generally refeπed to as an allosteric "off-switch" described by Gossen and Bujard (Proc. Natl. Acad. Sci. U.S.A. (1992) 89:5547) and in U.S. Patents 5,464,758; 5,650,298; and 5,589,362 by Bujard et al. Furthermore, depending on the concentration of the antibiotic in the culture medium (0-1 mu g/ml), target gene expression can be regulated over concentrations up to several orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in eukaryotic cells but also is suitable for creation of "on/off situations for such genes in a reversible way. This system provides low background and relatively high target gene expression in the absence of tetracycline or an analog. Thus, the invention described herein provides a method for obtaining even stronger transcriptional induction of a target gene, which is regulatable by the tetracycline system or other inducible DNA binding domain. For example, a TetR can be linked to a KIX binding peptide and one or more activation tags, such that high levels of transcription occur in the absence of tetracycline or analog thereof and that transcription is repressed in the presence of tetracycline.

In another embodiment, a "reverse" Tet system is used, again based on a DNA binding domain that is a mutant of the E. coli TetR, but which binds to TetO in the presence of Tet. Thus, the invention described herein provides a method for obtaining even stronger transcriptional induction of a target gene in the presence of tetracycline or an analog thereof from a very low background in the absence of tetracycline. (iv) Oligomerization domains

As set out above, in various embodiments of the transcriptional activators, the chimeric proteins can also include at least one oligomerization domain. Such a domain can be a constitutive oligomerization domain, or an inducible oligomerization domain, i.e., a domain mediating oligomerization only in the presence of a third molecule, such as a small organic molecule. Examples of constitutive oligomerization domains include leucine zippers.

Example of inducible oligomerization domains include FK506 and cyclosporin binding domains of FK506 binding proteins and cyclophilins, and the rapamycin binding domain of FRAP (tori). Such inducible oligomerization domains are refeπed to herein as "ligand binding domains" and are further described herein under the section entitled accordingly.

In one embodiment of the invention, at least one KIX binding peptide optionally with one or more activation tag or ligand binding domain or DNA binding domain is linked to a constitutive oligomerization domain, e.g., a dimerization or tetramerization domain. A dimerization domain is defined herein as a sequence of amino acids capable of forming homodimers or heterodimers. One example of a dimerization domain is the leucine zipper (LZ) element. Leucine zippers have been identified, generally, as stretches of about 35 amino acids containing 4-5 leucine residues separated from each other by six amino acids (Maniatis and Abel (1989) Nature 341:24-25). Exemplary leucine zippers occur in a variety of eukaryotic DNA binding proteins, such as GCN4, C/EBP, c-Fos, c- Jun, c-Myc and c-Max. Other dimerization domains include helix-loop-helix domains (Muπe, C. et al. (1989) Cell 58:537-544). Dimerization domains may also be selected from other proteins, such as the retinoic acid receptor, the thyroid hormone receptor or other nuclear hormone receptors (Kurokawa et al. (1993) Genes Dev. 7:1423-1435) or from the yeast transcription factors GAL4 and HAP1 (Marmonstein et al. (1992) Nature 356:408-414; Zhang et al. (1993) Proc. Natl. Acad. Sci. USA 90:2851-2855). Dimerization domains are further described in U.S. Patent No. 5,624,818 by Eisenman. In another embodiment, the oligomerization domain is a tetramerization domain.

For example, four activation units, e.g., KLX binding peptides, can be linked to a single DNA binding domain or a ligand binding domain by covalently linking the activation units to a tetramerization domain. In a prefeπed embodiment, the tetramerization domain is the E. coli lactose repressor tetramerization domain (amino acids 46-360; Chakerian et al. (1991) J. Biol. Chem. 266:1371; Alberti et al. (1993) EMBO J. 12:3227; and Lewis et al. (1996) Nature 271:1247), as described in the Examples. Thus, the inclusion of a tetramerization domain in a transcriptional activator allows four activation domains to be complexed together and form a transcriptional activator complex. Furthermore, more than one activation unit can be linked to one tetramerization domain, to thereby form a transcriptional activator complex comprising more than 4 activation units.

In another embodiment, the tetramerization domain is that from a p53 protein. The p53 tetramerization domain maps to residues 322-355 of p53 (Wang et al. (1994) Mol. Cell. Biol. 14:5182; Clore et al. (1994) Science 265:386) and is further described in U.S. Pat. No. 5,573,925 by Halazonetis. The invention also provides for transcriptional activators containing at least one modified oligomerization domain. Modifications in the oligomerization domain may increase the stability of tetramer formation, for example, substitutions that stabilize oligomerization driven by leucine zippers are known (Krylov et al. (1994) cited above; O'Shea et al. (1992) cited above). As an exemplary modification of this type, residues 174 or 175 of human p53 are substituted by glutamine or leucine, respectively, in a p53 chimeric protein of this invention.

In other embodiments, the oligomerization domain can be an altered p53 tetramerization domain which is incapable of forming hetero-tetramers with p53 proteins that have a wild-type p53 tetramerization domain, such as wild-type p53 or tumor- derived p53 mutants. Such altered p53 tetramerization domains are further described in

U.S. Pat. No. 5,573,925 by Halazonetis.

These altered p53 tetramerization domains are characterized by disruption of the native p53 tetramerization domain and insertion of a heterologous oligomerization domain in a way that preserves tetramerization. According to this invention, a disruption of the p53 tetramerization domain, involving residues 335-348 or a subset of these residues, sufficiently disrupts the function of this domain so that it can no longer drive tetramerization with wild-type p53 or tumor-derived p53 mutants. At the same time, however, introduction of a heterologous dimerization domain reestablishes the ability to form tetramers, which is mediated both by the heterologous dimerization domain and by the residual tetramerization domain of p53.

Other exemplary suitable tetramerization domains include artificial tetramerization domains, such as variants of the GCN4 leucine zipper that form tetramers (Alberti et al. (1993) EMBO J. 12:3227-3236; Harbury et al. (1993) Science 262:1401-1407; Krylov et al. (1994) (1994) EMBO J. 13:2849-2861). One of skill in the art could readily select alternate tetramerization domains. For example, the tetrameric variant of GCN4 leucine zipper described in Harbury et al. (1993), supra, has isoleucines at positions d of the coiled coil and leucines at positions a, in contrast to the original zipper which has leucines and valines, respectively.

The GCN4 leucine zipper drives parallel subunit assembly [Harbury et al. (1993), cited above], while the native p53 tetramerization domain drives antiparallel assembly [Clore et al. (1994) cited above; Sakamoto et al. (1994) Proc. Natl. Acad. Sci. USA

91 :8974-8978]. Thus, various conformations of activation unit complexes can be obtained by choosing various tetramerization domains.

In addition, the art also provides a variety of techniques for identifying other naturally occurring oligomerization domains, as well as oligomerization domains derived from mutant or otherwise artificial sequences. See, for example, Zeng et al. (1997) Gene 185:245; O'Shea et al. (1992) Cell 68:699-708; Krylov et al. [cited above].

The distance between the oligomerization domain and other components of the fusion proteins can be varied. In one embodiment, there is no linker between an activation unit and a tetramerization domain, e.g, an altered GCN4 leucine zipper. In other embodiments however, there are glutamic acid or asparagine or isoleucine linkers, respectively. Linkers may be present for cloning convenience or to confer some useful property. For example, residues that stabilize specific secondary structure elements, such as alpha -helices, are known (Richardson et al. (1988) Science 240:1648-1652]. Such residues can be introduced in the linkers to stabilize the oligomerization domains. For example the linkers glycine-asparagine, arginine-glycine- asparagine, arginine- glycine-glycine-asparagine-proline-glutamic acid, glycine-glycine- asparagine- glutamine-alanine, are all designed to stabilize the N-terminus of the alpha -helical oligomerization domain.

In one embodiment, the chimeric protein comprises an activation unit fused to an asparagine linker and then to a tetrameric variant of GCN4 residues 249-281.

Alternatively, the linker can be an arginine-glycine-asparagine linker, an arginine- glycine-glycine-asparagine- proline-glutamic acid linker, a glycine-glycine-asparagine- glutamine-alanine linker.

A variety of other amino acid or peptide linkers may be used for the reasons discussed above, provided they do not interfere with the function of the activation units and ligand binding domain or DNA binding domain.

(v) Ligand binding domain

In another embodiment of the invention, components of the subject system include one or more ligand binding domains for mediating oligomerization of fusion proteins in a ligand-dependent fashion. In a prefeπed embodiment, the ligand is capable of interacting with two ligand binding domains. In an exemplary embodiment, formation of transcriptional complexes is regulated by addition of a ligand, and comprises introducing into the cell a fusion protein including a KIX binding peptide and a ligand binding domain, as well as a second fusion protein including a DNA binding domain and a ligand binding domain, such that in the presence of the ligand, a transcriptional activator complex is formed between the two fusion proteins. Prefeπed ligands include macro lides such as rapamycin, cyclosporin A, FK506, FK1012, and analogs thereof, and other synthetic dimerizers or oligomerizers. Ligand binding domains include the FK506 binding domain of FKBP, the cyclosporin-binding domain of calcineurin, and the rapamycin-binding domain of FRAP. These binding domains and ligands are further disclosed, e.g., in PCT/US93/01617 and U.S. Patents 5,871,753; 5,869,337; and 5,834,266, all by Crabtree et al. Such fusion proteins permit control of the expression of a target gene to be dependent on addition of an appropriate ligand, e.g., one which is capable of interacting simultaneously with the two ligand binding domains.

In general, the ligand binding domain of a chimeric protein of this invention can be any convenient domain which will allow for ligand-dependent oligomerization of fusion proteins using a natural or unnatural ligand, preferably an unnatural synthetic ligand. Of particular interest are binding proteins for which ligands (preferably small organic ligands) are known or may be readily produced. These receptors or ligand binding domains include the FKBPs and cyclophilin receptors, the steriod receptors, the tetracycline receptor, the other receptors indicated above, and the like, as well as "unnatural" receptors, which can be obtained from antibodies, particularly the heavy or light chain subunit, mutated sequences thereof, random amino acid sequences obtained by stochastic procedures, combinatorial syntheses, and the like.

For the most part, the receptor domains will be at least about 50 amino acids, and fewer than about 350 amino acids, usually fewer than 200 amino acids, either as the natural domain or truncated active portion thereof. Preferably the binding domain will be small (<25 kDa, to allow efficient transfection in viral vectors), monomeric (this rules out the avidin-biotin system), nonimmunogenic, and should have synthetically accessible, cell permeable, nontoxic ligands that can be configured for dimerization.

Multimerizing ligands useful in practicing this invention are multivalent, i.e., capable of binding to, and thus multimerizing, two or more of chimeric protein molecules having a ligand binding domain. The multimerizing ligand may bind to the proteins containing such ligand-binding domains, in either order or simultaneously, preferably with a Kd value below about 10^-6, more preferably below about 10^-7, even more preferably below about 10^-8, and in some embodiments below about 10"⁹ M. The ligand preferably is not a protein or polypeptide and has a molecular weight of less than about 5 kDa, preferably below 2 kDa. The ligand-binding domains of the chimeric proteins so multimerized may be the same or different. Ligand binding domains include among others, various immunophilin domains. One example is the FKBP domain which is capable of binding to dimerizing ligands incorporating FK506 moieties or other FKBP-binding moieties. See e.g. PCT/US93/0161701617 and U.S. Patents 5,871,753; 5,869,337; and 5,834,266, all by Crabtree et al., the full contents of which are hereby incorporated by reference.

The portion of the construct encoding the ligand binding domain can be subjected to mutagenesis for a variety of reasons. The mutagenized domain can provide for higher binding affinity, allow for discrimination by a ligand between the mutant and naturally occurring forms of the ligand binding domain, provide opportunities to design a ligand-ligand binding domain pairs, or the like. The change in the ligand binding domain can involve changes in amino acids known to be at the binding site, random mutagenesis using combinatorial techniques, where the codons for the amino acids associated with the binding site or other amino acids associated with conformational changes can be subject to mutagenesis by changing the codon(s) for the particular amino acid, either with known changes or randomly, expressing the resulting proteins in an appropriate prokaryotic host and then screening the resulting proteins for binding The ability to employ in vitro mutagenesis or combinatorial modifications of sequences encoding proteins allows for the production of libraries of proteins which can be screened for binding affinity for different ligands. For example, one can totally randomize a sequence of 1 to 5, 10 or more codons, at one or more sites in a DNA sequence encoding a binding protein, make an expression construct and introduce the expression construct into a unicellular microorganism, and develop a library. One can then screen the library for binding affinity to one or desirably a plurality of ligands. The best affinity sequences which are compatible with the cells into which they would be introduced can then be used as the ligand binding domain. The ligand would be screened with the host cells to be used to determine the level of binding of the ligand to endogenous proteins. A binding profile could be defined weighting the ratio of binding affinity to the mutagenized binding domain with the binding affinity to endogenous proteins. Those ligands which have the best binding profile could then be used as the ligand. Phage display techniques, as a non-limiting example, can be used in carrying out the foregoing. In other embodiments, antibody subunits, e.g. heavy or light chain, particularly fragments, more particularly all or part of the variable region, or fusions of heavy and light chain to create single chain antibodies, can be used as the ligand binding domain. Antibodies can be prepared against haptenic molecules which are physiologically acceptable and the individual antibody subunits screened for binding affinity. The cDNA encoding the subunits can be isolated and modified by deletion of the constant region, portions of the variable region, mutagenesis of the variable region, or the like, to obtain a binding protein domain that has the appropriate affinity for the ligand. In this way, almost any physiologically acceptable haptenic compound can be employed as the ligand or to provide an epitope for the ligand. Instead of antibody units, natural receptors can be employed, where the binding domain is known and there is a useful ligand for binding. In yet another embodiment of the invention, the DNA binding unit is linked to more than one ligand binding domain. For example, a DNA binding domain can be linked to at least 2, 3, 4, or 5 ligand binding domains. A DNA binding domain can also be linked to at least 5 ligand binding domains or any number of ligand binding domains. In such embodiments, the ligand binding domains can be, by illustration, linked to each other in a linear array, by linking the NH2-terminus of one ligand binding domain to the COOH-terminus of another ligand binding domain. Thus, numerous composite activators can be linked to a single DNA binding domain in the presence of a ligand.

The invention further provides additional induction systems. In one embodiment, the invention uses an alternative allosteric on-switch for transcription which employs a deletion mutant of the human progesterone receptor, i.e., which no longer binds progesterone or any known endogenous steroid but can be activated by the orally active progesterone antagonist RU486, described, e.g, in Wang et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:8180. Activation was demonstrated, e.g, in cells transplanted into mice using doses of RU486 (5-50 μg/kg) considerably below the usual dose for inducing abortion in humans (10 mg/kg). However, according to the art describing this system, the induction ratio in culture and in animals was rather low. Applying the invention described herein in this system would provide an inducible system having a higher induction ratio. Thus, the invention provides a transcriptional activator comprising at least one subunit which is covalently linked to a mutant steroid binding domain to yield a transcriptional activator which transactivates in a RU486-dependent manner, resulting in high induction ratios.

The invention can be adapted to an ecdysone inducible system. Early work demonstrated that fusing the Drosophila steroid ecdysone (Ec) receptor (EcR) Ec- binding domain to heterologous DNA binding and activation domains, such as E. coli lex A and herpesvirus VP16 permits ecdysone-dependent activation of target genes downstream of appropriate binding sites (Christopherson et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:6314). An improved ecdysone regulation system has been developed, using the DNA binding domain of the EcR itself. In this system, the regulating transcription factor is provided as two proteins: (1) a truncated, mutant EcR fused to herpes VP16 and (2) the mammalian homolog (RXR) of Ultraspiracle protein (USP), which heterodimerizes with the EcR (No et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:3346). In this system, because the DNA binding domain was also recognized by a human receptor (the human farnesoid X receptor), it was altered to a site recognized only by the mutant EcR. Thus, the invention provides an ecdysone inducible system, in which a truncated mutant EcR is fused to at least one KLX binding peptide and optionally one or more activation tags of the invention. The transcriptional activator further comprises USP, thereby providing high level induction of transcription of a target gene having the EcR target sequence, dependent on the presence of ecdysone.

The invention can also be applied to any other inducible system, thereby providing an inducible system having a higher ratio of background to induction due to the potent transcriptional activity of the transcriptional activators of the invention.

(vi) Additional domains and linkers

Additional domains may be included in the transcriptional activators of this invention. For example, the transcriptional activators may contain a nuclear localization sequence which provides for the protein to be translocated to the nucleus. Accordingly, in one embodiment of the invention, at least one of the subunits of the transcriptional activator of the invention, e.g., activation unit, DNA binding domain, or ligand binding domain, further comprises a nuclear localization signal (NLS). A NLS can be fused to the N-terminus, or the C-terminus of a subunit, e.g., an activation unit, or can be inserted at the junction of one subunit with another subunit, e.g., between an activation domain and a DNA binding or ligand binding domain or oligomerization domain or elsewhere in the protein, as long as the function of the subunits is not disrupted by insertion of the NLS. Typically a nuclear localization sequence has a plurality of basic amino acids, refeπed to as a bipartite basic repeat (reviewed in Garcia-Bustos et al. (1991) Biochimica et Biophysica Acta 1071:83-101). The NLS may be that of SV40 large T antigen which is comprised of amino acids proline-lysine-lysine-lysine-arginine-lysine- valine (Kalderon et al. (1984) Cell 39:499-509). The NLS may also be from a p53 protein. Wild-type p53 contains three nuclear localization signals (NLS), all of which map to the C-terminus of wild-type p53 and specifically to residues 316-325, 369-375 and 379-384 of p53 (Shaulsky et al. (1990) Mol. Cell. Biol.l0:6565-6577). Additional heterologous NLS are described by Shaulsky et al (1990) supra and Shaulsky et al.(1991) Oncogene 6:2056. The chimeric proteins may include domains that facilitate their purification, e.g. "histidine tags" or a glutathione-S-transferase domain. They may include "epitope tags" encoding peptides recognized by known monoclonal antibodies for the detection of proteins within cells or the capture of proteins by antibodies in vitro. It may be necessary in some instances to introduce an unstructured polypeptide linker region between an activation tag or tags and other portions of the chimeric activator. Where the fusion protein also includes, for example, oligomerization sequences, it may be preferable to situate the linker between the oligomerization sequences and the activation tags. The linker can facilitate enhanced flexibility of the fusion protein, while the oligomerization sequences are relatively free to make other inter-protein contacts, e.g., with other chimeric activators. The linker can also reduce steric hindrance between any two fragments of the fusion protein. The linker can also facilitate the appropriate folding of each fragment to occur. The linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein. An exemplary linker sequence is the linker found between the C-terminal and N-terminal domains of the RNA polymerase a subunit. Other examples of naturally occurring linkers include linkers found in the lcl and LexA proteins. Alternatively, the linker can be of synthetic origin. For instance, the sequence (Gly4Ser)3 can be used as a synthetic unstructured linker. Linkers of this type are described in Huston et al. (1988) PNAS 85:4879; and U.S. Patent No. 5,091,513, both incoφorated by reference herein.

In some embodiments it is preferable that the design of a linker involve an aπangement of domains which requires the linker to span a relatively short distance, preferably less than about 10 Angstrom. However, in certain embodiments, depending, e.g., upon the selected DNA-binding domains and the configuration, the linker may span a distance of up to about 50 Angstrom.

Within the linker, the amino acid sequence may be varied based on the prefeπed characteristics of the linker as determined empirically or as revealed by modeling. For instance, in addition to a desired length, modeling studies may show that side groups of certain amino acids may interfere with the biological activity, e.g. DNA binding or transcriptional activation, of the protein. Considerations in choosing a linker include flexibility of the linker, charge of the linker, and presence of some amino acids of the linker in the naturally-occurring subunits. The linker can also be designed such that residues in the linker contact DNA, thereby influencing binding affinity or specificity, or to interact with other proteins. For example, a linker may contain an amino acid sequence which can be recognized by a protease so that the activity of the chimeric protein could be regulated by cleavage. In some cases, particularly when it is necessary to span a longer distance between subunits or when the domains must be held in a particular configuration, the linker may optionally contain an additional folded domain.

Most of the subject fusion proteins can be tested for activity in vivo using a simple assay (F.M. Ausubel et al. Eds. Cuπent Protocols in Molecular Biology, John Wiley & Sons, New York, 1994; de Wet et al. (1987) Mol. Cell Biol. 7:725). The in vivo assay requires an expression construct containing and capable of directing the expression of a recombinant DNA sequence encoding the transcriptional activator, and as appropriate, other proteins required for DNA localization of the activator. The assay also requires a plasmid containing a reporter gene , e.g., the luciferase gene, the chloramphenicol acetyl transferase (CAT) gene, secreted alkaline phosphatase or the human growth hormone (hGH) gene, linked to a binding site for the transcription factor. The expression constructs are introduced into host cells which normally do not produce interfering levels of the reporter gene product. A second group of cells, which lacks the transcriptional activator or the means for localizing the activator to the reporter gene can serve as the control. The production of mRNA or protein encoded by the reporter gene is measured.

An increase in reporter gene expression not seen in the controls indicates that the transcription factor is a positive regulator of transcription. If reporter gene expression is less than that of the control, the transcription factor is a negative regulator of transcription. Optionally, the assay may include a transfection efficiency control plasmid. This plasmid expresses a gene product independent of the test gene, and the amount of this gene product indicates roughly how many cells are taking up the plasmids and how efficiently the DNA is being introduced into the cells. Additional guidance on evaluating chimeric proteins of this invention is provided below.

B. Nucleic Acid Compositions

In another aspect of the invention, the proteins described herein are provided in expression vectors. For instance, expression vectors are contemplated which include a nucleotide sequence encoding a polypeptide containing a transcriptional activator of the present invention, which coding sequence is operably linked to at least one transcriptional regulatory sequence. Regulatory sequences for directing expression of the instant fusion proteins are art-recognized and are selected by a number of well understood criteria. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the fusion proteins of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, and the promoters of the yeast a-mating factors and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.

As will be apparent, the subject gene constructs can be used to cause expression of the subject fusion proteins in cells propagated in culture, e.g. to produce proteins or polypeptides, including fusion proteins, for purification.

This invention also pertains to a host cell transfected with a recombinant gene in order to express one of the subject polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For example, a fusion proteins of the present invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Accordingly, the present invention further pertains to methods of producing the subject fusion proteins. For example, a host cell transfected with an expression vector encoding a protein of interest can be cultured under appropriate conditions to allow expression of the protein to occur. The protein may be secreted, by inclusion of a secretion signal sequence, and isolated from a mixture of cells and medium containing the protein. Alternatively, the protein may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The proteins can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the protein.

Thus, a coding sequence for a fusion protein of the present invention can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Expression vehicles for production of a recombinant protein include plasmids and other vectors. For instance, suitable vectors for the expression of the instant fusion proteins include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incoφorated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used.

The prefeπed mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Ban virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant fusion proteins by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW- derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the beta-gal containing pBlueBac IH).

In yet other embodiments, the subject expression constructs are derived by insertion of the subject gene into viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and heφes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. As described in greater detail below, such embodiments of the subject expression constructs are specifically contemplated for use in various in vivo and ex vivo gene therapy protocols. Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transfeπed nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy puφoses (for a review see Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a fusion protein of the present invention, e.g., a transcriptional activator, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Cuπent Protocols in Molecular Biology, Ausubel, F.M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include SYMBOL 121 \f "SymboV'Crip, SYMBOL 121 \f "SymboV'Cre, SYMBOL 121 \f "Symbol"2 and SYMBOL 121 \f "SymboT'Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone maπow cells, in vitro and/or in vivo (see for example Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al., (1990) PNAS USA 87:6141-6145; Huber et al, (1991) PNAS USA 88:8039-8043; Ferry et al., (1991) PNAS USA 88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al., (1992) PNAS USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy 3:641-647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al., (1989) PNAS USA 86:9079-9083; Man et al., (1992) J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single- chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.

Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivate in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al., (1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand et al, (1992) PNAS USA 89:6482- 6486), hepatocytes (Herz and Gerard, (1993) PNAS USA 90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors cuπently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, E.J. Muπay, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression of the inserted chimeric gene can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the viral E3 promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery of the subject chimeric genes is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a heφes virus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Cuπ. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al., (1989) J. Virol. 63:3822-3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., (1984) PNAS USA 81 :6466-6470; Tratschin et al., (1985) Mol. Cell. Biol. 4:2072- 2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993) J. Biol. Chem. 268:3781-3790).

