NOVEL COMPOUNDS AND METHODS FOR INHIBITING P53 ACTIVITY

FIELD OF THE INVENTION

[0001] The present invention provides novel compounds that block p53 association with the co-activator CBP. This interaction is implicated in p53-induced transcription of the cell cycle inhibitor p21 in response to DNA damage.

BACKGROUND OF THE INVENTION

[0002] The human tumor suppressor ρ53 is a transcription factor that binds in a sequence- specific manner to particular sites in the genome and activates transcription of target genes Vousden, et al, (2002) Nat Rev Cancer 2, 594-604; Oren (2003) Cell Death Differ 10, 431-442; el-Deiry (1998) Semin Cancer Biol 8, 345-357). It plays a pivotal role in cellular response to stress signals in cell cycle arrest, senescence, DNA repair or apoptosis (Vogelstein, et al, (2000) Nature 408, 307-310; Levine (1997) Cell 88, 323-331 ; Prives et al. (1999) J. Pathol. 187, 112- 126; Ko et al, (1996) Genes Dev. 10, 1054-1072). The biological activity of p53 is tightly regulated by post-translational modifications in its N- and C-terminal regions (Alarcon- Vargas et al., (2002) Carcinogenesis 23, 541-547). Upon DNA damage, p53 is extensively phosphorylated within the N-terminal activation domain, which relieves it from association with the negative regulator Mdm2, resulting in p53 stabilization and activation (Haupt et al., (1997) Nature 387, 296-299; Kubbutat et al, (1997) Nature 387, 299-303; Momand et al, (1997) J. Cell. Biochem. 64, 343-352). In addition, phosphorylation occurs in the C-terminus of p53, which has been suggested to enhance its DNA binding in vitro (Hupp et al, (1994) Curr. Biol. 4, 865-875; Wang et al, (1995) Nature 376, 88-91).

[0003] In response to extra-cellular stress or DNA damage p53 becomes acetylated on multiple lysine residues at its C-terminus (Sakaguchi et al, (1998) Genes Dev 12, 2831-2841 ; Gu et al, (1997) Cell 90, 595-606; Liu et al, (1999) MoI Cell Biol 19, 1202-1209). Particularly, the transcriptional co-activator histone acetyltransferase p300/CBP (CREB-binding protein) has been shown to acetylate K373, K382 and to a lesser extent K372 and K381 , whereas another co- activator p300/CBP-asso dated factor (PCAF) acetylates K320. The dynamic inter-play between lysine acetylation and deacetylation of p53 has been directly linked to its ability to regulate p53 stability or protein level in cells as well as functional activation as a transcription factor in cell cycle arrest, apoptosis, and senescence (Ito et al., (2001) Embo J 20, 1331-1340; Luo et al., (2000) Nature 408, 377-381 ; Guo et al, (2000) Nat. Cell Biol 2, 730-736; Barlev et al, (2001) MoI Cell 8, 1243-1254; Li et al., (2002) /. Biol Chem. 50607-50611, Pearson et al, (2000) Nature 406, 207-210). It was hypothesized on the basis of in vitro data that p53 acetylation enhances its DNA binding through the relief of negative regulation of DNA binding exerted by the C-terminal region. However, more recent cell-based studies show that lysine acetylation of p53 may not result in direct enhancement of its DNA binding ability, but rather promotes its recruitment of transcriptional co -activators, which leads to subsequent histone acetylation of chromatin and transcriptional activation of its target genes. Acetylated K382 in p53 may serve as a binding site for the CBP bromodomain and that this bromodomain/acetyl-lysine binding is responsible for p53 acetylation-dependent coactivator recruitment after DNA damage, a step that is essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21 in Gl cell cycle arrest (Mujtaba et al, (2004) MoI Cell 13, 251-263).

[0004] Despite the fact that these post-translational modifications are known to play an important role in p53 function, specific effects of single or combinatorial modifications on p53 function remain elusive. Because many of these modifications are clustered within a relatively short stretch of the protein sequence, conventional point mutational analysis of one modification site can lead to masking of effects exerted by different neighboring modifications. To circumvent this problem, it would be desirable to develop small-molecule chemical ligands that are capable of selectively modulating molecular interactions and regulation of p53 function involving these modifications, particularly lysine acetylation. Such small molecules can be used to study endogenous p53 and its effector proteins in cell-based assays, which may help us gain mechanistic insights into the effects of single or combinatorial modifications on p53 activation in response to DNA damage.

SUMMARY OF THE INVENTION

[0005] The present invention features compounds of the following general formula(I) wherein:

O II

R-C-R' (I)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH ₂₁ NO _2, SO ₂ , CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH, CN ₃ halogen, carboxy, and alkoxy and their pharmaceutically acceptable salts of acids or bases. The general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

[0006] In preferred embodiments, R may be selected from the group consisting of

In other preferred embodiments, R' may be lower alkyl or hydrogen. In other preferred embodiments, each compound consists of one aromatic ring connected to an -NHCOCH ₃ group either directly or via a two-three carbon chain. In yet other preferred embodiments, each

compound consists of one aromatic ring fused to an alicyclic ring. In still other preferred embodiments, each compound consists of two fused aromatic rings. In still other preferred embodiments, each compound consists of one aromatic ring substituted with -(OHb) ₂ NHCOCH ₃ .

[0007] In a second aspect, the present invention features a pharmaceutical composition comprising a compound of formula I wherein

O II

R-C-R' (I)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH ₂₁ NO ₂ , SO ₂ , CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases, together with a pharmaceutically acceptable earner. The general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

[0008] In preferred embodiments, R may be selected from the group consisting of

In other preferred embodiments, R' may be lower alkyl or hydrogen. In other preferred embodiments, each compound consists of one aromatic ring connected to an -NHCOCH ₃ group either directly or via a two-three carbon chain. In yet other preferred embodiments, each compound consists of one aromatic ring fused to an alicyclic ring. In still other preferred

embodiments, each compound consists of two fused aromatic rings. In still other preferred embodiments, each compound consists of one aromatic ring substituted with -(CHa) ₂ NHCOCHs.

[0009] In a third aspect, the present invention features methods for inhibiting the biological activity of p53 comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (I) or a pharmaceutical composition comprising a compound of the following general formula(I) wherein:

O II

R-C-R' (I)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH ₂₁ NO _2, SO ₂ , CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH ₃ CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases. The general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

[0010] In preferred embodiments, R may be selected from the group consisting of

In other preferred embodiments, R' may be lower alkyl or hydrogen. In other preferred embodiments, each compound consists of one aromatic ring connected to an -NHCOCH ₃ group cither directly or via a two-three carbon chain. In yet other preferred embodiments, each compound consists of one aromatic πng fused to an alicyclic ring. In still other preferred

embodiments, each compound consists of two fused aromatic rings. In still other preferred embodiments, each compound consists of one aromatic ring substituted with -(CH ₂ ) ₂ NH COCH ₃ .