Other viral vector systems that may have application in gene therapy have been derived from heφes virus, vaccinia virus, and several RNA viruses. In particular, heφes virus vectors may provide a unique strategy for persistence of the recombinant gene in cells of the central nervous system and ocular tissue (Pepose et al., (1994) Invest Ophthalmol Vis Sci 35:2662-2666). Another prefeπed viral delivery system is the HIV- derived lentiviral vectors. See, for example, Klimatcheva et al. (1999) Front. Biosci. 4:D481-96.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a protein in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In prefeπed embodiments, non- viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding a transcriptional activator can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of neuroglioma cells can be carried out using liposomes tagged with monoclonal antibodies against glioma-associated antigen (Mizuno et al., (1992) Neurol. Med. Chir. 32:873-876). In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as poly-lysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, any of the subject gene constructs can be used to transfect specific cells in vivo using a soluble polynucleotide carrier comprising an antibody conjugated to a polycation, e.g. poly-lysine (see U.S. Patent 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via -mediated endocytosis can be improved using agents which enhance escape of the gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al., (1993) Science 260-926; Wagner et al., (1992) PNAS USA 89:7934; and Christiano et al., (1993) PNAS USA 90:2122).

In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the construct in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery of the recombmant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g. Chen et al., (1994) PNAS USA 91 : 3054-3057).

C. Exemplary Target genes

As used herein, the term "target gene" refers to a gene, whose transcription is modulated, e.g., stimulated according to the method of the invention. In a prefeπed embodiment, the gene is integrated in the chromosomal DNA of a cell. A cell comprising a target gene is refeπed to herein as a "target cell".

In a prefeπed embodiment of the invention, the target gene is an endogenous gene. As used herein, the term "endogenous gene" refers to a gene which is naturally present in a cell, in its natural environment, i.e., not a gene which has been introduced into the cell by genetic engineering. The endogenous gene can be any gene having a promoter that is recognized by at least one transcription factor. In a prefeπed embodiment, the promoter or any regulatory element thereof, of the endogenous gene ("endogenous promoter" and "endogenous regulatory element", respectively), is recognized by a known, preferably cloned, DNA binding protein, whether it is a transcriptional activator or repressor. Alternatively, if no DNA binding protein is known to interact with a target promoter, it is possible to clone such a factor using techniques well known in the art without undue experimentation, such as screening of expression libraries with at least a portion of the target promoter. Furthermore, the affinity of binding of a DNA binding domain to a target sequence can be improved according to methods known in the art. Such methods comprise, e.g., introducing mutations into the DNA binding domain and screening for mutants having increased DNA binding affinity.

In another embodiment of the invention, the target gene is an endogenous gene, which contains an exogenous target sequence. The exogenous target sequence can be inserted into the endogenous promoter or substitute at least a portion of the endogenous promoter. In prefeπed embodiments, the exogenous promoter or regulatory element introduced into the endogenous target promoter is recognized by a DNA binding protein, capable of binding with high affinity and specificity to a target sequence. In a preferred embodiment, the DNA binding protein is human. However, the DNA binding protein can be from any other species. For example, the DNA binding protein can be from the yeast GAL4 protein.

In yet another embodiment, the target gene is an exogenous gene. In a prefeπed embodiment, the exogenous gene is integrated into the chromosomal DNA of a cell. The exogenous gene can be inserted into the chromosomal DNA, or the exogenous gene can substitute for at least a portion of an endogenous gene. The target gene can be present in a single copy or in multiple copies. In view of the experimental results described herein, it is not necessary that the target gene be present in more than one copy. However, if even higher levels of protein encoded by the target gene is desired, multiple copies of the gene can be used.

In one embodiment, the target gene construct enables transcription of a target gene to be regulated by a transcription factor in accordance with this invention comprises a DNA molecule which includes a synthetic transcription unit typically consisting of: (1) one copy or multiple copies of a DNA sequence recognized with high-affinity by the DNA binding domain of a fusion protein which includes a composite activator, or of a protein which recruits the composite activator; (2) a promoter sequence consisting minimally of a TATA box and initiator sequence but optionally including other transcription factor binding sites; (3) a coding sequence for a desired gene product, including sequences that promote the initiation and termination of translation, if appropriate; (4) an optional sequence consisting of a splice donor, splice acceptor, and intervening intron DNA; and (5) a sequence directing cleavage and polyadenylation of the resulting RNA transcript.

A wide variety of genes can be employed as the target gene, including genes that encode a therapeutic protein, antisense sequence or ribozyme of interest. The target gene can be any sequence of interest which provides a desired phenotype. It can encode a surface membrane protein, a secreted protein, a cytoplasmic protein, or there can be a plurality of target genes encoding different products. The target gene may be an antisense sequence which can modulate a particular pathway by inhibiting a transcriptional regulation protein or turn on a particular pathway by inhibiting the translation of an inhibitor of the pathway. The target gene can encode a ribozyme which may modulate a particular pathway by interfering, at the RNA level, with the expression of a relevant transcriptional regulator or with the expression of an inhibitor of a particular pathway. The proteins which are expressed, singly or in combination, can involve homing, cytotoxicity, proliferation, immune response, inflammatory response, clotting or dissolving of clots, hormonal regulation, etc. The proteins expressed may be naturally-occurring proteins, mutants of naturally-occurring proteins, unique sequences, or combinations thereof.

Various secreted products include hormones, such as insulin, human growth hormone, glucagon, pituitary releasing factor, ACTH, melanotropin, relaxin, etc.; growth factors, such as EGF, IGF-1, TGF-α, -β, PDGF, G-CSF, M-CSF, GM-CSF, FGF, erythropoietin, thrombopoietin, megakaryocytic stimulating and growth factors, etc.; interleukins, such as IL-1 to -13; TNF-α and -β, etc.; and enzymes and other factors, such as tissue plasminogen activator, members of the complement cascade, performs, superoxide dismutase, coagulation factors, antithrombin-HI, Factor VHIc, Factor VπtvW, Factor IX, α-antitrypsin, protein C, protein S, endoφhins, dynoφhin, bone moφhogenetic protein, etc. The gene can encode a naturally-occurring surface membrane protein or a protein made so by introduction of an appropriate signal peptide and transmembrane sequence. Various such proteins include homing receptors, e.g. L-selectin (Mel- 14), blood-related proteins, particularly having a kringle structure, e.g. Factor VHIc, Factor VIHvW, hematopoietic cell markers, e.g. CD3, CD4, CD8, B-cell receptor, TCR subunits α, β, γ, δ, CD10, CD19, CD28, CD33, CD38, CD41, etc., receptors, such as the interleukin receptors IL-2R, IL-4R, etc., channel proteins for influx or efflux of ions, e.g. Ca+2, K+, Na+, Cl- and the like; CFTR, tyrosine activation motif, zap-70, etc.

Proteins may be modified for transport to a vesicle for exocytosis. By adding the sequence from a protein which is directed to vesicles, where the sequence is modified proximal to one or the other terminus, or situated in an analogous position to the protein source, the modified protein will be directed to the Golgi apparatus for packaging in a vesicle. This process in conjunction with the presence of the chimeric proteins for exocytosis allows for rapid transfer of the proteins to the extracellular medium and a relatively high localized concentration.

Also, intracellular proteins can be of interest, such as proteins in metabolic pathways, regulatory proteins, steroid receptors, transcription factors, etc., depending upon the nature of the host cell. Some of the proteins indicated above can also serve as intracellular proteins.

By way of further illustration, in T-cells, one may wish to introduce genes encoding one or both chains of a T-cell receptor. For B-cells, one could provide the heavy and light chains for an immunoglobulin for secretion. For cutaneous cells, e.g. keratinocytes, particularly stem cell keratinocytes, one could provide for protection against infection, by secreting α-, β- or γ-interferon, antichemotactic factors, proteases specific for bacterial cell wall proteins, etc.

In addition to providing for expression of a gene having therapeutic value, there will be many situations where one may wish to direct a cell to a particular site. The site can include anatomical sites, such as lymph nodes, mucosal tissue, skin, synovium, lung or other internal organs or functional sites, such as clots, injured sites, sites of surgical manipulation, inflammation, infection, etc. By providing for expression of surface membrane proteins which will direct the host cell to the particular site by providing for binding at the host target site to a naturally-occurring epitope, localized concentrations of a secreted product can be achieved. Proteins of interest include homing receptors, e.g. L-selectin, GMP140, CLAM-1, etc., or addressins, e.g. ELAM-1, PNAd, LNAd, etc., clot binding proteins, or cell surface proteins that respond to localized gradients of chemotactic factors. There are numerous situations where one would wish to direct cells to a particular site, where release of a therapeutic product could be of great value. For use in gene therapy, the target gene can encode any gene product that is beneficial to a subject. The gene product can be a secreted protein, a membraneous protein, or a cytoplasmic protein. Prefeπed secreted proteins include growth factors, differentiation factors, cytokines, interleukins, tPA, and erythropoietin. Prefeπed membraneous proteins include receptors, e.g, growth factor or cytokine receptors or proteins mediating apoptosis, e.g., Fas receptor. Other candidate therapeutic genes are disclosed in PCT/US93/01617.

In yet another embodiment, a "gene activation" construct which, by homologous recombination with a genomic DNA, alters the transcriptional regulatory sequences of an endogenous gene, can be used to introduce recognition elements for a DNA binding activity of one of the subject engineered proteins. A variety of different formats for the gene activation constructs are available. See, for example, the Transkaryotic Therapies, Inc PCT publications WO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.

D. Kits This invention further provides kits useful for the foregoing applications. One such kit contains one or more nucleic acids encoding a transcriptional activator or subunits thereof. The kit may further comprise an additional nucleic acid containing a target gene linked to a DNA sequence to which the transcriptional activator is capable of binding. Alternatively, the additional nucleic acid may contain a cloning site for insertion of a desired target gene by the practitioner. For regulatable applications, i.e., in cases in which the recombinant protein contains a ligand binding domain or inducible domain, the kit may further contain an oligomerizing agent, such as the macrolide dimerizers discussed above. Such kits may for example contain a sample of a dimerizing agent capable of dimerizing the two recombinant proteins and activating transcription of the target gene.

E Polypeptides and Peptides and Small Molecule Mimetics

As described in further detail below, the KLX binding ligands of the invention can be used for treating or preventing genetic conditions, such as those associated with an abnormal level of expression of a gene or with the presence of a mutated gene product. For example, in conditions in which an abnormally high level of protein is produced, or in situations in which an abnormal protein is produced in a subject, the production of such a protein can be decreased or inhibited by administering to the patient a pharmaceutically efficient amount of a KLX binding ligand to decrease or inhibit the transcription of the gene encoding the protein. In another prefeπed embodiment, KIX binding peptides are used to treat or prevent conditions associated with an abnormally low level of expression of a gene. Thus, in this embodiment, a KLX binding peptide fused to a DNA binding domain or an oligomerizing domain is administered to a subject to stimulate transcription of the gene. In the embodiment in which an oligomerizing domain is fused to a KLX binding peptide, the method further requires the presence in the target cells of the subject of a polypeptide comprising a DNA binding domain and an oligomerizing domain, such that oligomerization of the chimeric protein comprising the KIX binding peptide and the DNA binding protein results in the formation of a transcriptional activation complex on the DNA binding site and stimulation of transcription of the target gene.

In addition to the use of the invention for coπecting the expression of abnormally expressed genes in a subject, the invention can also be used to express any target gene, e.g., a gene encoding a cytokine, growth hormone, erythropoietin, or any other therapeutically beneficial protein. Since the level of transcriptional activity has been shown herein to be proportional to the affinity of the KIX binding peptide for the KIX domain of a histone acetyltransferase, e.g., p300 or CBP, the choice of KIX binding peptide and the number of KIX binding peptides in the chimeric transcriptional activator will depend from the level of transcriptional activation of the target gene that is desired.

(i) Exemplary Compositions

According to the present invention, there is provided compositions capable of binding to the KIX domain of a polypeptide. In prefeπed embodiments of the present invention, the subject composition can be a peptide, e.g., having a naturally occurring peptide backbone and amino acid side chains, though it may be N-terminally and or C- terminally protected.

In prefeπed embodiments, the peptidyl component of the subject compounds includes, in addition to the core sequences described below, no more than about 20 amino acid residues, more preferably no more than 10-15 amino acid residues and most preferably 8 amino acid residues. With the exception of certain chimeric KIX binding compositions described herein, such as fusion proteins, a prefeπed composition includes a peptide comprising a KLX binding core motif and having a molecular weight preferably less than 5000 amu, more preferably less than 2500 amu, and most preferably less than 1000 amu. The peptide, in addition to the KLX binding core motif, may include other amino acid residues, such as transcytosis peptide, and may be derivatized at one or more backbone or sidechain points with, e.g., peptides, nucleic acids, carbohydrates, etc. In certain embodiments, the peptide is derivatized with one or more functional groups that enhance cellular uptake and/or impair the half-life of the KLX binding core motif.

Additionally, as will be described in more detail below, the present invention contemplates compositions for binding to the KIX binding domain including small molecule peptidomimetics and non-peptidic small molecules.

This invention further contemplates a method of generating sets of combinatorial libraries of the subject KIX binding peptides, peptidomimetics or other small molecules which is especially useful for identifying potential variant sequences or other small molecules that are functional in either inhibiting KIX binding interactions with natural KIX binding ligands or promoting transactivation. Combinatorially-derived homologs can be generated which have, e.g., greater affinity, an enhanced potency relative to native KLX binding peptide sequences, or intracellular half-lives different than the coπesponding natural KIX binding motifs. For example, the altered peptide, peptidomimetic, or small molecule can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in the destruction of, or otherwise inactivation of the original composition. Such homologs can be utilized to alter the envelope of therapeutic application by modulating the half-life of the peptide. For instance, a short half-life can give rise to more transient biological effects and can allow tighter control of peptide levels within the cell. In particularly prefeπed embodiments of the present invention, there is provided compositions capable of binding to the KIX domain of a polypeptide comprising nucleic acids encoding polypeptides which include a KIX binding sequence, recombinant polypeptides which include a KIX binding sequence, and peptides, peptidomimetics, and libraries thereof of 6-20 amino acid residues in length which include a KIX binding sequence.

In one particularly prefeπed embodiment, each of these compositions includes a KIX binding sequence represented in the general formula (I):

X1-X2-X3-Y-X4-X5-L-F (I) wherein

X2 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably A or W; X3 represents an amino acid residue with a small hydrophobic sidechain, preferably A, G, or V, and more preferably A or V; X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably and acidic sidechain, such as D or E;

More generally, in another prefeπed embodiment, each of the compositions includes KIX binding sequence represented in the general formula (II):

X1-X2-X3-A1-X4-A2-A3-A4 (II)

wherein

It is prefeπed that X4 is selected from the group consisting of D, E, and a peptidomimetic thereof. It is also particularly prefeπed that Al, A2, A3 and A4 each independently comprise an aromatic or aliphatic moiety selected from the group consisting of A, V, L, I, F, W, Y, M, P, and a peptidomimetic thereof.

More generally, in still another prefeπed embodiment, each of the compositions includes a KIX binding sequence represented in the general formula (III)

A1-A2-A3-A4-X1-A5-A6-A7 (III)

wherein Al, A2, A3, A4, A5, A6 and A7 each independently comprise an aromatic or aliphatic natural or non-natural amino acid or peptidomimetic thereof; and

XI comprises a charged natural or non-natural amino acid or peptidomimetic thereof. It is particularly prefeπed that Al, A2, A3, A4, A5, A6 and A7 each independently comprise an aromatic or aliphatic moiety selected from the group consisting of A, V, L, F, W, Y, M, P, I, and a peptidomimetic thereof; and XI is selected from the group consisting of D, E and a peptidomimetic thereof. It is more particularly prefeπed that A4 comprises tyrosine or a peptidomimetic thereof; XI comprises D, E, or a peptidomimetic thereof; A6 comprises L or a peptidomimetic thereof; and A7 comprises F or a peptidomimetic thereof. It is still more particularly prefeπed that Al comprises W, L or a peptidomimetc thereof, A2 comprises W, A, or a peptidomimetic thereof; A3 comprises V, A, or a peptidomimetic thereof; A4 comprises Y or a peptidomimetic thereof; C comprises D, E, or a peptidomimetic thereof; A5 comprises L, V, or a peptidomimetic thereof; A6 comprises L or a peptidomimetic thereof; and A7 comprises F or a peptidomimetic thereof.

In still other embodiments, the subject peptide (or peptidomimetic analogs thereof) are cyclic, e.g., and have an amino acid sequence represented by formula VI:

S-C-Xi-X₂-X₃-X4-X5-X6-X7-X8-C-G-S (IV) X represents any amino acid residue, more preferably an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V), polar (e.g., R, N, D, C, E, Q, H, K, S or T), acidic (D or E) or basic (R, H or K) side chain, and more preferably is P, W, E, S, L, N or R;

X₂ represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, L V, K H, T or G;

X₃ represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is W, T, D or V; X4 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L,

M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, R, M, G, T, A, Y, L;

X6 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is D, S, L, P, G, R, K, V;

X7 represents an amino acid residue with an aromatic (F, Y, W) or acidic (D or E) sidechain, and more preferably is F, Y, W or D; X8 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably, preferably an aromatic side chain (F, Y, W), and more preferably S, E, V, Y, R, W, T or P.

(ii) Generating variants of KIX binding sequence

As mentioned above, the inventive peptide compositions, including other peptidomimetics, non-peptide small molecules, genes and recombinant polypeptides may be generated using combinatorial techniques using techniques which are available in the art for generating combinatorial libraries of small organic/peptide libraries. See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Patents 5,359,115 and 5,362,899; the Ellman U.S. Patent 5,288,514; the Still et al. PCT publication WO 94/08051; Chen et al. (1994) JACS 116:2661; Ken et al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCT publication WO93/20242).

In a prefeπed embodiment, the combinatorial peptide library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential KLX binding sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential KLX binding nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of KIX binding peptide sequences therein.

There are many ways by which the gene library of potential KIX binding homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression. The puφose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential KLX binding sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. X X :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815).

A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of KLX binding sequences. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Such illustrative assays are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.

In an illustrative embodiment of a screening assay, the KIX binding gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at very high concentrations, a large number of phage can be screened at one time. Second, since each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection.

The group of almost identical E.coli filamentous phages Ml 3, fd, and fl are most often used in phage display libraries, as either of the phage gHI or gVHI coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02909; Gaπard et al., PCT publication

WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffths et al.

(1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).

For example, the recombinant phage antibody system (RPAS, Pharmacia Catalog number 27-9400-01) can be easily modified for use in expressing and screening KIX binding motif combinatorial libraries of the present invention. For instance, the pCANTAB 5 phagemid of the RPAS kit contains the gene which encodes the phage gHI coat protein. The KIX binding combinatorial gene library can be cloned into the phagemid adjacent to the gHI signal sequence such that it will be expressed as a gHI fusion protein. After ligation, the phagemid is used to transform competent E. coli TGI cells. Transformed cells are subsequently infected with M13KO7 helper phage to rescue the phagemid and its candidate KIX binding gene insert. The resulting recombinant phage contain phagemid DNA encoding a specific candidate KIX binding peptide, and display one or more copies of the coπesponding fusion coat protein. The phage- displayed candidate proteins which are capable of, for example, binding a KLX domain, are selected or enriched by panning. For instance, the phage library can be panned on glutathione immobilized fusion proteins, and unbound phage washed away from the cells. The bound phage is then isolated, and if the recombinant phage express at least one copy of the wild type glH coat protein, they will retain their ability to infect E. coli. Thus, successive rounds of reinfection of E. coli, and panning will greatly enrich for KIX binding homologs which can then be screened for further PV inhibitory activities. Subsequent selection, e.g. of a reduced set of variants from the library, may then be based upon more meaningful criteria rather than simple -binding ability. For instance, intracellular half-life or selectivity can become selection criteria in secondary screens.

Combined with certain formulations, such peptides can be effective intracellular agents. However, in order to increase the efficacy of such peptides, the KLX binding peptide can be provided a fusion peptide along with a second peptide which promotes "transcytosis", e.g., uptake of the peptide by epithelial cells. To illustrate, the KIX binding peptide of the present invention can be provided as part of a fusion polypeptide with all or a fragment of the N-terminal domain of the HIV protein Tat, e.g., residues 1- 72 of Tat or a smaller fragment thereof which can promote transcytosis. In other embodiments, the KIX binding peptide can be provided a fusion polypeptide with all or a portion of the antenopedia HI protein.

To further illustrate, the KIX binding peptide (or peptidomimetic) can be provided as a chimeric peptide which includes a heterologous peptide sequence ("internalizing peptide") which drives the translocation of an extracellular form of a KLX binding peptide sequence across a cell membrane in order to facilitate intracellular localization of the KIX binding peptide. In this regard, the therapeutic KIX binding sequence is one which is active intracellularly. The internalizing peptide, by itself, is capable of crossing a cellular membrane by, e.g., transcytosis, at a relatively high rate. The internalizing peptide is conjugated, e.g., as a fusion protein, to the KIX binding peptide. The resulting chimeric peptide is transported into cells at a higher rate relative to the activator polypeptide alone to thereby provide an means for enhancing its introduction into cells to which it is applied, e.g., to enhance topical applications of the KIX binding peptide.

In one embodiment, the internalizing peptide is derived from the Drosophila antennapedia protein, or homologs thereof. The 60 amino acid long long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is couples. See for example Derossi et al. (1994) J Biol Chem 269:10444- 10450; and Perez et al. (1992) J Cell Sci 102:717-722. Recently, it has been demonstrated that fragments as small as 16 amino acids long of this protein are sufficient to drive internalization. See Derossi et al. (1996) J Biol Chem 271 :18188-18193. The present invention contemplates a KIX binding peptide or peptidomimetic sequence as described herein, and at least a portion of the Antennapedia protein (or homolog thereof) sufficient to increase the transmembrane transport of the chimeric protein, relative to the KIX binding peptide or peptidomimetic, by a statistically significant amount. Another example of an internalizing peptide is the HIV transactivator (TAT) protein. This protein appears to be divided into four domains (Kuppuswamy et al. (1989) Nucl. Acids Res. 17:3551-3561). Purified TAT protein is taken up by cells in tissue culture (Frankel and Pabo, (1989) Cell 55:1189-1193). and peptides, such as the fragment coπesponding to residues 37 -62 of TAT, are rapidly taken up by cell in vitro (Green and Loewenstein, (1989) Cell 55:1179-1188). The highly basic region mediates internalization and targeting of the internalizing moiety to the nucleus (Ruben et al., (1989) I Virol. 63:1-8).

Another exemplary transcellular polypeptide can be generated to include a sufficient portion of mastoparan (T. Higashijima et al., (1990) J. Biol. Chem. 265:14176) to increase the transmembrane transport of the chimeric protein.

While not wishing to be bound by any particular theory, it is noted that hydrophilic polypeptides may be also be physiologically transported across the membrane barriers by coupling or conjugating the polypeptide to a transportable peptide which is capable of crossing the membrane by receptor-mediated transcytosis. Suitable internalizing peptides of this type can be generated using all or a portion of, e.g., a histone, insulin, transferrin, basic albumin, prolactin and insulin-like growth factor I (IGF-I), insulin-like growth factor H (IGF-H) or other growth factors. For instance, it has been found that an insulin fragment, showing affinity for the insulin receptor on capillary cells, and being less effective than insulin in blood sugar reduction, is capable of transmembrane transport by receptor-mediated transcytosis and can therefor serve as an internalizing peptide for the subject transcellular peptides and peptidomimetics. Preferred growth factor-derived internalizing peptides include EGF (epidermal growth factor)-derived peptides, such as CMHIESLDSYTC and CMYIEALDKYAC; TGF- beta (transforming growth factor beta )-derived peptides; peptides derived from PDGF (platelet-derived growth factor) or PDGF-2; peptides derived from IGF-I (insulin-like growth factor) or IGF-H; and FGF (fibroblast growth factor)-derived peptides. Another class of translocating/internalizing peptides exhibits pH-dependent membrane binding. For an internalizing peptide that assumes a helical conformation at an acidic pH, the internalizing peptide acquires the property of amphiphilicity, e.g., it has both hydrophobic and hydrophilic interfaces. More specifically, within a pH range of approximately 5.0-5.5, an internalizing peptide forms an alpha-helical, amphiphilic structure that facilitates insertion of the moiety into a target membrane. An alpha-helix- inducing acidic pH environment may be found, for example, in the low pH environment present within cellular endosomes. Such internalizing peptides can be used to facilitate transport of KIX binding peptides and peptidomimetics, taken up by an endocytic mechanism, from endosomal compartments to the cytoplasm.