[0011] In a fourth aspect, the present invention features methods for treating a disease associated with p53 transcription comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (I) or a pharmaceutical composition comprising a compound of the following general formula(I) wherein:

O II

R-C-R' (I)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH _2, NO _2, SO ₂ , CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases. The general formula (I) includes every stereoisomer, epimer and diastereo isomer, as a mixture or in isolated form.

[0012] In preferred embodiments, R may be selected from the group consisting of

In other preferred embodiments, R' may be lower alkyl or hydrogen. In other preferred embodiments, each compound consists of one aromatic ring connected to an -NHCOCH ₃ group either directly or via a two-three carbon chain. In yet other preferred embodiments, each compound consists of one aromatic ring fused to an alicyclic ring. In still other preferred

embodiments, each compound consists of two fused aromatic rings. In still other preferred embodiments, each compound consists of one aromatic ring substituted with -(CHa) ₂ NHCOCH ₃ .

Likewise, in some embodiments, the disease may be selected from the group consisting of cancer, cell death caused all or in part by cancer treatments such as chemotherapy or radiation thereapy, neuron cell death, stroke, Alzheimer's Disease and Parkinson's Disease.

[0013] In a fifth aspect, the present invention features compounds of the following general formula(II) wherein:

R-X-R' (H)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH ₂₁ NO _2, SO _2>. CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH, CN, halogen, carboxy, and alkoxy and their pharmaceutically acceptable salts of acids or bases. X is selected from the group consisting of O, S, and N. The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

[0014] In preferred embodiments, the compound of formula (II) may be selected from the group consisting of

[0015] In a sixth aspect, the present invention features a pharmaceutical composition comprising a compound of formula II wherein

R-X-R' (II)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH ₂ , NO _2, SO ₂ , CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases, together with a pharmaceutically acceptable carrier. X is selected from the group consisting of O, S, and N. The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

10016] In preferred embodiments, the compound of formula (II) may be selected from the group consisting of

[0017] In a seventh aspect, the present invention features methods for inhibiting the biological activity of p53 comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (II) or a pharmaceutical composition comprising a compound of the following general formula(II) wherein:

R-X-R' (H)

R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO2, NH ₂ , NO ₂ , SO _2, CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases. X is selected from the group consisting of O, S, and N. The general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

[0018] In preferred embodiments, the compound of general formula (II) may be selected from the group consisting of

[0019] In an eighth aspect, the present invention features methods for treating a disease associated with p53 transcription comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (II) or a pharmaceutical composition comprising a compound of the following general formula{ll) wherein:

R-X-R' (II)

R and R" are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO ₂ , NH _2, NO _2, SO ₂ , CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCOCH ₃ , CH ₂ COCH ₃ , OH ₃ CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases. X is selected from the group consisting of O, S, and N. The general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.

[0020] In preferred embodiments, the compound of general formula (II) may be selected from the group consisting of

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1. Physicochemical properties of the acetyl-lysine binding sites in the CBP and PCAF bromodomains.

a. Ribbon diagram of the 3D structure of the CBP bromodomain in complex with lysine 382- acetylated p53 peptide (PDB USP). b. & c. Surface electrostatic potential representation of the bromodomains of CBP and PCAF, respectively. The electrostatic potential of the protein molecular surface was calculated using Delphi [47] and figures were produced using GRASP [49] and rendered using Pov4Grasp fhttp://pov4grasp.free. fiV). d. & e. 3D representations of electrostatic isopotential contours of the CBP and PCAF bromodomains. Contours are drawn at -1K _B /T (red) and +1K _B /T (blue). The electrostatic potential was calculated using Delphi [47] and figure was made using UCSF Chimera [48].

[0022] Figure 2. Discovery of initial lead compounds for the CBP bromodomain a. Small-molecule compounds that bind to the CBP bromodomain. b. Superposition of 2D ¹ H- ¹⁵ N-HSQC spectra of the CBP bromodomain showing changes of the protein NMR resonances from the free form (black) to the complex form with a representative compound MS2126. c. Weighted ¹ H and ¹⁵ N chemical shift changes (δδ) by the CBP bromodomain induced by binding to representative ligands from each group, i.e. MS2126, MS7972, MS9802 or MS0433. The AcK binding site lies between ZA and BC loops. Amino acid residues exhibiting major chemical shift perturbations are color-coded on the protein surface: red for 0.05 ppm < δδ <0.08 ppm and blue for δδ >0.08 ppm. The orientation of the protein structure is similar to that in Figure \a & Ib.

d. Stack plot of absolute changes in chemical shift (Aδ ^x = δ*, ^ - δp _ree , where x is ¹ H or ¹⁵ N) in

presence of four Group D compounds, along ¹ H or ¹⁵ N dimension. Note that directionality of chemical shift perturbations for each compound is same.

[0023] Figure 3. Structural analysis of CBP bromodomain interactions with small molecules α. Identification of binding locations of small molecules in the CBP bromodomain by Autodock 3.0 calculations (left panels) and j-surface calculations using NMR chemical shift perturbation data at 2σ (right panels). Ribbons diagrams depict best binding modes of ligands of each group, as determined by Autodock 3.0 calculations. The aromatic ring of each compound is color-coded according to the corresponding j-surface calculation. b. Three-dimensional structure of the CBP bromodomain bound to MS7972, as determined by NMR spectroscopy, illustrating the ligand binding site between the ZA and BC loops.

[0024] Figure 4. Small-molecule inhibition of CBP bromodomain and p53-AcK382 interaction α. Inhibition of CBP bromodomain and p53-AcK382 peptide binding by lead compounds in a competition assay detected by anti-GST Western blot. In this assay, a lead compound competes against the biotinylated p53-AcK382 peptide that was immobilized on strep tavidin agarose beads for binding to the GST CBP bromodomain. Concentration of the biotinylated or the non- biotinylated p53 peptide used in the assay is 10 μM and 25 μM, respectively, whereas compound concentration ranges from 0 to 100 μM, as indicated. b. Fluorescence titration of CBP bromodomain binding to MS7972. Superimposition of fluorescence spectra of the CBP bromodomain (~5 μM) with increasing amount of MS7972 (0-

80 μM). Binding affinity was determined by monitoring fluorescence intensity change at 450 nm as a function of ligand concentration (inset).