A prefeπed pH-dependent membrane-binding internalizing peptide includes a high percentage of helix-forming residues, such as glutamate, methionine, alanine and leucine. In addition, a prefeπed internalizing peptide sequence includes ionizable residues having pKa's within the range of pH 5-7, so that a sufficient uncharged membrane-binding domain will be present within the peptide at pH 5 to allow insertion into the target cell membrane.

A particularly prefeπed pH-dependent membrane-binding internalizing peptide in this regard is aal-aa2-aa3-EAALA(EALA)4-EALEALAA-amide, which represents a modification of the peptide sequence of Subbarao et al. (Biochemistry 26:2964. 1987). Within this peptide sequence, the first amino acid residue (aal) is preferably a unique residue, such as cysteine or lysine, that facilitates chemical conjugation of the internalizing peptide to a targeting protein conjugate. Amino acid residues 2-3 may be selected to modulate the affinity of the internalizing peptide for different membranes. For instance, if both residues 2 and 3 are lys or arg, the internalizing peptide will have the capacity to bind to membranes or patches of lipids having a negative surface charge. If residues 2-3 are neutral amino acids, the internalizing peptide will insert into neutral membranes.

Yet other preferred internalizing peptides include peptides of apo-lipoprotein A-l and B; peptide toxins, such as melittin, bombolittin, delta hemolysin and the pardaxins; antibiotic peptides, such as alamethicin; peptide hormones, such as calcitonin, corticotrophin releasing factor, beta endoφhin, glucagon, parathyroid hormone, pancreatic polypeptide; and peptides coπesponding to signal sequences of numerous secreted proteins. In addition, exemplary internalizing peptides may be modified through attachment of substituents that enhance the alpha-helical character of the internalizing peptide at acidic pH.

Yet another class of internalizing peptides suitable for use within the present invention include hydrophobic domains that are "hidden" at physiological pH, but are exposed in the low pH environment of the target cell endosome. Upon pH-induced unfolding and exposure of the hydrophobic domain, the moiety binds to lipid bilayers and effects translocation of the covalently linked polypeptide into the cell cytoplasm. Such internalizing peptides may be modeled after sequences identified in, e.g., Pseudomonas exotoxin A, clathrin, or Diphtheria toxin.

Pore- forming proteins or peptides may also serve as internalizing peptides herein.

Pore- forming proteins or peptides may be obtained or derived from, for example, C9 complement protein, cytolytic T-cell molecules or NK-cell molecules. These moieties are capable of forming ring-like structures in membranes, thereby allowing transport of attached polypeptide through the membrane and into the cell interior.

Mere membrane intercalation of an internalizing peptide may be sufficient for translocation of the KIX binding peptide or peptidomimetic, across cell membranes. However, translocation may be improved by attaching to the internalizing peptide a substrate for intracellular enzymes (i.e., an "accessory peptide"). It is prefeπed that an accessory peptide be attached to a portion(s) of the internalizing peptide that protrudes through the cell membrane to the cytoplasmic face. The accessory peptide may be advantageously attached to one terminus of a translocating/internalizing moiety or anchoring peptide. An accessory moiety of the present invention may contain one or more amino acid residues. In one embodiment, an accessory moiety may provide a substrate for cellular phosphorylation (for instance, the accessory peptide may contain a tyrosine residue).

An exemplary accessory moiety in this regard would be a peptide substrate for N- myristoyl transferase, such as GNAAAARR (Eubanks et al., in: Peptides. Chemistry and Biology, Garland Marshall (ed.), ESCOM, Leiden, 1988, pp. 566-69) In this construct, an internalizing peptide would be attached to the C-terminus of the accessory peptide, since the N-terminal glycine is critical for the accessory moiety's activity. This hybrid peptide, upon attachment to an E2 peptide or peptidomimetic at its C-terminus, is N- myristylated and further anchored to the target cell membrane, e.g., it serves to increase the local concentration of the peptide at the cell membrane. To further illustrate use of an accessory peptide, a phosphorylatable accessory peptide is first covalently attached to the C-terminus of an internalizing peptide and then incoφorated into a fusion protein with a KLX binding peptide or peptidomimetic. The peptide component of the fusion protein intercalates into the target cell plasma membrane and, as a result, the accessory peptide is translocated across the membrane and protrudes into the cytoplasm of the target cell. On the cytoplasmic side of the plasma membrane, the accessory peptide is phosphorylated by cellular kinases at neutral pH. Once phosphorylated, the accessory peptide acts to iπeversibly anchor the fusion protein into the membrane. Localization to the cell surface membrane can enhance the translocation of the polypeptide into the cell cytoplasm.

Suitable accessory peptides include peptides that are kinase substrates, peptides that possess a single positive charge, and peptides that contain sequences which are glycosylated by membrane-bound glycotransferases. Accessory peptides that are glycosylated by membrane-bound glycotransferases may include the sequence x-NLT-x, where "x" may be another peptide, an amino acid, coupling agent or hydrophobic molecule, for example. When this hydrophobic tripeptide is incubated with microsomal vesicles, it crosses vesicular membranes, is glycosylated on the luminal side, and is entrapped within the vesicles due to its hydrophilicity (C. Hirschberg et al., (1987) Ann- Rev. Biochem. 56:63-87). Accessory peptides that contain the sequence x-NLT-x thus will enhance target cell retention of coπesponding polypeptide.

In another embodiment of this aspect of the invention, an accessory peptide can be used to enhance interaction of the KIX binding peptide or peptidomimetic with the target cell. Exemplary accessory peptides in this regard include peptides derived from cell adhesion proteins containing the sequence "RGD", or peptides derived from laminin containing the sequence CDPGYIGSRC. Extracellular matrix glycoproteins, such as fibronectin and laminin, bind to cell surfaces through receptor-mediated processes. A tripeptide sequence, RGD, has been identified as necessary for binding to cell surface receptors. This sequence is present in fibronectin, vitronectin, C3bi of complement, von- Willebrand factor, EGF receptor, transforming growth factor beta , collagen type I, lambda receptor of E. Coli, fibrinogen and Sindbis coat protein (E. Ruoslahti, Ann. Rev. Biochem. 57:375-413, 1988). Cell surface receptors that recognize RGD sequences have been grouped into a superfamily of related proteins designated "integrins". Binding of "RGD peptides" to cell surface integrins will promote cell-surface retention, and ultimately translocation, of the polypeptide.

As described above, the internalizing and accessory peptides can each, independently, be added to the KIX binding peptide or peptidomimetic by either chemical cross-linking or in the form of a fusion protein. In the instance of fusion proteins, unstructured polypeptide linkers can be included between each of the peptide moieties.

In general, the internalization peptide will be sufficient to also direct export of the polypeptide. However, where an accessory peptide is provided, such as an RGD sequence, it may be necessary to include a secretion signal sequence to direct export of the fusion protein from its host cell. In prefeπed embodiments, the secretion signal sequence is located at the extreme N-terminus, and is (optionally) flanked by a proteolytic site between the secretion signal and the rest of the fusion protein. In an exemplary embodiment, a KIX binding peptide or peptidomimietic is engineered to include an integrin-binding RGD peptide/SV40 nuclear localization signal (see, for example Hart SL et al., 1994; J. Biol. Chem.,269:12468-12474), such as encoded by the nucleotide sequence provided in the Ndel-EcoRl fragment: catatgggtggctgccgtggcgatatgttcggttgcggtgctcctccaaaaaagaagagaaag-gtagctggattc, which encodes the RGD/SV40 nucleotide sequence: MGGCRGDMFGCGAPP- KKKRKVAGF. In another embodiment, the protein can be engineered with the HIV-1 tat(l-72) polypeptide, e.g., as provided by the Ndel-EcoRl fragment : catatggagccagtagatcctagactagagccc- tggaagcatccaggaagtcagcctaaaactgcttgtaccaattgctattgtaaaaagtgttgctttcattgccaagtttgtttcataac aaaagcccttggcatctcctatggcaggaagaagcggagacagcgacgaagacctcctcaaggcagtcagactcatcaagttt ctctaagtaagcaaggattc, which encodes the HIV-1 tat(l-72) peptide sequence: MEPVDPRLEPWKHPGSQPKT- ACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ. In still another embodiment, the fusion protein includes the HSV-1 VP22 polypeptide (Elliott G, O'Hare P (1997) Cell, 88:223-233) provided by the Ndel-EcoRl fragment: cat atg ace tet cgc cgc tec gtg aag teg ggt ccg egg gag gtt ccg cgc gat gag tac gag gat ctg tac tac ace ccg tet tea ggt atg gcg agt ccc gat agt ccg cct gac ace tec cgc cgt ggc gcc eta cag aca cgc teg cgc cag agg ggc gag gtc cgt ttc gtc cag tac gac gag teg gat tat gcc etc tac ggg ggc teg tea tec gaa gac gac gaa cac ccg gag gtc ccc egg acg egg cgt ccc gtt tec ggg gcg gtt ttg tec ggc ccg ggg cct gcg egg gcg cct ccg cca ccc get ggg tec gga ggg gcc gga cgc aca ccc ace ace gcc ccc egg gcc ccc cga ace cag egg gtg gcg act aag gcc ccc gcg gcc ccg gcg gcg gag ace ace cgc ggc agg aaa teg gcc cag cca gaa tec gcc gca etc cca gac gcc ccc gcg teg acg gcg cca ace cga tec aag aca ccc gcg cag ggg ctg gcc aga aag ctg cac ttt age ace gcc ccc cca aac ccc gac gcg cca tgg ace ccc egg gtg gcc ggc ttt aac aag cgc gtc ttc tgc gcc gcg gtc ggg cgc ctg gcg gcc atg cat gcc egg atg gcg gcg gtc cag etc tgg gac atg teg cgt ccg cgc aca gac gaa gac etc aac gaa etc ctt ggc ate ace ace ate cgc gtg acg gtc tgc gag ggc aaa aac ctg ctt cag cgc gcc aac gag ttg gtg aat cca gac gtg gtg cag gac gtc gac gcg gcc acg gcg act cga ggg cgt tet gcg gcg teg cgc ccc ace gag cga cct cga gcc cca gcc cgc tec get tet cgc ccc aga egg ccc gtc gag gaa ttc which encodes the HSV-1 VP22 peptide having the sequence:

MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSR QRGEVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPP PAGSGGAGRTPTTAPRAPRTGRVATKAPAAPAAETTRGRKSAQPESAALPDAPA STAPTRSKTPAQGLARKLHFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAM HARMAAVQLWDMSRPRTDEDLNELLGITTIRVTVCEGKNLLQRANELVNPDVV QDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE In still another embodiment, the fusion protein includes the C-terminal domain of the VP22 protein from, e.g., the nucleotide sequence (Ndel-EcoRl fragment): cat atg gac gtc gac gcg gcc acg gcg act cga ggg cgt tet gcg gcg teg cgc ccc ace gag cga cct cga gcc cca gcc cgc tec get tet cgc ccc aga egg ccc gtc gag gaa ttc which encodes the VP22 (C-terminal domain) peptide sequence: MDVDAATATRGRSA-ASRPTERPRAPARSASRPRRPVE

In certain instances, it may also be desirable to include a nuclear localization signal as part of the KLX binding peptide.

In the generation of fusion polypeptides including the subject KLX binding peptides, it may be necessary to include unstructured linkers in order to ensure proper folding of the various peptide domains. Many synthetic and natural linkers are known in the art and can be adapted for use in the present invention, including the (Gly₃Ser)₄ linker.

In other embodiments, the subject KIX binding therapeutics are peptidomimetics of the KIX binding peptide. Peptidomimetics are compounds based on, or derived from, peptides and proteins. The KIX binding peptidomimetics of the present invention typically can be obtained by structural modification of a known KIX binding peptide sequence using unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continuum of structural space between peptides and non-peptide synthetic structures; KIX binding peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent KLX binding peptides.

Moreover, as is apparent from the present disclosure, mimetopes of the subject KIX binding peptides can be provided. Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the coπesponding peptide), increased specificity and/or potency, and increased cell permeability for intracellular localization of the peptidomimetic. For illustrative pmposes, peptide analogs of the present invention can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in Peptides:

Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,

1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology,

G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, pi 23), C-7 mimics

(Huffman et al. in Peptides: Chemistry and Biologyy, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res CommunX26:4X9; and Dann et al. (1986) Biochem Biophys Res Commun 134:71), diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modifed (Roark et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, pi 34). Also, see generally, Session HI: Analytic and synthetic methods, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988)

In addition to a variety of sidechain replacements which can be carried out to generate the subject KIX binding peptidomimetics, the present invention specifically contemplates the use of conformationally restrained mimics of peptide secondary structure. Numerous suπogates have been developed for the amide bond of peptides. Frequently exploited suπogates for the amide bond include the following groups (i) trans-olefins, (ii) fluoroalkene, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.

Examples of Suπogates

trans olefin fluoroalkene methyleneamino

phosphonamide sulfonamide Additionally, peptidomimietics based on more substantial modifications of the backbone of the E2 peptide can be used. Peptidomimetics which fall in this category include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids).

Examples of analogs

retro-inverso N-alkyl glycine

Furthermore, the methods of combinatorial chemistry are being brought to bear, e,g„ by G.L. Verdine at Harvard University, on the development of new peptidomimetics. For example, one embodiment of a so-called "peptide moφhing" strategy focuses on the random generation of a library of peptide analogs that comprise a wide range of peptide bond substitutes.

dipeptide

peptide morphing

In an exemplary embodiment, the peptidomimetic can be derived as a retro- inverso analog of the peptide

Retro-inverso analogs can be made according to the methods known in the art, such as that described by the Sisto et al. U.S. Patent 4,522,752. As a general guide, sites which are most susceptible to proteolysis are typically altered, with less susceptible amide linkages being optional for mimetic switching The final product, or intermediates thereof, can be purified by HPLC.

In another illustrative embodiment, the peptidomimetic can be derived as a retro- enatio analog of the peptide, such as the exemplary retro-enatio peptide analog derived for the illustrative WAVYDLLF peptide:

NH₂-(d)Phe-(d)Leu-(d)Leu-(d)Asp-(d)Tyr-(d)Val-(d)Ala-(d)Tyr-COOH

Retro-enantio analogs such as this can be synthesized commercially available D- amino acids (or analogs thereof) and standard solid- or solution-phase peptide-synthesis techniques. For example, in a prefeπed solid-phase synthesis method, a suitably amino- protected (t-butyloxycarbonyl, Boc) D-tyr residue (or analog thereof) is covalently bound to a solid support such as chloromethyl resin. The resin is washed with dichloromethane (DCM), and the BOC protecting group removed by treatment with TFA in DCM. The resin is washed and neutralized, and the next Boc-protected D-amino acid (D-Ala) is introduced by coupling with diisopropylcarbodiimide. The resin is again washed, and the cycle repeated for each of the remaining amino acids in turn (D-Val, D-Tyr, etc).

When synthesis of the protected retro-enantio peptide is complete, the protecting groups are removed and the peptide cleaved from the solid support by treatment with hydrofluoric acid/anisole/dimethyl sulfide/thioanisole. The final product is purified by HPLC to yield the pure retro-enantio analog. In still another illustrative embodiment, trans-olefin derivatives can be made for any of the subject polypeptides. A trans olefin analog of KIX binding peptide can be synthesized according to the method of Y.K. Shue et al. (1987) Tetrahedron Letters 28:3225 and also according to other methods known in the art. It will be appreciated that variations in the cited procedure, or other procedures available, may be necessary according to the nature of the reagent used.

It is further possible couple the pseudodipeptides synthesized by the above method to other pseudodipeptides, to make peptide analogs with several olefinic functionalities in place of amide functionalities. For example, pseudodipeptides coπesponding to Ala- Val or Tyr-Asp, etc. could be made and then coupled together by standard techniques to yield an analog of the KLX binding peptide which has alternating olefinic bonds between residues. Still another class of peptidomimetic derivatives include phosphonate derivatives. The synthesis of such phosphonate derivatives can be adapted from known synthesis schemes. See, for example, Loots et al. in Peptides: Chemistry and Biology, (Escom Science Publishers, Leiden, 1988, p. 118); Petrillo et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co. Rockland, IL, 1985).

Many other peptidomimetic structures are known in the art and can be readily adapted for use in the subject KIX binding peptidomimetics. To illustrate, the KLX binding peptidomimetic may incoφorate the l-azabicyclo[4.3.0]nonane suπogate ( see Kim et al. (1997) J. Org. Chem. 62:2847), or an N-acyl piperazic acid (see Xi et al. (1998) J. Am. Chem. Soc. 720:80), or a 2-substituted piperazine moiety as a constrained amino acid analogue (see Williams et al. (1996) J. Med. Chem. 39:1345-1348). In still other embodiments, certain amino acid residues can be replaced with aryl and bi-aryl moieties, e.g., monocyclic or bicyclic aromatic or heteroaromatic nucleus, or a biaromatic, aromatic-heteroaromatic, or biheteroaromatic nucleus.

The subject KIX binding peptidomimetics can be optimized by, e.g., combinatorial synthesis techniques combined with such high throughput screening as described herein.

Moreover, other examples of mimetopes include, but are not limited to, protein- based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof. A mimetope can be obtained by, for example, screening libraries of natural and synthetic compounds for compounds capable of binding to the KIX binding domain or inhibiting the interaction between the KIX binding domain and the natural ligand. A mimetope can also be obtained, for example, from libraries of natural and synthetic compounds, in particular, chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks). A mimetope can also be obtained by, for example, rational drug design. In a rational drug design procedure, the three-dimensional structure of a compound of the present invention can be analyzed by, for example, nuclear magnetic resonance (ΝMR) or x-ray crystallography. The three- dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modeling, the predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DΝA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi). (iii) Methods for detecting binding to the KIX domain:

According to another aspect of this invention, the compositions and libraries described herein may be tested for their ability to bind to a KIX domain. In a prefeπed embodiment, the subject method used to determine the ability of an inventive composition to interact with the KIX domain of a polypeptide (e.g., p300/CBP) involves (1) generating a reaction mixture comprising a polypeptide including a KLX domain (e.g., p300/CBP) and a composition including a KIX binding sequence, which reaction mixture may be a whole cell, a cell lysate, or a reconstituted protein preparation; (2) contacting the reaction mixture with a desired composition, such as a peptide, peptidomimetic, or small molecule, as described herein; and (3) ascertaining the ability of the composition to interact with the KIX domain.

It will also be appreciated that the inventive method also encompasses determining the ability of an inventive composition to inhibit a binding interaction between the KLX domain of a polypeptide and a natural KLX domain ligand. Thus, the present invention includes the ability to determine the ability of small peptides, small peptidomimetics and small molecules to bind to the KLX binding domain, which previously were thought to be too small to participate in this binding interaction (Ptashne, 1997).

(iv) Pharmaceutical Preparations of KIX binding peptides and peptidomimetics

According to another aspect of this invention, KLX binding peptides, peptidomimetics, and small molecules may be administered directly to infected cells. Direct delivery of such KIX binding therapeutics maybe facilitated by formulation of the peptidyl compound in any pharmaceutically acceptable dosage form, e.g., for delivery orally, intratumorally, peritumorally, interlesionally, intravenously, intramuscularly, subcutaneously, periolesionally, or (preferably) topical routes, to exert local therapeutic effects.

Topical administration of the therapeutic is advantageous since it allows localized concentration at the site of administration with minimal systemic adsoφtion. This simplifies the delivery strategy of the agent to the disease site and reduces the extent of toxicological characterization. Furthermore, the amount of material to be applied is far less than that required for other administration routes. Effective delivery requires the agent to diffuse into the affected cells. Successful intracellular delivery of agents not naturally taken up by cells has been achieved by exploiting the natural process of intracellular membrane fusion, or by direct access of the cell's natural transport mechanisms which include endocytosis and pinocytosis (Duzgunes (1985) Subcellular Biochemistry 11 :195-286). Such processes are also useful in the direct delivery and uptake of the subject KIX binding peptides and peptidomimetic by papillomavirus- infected cells.

In one embodiment, the membrane barrier can be overcome by associating the KIX binding protein in complexes with lipid formulations closely resembling the lipid composition of natural cell membranes. In particular, the subject KIX binding peptidomimetics are encapsulated in liposomes to form pharmaceutical preparations suitable for administration to living cells and, in particular, suitable for topical administration to human skin. The Yarosh U.S. Patent 5,190,762 demonstrates that proteins can be delivered across the outer skin layer and into living cells, without receptor binding, by liposome encapsulation.

These lipids are able to fuse with the cell membranes on contact, and in the process, the associated KIX binding peptidomimetic is delivered intracellularly. Lipid complexes can not only facilitate intracellular transfers by fusing with cell membranes but also by overcoming charge repulsions between the cell membrane and the molecule to be inserted. The lipids of the formulations comprise an amphipathic lipid, such as the phospholipids of cell membranes, and form hollow lipid vesicles, or liposomes, in aqueous systems. This property can be used to entrap the KIX binding peptidomimetic within the liposomes. Liposomes offer several advantages: They are non-toxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues. Finally, cost effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.

Liposomes have been described in the art as in vivo delivery vehicles. The structure of various types of lipid aggregates varies, depending on composition and method of forming the aggregate. Such aggregates include liposomes, unilamellar vesicles, multilameller vesicles, micelles and the like, having particle sizes in the nanometer to micrometer range. Methods of making lipid aggregates are by now well- known in the art. For example, the liposomes may be made from natural and synthetic phospholipids, glycolipids, and other lipids and lipid congeners; cholesterol, cholesterol derivatives and other cholesterol congeners; charged species which impart a net charge to the membrane; reactive species which can react after liposome formation to link additional molecules to the liposome membrane; and other lipid soluble compounds which have chemical or biological activity. In one embodiment, pH sensitive liposomes are a prefeπed type of liposome for use with the present invention. One pathway for the entry of liposomes into cellular cytoplasm is by endocytosis into lysozymes of low pH. Accordingly, liposomes which are stable at neutral pH but release their contents at acidic pH can be used to deliver enzymes into the lysozymes of the cytoplasm, whereupon the contents are released.

Liposomes can be made sensitive to the low pH of the lysozymes by the lipid composition. In particular, pH sensitive liposomes can be prepared by using phospholipids which form lipid bilayers when charged but fail to stack in an ordered fashion when neutralized. An example of such a phospholipid is phosphatidylethanolamine, which is negatively charged above pH 9. The net charge of a phospholipid can be maintained at a pH which would otherwise neutralize the head groups by including charged molecules in the lipid bilayer which themselves can become neutralized. Examples of these charged molecules are oleic acid and cholesteryl hemisuccinate, which are negatively charged at neutral pH but become neutralized at pH 5. The effect of combining these together in a lipid bilayer is that at pH 9 all molecules are charged; at pH 7 the net negative charge of the oleic acid and cholesteryl hemisuccinate maintains the stability of the phosphatidylethanolamine, and at pH 5 all components are protonated and the lipid membrane is destabilized. Additional neutral molecules, such as phosphatidylcholine, can be added to the liposomes as long as they do not interfere with stabilization of the pH sensitive phospholipid by the charged molecules.

In another embodiment, the KIX binding peptidomimetic is formulated with a positively charged synthetic (cationic) lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), in the form of liposomes, or small vesicles, which can fuse with the negatively charged lipids of the cell membranes of mammalian cells, resulting in uptake of the contents of the liposome (see, for example, Feigner et al. (1987) PNAS 84:7413-7417; and U.S. Pat. No. 4,897,355 to Eppstein, D. et al.). Another cationic lipid which can be used to generate KIX binding peptidomimetic containing liposomes is the DOTMA analogue, l,2-bis(oleoyloxy)-3-(trimethyl-ammonio)propane (DOTAP) in combination with a phospholipid to form delivery vesicles.

Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) and/or LipofectAMINE™, commercially available reagents, can be used to deliver the E2 peptidomimetic directly into cells. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and can efficiently deliver functional KLX binding peptidomimetic into, for example, keratinocytes. Sells et al. (1995) Biotechniques 19:72-76 describe a procedure for delivery of purified proteins into a variety of cells using such ploycationic lipid preparations.

A significant body of information is emerging regarding the use of other cationic lipids for the delivery of macromolecules into cells. Other suitable lipid vesicles for direct delivery of the KLX binding peptidomimetic include vesicles containing a quaternary ammonium surfactant (Ballas et al. (1988) Biochim. Biophys Acta 939:8-18); lipophilic derivatives of spermine (Behr et al. (1989) PNAS 86:6982-6986).

The lipid formulations of the subject KLX binding peptidomimetic can be used in pharmaceutical formulations to deliver the KLX binding peptidomimetic by various routes and to various sites in the animal body to achieve the desired therapeutic effect. Local or systemic delivery of the therapeutic agent can be achieved by administration comprising application or insertion of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intradermal, peritoneal, subcutaneous and topical administration. Topical formulations are those advantageously applied to the skin or mucosa.

Target mucosa can be that of the vaginal, cervical, vulvar, penal or anorectal mucosa, or target mucosa can be that of the gastrointestinal tract, comprising the mouth, larynx, esophagous and stomach. Other target tissues can be the accessible epidermal tissues which are infected by HPV. Lipids present in topical formulations can act to facilitate introduction of therapeutic KIX binding peptidomimetic into the target tissue, such as the stratum or corneum of the skin, by perturbing the barrier properties of the protective membrane, or by introducing perturbing agents or penetration enhancers such as DMSO, Azone™ or by promoting the activity of these penetration enhancers.

Other pharmaceutical formulations comprising the cationic lipids of the invention are topical preparations containing an anesthetic or cytostatic agent, immunomodulators, bioactive peptides or oligonucleotides, sunscreens or cosmetics. Preparations for topical use are conveniently prepared with hydrophilic and hydrophobic bases in the form of creams, lotions, ointments or gels; alternatively, the preparation may be in the form of a liquid that is sprayed on the skin. The effect of the cationic lipids is to facilitate the penetration of the active antiviral agent through the stratum corneum of the dermis.

The composition and form of pharmaceutical preparations comprising the liposome, in combination with the KIX binding peptidomimetic, can vary according to the intended route of administration. Also, by suitable modifications of the liposome membranes, the liposomes can be made to bind to specific sub-populations of cells. In still another embodiment, the therapeutic KIX binding peptidomimetic can be delivered by way of an artificial viral envelope (AVE). The art as described a number of viral envelopes which exploit molecular recognition of cell surface receptors by viral surface proteins as a means for selective intracellular delivery of macromolecules, including proteins. According to the method of Schreier, et. al., U.S. Patent 5,252,348, a virtually unlimited number of artificial viral envelopes can be prepared and applied using recombinant or isolated surface determinants. For example, the AVEs be generated as viral mimetics of a number of human viruses including arboviruses; flaviviridae; bunyaviridae; hepatitis viruses; Epstein-Barr viruses; heφes viruses; paramyxoviruses; respiratory syncytical virus; retroviruses including human T-lymphotrophic virus type I and H (HTLV-I/H) and human immunodeficiency virus type 1 and 2 (HIV- 1/2 ); rhino viruses; orthopoxviruses; and human papilloma viruses.

In another embodiment, direct delivery of a therapeutic KIX binding peptidomimetic may be facilitated by chemical modification of the polypeptide itself. One such modification involves increasing the lipophilicity of the KIX binding peptidomimetic in order to increase binding to the cell surface, in turn, stimulating nonspecific endocytosis of the protein. Lipophilicity may be increased by adding a lipophilic moiety (e.g., one or more fatty acid molecules) to the KIX binding peptidomimetic. A wide variety of fatty acids may be employed. For example, the protein may be palmitoylated. Alternatively, a lipopeptide may be produced by fusion or cross-linking, to permit the KIX binding peptidomimetic to resemble the natural lipopeptide from E.coli, tripalmitoyl-S-glycerylcysteil-seryl-serine, at its amino terminus. This lipopeptide has been shown to increase the uptake of fused peptides (P. Hoffmann et al., (1988) Immunobiol. 177:158-70). Lipophilicity may also be increased by esterification of the protein at tyrosine residues or other amino acid residues. And uptake of the KLX binding peptidomimetic may be increased by addition of a basic polymer such as polyarginine or polylysine (Shen et al. (1978) PNAS 75:1872-76).

Direct delivery of KIX binding peptidomimetics according to this invention may also be effected by the use of transport moieties, such as protein carriers known to cross cell membranes. For example, a KLX binding peptide may be fused to a carrier protein, preferably by a genetic fusion which may be expressed in a system such as E.coli, barulo virus or yeast. According to one embodiment of this invention, the amino terminus of the KIX binding peptide may be fused to the carboxy terminus of a transport moiety using standard techniques. Nucleotide sequences encoding such carrier-KIX binding peptide fusion proteins, operatively linked to regulatory sequences, may be constructed and introduced into appropriate expression systems using conventional recombinant DNA procedures. The resulting fusion protein may then be purified and tested for its capacity to (1) enter intact eukaryotic cells and (2) inhibit viral DNA replication once inside the intact eukaryotic cells.

In choosing a useful carrier protein, those of skill in the art will recognize the desirability of appropriate control experiments designed, inter alia, to test the possibility that the carrier portion of the fusion protein itself interacts with elements of the E1/E2 DNA replication transcriptional regulation system. If the carrier portion of the fusion protein is found to have undesirable interactions, such as enhancement of papillomavirus DNA replication, the portions of the carrier sequence responsible for these interactions should be identified and deleted in a way which permits the sequence to retain its carrier capacity.

Useful carrier proteins include, for example, bacterial hemolysins or "blending agents", such as alamethicin or sulfhydryl activated lysins. Other carrier moieties which may be used include cell entry components of bacterial toxins, such as Pseudomonas exotoxin, tetanus toxin, ricin toxin, and diphtheria toxin. Also useful is melittin, from bee venom. Other useful carrier proteins include proteins which are viral receptors, cell receptors or cell ligands for specific receptors that are internalized, i.e., those which cross mammalian cell membranes via specific interaction with cell surface receptors, recognized and taken into the cell by cell surface receptors. Such cell ligands include, for example, epidermal growth factor, fibroblast growth factor, transferrin and platelet- derived growth factor. Alternatively, the ligand may be a non-peptide, such as mannose- 6-phosphate, which permits internalization by the mannose-6-phosphate receptor. The transport moiety may also be selected from bacterial immunogens, parasitic immunogens, viral immunogens, immunoglobulins or fragments thereof that bind to target molecules, cytokines, growth factors, colony stimulating factors and hormones. A transport moiety may also be derived from the tat protein of HIV-1.

As an alternative or addition to the above-described chemical modifications and protein carriers, which may be employed alone or in combination, other agents which allow penetration of the keratinized cell layer may be employed to facilitate delivery of the E2 peptidomimetics of this invention. In topical applications, for example, the KLX binding peptidomimetic may be administered in combination with dimethylsulfoxide, an agent which promotes penetration of cell membranes by substances mixed with it. Useful keratinolytic agents include, for example, salicylic acid, urea, and alpha - hydroxyacids. For such applications, the KLX binding peptidomimetic and any other agent may be administered topically, in cream or gel form.

According to an alternate embodiment of this invention, the therapeutic KLX binding peptidomimetic may be administered serially or in combination with other therapeutics used in the treatment of papillomavirus infections or diseases caused by them. Such therapeutics include interferons, such as IFN-γ , IFN-α and IFN-β derived from natural sources or produced by recombinant techniques, other cell mediators formed by leukocytes or produced by recombinant techniques such as for example, 5 interleukin-1, interleukin-2, tumor necrosis factor, macrophage colony stimulating factor, macrophage migration inhibitory factor, macrophage activation factor, lymphotoxin and fibroblast growth factor. Alternatively, the KIX binding peptidomimetic may be administered serially or in combination with conventional therapeutic agents or regimens such as, for example, salicylic acid, podophyllotoxin, 10 retinoic acid, surgery, laser therapy and cryotherapy. Such combination therapies may advantageously utilize less than conventional dosages of those agents, or involve less radical regimens, thus avoiding any potential toxicity or risks associated with those therapies.

It will also be understood by those skilled in the art that any of the above

15. enumerated delivery methods may be augmented, where topical application is being carried out, by the use of ultrasound or iontophoretic delivery devises which facilitate transdermal delivery of proteins. See, for example, Banga et al. (1993) Pharm Res

10:697-702; and Mitragotri et al. (1995) Science 269:850-853.

20 F. Exemplary Modes of Treatment

In one embodiment, the subject KIX binding peptides and analogs thereof can be provided as inhibitors of KlX-dependent gene expression, e.g., it inhibits transcriptional activation by CREB, c-Myb, c-Jun, Statla or SREBP. In other embodiments, the KIX binding peptides of the present invention can be provided as part of a chimeric construct 5 which includes, e.g., a DNA binding domain or other domain which recruits the KIX binding sequence to a gene for which expression is desired, e.g., the fusion protein is a constitutive or inducible transcriptional activator. In the instance of the latter, the fusion protein can be delivered to the cell in the form of a protein therapeutic or as a gene therapy construct.

30 In certain embodiments, the present invention contemplates the use of KIX therapeutics which inhibit KLX-dependent transcription of, for example, hemoglobin. Such inhibitors can be used in the treatment of disorders in which myb-dependent expression is a problem, such as erythrocythemia or polycythemia or other hemoglobinopathies resulting from over-expression of hemoglobin. 5 Another aspect of the present invention relates to a method of modulating a KLX- dependent differentiated state, survival, and/or proliferation of a cell by contacting the cells with a KIX therapeutic according to the subject method and as the circumstances may waπant.

For instance, it is contemplated by the invention that, in light of the findings of an apparently broad involvement of cAMP-dependent transcriptions in the formation of ordered spatial aπangements of differentiated tissues in vertebrates, the subject method could be used as part of a process for generating and/or maintaining an array of different vertebrate tissue both in vitro and in vivo. The KIX therapeutic, whether inductive or anti-inductive with respect proliferation or differentiation of a given tissue, can be, as appropriate, any of the preparations described above. For example, the present method of using a cAMP antagonist is applicable to cell culture techniques. In vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF). One use of the present method may be in cultures of neuronal stem cells, such as in the use of such cultures for the generation of new neurons and glia. In such embodiments of the subject method, the cultured cells can be contacted with a KIX therapeutic of the present invention in order to alter the rate of proliferation of neuronal stem cells in the culture and/or alter the rate of differentiation, or to maintain the integrity of a culture of certain terminally differentiated neuronal cells. In an exemplary embodiment, the subject method can be used to culture, for example, sensory neurons or, alternatively, motorneurons. Such neuronal cultures can be used as convenient assay systems as well as sources of implantable cells for therapeutic treatments.

According to the present invention, large numbers of non-tumorigenic neural progenitor cells can be peφetuated in vitro and their rate of proliferation and/or differentiation can be effected by contact with KLX therapeutics of the present invention. Generally, a method is provided comprising the steps of isolating neural progenitor cells from an animal, peφetuating these cells in vitro or in vivo, preferably in the presence of growth factors, and regulating the differentiation of these cells into particular neural phenotypes, e.g., neurons and glia, by contacting the cells with a KIX therapeutic.

Progenitor cells are thought to be under a tonic inhibitory influence which maintains the progenitors in a suppressed state until their differentiation is required. However, recent techniques have been provided which permit these cells to be proliferated, and unlike neurons which are terminally differentiated and therefore non- dividing, they can be produced in unlimited number and are highly suitable for transplantation into heterologous and autologous hosts with neurodegenerative diseases. By "progenitor" it is meant an oligopotent or multipotent stem cell which is able to divide without limit and, under specific conditions, can produce daughter cells which terminally differentiate such as into neurons and glia. These cells can be used for transplantation into a heterologous or autologous host. By heterologous is meant a host other than the animal from which the progenitor cells were originally derived. By autologous is meant the identical host from which the cells were originally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adult neural tissue from any animal. By any animal is meant any multicellular animal which contains nervous tissue. More particularly, is meant any fish, reptile, bird, amphibian or mammal and the like. The most preferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may be euthanized, and the brain and specific area of interest removed using a sterile procedure. Brain areas of particular interest include any area from which progenitor cells can be obtained which will serve to restore function to a degenerated area of the host's brain. These regions include areas of the central nervous system (CNS) including the cerebral cortex, cerebellum, midbrain, brainstem, spinal cord and ventricular tissue, and areas of the peripheral nervous system (PNS) including the carotid body and the adrenal medulla. More particularly, these areas include regions in the basal ganglia, preferably the striatum which consists of the caudate and putamen, or various cell groups such as the globus pallidus, the subthalamic nucleus, the nucleus basalis which is found to be degenerated in Alzheimer's Disease patients, or the substantia nigra pars compacta which is found to be degenerated in Parkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetal tissue obtained from elective abortion, or from a post-natal, juvenile or adult organ donor. Autologous neural tissue can be obtained by biopsy, or from patients undergoing neurosurgery in which neural tissue is removed, in particular during epilepsy surgery, and more particularly during temporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individual cells from the connecting extracellular matrix of the tissue. Dissociation can be obtained using any known procedure, including treatment with enzymes such as trypsin, collagenase and the like, or by using physical methods of dissociation such as with a blunt instrument or by mincing with a scalpel to a allow outgrowth of specific cell types from a tissue.

Dissociation of fetal cells can be carried out in tissue culture medium, while a preferable medium for dissociation of juvenile and adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mM NaCl, 5 mM KC1, 1.3 mM MgCl , 2 mM

CaCl2, 26 mM NaHCO3, and 10 mM D-glucose. Low Ca2⁺ aCSF contains the same ingredients except for MgCl2 at a concentration of 3.2 mM and CaCl2 at a concentration of 0.1 mM.

Dissociated cells can be placed into any known culture medium capable of supporting cell growth, including MEM, DMEM, RPMI, F-l 2, and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum derived from bovine, equine, chicken and the like. A particularly preferable medium for cells is a mixture of DMEM and F-12.

Conditions for culturing should be close to physiological conditions. The pH of the culture media should be close to physiological pH, preferably between pH 6-8, more preferably close to pH 7, even more particularly about pH 7.4. Cells should be cultured at a temperature close to physiological temperature, preferably between 30 °C-40 °C, more preferably between 32 °C-38 °C, and most preferably between 35 °C-37 °C.

Cells can be grown in suspension or on a fixed substrate, but proliferation of the progenitors is preferably done in suspension to generate large numbers of cells by formation of "neurospheres" (see, for example, Reynolds et al. (1992) Science 255:1070- 1709; and PCT Publications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In the case of propagating (or splitting) suspension cells, flasks are shaken well and the neurospheres allowed to settle on the bottom corner of the flask. The spheres are then transfeπed to a 50 ml centrifuge tube and centrifuged at low speed. The medium is aspirated, the cells resuspended in a small amount of medium with growth factor, and the cells mechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growth factor which allows for the proliferation of progenitor cells and seeded in any receptacle capable of sustaining cells, though as set out above, preferably in culture flasks or roller bottles. Cells typically proliferate within 3-4 days in a 37 °C incubator, and proliferation can be reinitiated at any time after that by dissociation of the cells and resuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask and continue to proliferate in suspension forming a hollow sphere of undifferentiated cells. After approximately 3-10 days in vitro, the proliferating clusters (neurospheres) are fed every 2-7 days, and more particularly every 2-4 days by gentle centrifugation and resuspension in medium containing growth factor. After 6-7 days in vitro, individual cells in the neurospheres can be separated by physical dissociation of the neurospheres with a blunt instrument, more particularly by triturating the neurospheres with a pipette. Single cells from the dissociated neurospheres are suspended in culture medium containing growth factors, and differentiation of the cells can be control in culture by plating (or resuspending) the cells in the presence of a KIX therapeutic.

To further illustrate other uses of the subject KIX therapeutics, it is noted that intracerebral grafting has emerged as an additional approach to central nervous system therapies. For example, one approach to repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) JExp Biol 123:265-289; and Freund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a variety of brain regions can be successfully incoφorated into the adult brain, and such grafts can alleviate behavioral defects. For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are impaired after lesions of the neocortex can also be partially restored by grafts of embryonic cortical cells. The subject method can be used to regulate the growth state in the culture, or where fetal tissue is used, especially neuronal stem cells, can be used to regulate the rate of differentiation of the stem cells. Stem cells useful in the present invention are generally known. For example, several neural crest cells have been identified, some of which are multipotent and likely represent uncommitted neural crest cells, and others of which can generate only one type of cell, such as sensory neurons, and likely represent committed progenitor cells. The role of KIX therapeutics employed in the present method to culture such stem cells can be to regulate differentiation of the uncommitted progenitor, or to regulate further restriction of the developmental fate of a committed progenitor cell towards becoming a terminally differentiated neuronal cell. For example, the present method can be used in vitro to regulate the differentiation of neural crest cells into glial cells, schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic neurons, as well as peptidergic and serotonergic neurons. The KIX therapeutics can be used alone, or can be used in combination with neurotrophic factors which act to more particularly enhance a particular differentiation fate of the neuronal progenitor cell.

In addition to the implantation of cells cultured in the presence of the subject

KIX therapeutics, yet another aspect of the present invention concerns the therapeutic application of a KLX therapeutic to regulate the growth state of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system. The ability of CREB to regulate neuronal differentiation during development of the nervous system and also presumably in the adult state indicates that, in certain instances, the subject KIX therapeutics can be expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and treatment of degeneration in certain pathological conditions. In light of this understanding, the present invention specifically contemplates applications of the subject method to the treatment protocol of (prevention and or reduction of the severity of) neurological conditions deriving from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vascular injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor- induced injury; (ii) aging of the nervous system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntingdon's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations; and (iv) chronic immunological diseases of the nervous system or affecting the nervous system, including multiple sclerosis.

As appropriate, the subject method can also be used in generating nerve prostheses for the repair of central and peripheral nerve damage. In particular, where a crushed or severed axon is intubulated by use of a prosthetic device, KIX therapeutics can be added to the prosthetic device to regulate the rate of growth and regeneration of the dendridic processes. Exemplary nerve guidance channels are described in U.S. patents 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatment of neoplastic or hypeφlastic transformations such as may occur in the central nervous system. For instance, the KIX therapeutics can be utilized to cause such transformed cells to become either post-mitotic or apoptotic. The present method may, therefore, be used as part of a treatment for, e.g., malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and ependymomas.

In a prefeπed embodiment, the subject method can be used as part of a treatment regimen for malignant medulloblastoma and other primary CNS malignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatment program for medulloblastoma. Medulloblastoma, a primary brain tumor, is the most common brain tumor in children. A medulloblastoma is a primitive neuroectodermal tumor arising in the posterior fossa. They account for approximately 25% of all pediatric brain tumors (Miller). Histologically, they are small round cell tumors commonly aπanged in true rosettes, but may display some differentiation to astrocytes, ependymal cells or neurons (Rorke; Kleihues). PNET's may arise in other areas of the brain including the pineal gland (pineoblastoma) and cerebrum. Those arising in the supratentorial region generally fare worse than their PF counteφarts.

Medulloblastoma/PNET's are known to recur anywhere in the CNS after resection, and can even metastasize to bone. Pretreatment evaluation should therefore include an examination of the spinal cord to exclude the possibility of "dropped metastases". Gadolinium-enhanced MRI has largely replaced myelography for this puφose, and CSF cytology is obtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of treatment program for ependymomas. Ependymomas account for approximately 10% of the pediatric brain tumors in children. Grossly, they are tumors that arise from the ependymal lining of the ventricles and microscopically form rosettes, canals, and perivascular rosettes. In the CHOP series of 51 children reported with ependymomas, ³/ were histologically benign. Approximately 2/3 arose from the region of the 4th ventricle. One third presented in the supratentorial region. Age at presentation peaks between birth and 4 years, as demonstrated by SEER data as well as data from CHOP. The median age is about 5 years. Because so many children with this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation in the art that CREB is involved in moφhogenic signals in other vertebrate organogenic pathways in addition to neuronal differentiation as described above, having apparent roles in other endodermal patterning, as well as both mesodermal and endodermal differentiation processes. Thus, it is contemplated by the invention that compositions comprising KIX therapeutics can also be utilized for both cell culture and therapeutic methods involving generation and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery that CREB is apparently involved in controlling the development of stem cells responsible for formation of the digestive tract, liver, lungs, and other organs which derive from the primitive gut. Regulations of cAMP-dependent transcriptions regulates the inductive signal from the endoderm to the mesoderm, which is critical to gut moφhogenesis. Therefore, for example, KIX therapeutics of the instant method can be employed for regulating the development and maintenance of an artificial liver which can have multiple metabolic functions of a normal liver. In an exemplary embodiment, the tube stem cells to form hepatocyte cultures which can be used to populate extracellular matrices, or which can be encapsulated in biocompatible polymers, to form both implantable and extracoφoreal artificial livers. In another embodiment, therapeutic compositions of KIX therapeutics can be utilized in conjunction with transplantation of such artificial livers, as well as embryonic liver structures, to regulate uptake of intraperitoneal implantation, vascularization, and in vivo differentiation and maintenance of the engrafted liver tissue. In yet another embodiment, the subject method can be employed therapeutically to regulate such organs after physical, chemical or pathological insult. For instance, therapeutic compositions comprising KIX therapeutics can be utilized in liver repair subsequent to a partial hepatectomy.

The generation of the pancreas and small intestine from the embryonic gut depends on intercellular signalling between the endodermal and mesodermal cells of the gut. In particular, the differentiation of intestinal mesoderm into smooth muscle has been suggested to depend on signals from adjacent endodermal cells. Candidate mediators of endodermally derived signals in the embryonic hindgut act, inter alia, via regulations of KLX-dependent transcription In the context of the present invention, it is contemplated therefore that the subject KIX therapeutics can be used to control or regulate the proliferation and/or differentiation of pancreatic tissue both in vivo and in vitro.

There are a wide variety of pathological cell proliferative and differentiative conditions for which the inhibitors of the present invention may provide therapeutic benefits, with the general strategy being, for example, the coπection of abeπant insulin expression, or modulation of differentiation. The ability to repress intracellular signal- induced response pathways is an important mechanism in negative control of gene expression. Selective disruption of such pathways would allow the development of therapeutic agents capable of treating a variety of disease states related to improper activation and/or expression of specific transcription factors. More generally, however, the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival and/or affecting proliferation of pancreatic cells, by contacting the cells with the subject inhibitors. For instance, it is contemplated by the invention that, in light of the apparent involvement of cAMP-dependent factors such as CREB and c-myb in the formation of ordered spatial aπangements of pancreatic tissues, the subject method could be used as part of a technique to generate and/or maintain such tissue both in vitro and in vivo. For instance, modulation of the function of hedgehog can be employed in both cell culture and therapeutic methods involving generation and maintenance β-cells and possibly also for non-pancreatic tissue, such as in controlling the development and maintenance of tissue from the digestive tract, spleen, lungs, and other organs which derive from the primitive gut. In an exemplary embodiment, the present method can be used in the treatment of hypeφlastic and neoplastic disorders effecting pancreatic tissue, particularly those characterized by abeπant proliferation of pancreatic cells. For instance, pancreatic cancers are marked by abnormal proliferation of pancreatic cells which can result in alterations of insulin secretory capacity of the pancreas. For instance, certain pancreatic hypeφlasias, such as pancreatic carcinomas, can result in hypoinsulinemia due to dysfunction of β-cells or decreased islet cell mass. To the extent that abeπant activations or inactivations of CREB or C-Myb may be indicated in disease progression, the subject regulators can be used to enhance regeneration of the tissue after anti-tumor therapy.

Furthermore, manipulation of the differentiative state of pancreatic tissue can be utilized in conjunction with transplantation of artificial pancreas so as to promote implantation, vascularization, and in vivo differentiation and maintenance of the engrafted tissue. For instance, manipulation of KLX-dependent gene transcriptions to affect tissue differentiation can be utilized as a means of maintaining graft viability.