[0025] Figure 5. Modulation of p53 function in response to DNA damage by small molecules a. Increased p53 expression in response to DNA damage agent doxorubicin treatment, as illustrated with U2OS cells. Wild-type p53 expressing U2OS cells were incubated with 0.1 μg/ml doxorubicin for the indicated time (up to 24 hours) and then subjected to immunoblotting analysis with specific anti-bodies. b. Small-molecule inhibition of the increase in p53 levels in response to DNA damage. Wild- type p53 expressing U2OS cells were either incubated with DMSO or treated with 20, 200 μM of each small-molecule compound for 16 hours. Cells were then further incubated with 0.1 μg/ml doxorubicin for additional 24 hours as shown. Cell lysates were then subjected to immunoblotting with the indicated antibodies. c & (L Modulation of p53 function in response to DNA damage by MS2126 or MS7972, respectively. Wild-type p53 expressing U2OS cells were either incubated with DMSO or treated with 200 μM of MS2126 or MS7972 for 16 hours. Cells were then further incubated with 0.1 μg/ml doxorubicin for the indicated times and then subjected to immunoblotting analysis with specific antibodies. e. Compound MS2126 does not affect the increase in HIFlα level in response to hypoxia. Wild-type p53 expressing U2OS cells were incubated in the absence or presence of 200 μM of MS2126 for 16 hours. Cells were then further incubated in either normoxic or hypoxic conditions for an additional 24 ^' hours as shown. Cell lysates were then subjected to immunoblotting with the indicated antibodies.

/. Compound MS5557 does not affect p53 function in response to DNA damage, as demonstrated in wild-type ρ53 expressing U2OS cells with experimental conditions similar to those in c and d. The cells were treated with DMSO, 200 μM of MS7972 or MS5557 for 16 hours, and then further incubated with 0.1 μg/ml doxorubicin for 24 hours and then subjected to immunoblotting analysis with specific antibodies.

[0026] Figure 6 demonstrates protection of radiation induced cell death by two p53 inhibitors MS7972 and MS5557.

[0027] Figure 7 demonstrates the effect of the p53 inhibitors MS2126 and MS7972 on TNFα inhibition of myogenic differentiation in myoblast cell cline C2C12.

[0028] Figure 8 demonstrates the effects of the p53 inhibitors MS2126 and MS7972 on reducing TNFα inhibition of myogenic differentiation in a myoblast cell line.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Compounds of the present in invention and pharmaceutical compositions comprising the same are useful for modulating, preventing, retarding the progression and treating diseases associated with p53 transcription. Lysine acetylation of human tumor suppressor p53 in response to cellular stress signals is required for its function as a transcription factor that regulates cell cycle arrest, senescence or apoptosis. Here, we report small molecules that block lysine 382-acetyIated p53 association with the bromodomain of the co-activator CBP, an interaction essential for p53-induced transcription of the cell cycle inhibitor p21 in response to DNA damage. These chemicals were discovered in target structure-guided nuclear magnetic

resonance spectroscopy screening of a focused chemical library constructed based on the structural knowledge of CBP bromodomain/p53-AcK382 binding. Structural characterization shows that these chemicals inhibit CBP/p53 association by binding to the acetyl-lysine binding site of the bromodomain. Cell-based functional assays demonstrate that the lead chemicals can modulate p53 stability and function in response to DNA damage.

[0030] The present invention features a series of small-molecule chemical compounds that can block, for instance, K382-acetylated p53 association with the bromodomain of CBP. These small molecules were indcntified in target structure-based nuclear magnetic resonance (NMR) spectroscopy screening of a focused library of chemical compounds that was constructed based on the structural knowledge of the CBP bromodomain/p53-AcK382 interaction. The structure- based characterization using combined experimental and computational methods demonstrates the surprising discovery that some of these small-molecule compounds can effectively inhibit CBP/p53 association by specifically binding to the acetyl-lysine binding pocket of the CBP bromodomain. Cell-based functional assays further demonstrate that these compounds can modulate p53 stability, protein level, modification patterns as well as transcriptional activation of downstream target gene p21 in response to DNA damage. The combined in-vitro and in-vivo data demonstrate the clinical efficacy of these small molecule compounds.

[0031] As used herein the following terms are defined as follows:

the terms "lower alkyl and lower alkoxy (see below)" are understood as meaning straight or branched alkyl and alkoxy groups having from 1 to 8 carbon atoms;

the term "aryl" is understood as meaning an aromatic group selected from phenyl and naphthyl groups;

the term "heteroaryl" is understood as meaning a mono- or bicyclic aromatic group, each cycle, or ring, comprising five or six atoms and said cycle, or ring, or both cycles, or rings, including in its carbon skeleton from one to three heteroatoms selected from nitrogen, oxygen and sulphur;

the terras "lower aralkyl" and "lower heteroaralkyl" are understood as meaning, in view of the definitions above, phenyl(Cj -Cs)alkyl or naphthyl(Ci -Cg)alkyl and heteroar(Ci -Cg)alkyl respectively;

the term "substituted" concerning the terms aryl, aralkyl, phenyl, radical (fϊve-membered, including Z), heteroaryl, heteroaralkyl, as defined above, signifies that the groups in question are substituted on the aromatic part with one or more identical or different groups selected from the groups: (Ci -C ₈ )alkyl, trifluoromethyl, (Ci -Cg)alkoxy, hydroxy, nitro, amino, (Ci - C8)alkylamino, di(Ci -Cs)alkylamino, sulphoxyl, sulphonyl, sulphonamide, sulpho(Ci -Cg)alkyl, carboxyl, carbalkoxyl, carbamide (it being possible for said (C] -Cg)alkyl groups to be linear or branched) or substituted with one or more halogen atoms; the term aminoacyl, which concerns the glutathionyl, cysteinyl, N-acetylcysteinyl or even the penicillaminyl group in the definition of X, signifies any natural aminoacid such as alanine, and leucine, for example.

[0032] As used herein a "bromodomain-acetyl-lysine binding complex" is a binding complex between a bromodomain or fragment thereof and either a peptide/polypeptide comprising an acetyl-lysine (or an analog of acetyl-lysine), or a free analog of acetyl-lysine, such as acetyl- histamine disclosed in the Example below. Preferably, the peptide comprises at least six amino acids in addition to the acetyl-lysine. A fragment of a bromodomain preferably comprises a ZA loop as defined below. The dissociation constant of a bromodomain-acetyl-lysine binding complex is dependent on whether the lysine residue or analog thereof is acetylated or not, such that the affinity for the bromodomain and the peptide comprising the lysine residue (for example) significantly decreases when that lysine residue is not acetylated. One example of a bromodomain-acetyl-lysine binding complex is that formed between P/CAF with Tat (the "Tat- P/CAF complex") as exemplified below.

[0033] As used herein the term "acetyl -lysine analog" is used interchangeably with the term "analog of acetyl-lysine" and is a compound that contains the acetyl-amine-like structure.