In certain embodiments, the subject peptides which function as CBP inhibitors can be used as part of a treatment for patients with non-insulin dependent diabetes mellitus (NIDDM). In NIDDM patients, hyperglycemia develops in part as a result of β cell failure secondary to chronic insulin resistance. This hyperglycemia appears to be exacerbated by hyperglucagonemia and increased hepatic gluconeogenesis. cAMP appears to be the major starvation state signal which triggers glucagon gene expression as well as transcription of PEPCK, the rate limiting enzyme in gluconeogenesis.

It has previously been reported that CBP cooperates with upstream activators involved in the activation of transcription by signal dependent transcription factors, such as c-Jun (responsive to phorbol ester), serum response factor, and the like. Accordingly, employing CBP antagonists can disrupt the ability of signal dependent transcription factors to activate transcription, e.g., which inhibit intracellular signal-induced response pathways in order to aid in the treatment of diabetes mellitus or other CBP-mediated alteration of the glucose response.. Accordingly, there are provided methods for treating diabetes mellitus, said method comprising contacting a biological system with an effective amount of a compound which inhibits binding of CREB to CBP, e.g., a KIX peptide or peptidomimetic thereof or other small organic molecule. Such methods ameliorate hyperglycemia associated with diabetes mellitus by modulating gluconeogenesis and/or hyperglucagonemia. Particularly, such methods employ compounds which disrupt the formation of CREB:CBP complexes, thus inhibiting transcription of PEPCK or glucagon gene.

In addition, many tumors may, based on evidence such as involvement of the CREB, c-Myb and other KLX-dependent pathways in these tumors be affected by treatment with the subject KLX therapeutics. Such tumors include, but are by no means limited to, tumors related to Gorlin's syndrome (e.g., basal cell carcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in pet knock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumors resulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.), tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma, thyroid carcinoma, etc.), Ext-7-related tumors (e.g., bone cancer, etc.), Shh- induced tumors (e.g., lung cancer, chondrosarcomas, etc.), and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney, bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinal cancer (e.g., stomach, intestine, etc.), etc.).

In still another embodiment of the present invention, compositions comprising KIX therapeutics can be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment of skeletal tissue deficiencies. The present invention particularly contemplates the use of KIX therapeutics to regulate the rate of chondrogenesis and/or osteogenesis. By "skeletal tissue deficiency", it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.

For instance, the method of the present invention can be used as part of a regimen for restoring cartilage function to a connective tissue. Such methods are useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechanical derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a laxation of a joint by a torn ligament, malignment of joints, bone fracture, or by hereditary disease. The present reparative method is also useful for remodeling cartilage matrix, such as in plastic or reconstructive surgery, as well as periodontal surgery. The present method may also be applied to improving a previous reparative procedure, for example, following surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or exacerbation of degenerative disease if applied early enough after trauma.

In one embodiment of the present invention, the subject method comprises treating the afflicted connective tissue with a therapeutically sufficient amount of a KIX therapeutic to regulate a cartilage repair response in the connective tissue by managing the rate of differentiation and/or proliferation of chondrocytes embedded in the tissue. Such connective tissues as articular cartilage, interarticular cartilage (menisci), costal cartilage (connecting the true ribs and the sternum), ligaments, and tendons are particularly amenable to treatment in reconstructive and/or regenerative therapies using the subject method. As used herein, regenerative therapies include treatment of degenerative states which have progressed to the point of which impairment of the tissue is obviously manifest, as well as preventive treatments of tissue where degeneration is in its earliest stages or imminent.

In an illustrative embodiment, the subject method can be used as part of a therapeutic intervention in the treatment of cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist, a knuckle of either a finger or toe, or a tempomandibular joint. The treatment can be directed to the meniscus of the joint, to the articular cartilage of the joint, or both. To further illustrate, the subject method can be used to treat a degenerative disorder of a knee, such as which might be the result of traumatic injury (e.g., a sports injury or excessive wear) or osteoarthritis. The subject regulators may be administered as an injection into the joint with, for instance, an arthroscopic needle. In some instances, the injected agent can be in the form of a hydrogel or other slow release vehicle described above in order to permit a more extended and regular contact of the agent with the treated tissue. The present invention further contemplates the use of the subject method in the field of cartilage transplantation and prosthetic device therapies. However, problems arise, for instance, because the characteristics of cartilage and fibrocartilage varies between different tissue: such as between articular, meniscal cartilage, ligaments, and tendons, between the two ends of the same ligament or tendon, and between the superficial and deep parts of the tissue. The zonal aπangement of these tissues may reflect a gradual change in mechanical properties, and failure occurs when implanted tissue, which has not differentiated under those conditions, lacks the ability to appropriately respond. For instance, when meniscal cartilage is used to repair anterior cruciate ligaments, the tissue undergoes a metaplasia to pure fibrous tissue. By regulating the rate of chondrogenesis, the subject method can be used to particularly address this problem, by helping to adaptively control the implanted cells in the new environment and effectively resemble hypertrophic chondrocytes of an earlier developmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing both the generation of prosthetic cartilage devices and to their implantation. The need for improved treatment has motivated research aimed at creating new cartilage that is based on collagen-glycosaminoglycan templates (Stone et al. (1990) Clin Orthop Relat Red 252:129), isolated chondrocytes (Grande et al. (1989) J Orthop Res 7:208; and Takigawa et al. (1987) Bone Miner 2:449), and chondrocytes attached to natural or synthetic polymers (Walitani et al. (1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg 88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed et al. (1993) J Biomed Mater Res 27: 11 ; and the Vacanti et al. U.S. Patent No. 5,041 , 138). For example, chondrocytes can be grown in culture on biodegradable, biocompatible highly porous scaffolds formed from polymers such as polyglycolic acid, polylactic acid, agarose gel, or other polymers which degrade over time as function of hydrolysis of the polymer backbone into innocuous monomers. The matrices are designed to allow adequate nutrient and gas exchange to the cells until engraftment occurs. The cells can be cultured in vitro until adequate cell volume and density has developed for the cells to be implanted. One advantage of the matrices is that they can be cast or molded into a desired shape on an individual basis, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices can be used which allow for manipulation at the time of implantation, as in a joint.

In one embodiment of the subject method, the implants are contacted with a KLX therapeutic during certain stages of the culturing process in order to manage the rate of differentiation of chondrocytes and the formation of hypertrophic chrondrocytes in the culture. In another embodiment, the implanted device is treated with a KIX therapeutic in order to actively remodel the implanted matrix and to make it more suitable for its intended function. As set out above with respect to tissue transplants, the artificial transplants suffer from the same deficiency of not being derived in a setting which is comparable to the actual mechanical environment in which the matrix is implanted. The ability to regulate the chondrocytes in the matrix by the subject method can allow the implant to acquire characteristics similar to the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhance attachment of prosthetic devices. To illustrate, the subject method can be used in the implantation of a periodontal prosthesis, wherein the treatment of the suπounding connective tissue stimulates formation of periodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as part of a regimen for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient. Indian hedgehog is particularly associated with the hypertrophic chondrocytes that are ultimately replaced by osteoblasts. For instance, administration of a KLX therapeutic of the present invention can be employed as part of a method for regulating the rate of bone loss in a subject. For example, preparations comprising KIX therapeutics can be employed, for example, to control endochondral ossification in the formation of a "model" for ossification.

The subject method also has wide applicability to the treatment or prophylaxis of disorders afflicting epithelial tissue, as well as in cosmetic uses. In general, the method can be characterized as including a step of administering to an animal an amount of a KIX therapeutic effective to alter the growth state of a treated epithelial tissue. The mode of administration and dosage regimens will vary depending on the epithelial tissue(s) which is to be treated. For example, topical formulations will be prefeπed where the treated tissue is epidermal tissue, such as dermal or mucosal tissues. A method which "promotes the healing of a wound" results in the wound healing more quickly as a result of the treatment than a similar wound heals in the absence of the treatment. "Promotion of wound healing" can also mean that the method regulates the proliferation and/or growth of, inter alia, keratinocytes, or that the wound heals with less scarring, less wound contraction, less collagen deposition and more superficial surface area. In certain instances, "promotion of wound healing" can also mean that certain methods of wound healing have improved success rates, (e.g., the take rates of skin grafts,) when used together with the method of the present invention.

Despite significant progress in reconstructive surgical techniques, scarring can be an important obstacle in regaining normal function and appearance of healed skin. This is particularly true when pathologic scarring such as keloids or hypertrophic scars of the hands or face causes functional disability or physical deformity. In the severest circumstances, such scarring may precipitate psychosocial distress and a life of economic deprivation. Wound repair includes the stages of hemostasis, inflammation, proliferation, and remodeling. The proliferative stage involves multiplication of fibroblasts and endothelial and epithelial cells. Through the use of the subject method, the rate of proliferation of epithelial cells in and proximal to the wound can be controlled in order to accelerate closure of the wound and/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeutic regimen for treating oral and paraoral ulcers, e.g., resulting from radiation and/or chemotherapy. Such ulcers commonly develop within days after chemotherapy or radiation therapy. These ulcers usually begin as small, painful iπegularly shaped lesions usually covered by a delicate gray necrotic membrane and suπounded by inflammatory tissue. In many instances, lack of treatment results in proliferation of tissue around the periphery of the lesion on an inflammatory basis. For instance, the epithelium bordering the ulcer usually demonstrates proliferative activity, resulting in loss of continuity of surface epithelium. These lesions, because of their size and loss of epithelial integrity, dispose the body to potential secondary infection. Routine ingestion of food and water becomes a very painful event and, if the ulcers proliferate throughout the alimentary canal, diarrhea usually is evident with all its complicating factors. According to the present invention, a treatment for such ulcers which includes application of a KIX therapeutic can reduce the abnormal proliferation and differentiation of the affected epithelium, helping to reduce the severity of subsequent inflammatory events.

The subject method and compositions can also be used to treat wounds resulting from dermatological diseases, such as lesions resulting from autoimmune disorders such as psoriasis. Atopic dermititis refers to skin trauma resulting from allergies associated with an immune response caused by allergens such as pollens, foods, dander, insect venoms and plant toxins.

In other embodiments, antiproliferative preparations of KIX therapeutics can be used to inhibit lens epithelial cell proliferation to prevent post-operative complications of extracapsular cataract extraction. Cataract is an intractable eye disease and various studies on a treatment of cataract have been made. But at present, the treatment of cataract is attained by surgical operations. Cataract surgery has been applied for a long time and various operative methods have been examined. Extracapsular lens extraction has become the method of choice for removing cataracts. The major medical advantages of this technique over intracapsular extraction are lower incidence of aphakic cystoid macular edema and retinal detachment. Extracapsular extraction is also required for implantation of posterior chamber type intraocular lenses which are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the high incidence of posterior lens capsule opacification, often called after-cataract, which can occur in up to 50% of cases within three years after surgery. After-cataract is caused by proliferation of equatorial and anterior capsule lens epithelial cells which remain after extracapsular lens extraction. These cells proliferate to cause Sommerling rings, and along with fibroblasts which also deposit and occur on the posterior capsule, cause opacification of the posterior capsule, which interferes with vision. Prevention of after- cataract would be preferable to treatment. To inhibit secondary cataract formation, the subject method provides a means for inhibiting proliferation of the remaining lens epithelial cells. For example, such cells can be induced to remain quiescent by instilling a solution containing an KIX therapeutic preparation into the anterior chamber of the eye after lens removal. Furthermore, the solution can be osmotically balanced to provide minimal effective dosage when instilled into the anterior chamber of the eye, thereby inhibiting subcapsular epithelial growth with some specificity.

The subject method can also be used in the treatment of corneopathies marked by corneal epithelial cell proliferation, as for example in ocular epithelial disorders such as epithelial downgrowth or squamous cell carcinomas of the ocular surface.

Yet another aspect of the present invention relates to the use of the subject method to control hair growth. Hair is basically composed of keratin, a tough and insoluble protein; its chief strength lies in its disulphide bond of cystine. Each individual hair comprises a cylindrical shaft and a root, and is contained in a follicle, a flask-like depression in the skin. The bottom of the follicle contains a finger-like projection termed the papilla, which consists of connective tissue from which hair grows, and through which blood vessels supply the cells with nourishment. The shaft is the part that extends outwards from the skin surface, whilst the root has been described as the buried part of the hair. The base of the root expands into the hair bulb, which rests upon the papilla. Cells from which the hair is produced grow in the bulb of the follicle; they are extruded in the form of fibers as the cells proliferate in the follicle. Hair "growth" refers to the formation and elongation of the hair fiber by the dividing cells. As is well known in the art, the common hair cycle is divided into three stages: anagen, catagen and telogen. During the active phase (anagen), the epidermal stem cells of the dermal papilla divide rapidly. Daughter cells move upward and differentiate to form the concentric layers of the hair itself. The transitional stage, catagen, is marked by the cessation of mitosis of the stem cells in the follicle. The resting stage is known as telogen, where the hair is retained within the scalp for several weeks before an emerging new hair developing below it dislodges the telogen-phase shaft from its follicle. From this model it has become clear that the larger the pool of dividing stem cells that differentiate into hair cells, the more hair growth occurs. Accordingly, methods for increasing or reducing hair growth can be carried out by potentiating or inhibiting, respectively, the proliferation of these stem cells.

In certain embodiments, the subject method can be employed as a way of reducing the growth of human hair as opposed to its conventional removal by cutting, shaving, or depilation. For instance, the present method can be used in the treatment of trichosis characterized by abnormally rapid or dense growth of hair, e.g. hypertrichosis. In an exemplary embodiment, KLX therapeutics can be used to manage hirsutism, a disorder marked by abnormal hairiness. The subject method can also provide a process for extending the duration of depilation.

Moreover, because a certain KLX therapeutic can often be cytostatic to epithelial cells, rather than cytotoxic, such agents can be used to protect hair follicle cells from cytotoxic agents which require progression into S-phase of the cell-cycle for efficacy, e.g. radiation-induced death. Treatment by the subject method can provide protection by causing the hair follicle cells to become quiescent, e.g., by inhibiting the cells from entering S phase, and thereby preventing the follicle cells from undergoing mitotic catastrophe or programmed cell death. For instance, KIX therapeutics can be used for patients undergoing chemo- or radiation-therapies which ordinarily result in hair loss. By inhibiting cell-cycle progression during such therapies, the subject treatment can protect hair follicle cells from death which might otherwise result from activation of cell death programs. After the therapy has concluded, the instant method can also be removed with concommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis, such as folliculitis decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis. For example, a cosmetic preparation of an KIX therapeutic can be applied topically in the treatment of pseudofolliculitis, a chronic disorder occurring most often in the submandibular region of the neck and associated with shaving, the characteristic lesions of which are erythematous papules and pustules containing buried hairs.

In another aspect of the invention, the subject method can be used to induce differentiation and/or inhibit proliferation of epithelially derived tissue. Such forms of these molecules can provide a basis for differentiation therapy for the treatment of hypeφlastic and/or neoplastic conditions involving epithelial tissue. For example, such preparations can be used for the treatment of cutaneous diseases in which there is abnormal proliferation or growth of cells of the skin. For instance, the pharmaceutical preparations of the invention are intended for the treatment of hypeφlastic epidermal conditions, such as keratosis, as well as for the treatment of neoplastic epidermal conditions such as those characterized by a high proliferation rate for various skin cancers, as for example basal cell carcinoma or squamous cell carcinoma. The subject method can also be used in the treatment of autoimmune diseases affecting the skin, in particular, of dermatological diseases involving morbid proliferation and/or keratinization of the epidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cell carcinoma, keratoacanthoma and actinic keratosis are characterized by localized abnormal proliferation and growth. For example, in psoriasis, which is characterized by scaly, red, elevated plaques on the skin, the keratinocytes are known to proliferate much more rapidly than normal and to differentiate less completely.

In one embodiment, the preparations of the present invention are suitable for the treatment of dermatological ailments linked to keratinization disorders causing abnormal proliferation of skin cells, which disorders may be marked by either inflammatory or non-inflammatory components. To illustrate, therapeutic preparations of a KLX therapeutic, e.g., which promotes quiescense or differentiation can be used to treat varying forms of psoriasis, be they cutaneous, mucosal or ungual. Psoriasis, as described above, is typically characterized by epidermal keratinocytes which display marked proliferative activation and differentiation along a "regenerative" pathway. Treatment with an antiproliferative embodiment of the subject method can be used to reverse the pathological epidermal activation and can provide a basis for sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatment with the subject method. Actinic keratoses, for example, are superficial inflammatory premalignant tumors arising on sun-exposed and iπadiated skin. The lesions are erythematous to brown with variable scaling. Cuπent therapies include excisional and cryosurgery. These treatments are painful, however, and often produce cosmetically unacceptable scarring. Accordingly, treatment of keratosis, such as actinic keratosis, can include application, preferably topical, of a KLX therapeutic composition in amounts sufficient to inhibit hypeφroliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated by the subject method. Acne vulgaris, for instance, is a multi factorial disease most commonly occurring in teenagers and young adults, and is characterized by the appearance of inflammatory and noninflammatory lesions on the face and upper trunk. The basic defect which gives rise to acne vulgaris is hypercornification of the duct of a hyperactive sebaceous gland. Hypercornification blocks the normal mobility of skin and follicle microorganisms, and in so doing, stimulates the release of lipases by Propinobacterium acnes and Staphylococcus epidermidis bacteria and Pitrosporum ovale, a yeast. Treatment with an antiproliferative cAMP antagonist, particularly topical preparations, may be useful for preventing the transitional features of the ducts, e.g. hypercornification, which lead to lesion formation. The subject treatment may further include, for example, antibiotics, retinoids and antiandrogens.

The present invention also provides a method for treating various forms of dermatitis. Dermatitis is a descriptive term referring to poorly demarcated lesions which are either pruritic, erythematous, scaly, blistered, weeping, fissured or crusted. These lesions arise from any of a wide variety of causes. The most common types of dermatitis are atopic, contact and diaper dermatitis. For instance, seboπheic dermatitis is a chronic, usually pruritic, dermatitis with erythema, dry, moist, or greasy scaling, and yellow crusted patches on various areas, especially the scalp, with exfoliation of an excessive amount of dry scales. The subject method can also be used in the treatment of stasis dermatitis, an often chronic, usually eczematous dermatitis. Actinic dermatitis is dermatitis that due to exposure to actinic radiation such as that from the sun, ultraviolet waves or x- or gamma-radiation. According to the present invention, the subject method can be used in the treatment and/or prevention of certain symptoms of dermatitis caused by unwanted proliferation of epithelial cells. Such therapies for these various forms of dermatitis can also include topical and systemic corticosteroids, antipuritics, and antibiotics.

Ailments which may be treated by the subject method are disorders specific to non-humans, such as mange.

In still another embodiment, the subject method can be used in the treatment of human cancers, particularly basal cell carcinomas and other tumors of epithelial tissues such as the skin. For example, cAMP antagonists can be employed, in the subject method, as part of a treatment for basal cell nevus syndrome (BCNS), and other human carcinomas, adenocarcinomas, sarcomas and the like.

In a prefeπed embodiment, the subject method is used as part of a treatment to prophylaxis regimen for treating (or preventing) basal cell carcinoma. The deregulation of the hedgehog signaling pathway may be a general feature of basal cell carcinomas caused by ptc mutations and implicating cAMP-dependent response, such as VIA CREB or c-Myb. Consistent overexpression of human ptc mRNA has been described in tumors of familial and sporadic BCCs, determined by in situ hybridization. Mutations that inactivate ptc may be expected to result in overexpression of mutant Ptc, because ptc displays negative autoregulation. Prior research demonstrates that overexpression of hedgehog proteins can also lead to tumorigenesis. That sonic hedgehog {Shh) has a role in tumorigenesis in the mouse has been suggested by research in which transgenic mice overexpressing Shh in the skin developed features of BCNS, including multiple BCC- like epidermal proliferations over the entire skin surface, after only a few days of skin development. A mutation in the Shh human gene from a BCC was also described; it was suggested that Shh or other Hh genes in humans could act as dominant oncogenes in humans. Sporadic ptc mutations have also been observed in BCCs from otherwise normal individuals, some of which are UV-signature mutations. In one recent study of sporadic BCCs, five UV-signature type mutations, either CT or CCTT changes, were found out of fifteen tumors determined to contain tc mutations. Another recent analysis of sporadic ptc mutations in BCCs and neuroectodermal tumors revealed one CT change in one of three ptc mutations found in the BCCs. See, for example, Goodrich et al. (1997) Science 277:1109-13; Xie et al. (1997) Cancer Res 57:2369-72; Oro et al. (1997) Science 276:817-21; Xie et al. (1997) Genes Chromosomes Cancer 18:305-9; Stone et al. (1996) Nature 384:129-34; and Johnson et al. (1996) Science 272:1668-71.

The subject method can also be used to treatment patients with BCNS, e.g., to prevent BCC or other effects of the disease which may be the result of ptc loss-of- function, hedgehog gain-of-function, or smoothened gain-of-function. Basal cell nevus syndrome is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age. BCNS patients are very susceptible to the development of these tumors; in the second decade of life, large numbers appear, mainly on sun-exposed areas of the skin. This disease also causes a number of developmental abnormalities, including rib, head and face alterations, and sometimes polydactyfy, syndactyly, and spina bifida. They also develop a number of tumor types in addition to BCCs: fibromas of the ovaries and heart, cysts of the skin and jaws, and in the central nervous system, medulloblastomas and meningiomas. The subject method can be used to prevent or treat such tumor types in BCNS and non-BCNS patients. Studies of BCNS patients show that they have both genomic and sporadic mutations in the ptc gene, suggesting that these mutations are the ultimate cause of this disease.

In another aspect, the present invention provides pharmaceutical preparations and methods for controlling the formation of megakaryocyte-derived cells and/or controlling the functional performance of megakaryocyte-derived cells. For instance, certain of the compositions disclosed herein may be applied to the treatment or prevention of a variety hypeφlastic or neoplastic conditions affecting platelets.

KLX-dependent interactions have also been implicated in various viral infections and viral pathogenesis. For instance, the Epstein-Ban virus (EBV) immediate-early protein BZLF1 (Z) is a key regulator of the EBV latent-to-lytic switch. Z is a transcriptional activator which induces EBV early gene expression. It has been demonstrated that Z interacts with CBP. See Adamson et al. (1999) J Virol 73:6551. It is proposed that Z interacts with CBP to enhance viral early gene transcription. In addition, the Z-CBP interaction may control host cellular transcription factor activity through competition for limiting amounts of cellular CBP.

The HTLV-I oncoprotein Tax is required for high level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Tax stimulates HTLV-I transcription through high affinity binding to the KLX domain of CBP, a pleiotropic coactivator. Several cellular proteins, including c-jun, also bind to KIX and utilize CBP as a coactivator. Van Orden (1999) Oncogene 18:3766. Likewise, it has been reported that HIV-1 TAT transactivator recruits p300 and CBP to the viral promoter. Marzio et al. (1998) PNAS 95:13519.

Given the significant implications of KLX-mediated events for cellular homeostasis and transformation in such virally infected cells, it is contemplated that the subject KLX.therapeutics, particularly the antagonists, may be used to treat (including lessening the severity of) viral infections, especially EBV and HIV infection.

In still another embodiment, the subject KIX therapeutics can be used as part of a treatment for various cancers, such as melanomas and leukemias.

The pleiotropic cellular coactivator CREB binding protein (CBP) plays a critical role in supporting p53-dependent tumor suppressor functions. p53 has been shown to directly interact with a carboxyl-terminal region of CBP for recruitment of the coactivator to p53-responsive genes. The KLX domain is a p53 contact point on CBP. Van Orden et al. (1999) J Biol Chem 274:26321. Accordingly, the subject KIX therapeutics can be used to inhibit p53/KIX domain interactions. Thus, in certain embodiments, the KLX therapeutic is used to sensitize the tumor for radiation therapy or chemotherapy with apoptisis-inducing compounds.

Moreover, in certain leukemias, especially myeloid leukemias, it has been reported that a chromosomal translocation occurs which creates a fusion protein including a portion of the MOZ protein, e.g., the MOZ finger motifs and putative acetyltransferase domain, and at least the KIX domain of CBP. See, e.g., Giles et al. (1997) Leukemia 11 :2087; and Chaffanet et al. (1999) Genes Chromosomes Cancer 26:161.

The subject compositions may be used alone, or as part of a conjoint therapy with other chemotherapeutic compounds. The KLX therapeutics for use in the subject method may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the KIX therapeutics, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Pharmaceutical formulations of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of the KIX therapeutics suitable for veterinary uses, e.g., for the treatment of live stock or domestic animals, e.g., dogs.

Other formulations of the present invention include agricultural formulations, e.g., for application to plants. Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a KIX therapeutic at a particular target site.

The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, controlled release patch, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral and topical administrations are prefeπed.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracistemally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular KIX therapeutic employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

The term "treatment" is intended to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.

G. Application to other domain-specific transcription activators

Still another aspect of the present invention relates to a generalized approach for identifying minimal domain-specific transcriptional activation sequences. The results of the present studies with KIX binding domains demonstrate that molecular diversity approaches can be used to discover minimal, co-activator domain specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment.

In general, the present invention provides a method for identify optimal activator sequences by employing a partially or completely degenerate peptide display library with peptides of, e.g., 3-20 residues in length, more preferably 4-10, and even more preferably 4-8. The peptides can be displayed in virtually any format, e.g., as encoded combinatorial libraries or as part of genetic packages. In certain embodiments, the peptides are provided as part of a fusion protein.

To further illustrate, a library of test peptides is expressed by a population of display packages to form a peptide display library. With respect to the display package on which the variegated peptide library is manifest, it will be appreciated from the discussion provided herein that the display package will preferably be able to be (i) genetically altered to encode heterologous peptide, (ii) maintained and amplified in culture, (iii) manipulated to display the peptide-containing gene product in a manner permitting the peptide to interact with a target during an affinity separation step, and (iv) affinity separated while retaining the nucleotide sequence encoding the test peptide (herein "peptide gene") such that the sequence of the peptide gene can be obtained. In prefeπed embodiments, the display remains viable after affinity separation.

Ideally, the display package comprises a system that allows the sampling of very large variegated peptide display libraries, rapid sorting after each affinity separation round, and easy isolation of the peptide gene from purified display packages or further manipulation of that sequence in the secretion mode. The most attractive candidates for this type of screening are prokaryotic organisms and viruses, as they can be amplified quickly, they are relatively easy to manipulate, and large number of clones can be created. Prefeπed display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses). However, the present invention also contemplates the use of eukaryotic cells, including yeast and their spores, as potential display packages.

In addition to commercially available kits for generating phage display libraries (e.g. the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating the variegated peptide display library of the present invention can be found in, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. international Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication WO 92/20791; the Markland et al. international Publication No. WO 92/15679; the Breitling et al. international Publication WO 93/01288; the McCafferty et al. International Publication No. WO 92/01047; the Gaπard et al. International Publication No. WO 92/09690; the Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) JMol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Gaπad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133- 4137; and Barbas et al. (1991) PNAS 88:7978-7982. These systems can, with modifications described herein, be adapted for use in the subject method. When the display is based on a bacterial cell, or a phage which is assembled periplasmically, the display means of the package will comprise at least two components. The first component is a secretion signal which directs the recombinant peptide to be localized on the extracellular side of the cell membrane (of the host cell when the display package is a phage). This secretion signal can be selected so as to be cleaved off by a signal peptidase to yield a processed, "mature" peptide. The second component is a display anchor protein which directs the display package to associate the test peptide with its outer surface. As described below, this anchor protein can be derived from a surface or coat protein native to the genetic package.

When the display package is a bacterial spore, or a phage whose protein coating is assembled intracellularly, a secretion signal directing the peptide to the inner membrane of the host cell is unnecessary. In these cases, the means for aπaying the variegated peptide library comprises a derivative of a spore or phage coat protein amenable for use as a fusion protein.

In some instances it may be necessary to introduce an unstructured polypeptide linker region between portions of the chimeric protein, e.g., between the test peptide and display polypeptide. This linker can facilitate enhanced flexibility of the chimeric protein allowing the test peptide to freely interact with a target by reducing steric hindrance between the two fragments, as well as allowing appropriate folding of each portion to occur. The linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein. Alternatively, the linker can be of synthetic origin. For instance, the sequence (Gly4Ser)3 can be used as a synthetic unstructured linker. Linkers of this type are described in Huston et al. (1988) PNAS 85:4879; and U.S. Patent Nos. 5,091,513 and 5,258,498. Naturally occurring unstructured linkers of human origin are prefeπed as they reduce the risk of immunogenicity.

In the instance wherein the display package is a phage, the cloning site for the test peptide gene sequences in the phagemid should be placed so that it does not substantially interfere with normal phage function. One such locus is the intergenic region as described by Zinder and Boeke, (1982) Gene 19:1-10.

The number of possible combinations in a peptide library can get large as the length is increased and selection criteria for degenerating at each position is relaxed. To sample as many combinations as possible depends, in part, on the ability to recover large numbers of transformants. For phage with plasmid-like forms (as filamentous phage), electrotransformation provides an efficiency comparable to that of phage-transfection with in vitro packaging, in addition to a very high capacity for DNA input. This allows large amounts of vector DNA to be used to obtain very large numbers of transformants. The method described by Dower et al. (1988) Nucleic Acids Res., 16:6127-6145, for example, may be used to transform fd-tet derived recombinants at the rate of about 10⁷ transformants/ug of ligated vector into E. coli (such as strain MCI 061), and libraries may be constructed in fd-tet Bl of up to about 3 x 10⁸ members or more, increasing DNA input and making modifications to the cloning protocol within the ability of the skilled artisan may produce increases of greater than about 10- fold in the recovery of transformants, providing libraries of up to 10¹⁰ or more recombinants.

As will be apparent to those skilled in the art, in embodiments wherein high affinity peptides are sought, an important criteria for the present selection method can be that it is able to discriminate between peptides of different affinity for a particular target, and preferentially enrich for the peptides of highest affinity. Applying the well known principles of peptide affinity and valence (i.e. avidity), it is understood that manipulating the display package to be rendered effectively monovalent can allow affinity enrichment to be carried out for generally higher binding affinities (i.e. binding constants in the range of 10⁶ to 10¹⁰ M^_1) as compared to the broader range of affinities isolable using a multivalent display package. To generate the monovalent display, the natural (i.e. wild- type) form of the surface or coat protein used to anchor the peptide to the display can be added at a high enough level that it almost entirely eliminates inclusion of the peptide fusion protein in the display package. Thus, a vast majority of the display packages can be generated to include no more than one copy of the peptide fusion protein (see, for example, Gaπad et al. (1991) Bio/Technology 9:1373-1377). In a prefeπed embodiment of a monovalent display library, the library of display packages will comprise no more than 5 to 10% polyvalent displays, and more preferably no more than 2% of the display will be polyvalent , and most preferably, no more than 1% polyvalent display packages in the population. The source of the wild-type anchor protein can be, for example, provided by a copy of the wild-type gene present on the same construct as the peptide fusion protein, or provided by a separate construct altogether. However, it will be equally clear that by similar manipulation, polyvalent displays can be generated to isolate a broader range of binding affinities. Such peptides can be useful, for example, in purification protocols where avidity can be desirable.

i) Phages As Display Packages Bacteriophage are attractive prokaryotic-related organisms for use in the subject method. Bacteriophage are excellent candidates for providing a display system of the variegated peptide library as there is little or no enzymatic activity associated with intact mature phage, and because their genes are inactive outside a bacterial host, rendering the mature phage particles metabolically inert. In general, the phage surface is a relatively simple structure. Phage can be grown easily in large numbers, they are amenable to the practical handling involved in many potential mass screening programs, and they carry genetic information for their own synthesis within a small, simple package. As the peptide gene is inserted into the phage genome, choosing the appropriate phage to be employed in the subject method will generally depend most on whether (i) the genome of the phage allows introduction of the peptide gene either by tolerating additional genetic material or by having replaceable genetic material; (ii) the virion is capable of packaging the genome after accepting the insertion or substitution of genetic material; and (iii) the display of the peptide on the phage surface does not disrupt virion structure sufficiently to interfere with phage propagation. One concern presented with the use of phage is that the moφhogenetic pathway of the phage determines the environment in which the peptide will have opportunity to fold. Periplasmically assembled phage are prefeπed as the displayed peptides may contain essential disulfides, and such peptides may not fold coπectly within a cell. However, in certain embodiments in which the display package forms intracellularly (e.g., where λ phage are used), it has been demonstrated in other instances that disulfide- containing peptides can assume proper folding after the phage is released from the cell. Another concern related to the use of phage, but also pertinent to the use of bacterial cells and spores as well, is that multiple infections could generate hybrid displays that carry the gene for one particular test peptide yet have two or more different test peptides on their surfaces. Therefore, it can be preferable, though optional, to mini- mize this possibility by infecting cells with phage under conditions resulting in a low multiple-infection.

For a given bacteriophage, the prefeπed display means is a protein that is present on the phage surface (e.g. a coat protein). Filamentous phage can be described by a helical lattice; isometric phage, by an icosahedral lattice. Each monomer of each major coat protein sits on a lattice point and makes defined interactions with each of its neighbors. Proteins that fit into the lattice by making some, but not all, of the normal lattice contacts are likely to destabilize the virion by aborting formation of the virion as well as by leaving gaps in the virion so that the nucleic acid is not protected. Thus in bacteriophage, unlike the cases of bacteria and spores, it is generally important to retain in the peptide fusion proteins those residues of the coat protein that interact with other proteins in the virion. For example, when using the Ml 3 cpVHI protein, the entire mature protein will generally be retained with the peptide fragment being added to the N-terminus of cpVLfl, while on the other hand it can suffice to retain only the last 100 carboxy terminal residues (or even fewer) of the M13 cpHI coat protein in the peptide fusion protein.

Under the appropriate induction, the test peptide library is expressed and exported, as part of the fusion protein, to the bacterial cytoplasm, such as when the λ phage is employed. The induction of the fusion protein(s) may be delayed until some replication of the phage genome, synthesis of some of the phage structural-proteins, and assembly of some phage particles has occuπed. The assembled protein chains then interact with the phage particles via the binding of the anchor protein on the outer surface of the phage particle. The cells are lysed and the phage bearing the library- encoded test peptide (that coπesponds to the specific library sequences carried in the DNA of that phage) are released and isolated from the bacterial debris. To enrich for and isolate phage which encodes a selected test peptide, and thus to ultimately isolate the nucleic acid sequences (the peptide gene) themselves, phage harvested from the bacterial debris are affinity purified. As described below, when a test peptide which specifically binds a particular target is desired, the target can be used to retrieve phage displaying the desired test peptide. The phage so obtained may then be amplified by infecting into host cells. Additional rounds of affinity enrichment followed by amplification may be employed until the desired level of enrichment is reached.

The enriched peptide-phage can also be screened with additional detection- techniques such as expression plaque (or colony) lift (see, e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeled target is used as a probe.

a) Filamentous Phage Filamentous bacteriophages, which include Ml 3, fl, fd, Ifl, D e, Xf, Pfl, and PB, are a group of related viruses that infect bacteria. They are termed filamentous because they are long, thin particles comprised of an elongated capsule that envelopes the deoxyribonucleic acid (DNA) that forms the bacteriophage genome. The F pili filamentous bacteriophage (Ff phage) infect only gram-negative bacteria by specifically adsorbing to the tip of F pili, and include fd, fl and Ml 3.

Compared to other bacteriophage, filamentous phage in general are attractive and Ml 3 in particular is especially attractive because: (i) the 3-D structure of the virion is known; (ii) the processing of the coat protein is well understood; (iii) the genome is expandable; (iv) the genome is small; (v) the sequence of the genome is known; (vi) the virion is physically resistant to shear, heat, cold, urea, guanidinium chloride, low pH, and high salt; (vii) the phage is a sequencing vector so that sequencing is especially easy; (viii) antibiotic-resistance genes have been cloned into the genome with predictable results (Hines et al. (1980) Gene 11 :207-218); (ix) it is easily cultured and stored, with no unusual or expensive media requirements for the infected cells, (x) it has a high burst size, each infected cell yielding 100 to 1000 M13 progeny after infection; and (xi) it is easily harvested and concentrated (Salivar et al. (1964) Virology 24: 359-371). The entire life cycle of the filamentous phage Ml 3, a common cloning and sequencing vector, is well understood. The genetic structure of M13 is well known, including the complete sequence (Schaller et al. in The Single-Stranded DNA Phages eds. Denhardt et al. (NY: CSHL Press, 1978)), the identity and function of the ten genes, and the order of transcription and location of the promoters, as well as the physical structure of the virion (Smith et al. (1985) Science 228:1315-1317; Raschad et al. (1986) Microbiol Dev 50:401-427; Kuhn et al. (1987) Science 238:1413-1415; Zimmerman et al. (1982) JBiol Chem 257:6529-6536; and Banner et al. (1981) Nature 289:814-816). Because the genome is small (6423 bp), cassette mutagenesis is practical on RF M13 {Current Protocols in Molecular Biology, eds. Ausubel et al. (NY: John Wiley & Sons, 1991)), as is single-stranded oligonucleotide directed mutagenesis (Fritz et al. in DNA Cloning, ed by Glover (Oxford, UK: IRC Press, 1985)). Ml 3 is a plasmid and transformation system in itself, and an ideal sequencing vector. Ml 3 can be grown on Rec- strains of E. coli. The M13 genome is expandable (Messing et al. in The Single-Stranded DNA Phages, eds Denhardt et al. (NY: CSHL Press, 1978) pages 449-453; and Fritz et al., supra) and Ml 3 does not lyse cells. Extra genes can be inserted into Ml 3 and will be maintained in the viral genome in a stable manner.

The mature capsule or Ff phage is comprised of a coat of five phage-encoded gene products: cpVLfl, the major coat protein product of gene VHI that forms the bulk of the capsule; and four minor coat proteins, cpHI and cpPV at one end of the capsule and cpVH and cpIX at the other end of the capsule. The length of the capsule is formed by 2500 to 3000 copies of cpVHI in an ordered helix aπay that forms the characteristic filament structure. The gene IH-encoded protein (cpIH) is typically present in 4 to 6 copies at one end of the capsule and serves as the receptor for binding of the phage to its bacterial host in the initial phase of infection. For detailed reviews of Ff phage structure, see Rasched et al., Microbiol. Rev., 50:401-427 (1986); and Model et al.,in The Bacteriophages, Volume 2, R. Calendar, Ed., Plenum Press, pp. 375-456 (1988).

The phage particle assembly involves extrusion of the viral genome through the host cell's membrane. Prior to extrusion, the major coat protein cpVLH and the minor coat protein cpIH are synthesized and transported to the host cell's membrane. Both cpVHI and cpIH are anchored in the host cell membrane prior to their incoφoration into the mature particle. In addition, the viral genome is produced and coated with cpV protein. During the extrusion process, cpV-coated genomic DNA is stripped of the cpV coat and simultaneously recoated with the mature coat proteins.

Both cpIH and cpVHI proteins include two domains that provide signals for assembly of the mature phage particle. The first domain is a secretion signal that directs the newly synthesized protein to the host cell membrane. The secretion signal is located at the amino terminus of the polypeptide and targets the polypeptide at least to the cell membrane. The second domain is a membrane anchor domain that provides signals for association with the host cell membrane and for association with the phage particle during assembly. This second signal for both cpVHI and cpFH comprises at least a hydrophobic region for spanning the membrane.

The 50 amino acid mature gene VHI coat protein (cpVIH) is synthesized as a 73 amino acid precoat (Ito et al. (1979) PNAS 76:1199-1203). cpVHI has been extensively studied as a model membrane protein because it can integrate into lipid bilayers such as the cell membrane in an asymmetric orientation with the acidic amino terminus toward the outside and the basic carboxy terminus toward the inside of the membrane. The first 23 amino acids constitute a typical signal-sequence which causes the nascent polypeptide to be inserted into the inner cell membrane. An E. coli signal peptidase (SP-I) recognizes amino acids 18, 21, and 23, and, to a lesser extent, residue 22, and cuts between residues 23 and 24 of the precoat (Kuhn et al. (1985) J. Biol. Chem. 260:15914- 15918; and Kuhn et al. (1985) J. Biol. Chem. 260:15907-15913). After removal of the signal sequence, the amino terminus of the mature coat is located on the periplasmic side of the inner membrane; the carboxy terminus is on the cytoplasmic side. About 3000 copies of the mature coat protein associate side-by-side in the inner membrane.

The sequence of gene VHI is known, and the amino acid sequence can be encoded on a synthetic gene. Mature gene VHI protein makes up the sheath around the circular ssDNA. The gene VHI protein can be a suitable anchor protein because its location and orientation in the virion are known (Banner et al. (1981) Nature 289:814- 816). Preferably, the peptide is attached to the amino terminus of the mature Ml 3 coat protein to generate the phage display library. As set out above, manipulation of the concentration of both the wild-type cpVHI and Ab/cpVHI fusion in an infected cell can be utilized to decrease the avidity of the display and thereby enhance the detection of high affinity peptides directed to the target(s).

Another vehicle for displaying the peptide is by expressing it as a domain of a chimeric gene containing part or all of gene HI, e.g., encoding cpHI. When monovalent displays are required, expressing the peptide as a fusion protein with cpIH can be a prefeπed embodiment, as manipulation of the ratio of wild-type cpIH to chimeric cpIH during formation of the phage particles can be readily controlled. This gene encodes one of the minor coat proteins of Ml 3. Genes VI, VH, and LX also encode minor coat proteins. Each of these minor proteins is present in about 5 copies per virion and is related to moφhogenesis or infection. In contrast, the major coat protein is present in more than 2500 copies per virion. The gene VI, VH, and IX proteins are present at the ends of the virion; these three proteins are not posttranslationally processed (Rasched et al. (1986) Ann Rev. Microbiol. 41 :507-541). In particular, the single-stranded circular phage DNA associates with about five copies of the gene HI protein and is then extruded through the patch of membrane-associated coat protein in such a way that the DNA is encased in a helical sheath of protein (Webster et al. in The Single-Stranded DNA Phages, eds Dressier et al. (NY:CSHL Press, 1978).

Manipulation of the sequence of cpHI has demonstrated that the C-terminal 23 amino acid residue stretch of hydrophobic amino acids normally responsible for a membrane anchor function can be altered in a variety of ways and retain the capacity to associate with membranes. Ff phage-based expression vectors were first described in which the cpIH amino acid residue sequence was modified by insertion of polypeptide "targets" (Parmely et al., Gene (1988) 73:305-318; and Cwirla et al., PNAS (1990) 87:6378-6382) or an amino acid residue sequence defining a single chain peptide domain (McCafferty et al., Science (1990) 348:552-554). It has been demonstrated that insertions into gene HI can result in the production of novel protein domains on the virion outer surface. (Smith (1985) Science 228:1315-1317; and de la Cruz et al. (1988) J. Biol. Chem. 263:4318-4322). The peptide gene may be fused to gene HI at the site used by Smith and by de la Cruz et al., at a codon coπesponding to another domain boundary or to a surface loop of the protein, or to the amino terminus of the mature protein.

Generally, the successful cloning strategy utilizing a phage coat protein, such as cpHI of filamentous phage fd, will provide expression of a peptide chain fused to the N- terminus of a coat protein (e.g., cpFH) and transport to the inner membrane of the host where the hydrophobic domain in the C-terminal region of the coat protein anchors the fusion protein in the membrane, with the N-terminus containing the peptide chain protruding into the periplasmic space. Similar constructions could be made with other filamentous phage. Pf3 is a well known filamentous phage that infects Pseudomonos aerugenosa cells that harbor an IncP-I plasmid. The entire genome has been sequenced ((Luiten et al. (1985) J. Virol. 56:268-276) and the genetic signals involved in replication and assembly are known (Luiten et al. (1987) DNA 6:129-137). The major coat protein of PF3 is unusual in having no signal peptide to direct its secretion. The sequence has charged residues ASP- 7, ARG-37, LYS-40, and PHE44 which is consistent with the amino terminus being exposed. Thus, to cause a peptide to appear on the surface of Pf3, a tripartite gene can be constructed which comprises a signal sequence known to cause secretion in P. aerugenosa, fused in-frame to a gene fragment encoding the peptide sequence, which is fused in-frame to DNA encoding the mature Pf3 coat protein. Optionally, DNA encoding a flexible linker of one to 10 amino acids is introduced between the peptide gene fragment and the PO coat-protein gene. This tripartite gene is introduced into Pf3 so that it does not interfere with expression of any Pf3 genes. Once the signal sequence is cleaved off, the peptide is in the periplasm and the mature coat protein acts as an anchor and phage-assembly signal.

b) Bacteriophage φXl 74

The bacteriophage φX174 is a very small icosahedral virus which has been thoroughly studied by genetics, biochemistry, and electron microscopy (see The Single Stranded DNA Phages (eds. Den hardt et al. (NY:CSHL Press, 1978)). Three gene products of φX174 are present on the outside of the mature virion: F (capsid), G (major spike protein, 60 copies per virion), and H (minor spike protein, 12 copies per virion). The G protein comprises 175 amino acids, while H comprises 328 amino acids. The F protein interacts with the single-stranded DNA of the virus. The proteins F, G, and H are translated from a single mRNA in the viral infected cells. As the virus is so tightly constrained because several of its genes overlap, φX174 is not typically used as a cloning vector due to the fact that it can accept very little additional DNA. However, mutations in the viral G gene (encoding the G protein) can be rescued by a copy of the wild-type G gene carried on a plasmid that is expressed in the same host cell (Chambers et al. (1982) Nuc Acid Res 10:6465-6473). In one embodiment, one or more stop codons are introduced into the G gene so that no G protein is produced from the viral genome. The variegated peptide gene library can then be fused with the nucleic acid sequence of the H gene. An amount of the viral G gene equal to the size of peptide gene fragment is eliminated from the φX174 genome, such that the size of the genome is ultimately unchanged. Thus, in host cells also transformed with a second plasmid expressing the wild-type G protein, the production of viral particles from the mutant virus is rescued by the exogenous G protein source. Where it is desirable that only one test peptide be displayed per φX174 particle, the second plasmid can further include one or more copies of the wild-type H protein gene so that a mix of H and test peptide/H proteins will be predominated by the wild-type H upon incoφoration into phage particles.

c) Large DNA Phage

Phage such as λ or T4 have much larger genomes than do Ml 3 or φX174, and have more complicated 3-D capsid structures than M13 or φPX174, with more coat proteins to choose from. In embodiments of the invention whereby the test peptide library is processed and assembled into a functional form and associates with the bacteriophage particles within the cytoplasm of the host cell, bacteriophage λ and derivatives thereof are examples of suitable vectors. The intracellular moφhogenesis of phage λ can potentially prevent protein domains that ordinarily contain disulfide bonds from folding coπectly. However, variegated libraries expressing a population of functional peptides, which include such bonds, have been generated in λ phage. (Huse et al. (1989) Science 246:1275-1281; Mullinax et al. (1990) PNAS 87:8095-8099; and Pearson et al. (1991) PNAS 88:2432-2436). Such strategies take advantage of the rapid construction and efficient transformation abilities of λ phage.