[0034] As used herein a "ZA loop" of a bromodomain is a key protion of a bromodomain that is involved in the binding of the bromodomain to the acetyl-lysine. The structure of the

actual ZA loop of the bromodonϊain of P/CAF is depicted in Fig. 2A. As used herein, however, a ZA loop has between about 20 and 40 amino acids and preferably comprises the amino acid sequence set forth in United States Patent Application 09/784,553 and United States Patent Application 10/209/201, now published as United States Patent Publication No. 2004/0009613, the disclosure of which is hereby incorporated by reference in its entirety. More preferably the ZA loop comprises between about 23 to 34 amino acids.

(0035] A "polypeptide" or "peptide" comprising a fragment of a bromodomain, such as the ZA loop, or a peptide or polypeptide comprising an acetyl- lysine, as used herein can be the "fragment" alone, or a larger chimeric or fusion peptide/protein which contains the "fragment".

[0036] As used herein the terms "fusion protein" and "fusion peptide" are used interchangeably and encompass "chimeric proteins and/or chimeric peptides" and fusion "intein proteins/peptides". A fusion protein comprises at least a portion of a protein or peptide of the present invention, e.g., a bromodomain, joined via a peptide bond to at least a portion of another protein or peptide including e.g., a second bromodomain in a chimeric fusion protein. In a particular embodiment the portion of the bromodomain is antigenic. Fusion proteins can comprise a marker protein or peptide, or a protein or peptide that aids in the isolation and/or purification of the protein, for example.

[0037] As used herein, and unless otherwise specified, the terms "agent", "potential drug", "compound", "test compound" or "potential compound" are used interchangeably, and refer to chemicals which potentially have a use as an inhibitor or activator/stabilizer of bromodomain- acetyl-lysine binding. Therefore, such "agents", "potential drugs", "compounds" and "potential compounds" may be used, as described herein, in drug assays and drug screens and the like.

[00381 As used herein a "small organic molecule" or "small molecule" is an organic compound, including a peptide or organic compound complexed with an inorganic compound (e.g., metal) that has a molecular weight of less than 3 Kilodaltons. Such small organic molecules can be included as agents, etc. as defined above.

[0039] As used herein the term "binds to" is meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule. This includes processes such as covalent, ionic, hydrophobic and hydrogen bonding but does not include non-specific associations such as solvent preferences.

[0040J As used herein, the term "homologous" in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies {e.g., the immunoglobulin superfamily) and homologous proteins from different species {e.g., myosin light chain, etc.) (Reeck et al., Cell, 50:667 (1987)).- Such proteins have sequence homology as reflected by their high degree of sequence similarity.

[0041] Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin {see Reeck et al. , supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not a common evolutionary origin.

[0042] Two DNA sequences are "substantially homologous" when at least about 60% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art {See, e.g., Sambrook et al, 1989 supra; DNA Cloning, VoIs. I & II, supra; Nucleic Acid Hybridization, supra., and Sambrook and Russell, 2001).

[0043] As used herein an amino acid sequence is 100% "homologous" to a second amino acid sequence if the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions as defined below. Accordingly, an amino acid sequence is 50% "homologous" to a second amino acid sequence if 50% of the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions. As used herein, DNA and

protein sequence percent identity can be determined using Mac Vector 6.0.1 , Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters.

[0044] As used herein "biological activity of p53" refers not only to the binding affinity or activity of a single molecule but also to the pathological or clinical sequelae of the molecule as it functions in various interactions, pathways and cascades in vivo in an organism. Specifically, the term refers to p53 function as a transcriptional activator for its target genes. Similarly, the term "disease associated with p53 transcription" refers to any disease or pathology caused all or in part by any biological activity of p53 that is altered relative to its normal or homeostatic level.

[0045] Knowledge-based Design of a Focused Library. NMR-based screening of chemical compounds for a given target protein is considered to be reliable and target site-specific, making it preferable over random high-throughput screening (Shuker et al., (1996) Science 274, 1531- 1534; Hajduk et al, (1997) Science 278, 498-499; Hajduk et al., (1999) Q Rev Biophys 32, 211- 240; Moore (1999) Curr. Opin. in Biotech. 10, 54-58). However, because of relatively slow speed of NMR spectral acquisition and requirement of significant amount of protein samples, NMR is generally not best suited for chemical screening in a high-throughout fashion. To circumvent this shortcoming, we employed a strategy that combines a target structural knowledge-based construction of a "focused" library and NMR screening for lead identification. In designing a library, diversity of chemical compounds plays an important role in successful outcome of screening. Although a desirable property, chemical diversity is not the sole criterion; and pure diversity emphasis may bias libraries away from preferred drug properties (Lepre (2001) Drug Discov Today 6, 133-140; Gorse et al., (1999) Drug Discov Today 4, 257-264; Martin et al., (1999) J Comb Chem 7, 32-45). Need of chemical diversity coverage for

successful lead identification, however, is inversely proportional to available structural and functional knowledge for a given target.

[0046] The bromodomain (BRD) is found in a large number of chromatin associated proteins and nuclear histone lysine acetyltransferases (HATs), and has been recently shown to function as an acetyl-lysine (AcK) binding domain. Bromodomain/AcK. binding plays a pivotal role in regulation of chromatin remodeling and gene transcription (Zeng et al., (2002) FEBS Lett 513, 124-128; Winston et al., (1999) Nature Struct. Biol. 6, 601-604; Marmorstein et al., (2001) Gene 272, 1-9). BRDs adopt a conserved structural fold of a left-handed four-helix bundle (αZ, αA, αB and αC), as first shown in the PCAF BRD (Dhalluin et al., (1999) Nature 399, 491-496). The ZA and BC loops at one end of the bundle form a hydrophobic pocket for AcK binding. The structure of the CBP BRD bound to a p53-AcK382 peptide shows that AcK382 intercalates into the protein hydrophobic cavity and interacts with residues of the ZA and BC loop (Figure Ia). The structures of PCAF BRD/HIV Tat-AcK50 and GCN5p BRD/H4-AcK16 complexes confirm that the residues in BRDs important for AcK recognition arc largely conserved, whereas sequence variations in the ZA and BC loops with amino acid deletion or insertion enable discrimination of different binding targets (Mujtaba et al., (2002) MoI Cell 9, 575-586; Owen et al., (2000) Embo J19, 6141-6149).

[0047] The AcK binding pocket is hydrophobic in nearly all BRDs, whereas electrostatics at the opening of the AcK binding pocket displays significant variations in different BRDs. For example, in CBP BRD, the opening is slightly positively charged, while it is more negatively charged in PCAF BRD (Figure Ib vs. Ic and Id vs. Ie). These differences at the ligand binding site serve as the basis for selectivity of chemical ligands targeting a particular BRD. Given these

structural feature differences at the AcK binding pocket and the fact that most of known drug molecules contain one aromatic ring, we constructed a knowledge-based library of about 200 compounds from a collection of about 14,000 small molecules (ChemBridge, Inc.). The following criteria were used for the compound selection: (1) each compound consists of one aromatic ring connected to an -NHCOCH ₃ group either directly or via a two-three carbon chain; and (2) drug-like properties of compounds are evaluated according to the Lipinsky's Rule of Five (Lipinski et al., (1997) Advanced Drug Delivery Reviews 23, 3-25).

[0048] Lead Identification by NMR. We divided the library of 200 compounds in 25 screening mixtures with each containing eight compounds. Ligand binding to the protein was detected by monitoring chemical shift changes of protein backbone amide resonances in 2D ¹ H- ¹⁵ N-HSQC spectra acquired in presence and absence of screening mixture; and positive mixtures were then deconvoluted to identify individual binding ligands. From this knowledge-based library, we identified 14 compounds that bind to the CBP BRD (Figure 2a). All of these ligands (except the MS561 1) showed selective binding to CBP BRD as they did not show any binding to the structurally similar PCAF BRD (data not shown). A weighted chemical shift perturbation δδ,

(δδ ) of protein residues, was used to characterize small-molecule binding to the protein. As the protein residues that exhibited the most significant ligand-induced chemical shift perturbations are largely located in the ZA and BC loops (Figure 2c, lower panel), it is possible that all these compounds bind near the AcK binding site (Figure 2c, upper panel).

[0049] We classified these 14 compounds into four groups on the basis of their chemical structures (Figure 2a): (1) Group A compounds contain one aromatic ring fused to an alicyclic ring; (2) Group B compounds have two fused aromatic rings; (3) Group C compounds have only one aromatic ring attached to an -NHCOCH ₃ group; and (4) Group D compounds have one aromatic ring substituted with -(CH ₂ ) ₂ NHCOCH ₃ .

[0050] The proper grouping of compounds provides valuable insights into structure-activity relationships (SAR) in their interaction with the CBP bromodomain. Consistent with their similarity in chemical structures, directionality of absolute changes in chemical shift perturbations along ¹ H or ¹⁵ N dimension in the 2D HSQC spectra was similar for compounds in a given group, e.g. Group D (Figure 2d). Frequency of protein residues perturbed by different ligands in a given group highlights the importance of certain residues in ligand recognition (Figure Sl). For example, there are 1 1 residues in the BRD, mostly located in the ZA or BC loop, perturbed by all the ligands in Group A. While overall residues perturbed by ligands crossing different groups are different, information of residues commonly perturbed by binding to different ligands reveal similarity and/or difference in modes of ligand binding, and thus help in lead optimization. Taken together, these results indicate that the mode by which the protein interacts with ligands within one group is similar, thus justifying the classification of these compounds.

[0051] Identification of Ligand Binding Sites. Localization of ligand binding site in a target protein by chemical shift perturbation data alone is difficult, as a direct correlation between chemical-shift perturbations and spatial proximity to a binding ligand can be masked by ligands with large shielding and anisotropics, as well as by effects of indirect conformational changes of

the protein. Two alternative computational approaches can provide invaluable insights into the structural basis of protein/ligand recognition by localizing the ligand binding site in the target protein. First, computational docking calculation of 3D structure model of a ligand bound in a given target protein; the resulting structure model can be evaluated with chemical shift perturbation data obtained in NMR binding. Second, calculation of j-surface — a method that is based on the concept that the flow of electrons (electron current density, j) is responsible for NMR chemical shifts and chemical shift perturbations (McCoy et al., (2002) J Am Chem Soc 124, 11758-11763).

[0052] Ligand docking into a target protein can be performed using Autodock 3.0 that uses a genetic search algorithm as a global optimizer and energy minimization as a local search method (Morris et al., (1998) Journal of Computational Chemistry 19, 1639-1662). Although as a grid- based method, Autodock limits itself to a rigid model of a target protein, ligand flexibility is allowed. To predict the best docking mode for a given ligand, docking calculations generate a number of clusters (i.e. solutions with pair-wise RMSD of all atoms of 1.0 A) and rank of each docking mode (cluster rank). Docking mode is selected from the lowest-energy solution of a cluster corresponding to the minimum docking energy. The Autodock calculations show that the aromatic ring of almost all ligands is located in the hydrophobic AcK binding site (Figure 3a, left column). Most of the residues predicted to be interacting with ligands in the ZA and BC loops were also shown to be perturbed by NMR data. Notably, ligands that cause major chemical shift perturbations in the protein (i.e. "good binders") generate fewer clusters than ligands that cause minor chemical shift perturbations (i.e. "poor-binders"), consistent with the notion that the foπner has a consensus of a single binding mode. While for a given ligand, ligand binding site in the BRD protein predicted by Autodock calculation generally correlates well with NMR

chemical shift perturbation data, limitations of the docked structure models likely exist due to high mobility of the ZA and BC loops, as assessed by the predicted NMR S order parameters of N-H ^N vectors of the protein backbone amide that were calculated with its structure using contact model (Figure S2) (Zhang et al., (2002) JAm Chem Soc 124, 12654-12655). Because the ZA and BC loops comprise the AcK binding site in the CBP BRD, such high structural dynamics may cause major conformational changes of the ligand binding site when bound to different ligands.

[0053] Calculation of electron current density surface using chemical shift perturbation data can also help localize a ligand when bound to a target protein. This method calculates the center of electron current density for a ligand aromatic ring using point-dipole that is represented as dot density (j-surface), where the highest dot density correlates to the center of the ligand aromatic ring. The surface can, therefore, guide to locate the binding site for the aromatic ring of the ligand. When chemical shift perturbations are not caused by direct ligand binding, inconsistency between calculated j-surface and chemical shift perturbation, i.e. diffused dot-density, may be observed. Possibly due to likely major conformational changes of the ZA and BC loops, induced by ligand binding, many ligands from different groups show diffused dot-density (Figure 3a, right column). Nevertheless, the Group B ligands exhibit excellent consistency between calculated j-surface and the observed chemical shift perturbation.

[0054] To validate the predicted ligand binding site for the Group B ligands, we solved the three- dimensional structure of the CBP BRD in complex with MS7972 (9-acetyl-2,3,4,9-tetrahydro- carbazol-1-one) by NMR. As revealed by the structure (Figure 3b, Table 1), the ligand is bound in a site formed by residues largely in the ZA loop at the entrance of the AcK binding pocket,

confirming the computational prediction. Moreover, the structure also shows that the ligand forms a network of inter-molecular hydrophobic and aromatic interactions with VaIl 1 15, Leul l20, Ilel l22, Tyrl l25 and Tyrl 167, and that the acetyl and ketone groups with VaIl 115 and Leul 120, and Glnl 113, respectively. As many of these residues are involved in interactions with the p53-AcK382 peptide, binding of MS7972 to CBP BRD likely block the protein interaction with an acetyl-lysine-containing binding partner such as p53 (see below). Taken together, these data demonstrate that combined use of NMR chemical shift perturbation mapping with Autodock and j-surface calculations may rapidly establish the most probable structures of a protein bound to a ligand. Such model structures of protein/ligand complexes are important for rational design of pharmacophore and combinatorial library for lead improvement to optimize potency and ligand binding selectivity of initial leads for a specific target protein.