When used for expression of peptide sequences (ixogenous nucleotide sequences), may be readily inserted into a λ vector. For instance, variegated peptide libraries can be constructed by modification of λ ZAP H through use of the multiple cloning site of a λ ZAP H vector (Huse et al. supra).

ii) Bacterial Cells as Display Packages Recombinant peptides are able to cross bacterial membranes after the addition of appropriate secretion signal sequences to the N-terminus of the protein (Better et al (1988) Science 240:1041-1043; and Skeπa et al. (1988) Science 240:1038-1041). In addition, recombinant peptides have been fused to outer membrane proteins for surface presentation. For example, one strategy for displaying peptides on bacterial cells comprises generating a fusion protein by inserting the peptide into cell surface exposed portions of an integral outer membrane protein (Fuchs et al. (1991) Bio/Technology 9:1370-1372). In selecting a bacterial cell to serve as the display package, any well-characterized bacterial strain will typically be suitable, provided the bacteria may be grown in culture, engineered to display the test peptide library on its surface, and is compatible with the particular affinity selection process practiced in the subject method. Among bacterial cells, the prefeπed display systems include Salmonella typhirnurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli. Many bacterial cell surface proteins useful in the present invention have been characterized, and works on the localization of these proteins and the methods of determining their structure include Benz et al. (1988) Ann Rev Microbiol 42: 359-393; Balduyck et al. (1985) Biol Chem Hoppe-Seyler 366:9-14; Ehrmann et al (1990) PNAS 81:1514-151%; Heijne et al. (1990) Protein Engineering 4:109-112; Ladner et al. U.S. Patent No. 5,223,409; Ladner et al. WO88/06630; Fuchs et al. (1991) Bio/technology 9:1370-1372; and Goward et al. (1992) TIBS 18:136-140. To further illustrate, the LamB protein of E coli is a well understood surface protein that can be used to generate a variegated library of test peptides on the surface of a bacterial cell (see, for example, Ronco et al. (1990) Biochemie 72:183-189; van der Weit et al. (1990) Vaccine 8:269-277; Charabit et al. (1988) Gene 70:181-189; and Ladner U.S. Patent No. 5,222,409). LamB of E. coli is a porin for maltose and maltodex- trin transport, and serves as the receptor for adsoφtion of bacteriophages λ and K10. LamB is transported to the outer membrane if a functional N-terminal signal sequence is present (Benson et al. (1984) PNAS 81:3830-3834). As with other cell surface proteins, LamB is synthesized with a typical signal-sequence which is subsequently removed. Thus, the variegated peptide gene library can be cloned into the LamB gene such that the resulting library of fusion proteins comprise a portion of LamB sufficient to anchor the protein to the cell membrane with the test peptide fragment oriented on the extracellular side of the membrane. Secretion of the extracellular portion of the fusion protein can be facilitated by inclusion of the LamB signal sequence, or other suitable signal sequence, as the N-terminus of the protein. The E. coli LamB has also been expressed in functional form in S. typhirnurium

(Harkki et al. (1987) Mol Gen Genet 209:607-611), V. cholerae (Harkki et al. (1986) Microb Pathol 1 :283-288), and K pneumonia (Wehmeier et al. (1989) Mol Gen Genet 215:529-536), so that one could display a population of test peptides in any of these species as a fusion to E. coli LamB. Moreover, K. pneumonia expresses a maltoporin similar to LamB which could also be used. In P. aeruginosa, the Dl protein (a homologue of LamB) can be used (Trias et al. (1988) Biochem Biophys Acta 938:493- 496). Similarly, other bacterial surface proteins, such as PAL, OmpA, OmpC, OmpF, PhoE, pilin, BtuB, FepA, FhuA, IutA, FecA and FhuE, may be used in place of LamB as a portion of the display means in a bacterial cell.

In another exemplary embodiment, the fusion protein can be derived using the FliTrx™ Random Peptide Display Library (Invitrogen). That library is a diverse population of random dodecapeptides inserted within the thioredoxin active-site loop inside the dispensable region of the bacterial flagellin gene (fliC). The resultant recombinant fusion protein (FLITRX) is exported and assembled into partially functional flagella on the bacterial cell surface, displaying the random peptide library.

Peptides are fused in the middle of thioredoxin, therefore, both their N- and C- termini are anchored by thioredoxin's tertiary structure. This results in the display of a constrained peptide. By contrast, phage display proteins are fused to the N-terminus of phage coat proteins in an unconstrained manner. The unconstrained molecules possess many degrees of conformational freedom which may result in the lack of proper interaction with the target molecule. Without proper interaction, many potential protein- protein interactions may be missed.

Moreover, phage display is limited by the low expression levels of bacteriophage coat proteins. FliTrx™ and similar methods can overcome this limitation by using a strong promoter to drive expression of the test peptide fusions that are displayed as multiple copies. According to the present invention, it is contemplated that the FliTrx vector can be modified to provide, similar to the illustrated vectors of the attached figures, a vector which is differentially spliced in mammalian cells to yield a secreted, soluble test peptide.

iii) Bacterial Spores as Display Packages

Bacterial spores also have desirable properties as display package candidates in the subject method. For example, spores are much more resistant than vegetative bacterial cells or phage to chemical and physical agents, and hence permit the use of a great variety of affinity selection conditions. Also, Bacillus spores neither actively metabolize nor alter the proteins on their surface. However, spores have the disadvantage that the molecular mechanisms that trigger sporulation are less well worked out than is the formation of Ml 3 or the export of protein to the outer membrane of E. coli, though such a limitation is not a serious detractant from their use in the present invention.

Bacteria of the genus Bacillus form endospores that are extremely resistant to 5 damage by heat, radiation, desiccation, and toxic chemicals (reviewed by Losick et al. (1986) Ann Rev Genet 20:625-669). This phenomenon is attributed to extensive intermolecular cross-linking of the coat proteins. In certain embodiments of the subject method, such as those which include relatively harsh affinity separation steps, Bacillus spores can be the prefeπed display package. Endospores from the genus Bacillus are 10 more stable than are, for example, exospores from Streptomyces. Moreover, Bacillus subtilis forms spores in 4 to 6 hours, whereas Streptomyces species may require days or weeks to sporulate. In addition, genetic knowledge and manipulation is much more developed for B. subtilis than for other spore- forming bacteria.

Viable spores that differ only slightly from wild-type are produced in B. subtilis X 5 even if any one of four coat proteins is missing (Donovan et al. (1987) JMol Biol 196:1- 10). Moreover, plasmid DNA is commonly included in spores, and plasmid encoded proteins have been observed on the surface of Bacillus spores (Debro et al. (1986) J Bacteriol 165:258-268). Thus, it can be possible during sporulation to express a gene encoding a chimeric coat protein comprising a peptide of the variegated gene library, 0 without interfering materially with spore formation.

To illustrate, several polypeptide components of B. subtilis spore coat (Donovan et al. (1987) J Mol Biol 196:1-10) have been characterized. The sequences of two complete coat proteins and amino-terminal fragments of two others have been determined. Fusion of the test peptide sequence to cotC or cotD fragments is likely to 5 cause the peptide to appear on the spore surface. The genes of each of these spore coat proteins are prefeπed as neither cotC or cotD are post-translationally modified (see Ladner et al. U.S. Patent No. 5,223,409).

0 iv) Selecting Peptides from the display libraiy

Upon expression, the variegated peptide display is subjected to affinity enrichment in order to select for test peptides which bind preselected targets. The term "affinity separation" or "affinity enrichment" includes, but is not limited to: (1) affinity chromatography utilizing immobilized targets, (2) immunoprecipitation using soluble 5 targets, (3) fluorescence activated cell sorting, (4) agglutination, and (5) plaque lifts. In each embodiment, the library of display packages are ultimately separated based on the ability of the associated test peptide to bind the target of interest, e.g., a transcription factor of interest. See, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication WO 92/20791; the Markland et al. International Publication No. WO 92/15679; the Breitling et al. International Publication WO 93/01288; the McCafferty et al. international Publication No. WO 92/01047; the Gaπard et al. International Publication No. WO 92/09690; and the Ladner et al. International Publication No. WO 90/02809. In most prefeπed embodiments, the display library will be pre-enriched for peptides specific for the target by first contacting the display library with any negative controls or other targets for which differential binding by the test peptide is desired. Subsequently, the non-binding fraction from that pre-treatment step is contacted with the target and peptides from the display which are able to specifically bind the target are isolated.

With respect to affinity chromatography, it will be generally understood by those skilled in the art that a great number of chromatography techniques can be adapted for use in the present invention, ranging from column chromatography to batch elution, and including ELISA and biopanning techniques. Typically, where possible, the target transcription factor (or relevant domain thereof) is immobilized on an insoluble carrier, such as sepharose or polyacrylamide beads, or, alternatively, the wells of a microtitre plate. The target factor can be expressed as part of a fusion protein so as to include one or more domains which confer immobilization (e.g., GST fusions).

The population of display packages is applied to the affinity matrix under conditions compatible with the binding of the test peptide to a target polypeptide. The population is then fractionated by washing with a solute that does not greatly effect specific binding of peptides to the target, but which substantially disrupts any nonspecific binding of the display package to the target or matrix. A certain degree of control can be exerted over the binding characteristics of the peptides recovered from the display library by adjusting the conditions of the binding incubation and subsequent washing. The temperature, pH, ionic strength, divalent cation concentration, and the volume and duration of the washing can select for peptides within a particular range of affinity and specificity. Selection based on slow dissociation rate, which is usually predictive of high affinity, is a very practical route. This may be done either by continued incubation in the presence of a saturating amount of free target polypeptide, or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated pepti de-display package is prevented, and with increasing time, peptide- display packages of higher and higher affinity are recovered. Moreover, additional modifications of the binding and washing procedures may be applied to find peptides with special characteristics. The affinities of some peptides are dependent on ionic strength or cation concentration. This is a useful characteristic for peptides to be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptide are required. Specific examples are peptides which depend on Ca⁺⁺ for binding activity and which lose or gain binding affinity in the presence of EGTA or other metal chelating agent. Such peptides may be identified in the recombinant peptide library by a double screening technique isolating first those that bind the target in the presence of Ca⁺⁺, and by subsequently identifying those in this group that fail to bind in the presence of EGTA. After "washing" to remove non-specifically bound display packages, when desired, specifically bound display packages can be eluted by either specific desoφtion (using excess target) or non-specific desoφtion (using pH, polarity reducing agents, or chaotropic agents). In prefeπed embodiments, the elution protocol does not kill the organism used as the display package such that the enriched population of display packages can be further amplified by reproduction. The list of potential eluants includes salts (such as those in which one of the counter ions is Na⁺, NH4⁺, Rb⁺, SO4 -,

H₂PO₄-, citrate, K+ Li+, Cs⁺, HSO4-, CO₃ ²-, Ca +, Sr2+, CL, PO₄ ²-, HCO3-, Mg₂ ⁺,

Ba2⁺, Br, HPO4 -, or acetate), acid, heat, and, when available, soluble forms of the target (or analogs thereof). Because bacteria continue to metabolize during the affinity separation step and are generally more susceptible to damage by harsh conditions, the choice of buffer components (especially eluates) can be more restricted when the display package is a bacteria rather than for phage or spores. Neutral solutes, such as ethanol, acetone, ether, or urea, are examples of other agents useful for eluting the bound display packages. In prefeπed embodiments, affinity enriched display packages are iteratively amplified and subjected to further rounds of affinity separation until enrichment of the desired binding activity is detected. In certain embodiments, the specifically bound display packages, especially bacterial cells, need not be eluted per se, but rather, the matrix bound display packages can be used directly to inoculate a suitable growth media for amplification.

IV. Exemplification

Human gene therapy approaches involving transcription factors often rely on artificial activation domains for transcriptional activation. These domains are often large, e.g., eighty amino acids for VP16, recruit multiple co-activation complexes at once, and offer no fine control over the level of transcription. In an attempt to understand the sequence and structural requirements of a minimal mammalian activator, we employed a molecular diversity approach with a peptide phage display library comprised of random eight amino acid peptides. Using the KIX domain of the mammalian co-activators p300 and CBP as target, we discovered a family of synthetic binding peptides. These peptides share significant homology with natural KIX domain ligands, and are shown to bind an overlapping, yet distinct, surface of p300/CBP. When fused to a heterologous DNA binding domain, these synthetic peptides function as titratable, modular, and potent transcriptional activators in living cells through specific recruitment of p300/CBP, with the level of transcriptional activation proportional to the affinity of the synthetic peptide for the KIX domain.

A. Introduction

Human diseases such as cancer¹, ischemic heart disease², hypertension³, and autoimmune disorders⁴ are targets for transcription factor-mediated gene therapy (reviewed in Ref. 5). In most systems, conventional activation domains such as the VP16^6,7 are used to modulate cellular gene expression. Unfortunately, such activators are often large (80 amino acids for VP16) and provide only a digital signal for transactivation, that is, maximally on.

Activators are cuπently thought to consist of rather featureless peptide tracts of 40-80 amino acids that promote target gene expression via multiple low affinity contacts with proteins in the transcriptional apparatus⁸. These peptide tracts are notable only for the prevalence of certain amino acids such as glutamine, proline, and acidic residues. VP16, for example, is an acidic activator that has been shown to form multiple low- affinity contacts with co-activator complexes⁹, potentially bypassing higher levels of control that may be present in the natural signal transduction system.

Little is known about what constitutes the sequence and structural requirements of a minimal mammalian activation domain. Recent analysis of some mammalian transactivator/co-activator complexes suggests that specific sequences and structures are required for gene activation. The cAMP responsive factor CREB has been shown to promote cellular gene expression following its PKA mediated phosphorylation at Serl33 via recruitment of the co-activator paralogs p300 and CBP. NMR studies on the CREB: CBP complex, using relevant interaction domains refeπed to as KID and KLX, respectively, reveal that the KID domain undergoes a random coil to helix transition upon binding to KIX¹⁰. The transition in KID, in turn, stabilizes complex formation via hydrophobic interactions with a shallow groove in KIX¹¹. In addition to its interaction with phospo-CREB, the KIX domain also integrates signals from a variety of other molecules including c-Myb, c-Jun, Statlα, the SREBPs, and the HTLV-1 Tax protein. Sequence alignments of these binding partners suggest a potential consensus motif (φ X X φ φ X X φ; where φ is a hydrophobic residue) for KIX binding. Structurally, the isolated KIX domain is composed of three α-helices, two of which are parallel and form a hydrophobic groove¹⁰. Since the BOX domain of p300/CBP has a potential consensus sequence for binding and a well-formed structure in solution, we hypothesized that it might be an ideal target for selecting synthetic binding peptides from a molecular diversity library. Peptide phage display has been used previously to discover modulators of transcription such as nuclear hormone receptor binding peptides¹²'¹³ and optimal zinc finger dimerization domains¹⁴. Synthetic KIX domain binding peptides would be a unique tool to study domain-specific co-activator recruitment in isolation from the complex interactions of p300/CBP with natural binding proteins, and would potentially yield synthetic transcriptional activators and/or repressors for use in gene therapy applications.

B. Results

In Vitro Selection of Optimal KIX Domain Binding Peptides by Phage Display

To identify optimal KIX binding peptides, we employed a completely degenerate peptide phage display library with peptides of eight amino acids in length. Following one round of screening (see Experimental Protocol), greater than 95% of phage clones were specific for the KLX domain. After ranking over 100 clones by apparent affinity, the ten best binders were sequenced (Table 1). A weak consensus sequence emerged with acidic residues (aspartate or glutamate) suπounded by aliphatic/aromatic residues in the -1 and +1 o+3 positions. The optimal first round KIX domain binding peptide (KBP) 1.66 had a modest affinity (55.1 ± 1.1 μM) for the KLX domain when measured by fluorescence anisotropy (Table 1).

To further optimize KIX binding affinity, we constructed a partially degenerate eight amino acid library based on the first round consensus sequence (Table 1 and Experimental Protocol). Following a second round of screening, a strong selection was observed, even in positions that were completely degenerate in the starting library. Of ten clones with the highest affinity, one eight amino acid sequence was selected five times, and another was selected three times.

Sequence and Structural Similarities of Synthetic and Natural p300/CBP Binding

Peptides The optimal second round eight amino synthetic peptide KBP 2.20 has an affinity (16.1 ± 1.1 μM; Table 1) comparable to that of the twenty six amino acid minimal KIX binding domain of c-Myb (2.0 ± 0.3 μM; Ref. 15). KBP 2.20 also shares significant homology with c-Myb, and other natural KIX domain ligands including phospho-CREB, SREBP la, SREBP2 and Drosophila cubitus interruptus {Ci; Fig. IA). The homologous peptide sequences from these molecules require an α-helical conformation for binding to the KIX domain, and the sequence from SREBP la has been shown recently to participate in p300/CBP recruitment¹⁶. In the case of c-Myb, helicity, and hence KIX domain binding, is constitutive¹⁵, whereas with CREB, the helical conformation is dependent on phosphorylation of Serl33¹⁰. When projected as an α-helix, KBP 2.20 has hydrophobic residues positioned in an identical manner as the homologous α-helices of phospho-CREB and c-Myb (Fig. IB). This might explain why the first round synthetic peptides, most of which lack a comparable hydrophobic residue at position 1, have lower binding affinities and decreased biologic activity (see below). Indeed, molecular modeling of KBP 2.20 as α-helical results in a range of energies, the lowest of which positions the hydrophobic residues along a very naπow ridge (Fig. IB and 1C), analogous to the known structures of phospho-KLD and c-Myb.

Phage Display-Derived Peptides Bind an Overlapping, Yet Distinct Surface of P300/CBP as Compared to Natural KLX Domain Ligands

To determine whether synthetic KBPs recognize a comparable binding surface on KIX relative to natural ligands, we performed binding assays with a number of mutant KIX polypeptides (Fig. 2A). Residues lining the shallow hydrophobic groove in KLX (Leu599, Leu603, Lys606, Tyr650, Leu652, Leu653, Ile657, and Gln661; Fig. 2B) were critical for complex formation with KBP; mutagenesis of these residues strongly inhibited complex formation in vitro. The importance of Lys606 in KIX for complex formation may also reflect formation of a salt bridge with the selected acidic residue (Glu5/Asp5) of the synthetic KBPs. Taken together, these data support the notion that synthetic KBPs bind to an overlapping, but distinct and minimal, surface of KLX as compared to phospho-CREB¹⁰ and c-Myb¹⁵. Specifically, KBP 2.20 appears to penetrate deeper into the hydrophobic groove, as evidenced by critical binding to Tyr650 (see Fig. 2 A and Ref. 10).

Overlap of natural and synthetic KLX domain ligand binding surfaces was confirmed in two separate experiments. Firstly, a phospho-KLD peptide was able to effectively compete for KBP 2.20 binding to the KIX domain, whereas the unphosphorylated peptide was not (Fig. 3A). Secondly, the KBPs could act as repressors of c-Myb-dependent transcriptional activation, with the degree of repression coπelating with KIX domain binding affinity (Fig. 3B; see also below). The ability of KBP 2.20 to block c-Myb activity in transfection assays suggests that it binds directly to cellular p300/CBP protein, and can act as a transcriptional repressor under some circumstances.

Synthetic KIX Domain Binding Peptides Precipitate Full-Length p300/CBP from a Crude Lysate

To complement data obtained with the isolated KIX domain, we attempted to precipitate full-length p300/CBP from a whole cell lysate using just the synthetic peptides coupled to agarose. As shown in Fig. 3C, CBP was easily precipitated from a 293 cell lysate by both KBP 1.66 and KBP 2.20, with the amount precipitated coπelating with the KLX domain affinity measured in vitro. Of note, an extremely hydrophobic control peptide, not fitting the consensus sequence shown in Table 1, was not able to precipitate CBP (Fig. 3C).

Synthetic KIX Domain Binding Peptides are Potent Transcriptional Activators in

Living Cells, with the Level of Transcriptional Activation Proportional to Peptide Affinity

The ability of synthetic KBPs to bind full length p300/CBP prompted us to test whether these octapeptides can function as p300/CBP-dependent activators in living cells. When fused to the GAL4 DNA binding domain (GAL4 DBD), the optimal first round peptide KBP 1.66 (K_<j 55.1 ± 1.1 μM) stimulated target gene expression approximately 10-fold (Fig. 4A). The optimal second round peptide KBP 2.20 (K 16.1 ± 1.1 μM) induced reporter activity approximately 40-fold over GAL4 alone (Fig. 4A).

The Potency of Optimal KIX Domain Binding Peptides is Similar to Natural

Activators

Activation potency of KBPs was compared directly to several natural activators who themselves either require (c-Myb, c-Fos) or not require (Spl) p300/CBP for co- activation (Fig. 4A). As is shown, the minimal activation domain of c-Myb (amino acids 290-315) was a relatively weak activator, with potency approximately 3-fold less than the first round phage display-derived peptide KBP 1.66, and 10-fold less than the optimal second round peptide KBP 2.20. Full-length Spl and full-length c-Fos were only approximately 2- to 2.5-fold more potent than the 8 amino acid KBP 2.20 peptide. The activation domains of PDX (amino acids 1-138 Ref. 17) and VP16 (80 amino acids) were approximately 5- and 10-fold more potent than KBP 2.20, respectively (data not shown).

KBP-Mediated Transcriptional Activation is Dependent on p300/CBP The El A oncoprotein has been shown to block expression of certain genes in part by sequestering cellular p300/CBP activity. To test whether GAL4-KBP promoted target gene expression via a p300/CBP-dependent mechanism, we performed transfection assays with wild type (E1A) and p300 interaction-defective (ElAΔ) E1A expression vectors (Fig. 4B). Reporter induction by the GAL4 KBP 2.20 fusion protein was inhibited in a dose-dependent fashion by El A. Expression of ElAΔ had no significant effect on reporter activity until a 5-fold excess was used, and at this level, wild type E1A inhibited KBP 2.20-induced activation nearly completely. These data suggest that GAL4 KBP stimulates transcription via a p300/CBP-dependent mechanism. Indeed, over-expression of p300 in 293 cells potentiated both GAL4/KBP 1.66 and GAL4-KBP 2.20 activity (Fig. 4C).

C. Discussion

Our data suggest that it is possible to develop synthetic co-activator domain- specific binding peptides, without any assumptions about target or product, by using a molecular diversity approach. In doing so, we appear to have found the shortest modular peptide activators described to date in mammalian cells. Our data also support the conclusion of a recent report¹⁶ that p300/CBP recruitment by CREB (or in our case a synthetic peptide) is sufficient for transcriptional activation in mammalian cells. Hence, p300/CBP is basally in a transcriptionally competent complex containing RNA polymerase H.

Importantly, our peptides appear to function when fused as a single copy to a heterologous DNA binding domain. This is in contrast to the VP16-derived minimal aspartate/leucine-containing peptide sequence DDFDL described previously¹⁸, which has essentially no activity until multimerized as four tandem copies. By using a specific co-activator as the target for phage display screening, we have also shown that peptide binding affinity coπelates with the potency of transcriptional activation, hence, binding affinity measured in vitro can be used as a suπogate for transcriptional activation in living cells.

The technology we describe herein effectively converts a function normally attributed to entire proteins, or protein domains, to the level of peptide motifs (see Ref. 19 for a discussion). Since the synthetic peptides, derived from complete degeneracy and selected in vitro, share such striking homology to natural ligands, we propose that our data define a minimal consensus sequence (eight amino acids; Table 1, bottom) and structure (α-helical; Figs. IB, C) for p300/CBP KIX domain binding. The synthetic peptides differ from the natural ligands in that the latter require additional flanking amino acids for improved affinity, and hence, biologic potency. It is likely that our molecular diversity approach can be applied to other components of the transcriptional machinery since short α-helical peptide motifs have been shown previously to mediate important interactions of transcπption ' . One of the inherent advantages to phage display screening is that peptides are selected in the context of being tethered to a large, heterologous structure (i.e., the pIH coat protein and bacteriophage Ml 3 itself). Although non-peptide structures can certainly contribute to overall peptide affinity²², phage display-derived peptides tend to function well out of their original context. In this report, we demonstrate that KBPs are truly modular in that they function at the N terminus of pIH (Table 1), the C terminus of secreted alkaline phosphatase (Fig. 2A), the C terminus of GST (Fig. 3A), the C terminus of GAL4 DBD (Figs. 3B, 4A-C), and when coupled covalently to agarose (Fig. 3C). An inherent drawback to short peptides selected by phage display screening, however, is that they are often extremely hydrophobic owing to the limited number of contacts achievable with the target. This is highlighted by the fact that two tandem copies of KBP 2.20 fused to the C terminus of the GAL4 DBD had no bioactivity (data not shown), presumably due to aggregation.

Their inherent modularity and titratability make these peptides attractive reagents for human gene therapy. By fusing a particular synthetic peptide family member to a transcription factor of interest, the level of transcription can be fine-tuned with minimal energetic cost. Of course, this strategy will only work in systems where p300/CBP is not already part of the transcriptional complex, or is not already recruited maximally. The coπelation of KIX domain binding affinity with transactivation potency makes it possible, for the first time, to control the level of gene activation simply by choosing among synthetic peptides with different co-activator binding affinities. Also, since specific co-activators can now be recruited in living cells, one can match the transcriptional activation sequence with the physiological co-activator such that the cell can assert higher order control over the transcriptional event.

Although much effort has focused on the genetic expression of transcription factors in human disease, our report makes it possible to speculate about entirely synthetic, minimally sized transcription factors that can be administered as drugs to affect gene expression in a dose-dependent fashion (see also Refs. 23,24). Such synthetic transcription factors must have a DNA binding domain, a transactivation sequence, and the ability to freely accumulate intracellularly. We describe minimal mammalian transactivation sequences of 8 amino acids in length. Recent reports on the HIV TAT peptide suggest that only 11 additional amino acids are required for transduction of even large molecules intracellularly in a living animal²⁵'²⁶. Hence, with only 19 amino acids, two of the three essential functions of a synthetic transcription factor have been realized. When considered with previously published reports that non- hydrolyzable D-amino acids can substitute for L-amino acids in certain activation domains²³ , and that sequence-specific DNA binding polyamides²⁴ can substitute for DNA binding domains, the realization of minimally sized synthetic transcription factors may be in reach.