[0055] Inhibition of CBP BRD/p53-AcK382 Interactions. To evaluate whether these compounds are capable of blocking CBP BRD binding to lysine 382-acetylated p53, we perform an inhibition study. In this assay, a chemical ligand in a concentration-dependent manner competes against binding of the biotinylated p53-AcK382 peptide immobilized on streptavidin agarose to the GST-fusion CBP BRD, as assessed by anti-GST Western blot. As shown in Figure 4a, while MS9802 and MS0433 showed relatively little inhibition activity against CBP BRD/p53-AcK382 association in a ligand concentration of 5-100 μM, MS2126 and MS7972 can

almost completely block this BRD interaction at 100 μM and 50 μM, respectively. The inhibition activity of MS7972 is about 3-fold higher than that of MS2126. We further characterized binding affinity of compound MS7972 binding to the protein using fluorescence spectroscopy. Of three tryptophan residues in this BRD, Trpl l65 in the BC loop is near the AcK binding site. The intrinsic tryptophan fluorescence shows emission maxima at 350 run indicating that the

tryptophans are likely completely solvent exposed. Addition of MS7972 to the CBP BRD results in emission at 445 nm and quenching of the CBP BRD due to resonance energy transfer (Figure 4b). Upon saturation with MS7972, the protein tryptophan fluorescence undergoes a red shift at 350 nm, suggesting the possible change in protein local conformation upon ligand binding. Change of fluorescent intensity at 445 nm as a function of ligand concentration was used to determine binding constant Kp to be 19.6 ± 1.9 μM, which is consistent with its inhibition activity (Figure 4a).

[0056] Modulation of p53 function via inhibition of p53/CBP binding by small molecules.

We assessed the effects of these compounds on p53 function as transcription activator in response to DNA damage in a cell-based assay. Consistent with what is reported in the literature, p53 expression in U2OS cells is low in a resting state, likely due to its negative regulation by Mdm2 through interaction with the N-terminal region of p53 (Figure 5a). Upon DNA damage stimulation by doxorubicin treatment, p53 becomes phosphorylated within the N-terminal activation domain including serine 15, relieving it from association with the negative regulator Mdm2 and resulting in p53 stabilization and increase in protein level in the cell, as assessed by anti-p53 and anti-p53-pSerl5 Western blots. In response to DNA damage, p53 also becomes acetylated on its C-terminal lysine residues including lysine 382, promoting its recruitment of the transcriptional coactivator CBP/p300 via BRD/AcK binding, which leads to histone acetylation and transcriptional activation of target genes such as the cyclin-dependent kinase inhibitor p21 in cell cycle arrest. As shown in Figure 5b, treatment of U2OS cells with MS2126 or MS7972 at 200 μM, prior to the doxorubicin stimulation, results in a dramatic decrease of the doxorubicin- induced p53 increase as compared to the DMSO control. The lysine-acetyl atcd p53 in a free state is not stable in the cell, as it is subject to rapid de-acetylation by histone deacetylases and

subsequent υbiqutinalion and protein degradation by Mdm2. This effect is consistent with the corresponding decrease in p53-mediated p21 activation in response to doxorubicin-induced DNA damage. Treatment of U2OS cells with compound MS9802 or MS0433 showed much less effects, if any, on p53 protein level and activation in cells, which is consistent with their inability to inhibit CBP BRD/p53-AcK382 association in vitro (Figure 4a). Further studies at different time points post doxorubicin treatment show that treatment of MS2126 (Figure 5c) as well as MS7972 (Figure 5d) results in loss of p53 phosphorylation at Serl 5 as well as acetylation at Lys382 (Figure 5c).

[00571 D ^ue to differences in chemical stability and cell permeability for individual compounds, the ligand concentration required for effects on p53 function in the cell-based assay appears higher than that for in vitro inhibition activity (Figure 4a). Down-regulation of p53 protein level and functional activation in the presence of MS2126 or MS7972 that was exerted in ligand concentration-dependent and doxorubicin-exposure time-dependent manner results from their inhibitory activity in blocking CBP BRD/p53-AcK382 binding. Particularly, MS2126 does not seem to affect the up-regulation of HIFl α under hypoxic conditions (Figure 5e), and MS5557, which is a structural analog of MS7972 but does not bind to the CBP BRD (Figure S3), does not modulate p53 function under DNA damage condition (Figure 5f), thus further highlighting the specificity of MS2126 and MS7972 for p53 function. Taken together, these results demonstrate that inhibition of CBP BRD/p53 at AcK.382 by small-molecule compounds causes a dramatic inactivation of p53 transcriptional activity through promoting its protein instability by changes of its post-translational modification states. Moreover, these cell-based assays provide a valuable assessment of cell permeability and in vivo efficacy of these small-molecule compounds on p53

function as a transcriptional activator for its target genes, which is essential for further lead optimization through SAR-guided chemical modifications.

[00581 Significance. Molecular mechanisms underlying p53 function that direct cellular responses to external stress signals are undoubtedly complex. Small molecules designed to modulate specific molecular function of p53 can be used to explore mechanistic underpinnings of molecular interactions and regulation of p53 in physiological conditions. The knowledge of the structural and molecular basis of lysine 382-acetylated p53 interaction with the bromodomain of the coactivator CBP greatly facilitates the ability to identify small-molecule ligands for the target CBP bromodomain from NMR-based screening of a "focused" library constructed using a target structure-based approach. A structure-based understanding of target interactions with different classes of the lead compounds lays a foundation for rational design of pharmacophore and chemical modifications for lead optimization of potency and binding selectivity. The emerging results from the cell-based study of p53 protein level and functional activation using these small molecules can feed back to the rational design to address issues of cell permeation and stability of lead chemicals. The approach reported here is applicable to rational design of small-molecule ligands for other protein modular domains that play an important role in regulation of a wide variety of cellular processes.

[0059] Bromodomain. The present invention utilizes detailed structural information regarding a bromodomain and a bromodomain complexed with its acetylated binding partner. The present invention further utilizes knowledge of the three-dimensional structure of the bromodomain and a bromodomain acetylated binding partner complex. Since the interaction of the bromodomain with a ligand can play a significant role in remodeling/regulation/activation, the structural information can be employed in methods of identifying drugs that can modulate basic cell processes by modulating the transcription. In a particular, the three-dimensional

structural information is used in the design of a small organic molecule for the treatment of disease.

[0060] Indeed, the bromodomain and lysine-acetylated protein interaction can now be implicated to play a causal role in the development of a number of diseases. The resulting fusion protein MLL-CBP contains the tandem bromodomain-PHD fϊnger-HAT domain of CBP. It also has been shown that both the bromodomain and HAT domain of CBP are required for leukomogenesis, because deletion of either the bromodomain or the HAT domain results in loss of the MLL-CBP fusion protein's ability for cell transform. These results indicate that the CBP bromodomain, and more particularly, the ZA loop of the CBP bromodomain, is an excellent target for developing drugs that interfere with the bromodomain acetyl-lysine interaction that can be used in the treatment of disease. In addition, an antibody (e.g., a humanized antibody) raised specifically against a peptide from the ZA loop of the CBP bromodomain could also be effective for treating these conditions.

[00611 The key amino acid residues for the binding of a given bromodomain and its binding partner can be identified and further elucidated using basic mutagenesis and standard isothermal titration calorimetry, for example. Indeed, both the critical amino acids for the bromodomain and the binding partner (i.e., apart from the acetyl-lysine) can be readily determined and are also part of the present invention.

[0062] Compounds may be active to bind to two nearby sites on the bromodomain. In this case, a compound that binds a first site of the bromodomain does not bind a second nearby site. Binding to the second site can be determined by monitoring changes in a different set of amide chemical shifts in either the original screen or a second screen conducted in the presence of a ligand (or potential ligand) for the first site. From an analysis of the chemical shift changes the approximate location of a potential ligand for the second site is identified. Optimization of the second ligand for binding to the site is then carried out by screening structurally related compounds (e.g., analogs as described above). When ligands for the first site and the second site are identified, their location and orientation in the ternary complex can be determined experimentally either by NMR spectroscopy or X-ray crystallography. On the basis of this

structural information, a linked compound is synthesized in which the ligand for the first site and the ligand for the second site are linked. In a preferred embodiment of this type the two ligands are covalently linked. This linked compound is tested to determine if it has a higher binding affinity for the bromodomain than either of the two individual ligands. A linked compound is selected as a ligand when it has a higher binding affinity for the bromodomain than either of the two ligands. In a preferred embodiment the affinity of the linked compound with the bromodomain is determined monitoring the ¹⁵ N- or η-amide chemical shift changes in two dimensional ' ⁵ N-heteronuclear single-quantum correlation ( ¹⁵ N-HSQC) spectra upon the addition of the linked compound to the ^l5 N-labeled bromodomain as described above. A larger linked compound can be constructed in an analogous manner, e.g., linking three ligands which bind to three nearby sites on the bromodomain to form a multilinked compound that has an even higher affinity for the bromodomain than the linked compound.

[0063] Pharmaceutical Compositions. In yet another aspect of the present invention, pharmaceutical compositions of the compounds of formulae I and II are provided. Such pharmaceutical compositions maybe for administration for injection, or for oral, pulmonary, nasal or other forms of administration. In general, comprehended by the invention are pharmaceutical compositions comprising effective amounts of a low molecular weight component or components, or derivative products, of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.

[0064] Oral Delivery. Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and CT. Rhodes Chapter 10, 1979, herein incorporated by reference. In general, the formulation will include an agent of the present invention (or chemically modified forms thereof) and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. Also specifically contemplated are oral dosage forms of the above derivatized component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine. The therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The foπnulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression. One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Binders also may be used to hold the therapeutic agent

together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression also might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate. In addition, to aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Additives which potentially enhance uptake of the protein (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

[0065] Transdermal administration. Various and numerous methods are known in the art for transdermal administration of a drug, e.g., via a transdermal patch. Transdermal patches are described in for example, U.S. Patent No. 5,407,713, issued April 18, 1995 to Rolando et al.; U.S. Patent No. 5,352,456, issued October 4, 1004 to Fallon et al.; U.S. Patent No. 5,332,213 issued August 9, 1994 to D'Angelo et al.; U.S. Patent No. 5,336,168, issued August 9, 1994 to Sibalis; U.S. Patent No. 5,290,561, issued March 1, 1994 to Farhadieh et al; U.S. Patent No. 5,254,346, issued October 19, 1993 to Tucker et al; U.S. Patent No. 5,164,189, issued November 17, 1992 to Berger et al; U.S. Patent No. 5,163,899, issued November 17, 1992 to Sibalis; U.S. Patent Nos. 5,088,977 and 5,087,240, both issued February 18, 1992 to Sibalis; U.S. Patent No. 5,008,1 10, issued April 16, 1991 to Benecke et al; and U.S. Patent No. 4,921 ,475, issued May 1, 1990 to Sibalis, the disclosure of each of which is incorporated herein by reference in its entirety. It can be readily appreciated that a transdermal route of administration may be enhanced by use of a dermal penetration enhancer, e.g., such as enhancers described in U.S. Patent No. 5,164,189 {supra), U.S. Patent No. 5,008,110 {supra), and U.S. Patent No. 4,879,119, issued November 7, 1989 to Aruga et al, the disclosure of each of which is incorporated herein by reference in its entirety.

[0066] Pulmonary Delivery. Also contemplated herein is pulmonary delivery of the pharmaceutical compositions of the present invention. A pharmaceutical composition of the present invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of this include Adjei et al.

[Pharmaceutical Research, 7:565-569 (1990); Adjei et al., InternationalJournal of Pharmaceutics, 63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine, Vol. Ill, pp. 206-212 (1989) (αl -antitrypsin); Smith et al, J. Clin. Invest., 84:1145-1146 (1989) (α-1 -proteinase); Oswein ef al., "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (1990) (recombinant human growth hormone); Debs et al., J. Immunol., 140:3482-3488 (1988) (interferon-γ and tumor necrosis factor alpha); Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor)]. A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Patent No. 5,451,569, issued September 19, 1995 to Wong et al.

EXAMPLES

[0067J The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used {e.g.,. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. All publications referred to herein are specifically incorporated by reference in their entirety.

Example 1

[0068] Protein Sample Preparation. The bromodomain of CBP (residues 1082-1197) was expressed in Escherichia coli BL21(DE3) cells in the p ET 15b vector (Novagen) as described previously. Isotope-labeled proteins were prepared from cells grown on a minimal medium containing ¹⁵ NH ₄ Cl with or without ^l3 C ₆ -glucose in H ₂ O. The protein was purified by nickel-

IDA affinity chromatography, followed by thrombin cleavage to remove an N-terminal poly-His- tag. GST-fusion bromodomain of CBP (residues 1082-1197) was expressed in E. coli in the pGEX4T3 vector (Pharmacia), and purified with a glutathione-sepharose column.