D. Experimental Protocol

Peptide Phage Library Construction. The eight amino acid degenerate library was constructed essentially as described previously²⁷ using a novel peptide phage display vector mJi. mJi was constructed by inserting the Amp^R gene into the EcoRI site of mBAX²⁷, a derivative of M13mpl8. Using the polymerase chain reaction, the pLH signal sequence cleavage site and N- terminus of pLfl was replaced with a new cloning site composed of a Xhol site and BamHI site flanking an amber stop codon. Recombinants have a single serine residue fixed at the N terminus of the mature plfl protein to ensure minimal bias during signal sequence cleavage, and a (Gly-Ser) spacer between the peptide and full-length pIH protein. The complexity of the library was 1.8 x 10⁸ and the titer was 2 x 10¹³ PFU per ml. The partially degenerate library constructed for second round optimization used the codon scheme (G/T)(A G/T)(G/T) for biasing towards hydrophobic amino acids. This library had a complexity of 2.5 x 10⁷ and the titer was 5 x 10¹³ PFU per ml.

Differential Peptide Phage Display Selection.

Initially, PFUs containing 2000X the library complexity (3.6 x 10¹¹ PFU for Round 1, 5 x 10¹⁰ for Round 2) were applied to target protein in a total volume of 200 μL PBS + 0.1% Tween-20. Each round of phage library screening was composed of 3 cycles of affinity purification using GST/KIX 10-672 (100 μg cycle 1, 50 μg cycles 2,3) on glutathione agarose. GST-specific phage were removed by immunodepletion with 100 μg of agarose-bound GST after cycles 1 and 2 of affinity purification (differential phage display). ELISA Screening.

ELISA screening of plaque-purified phage, performed as described previously²⁷, used 100 ng of GST fusion proteins per well. After development with 2,2'-azino-bis (3- ethylbenzthiazoline 6-sulfonic acid) diammonium (ABTS; Sigma), results were quantitated at 405 nm using a Molecular Devices Emax microplate reader. Desired phage clones were grown as 5 ml overnight cultures in LB supplemented with 100 μg/ml ampicillin and RF (double stranded) DNA was purified using Qiagen miniprep spin columns. Automated DNA sequencing was performed on a Prism 377 sequencer (Applied Biotechnology).

Measurement of Apparent Peptide Affinities.

Apparent affinity is defined as the affinity of the KLX domain binding peptide when expressed as a pLH fusion on Ml 3 bacteriophage. The effective concentration of GST/KLX 10-672 in each ELISA well was estimated to be 12.2 nM by competition experiments using GST antibody and eluted GST (data not shown). The numbers of applied (free) and recovered (bound) phage particles from each well were counted by plate titering²⁷ and the apparent peptide affinity was calculated using the equilibrium binding equation. The obtained value was multiplied by five to approximate the contribution of five identical peptide/pIH fusions per phage particle.

Fluorescence Polarization.

The optimal first round (SSLVDLfLFGS) and second round (SWAVYELLFGS) KIX domain binding peptides were synthesized by standard Fmoc chemistry and purified by HPLC. The extreme hydrophobicity of the KIX binding peptides required development of a general puφose method for fluorescein labeling of hydrophobic peptides. The final fluorescent product was resuspended in polarization-grade DMSO (Pan Vera) and fluorescence polarization was used to measure the actual peptide affinity¹¹.

Bacterial Protein Expression.

GST/KLX 10-672, comprising amino acids 586-672 of mouse CBP, and mutations of the KIX domain have been described previously¹¹. Fusion of GST with KBP 2.20 (GST/KBP 2.20) was accomplished by kinasing, annealing, and ligating oligonucleotides coπesponding to the peptide sequence into pGEX-4T-3 (Pharmacia). GST proteins were purified as described previously²⁸.

Secreted Alkaline Phosphatase Fusion Proteins. Fusion of the KLX domain or KLX binding peptides to a secreted form of human placental alkaline phosphatase type 1 (sPLAP) was accomplished using a novel mammalian expression vector pAPi. pAPi was constructed by replacing the EGFP coding sequence of pEGFP-N3 (Clontech) with the coding sequence for sPLAP. Using the polyymerase chain reaction, a multiple cloning site was inserted at the C-terminus of GAL4 DBD, which resulted in the following spacing element between sPLAP and the peptide of interest: (Gly-Ser)₂-Ala-Val-Asp-Ala-Ser. pAPi fusion proteins were expressed in 293 -T cells and supernatants 66 hours post-transfection were adjusted to 10% glycerol and flash- frozen in LN₂ prior to use.

KIX Domain Mutation Analysis and BUD Peptide Competition.

4 μg of target protein on glutathione agarose was added to 100 μL of the appropriate sPLAP-containing cell supernatant. After one hour incubation at room temperature, agarose was washed three times with PBS + 0.1% Tween-20 and bound alkaline phosphatase activity was quantitated using the hydrolysis of p-nitrophenyl phosphate (pNPP; Sigma). For competition experiments, the KID-29 peptide (amino acids 119-147 of rat CREB) was phosphorylated on Serl33 (phospho-KLD-29) with protein kinase A¹⁰. Equimolar amounts of KID-29 or phospho-KLD-29 were added to the sPLAP-containing supernatants immediately before application to the agarose.

Cell Culture. Transfection, and Luciferase Assays.

293 cells (ATCC CRL-1573) grown in 24 well plates were transfected by the calcium phosphate method with 800 ng of total DNA including 400 ng of Firefly luciferase reporter plasmid, 20 ng of a promoterless Renilla luciferase plasmid for normalization and pBluescript SK+ carrier DNA. Reporter plasmids included 5X GAL4 Firefly luciferase (G5B-Luciferase) or -411 CD 13/APN Firefly luciferase¹⁵ (Myb- Luciferase). The mammalian expression vector pALi accepts peptide-coding inserts directly from phage vector mJ] and fuses them to the C terminus of the GAL4 DBD (amino acids 1-148). pALj was constructed by replacing the EGFP coding sequence of pEGFP-N3 (Clontech) with the coding sequence for the GAL4 DBD. Using the polymerase chain reaction, a multiple cloning site was inserted at the C-terminus of GAL4 DBD, which resulted in a (Gly-Ser)₂ spacer between the GAL4 DBD and peptide of interest. Cells were assayed 36 hours after transfection with a dual-luciferase reporter assay system (Promega).

Peptide Pulldowns.

For production of affinity resins, Fmoc-synfhesized, HPLC-purified peptides were resuspended in absolute methanol and coupled to Affi-Gel 10 (Bio-Rad) at a final concentration of 1 mg of peptide per ml of resin. One 10 cm plate of exponentially growing 293 cells at 90% confluence was lysed in 1 ml of NP-40 lysis buffer with protease inhibitors and clarified. Peptide-coupled resin (400 μM final peptide concentration) was added to 200 μL of cell lysate, incubated one hour at room temperature, washed three times with PBS + 0.1% Tween-20, and resuspended in SDS- PAGE sample buffer.

Western Blotting.

After normalization, cell lysates were Western blotted as indicated with: Mouse anti- GAL4 DBD (Santa Cruz #sc-510, 1 μg/ml) or Rabbit anti-CBP (Santa Cruz #sc- 583, 1 μg/ml).

E. References cited in Exemplification

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(1998).

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Albericio, F.) (Marcel Dekker, New York, 2000). 20. Uesugi, M., Nyanguile, O., Lu, H., Levine, A.J. & Verdine, G.L. Induced alpha helix in the VP16 activation domain upon binding to a human TAF. Science 277,

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22. Briesewitz, R., Ray, G.T., Wandless, T.J. & Crabtree, G.R. Affinity modulation of small -molecule ligands by boπowing endogenous protein surfaces. Proc. Natl. Acad. Sci. U.S.A. 96, 1953-1958 (1999).

23. Nyanguile, O., Uesugi, M., Austin, D.J. & Verdine, G.L. A nonnatural transcriptional coactivator. Proc. Natl. Acad. Sci. U.S.A. 94, 13402-13406 (1997).

24. Mapp, A.K., Ansari, A.Z., Ptashne, M. & Dervan, P.B. Activation of gene expression by small molecule transcription factors. Proc. Natl. Acad. Sci. U.S.A. 97, 3930-3935 (2000).

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All references, publications and patents cited in the specification above are herein incoφorated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Table 1 - In Vitro Selection of an Optimized KIX Domain Binding

Peptide

First Round Peptide Phage Display Screening

Apparent Actual

Clone # Aligned Sequence Affinity (μM)

Affinity (μM)

1. .45 S W T L G E R W W (G S)₄ 3.6

1. .50

1. .32 S V P W D I V W G (G S)₄ 3.5

1. .38 S W T L G D S (G S)₄ 3.2

1, .58

1. .37 S I E Y W L D W L (G S)₄ 3.1

1. .04 S D R E T L (G S)₄ 3.0

1, .70 S F G V D F I L (G S)₄ 2.7

1, .66 S S L V D L I L F (G S)₄2.4 55.1+1.1

1. .73

Consensus : X X X a D a a a

E

Second Round Peptide Phage Display Screening

Apparent Actual

Clone # Aligned Sequence Affinity (μM) Affinity (μM) 2.54 S L V Y D L L F (G S)₄ 1.5

2.05 S W W A Y E V L F (G S)₄ 1.4

2.42 \\ «

2.82 \\ \\

2.50 S L W V Y D Y L F (G S)₄ 1.1

2.20 S W A V Y E L L F (G S)₄ 1.0 16.1±1.1

2.22 \\ w \\ \\

2.62 \\ \\

2.64 \\ \\

2.75 « \\ \\ \\

Consensus i: W V Y D L L F

L A A E V

Differential peptide phage display library screening of the KIX domain of p300/CBP was performed as described in Methods. Shown are the results of first round screening with a completely degenerate 8 amino acid library (S-X-X-X-X-X-X-X-X; top), and second round screening with an optimized degenerate library (S-X-X-X-a-D/E-a-a-a; bottom) where X=all 20 natural amino acids and a=aliphatic/aromatic weighting as described in Methods. Apparent and actual peptide affinities were measured as described in Experimental Protocol.

Claims

ClaimsWe claim:

1. A method for identifying co-activator domain specific synthetic activators comprising:

(iv) contacting a target domain of a selected transcription factor with a peptide display library comprising a variegated population of test peptide sequences of 3-20 amino residues in length;

(v) identifying binding peptide sequences from the peptide display library which bind to the target domain; and

(vi) identifying active peptide sequences from binding peptide sequences of step (ii) which have the further characteristic(s):

(a) inhibit transcriptional activation by the selected transcription factor, and/or (b) activates transcription of a target gene when provided as a fusion protein with a DNA binding sequence or other domain which promotes localization of the peptide to the target gene.

2. The method of claim 1, where peptide library is provided as a fusion protein.

3. The method of claim 2, wherein the peptide display library is a phage display library.

4. A transcriptional inhibitor comprising an active peptide sequence identified according to the method of claim 1, or a peptidomimetic thereof, wherein the inhibitor reduces the rate of transcription of a gene in a manner dependent on the selected transcription factor.

5. A chimeric transcriptional activator comprising an active peptide sequence identified according to the method of claim 1 , and a DNA binding sequence or other domain which promotes localization of the activator to a target gene.

6. A method for identifying a compound which inhibits interaction of the selected transcription factor with a natural ligand therefore, comprising: a. generating a reaction mixture comprising a polypeptide including the target domain of the selected transcription factor and a peptide or polypeptide including an active peptide sequence identified in claim 1 ; b. contacting the reaction mixture with one or more test compounds; c. ascertaining the ability of the test compound to inhibit the interaction of the target domain and the active peptide sequence.

7. A nucleic acid encoding a polypeptide which includes a KLX binding sequence represented in the general formula: X1-X2-X3-Y-X4-X5-L-F (I) wherein

X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably and acidic sidechain, such as D or E; X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the polypeptide binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence.

8. The nucleic acid of claim 7, wherein KIX binding sequence is represented in the general formula (W/L)(W/A)(V/A)Y(D/E)(L/V)LF.

9. The nucleic acid of claim 7, wherein the KLX binding sequence mediates binding of the polypeptide to the CREB-binding protein (CBP) or p300 transcriptional coactivators.

10. The nucleic acid of claim 7, wherein the KIX binding sequence mediates binding of the polypeptide to the KIX domain with a Kd of 10^~5M or less.

11. The nucleic acid of claim 7, wherein the polypeptide, upon association with a transcriptional regulatory sequence of a target gene, upregulates expression of the target gene in a manner dependent upon the presence of the KLX binding sequence.

12. The nucleic acid of claim 11, wherein the polypeptide upregulates expression of the target gene by at least 2 fold relative to the absence of the polypeptide, and more preferably at least one, two or three orders of magnitude.

13. The nucleic acid of claim 7, wherein the polypeptide further includes a DNA binding domain.

14. The nucleic acid of claim 7, wherein the polypeptide further includes one or more additional activation domains and/or repressor domains.

15. The nucleic acid of claim 7, wherein the polypeptide further includes at least one additional KLX binding sequence, and preferably 3-10.

16. A recombinant polypeptide which includes a KLX binding sequence represented in the general formula:

X1-X2-X3-Y-X4-X5-L-F (I) wherein

R, D, E, H or K, and more preferably and acidic sidechain, such as D or E;

17. A peptide or peptidomimetic of 6-20 amino acid residues in length, which includes a KIX binding sequence represented in the general formula:

X1-X2-X3-Y-X4-X5-L-F (I) wherein

X3 represents an amino acid residue with a small hydrophobic sidechain, preferably A, G, or V, and more preferably A or V; X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably and acidic sidechain, such as D or E;

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the peptide binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence.

18. The peptide or peptidomimetic of claim 17, which inhibits binding of cyclic AMP-responsive factor (CREB) or c-Myb to the CBP or p300 transcriptional coactivators, preferably with a Ki of lOOμM or less, and more preferably with a

Ki less than lOμM, lμM, lOOnM, lOnM or even lnM.

19. The peptide or peptidomimetic of claim 17, which inhibits expression of CREB or c-Myb dependent gene transcription.

20. The peptide or peptidomimetic of claim 17, which is formulated in a pharmaceutically acceptable excipient.

21. A cyclic peptide or peptidomimetic of 6-20 amino acid residues in length, which includes a KIX binding sequence represented in the general formula:

S-C-Xi -X₂-X₃-X4-X₅-X₆-X₇-X₈-C-G-S (IV) wherein,

X represents any amino acid residue, more preferably an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V), polar (e.g., R, N, D, C, E, Q, H, K, S or T), acidic (D or E) or basic (R, H or K) side chain, and more preferably is P, W, E, S, L, N or R;

X₂ represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, L V, K H, T or G; X₃ represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H,

I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is W, T, D or V;

X4 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, R, M, G, T, A, Y, L; X5 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is V, Y, L, N, W, E, F, G;

X6 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or

T) side chain, and more preferably is D, S, L, P, G, R, K, V;

X7 represents an amino acid residue with an aromatic (F, Y, W) or acidic (D or E) sidechain, and more preferably is F, Y, W or D;

X8 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or

T) side chain, and more preferably, preferably an aromatic side chain (F, Y, W), and more preferably S, E, V, Y, R, W, T or P.

22. A method for stimulating transcription of a target gene, comprising (i) transfecting the cell with an expression construct encoding a polypeptide which includes a KIX binding sequence represented in the general

. formula: . _

X4 represents an amino acid residue with a charged sidechain, preferably R, D, E, H or K, and more preferably and acidic sidechain, such as D or E; X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V, wherein the polypeptide binds to a KIX domain in a manner dependent upon the presence of the KIX binding sequence. (ii) causing the cell to express the polypeptide;

(iii) causing the polypeptide to become associated with a transcriptional regulatory sequence of the target gene.

23. A peptide or peptidomimetic of 6-20 amino acid residues in length, which includes a KLX binding sequence represented in the general formula (II):

X1-X2-X3-A1-X4-A2-A3-A4 (II)

wherein

X4 comprises a charged natural or non-natural amino acid or peptidomimetic thereof, wherein the peptide or peptidomimetic binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence.

24. The composition of claim 23, wherein X4 is selected from the group consisting of D, E, and a peptidomimetic thereof.

25. The composition of claim 23, wherein Al, A2, A3 and A4 each independently comprise an aromatic or aliphatic moiety selected from the group consisting of A, V, L, I, F, W, Y, M, P, and a peptidomimetic thereof.

26. A peptide or peptidomimetic of 6-20 amino acid residues in length, which includes a KLX binding sequence represented in the general formula (III):

A1-A2-A3-A4-X1-A5-A6-A7 (III)

XI comprises a charged natural or non-natural amino acid or peptidomimetic thereof, wherein the peptide or peptidomimetic binds to a KLX domain in a manner dependent upon the presence of the KLX binding sequence.

27. The composition of claim 26, wherein Al, A2, A3, A4, A5, A6 and A7 each independently comprise an aromatic or aliphatic moiety selected from the group consisting of A, V, L, F, W, Y, M, P, I, and a peptidomimetic thereof; and wherein XI is selected from the group consisting of D, E and a peptidomimetic thereof.

28. The composition of claim 26, wherein A4 comprises tyrosine or a peptidomimetic thereof; wherein XI comprises D, E, or a peptidomimetic thereof; wherein A6 comprises L or a peptidomimetic thereof; and wherein A7 comprises F or a peptidomimetic thereof.

29. The composition of claim 26, wherein Al comprises W, L or a peptidomimetc thereof, wherein A2 comprises W, A, or a peptidomimetic thereof; wherein A3 comprises V, A, or a peptidomimetic thereof; wherein A4 comprises Y or a peptidomimetic thereof; wherein C comprises D, E, or a peptidomimetic thereof; wherein A5 comprises L, V, or a peptidomimetic thereof; wherein A6 comprises L or a peptidomimetic thereof; and wherein A7 comprises F or a peptidomimetic thereof.

30. A small molecule capable of binding to the KIX domain of a polypeptide (e.g., p300/CBP).

31. The small molecule of claim 30, wherein said small molecule is preferably less than 5000 amu, more preferably less than 2500 amu, and most preferably less than 1000 amu.

32. The small molecule of claim 30, wherein said small molecule is a small organic molecule.

33. A pharmaceutical preparation comprising a small molecule capable of binding to the KIX domain of a polypeptide (e.g. p300/CBP).

34. A library of peptides or peptidomimetics of 6-20 amino acid residues in length, wherein each peptide or peptidomimetic in said library includes a KIX binding sequence represented in the general formula (I):

X5 represents an amino acid residue with a hydrophobic sidechain, preferably A, G, I, L, M, F, P, W, Y or V, and more preferably L or V

35. A library of peptides or peptidomimetics of 6-20 amino acid residues in length, wherein each peptide or peptidomimetic in said library includes a KLX binding sequence represented in the general formula (H)

X1-X2-X3-A1-X4-A2-A3-A4 (II)

wherein,

XI, X2 and X3 each independently comprise a natural or non-natural amino acid or a peptidomimetic thereof;

Al, A2, A3 and A4 each independently comprise an aromatic or aliphatic natural or non-natural amino acid or peptidomimetic thereof; and

X4 comprises a charged natural or non-natural amino acid or peptidomimetic thereof, wherein each peptide or peptidomimetic binds to a KLX domain in a manner dependent upon the presence of the KIX binding sequence.

36. A method for identifying a compound which inhibits interaction of the KLX domain of a polypeptide (e.g., of p300/CBP) with a natural KIX domain ligand comprising: a. generating a reaction mixture comprising a polypeptide including a KLX domain (e.g., p300/CBP) and a polypeptide including a KLX binding sequence (as set forth in claim 11), e.g., which reaction mixture may be a whole cell, a cell lysate, or a reconstituted protein preparation; b. contacting the reaction mixture with a small molecule, e.g., a small organic molecule, e.g., a small molecule has a molecular weight of less than 10,000 amu; c. ascertaining the ability of the small molecule to inhibit the interaction of the KIX domain and he KIK binding sequence.

37. The method of claim 36, wherein the steps are repeated for a library of different test compounds.

38. The method of claim 36, wherein the steps are repeated for the library of compounds in claim 33, 34 or 35.

39. The method of claim 36, comprising the further steps of formulating a pharmaceutical preparation comprising one or more small molecules which inhibit interaction of the KLX domain of p300/CBP and natural KLX domain ligands.

40. A method of treating a cell affected by a condition caused by KlX-mediated transcription, comprising contacting the cell with a preparation comprising an effective amount of a small organic compound which can competitively inhibits interaction of a KIX domain, e.g., p300 or CBP, and a natural KLX domain ligand, e.g., myb, CREB, p53 or HIV Tat.

41. A method for reducing hyperglycemia comprising administering to a patient suffering therefrom an effective amount of a small organic compound which can competitively inhibits KLX-domain dependent interaction of CBP and CREB.

42. The method of claim 41 , wherein the patient suffers from diabetes mellitus.

43. A method of treating a eukaryote (e.g., an animal, a plant or a fungus) for a condition resulting from interaction of the KLX domain of p300/CBP and natural KLX domain ligands, or preventing said condition, comprising:

a. identifying a small molecule which inhibits interaction of the KLX domain of p300/CBP and natural KLX domain ligands, which small molecule has a molecular weight of less than 10,000 amu; b. administering, to the mammal, a pharmaceutical preparation comprising a therapeutically effective amount of a small molecule which has identified as an inhibitor, which compound is formulated in the pharmaceutical preparation for delivery into infected cells of the mammal.

44. The method of claim 43, wherein the small molecule is a peptide, or peptidomimetic thereof, comprising a core sequence having the formula (I):

X1-X2-X3-A1-C-A2-A3-A4 (I)

wherein

XI, X2 and X3 each independently comprise a natural or non-natural amino acid or a peptidomimetic thereof; Al, A2, A3 and A4 each independently comprise an aromatic or aliphatic natural or non-natural amino acid or peptidomimetic thereof; and C comprises a charged natural or non-natural amino acid or peptidomimetic thereof.

45. The method of claim 43, wherein the small molecule is a peptide, or peptidomimetic thereof, comprising a core sequence having the formula (II):

A1-A2-A3-A4-C-A5-A6-A7 (II)

wherein Al, A2, A3, A4, A5, A6 and A7 each independently comprise an aromatic or aliphatic natural or non-natural amino acid or peptidomimetic thereof; and wherein C comprises a charged natural or non-natural amino acid or peptidomimetic thereof.

46. The method of claim 43, wherein the small molecule is a peptide, or peptidomimetic thereof, comprising a core sequence having the formula (I):

X1-X2-X3-Y-X4-X5-L-F (I) wherein

R, D, E, H or K, and more preferably and acidic sidechain, such as D or E;

47. The method of claim 43, wherein the small molecule is a cyclic peptide, or cyclic peptidomimetic thereof, comprising a core sequence having the formula (IV): S-C-Xι-X₂-X₃-X4-X₅-X₆-X7-X₈-C-G-S (IV) wherein,

Xi represents any amino acid residue, more preferably an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V), polar (e.g., R, N, D, C, E, Q, H, K, S or T), acidic (D or E) or basic (R, H or K) side chain, and more preferably is P, W, E, S, L, N or R;

X4 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is S, R, M, G, T, A, Y, L; X5 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G,

H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably is V, Y, L, N, W, E, F, G;

X8 represents an amino acid residue with a neutral (e.g., A, N, C, Q, G, H, I, L, M, F, P, S, T, W, Y or V) or polar (e.g., e.g., R, N, D, C, E, Q, H, K, S or T) side chain, and more preferably, preferably an aromatic side chain (F, Y, W), and more preferably S, E, V, Y, R, W, T or P.

48. A library of peptides or peptidomimetics of 6-20 amino acid residues in length, wherein each peptide or peptidomimetic in said library includes a KLX binding sequence represented in the general formula (III): [51

A1-A2-A3-A4-X1-A5-A6-A7 (III)

XI comprises a charged natural or non-natural amino acid or peptidomimetic thereof, wherein each peptide or peptidomimetic binds to a KLX domain in a manner dependent upon the presence of the KIX binding sequence.