[0069] Chemical Screening by NMR Spectroscopy. All spectra were recorded at 25°C on a Bruker 500 or 600 HMz NMR spectrometer. The NMR data were processed and analyzed using NMRPipe and NMRView (Delaglio et al., (1995) J Biomol NMR 6, 277-293; Johnson et al., (1994) Journal of Biomolecular Nmr 4, 603-614). Typical protein solution for NMR study contained the CBP BRD of 0.2 mM in 100 mM phosphate buffer, pH 6.5, containing 5 mM perdeuterated DTT and 10% ² H ₂ O. All chemical stocks are prepared at —0.35 M in predeuterated DMSO. Initial protein and compound binding by NMR was conducted with mixtures of eight compounds, and each compound was 1-2 mM and deuterated DMSO was —3% (v/v). The ligand binding was detected by observing ligand-induced chemical shift perturbations of residues of the ^{l 5} N-labeled protein recorded in 2D ¹ H- ¹⁵ N-HSQC spectra. The positive mixtures were deconvoluted by screening individual compounds to identify the active compound. Deuterated DMSO in the latter protein/ligand solution was —0.8% (v/v), and a reference spectrum of the protein with DMSO alone with the corresponding concentration was collected for data analysis.

[0070] Protein Structure Determination by NMR. NMR samples contained the CBP BRD (0.5 mM) in complex with a chemical ligand MS7972 (—2 mM) in 100 mM phosphate buffer of pH 6.5, containing 5 mM perdeuterated DTT and 0.5 mM EDTA in H ₂ O/ ² H ₂ O (9/1) or ² H ₂ O. AU NMR spectra were acquired at 30 ⁰ C on a Bruker 500 or 600 MHz NMR spectrometer. The backbone ¹ H, ¹³ C and ¹⁵ N resonances were assigned using 3D HNCACB and HN(CO)CACB spectra. The side-chain atoms were assigned from 3D HCCH-TOCSY and (H)C(CO)NH-

TOCSY data. The NOE-derived distance restraints were obtained from ¹⁵ N- or ^{l 3} C-edited 3D NOESY spectra. The ³ J _HN1Ha coupling constants measured from 3D HNHA data were used to determine cA-angle restraints. Slowly exchanging amide protons were identified from a series of 2D ¹⁵ N-HSQC spectra recorded after H ₂ O/ ² H2O exchange. The intermolecular NOEs used in defining the structure of the CBP bromodomain/ligand complex were detected in ^l3 C-edited (F _/ ), ^I3 C/ ^I5 N- filtered (Fj) 3D NOESY spectra (Clore et al., (1994) Meth. Enzymol. 239, 249-363). Protein structures were calculated with a distance geometry-simulated annealing protocol with X-PLOR (Brunger (1993) X-PLOR Version 3.1: A system for X-Ray crystallography and NMR, version 3.1 Edition (New Haven, CT: Yale University Press). Initial structure calculations were performed with manually assigned NOE-derived distance restraints. Hydrogen-bond distance restraints, generated from the H/D exchange data, were added at a later stage of structure calculations for residues with characteristic NOEs. The converged structures were used for iterative automated NOE assignment by ARIA for refinement (Nilges et al., (1998) Prog. NMR Spectroscopy 32, 107-139). Structure quality was assessed by Procheck-NMR (Laskowski et al., J. Biomol. NMR 8, 477-486). The structure of the protein/ligand complex was determined using intermolecular NOE-derived distance restraints.

[0071] Calculation of Electrostatic Potential. The DelPhi program was used to calculate electrostatic potential by solving the linear form of the PBE (Nicholls et al. (1991) Journal of Computational Chemistry 12, 435-445). Manipulation of potential map was done using Delphi viewer module of UCSF Chimera (Pettersen et al., (2004) Journal of Computational Chemistry 25, 1605-1612) and Grasp (Nicholls et al. (1993) Biophys. J. 64, 166-170). The van der Waals redii and atomic charges were taken from CHARMM22 parameter set. The program parameters

used were as follows: interior dielectric 2, exterior dielectric 80, solvent probe radius 1.4A and ionic strength 0.150 M.

[0072] Computational Docking. Docking of ligands to the CBP BRD was performed using AutoDock v3.0.5 which uses a genetic algorithm as a global optimizer combined with energy minimization as a local search method. In this method the target protein is kept to be rigid and represented as grid while torsional flexibility is allowed in the ligand. Mass-centered (blind- docking) as well as binding-site centered affinity grid maps were generated with 0.375A spacing using the Autogrid program for the protein target. Blind-docking was used to evaluate the accuracy of prediction of correct binding site, which was determined based on NMR binding results. The Lamarckian genetic algorithm (LGA) and pscudo-Solis and ^" Wets method were used for minimization. Default parameters for Autodock calculations were used except for what otherwise stated. Particularly, for blind docking, each grid map consisted of a 126x126x126 grid with the centre of the map assigned to the geometric centre of the protein. Each LGA job consists of 50 runs with 270,000 generations in each run and maximum number of energy evaluations set to 2.5x10 . Moreover, for binding-site centred docking, a 62x76x80 grid was generated with each LGA job that consisted of 200 runs with 270,000 generations in each run and maximum number of energy evaluations of 5.OxIO ⁶ . Resulting docked orientations within 1.0 A RMSD tolerance of each other were clustered together. Docked conformations were analyzed using AutoDockTools (http://www.scripps.edu/~sanner) and LIGPLOT (Wallace et al., (1995) Protein Eng <5, 127-134).

[0073] Structures of chemical ligands were generated using molecular modeling software

SYBYL v6.7 (Tripos Associated, Inc. St. Louis, MO). Partial atomic charges were assigned

using the Gasteiger-Marsili method (Gasteiger et al., (1980) Tetrahedron 36, 3219-3228). The program Autotors was used to define torsional degrees of freedom in ligands. All the possible torsions were allowed. The coordinates used in Autodock calculation were taken from the first model of NMR structures of CBP bromodomain/p53-AcK382 peptide complex (PDB IJSP). For the docking purpose, the peptide coordinates were removed. Kollman united-atom partial charges and solvation parameters were added to protein file using AutoDockTools.

[0074J Electron Current Density Surface Calculation. Electron current density surface (J- surface) calculation was performed using the program, Jsurf, originally developed by McCoy and co-workers and latter modified and kindly provided by G. Moyna (The University of the Sciences in Philadelphia). This program quantifies effect of spatial dependence of aromatic ring current field (from a ligand) on local magnetic fields of neighboring spins at the ligand binding site within the target protein. Assuming chemical shift perturbation observed at a protein proton i, δ,, where δ ₍ = δ _b ' _ound — δ _f ' _ree , is generated mainly by aromatic ring current effect from the ligand,

the origin of δ, can be approximated by a single point-dipole located at the centre of the ligand

aromatic ring, and therefore δ, , where R; is the length of the vector from the centre of the ligand ring to the perturbed protein atom i, θ; is angle between the ring plane normal and R ₁ , and Bd _φ is a proportionality constant. The calculation of J-surface requires an input file containing absolute change in chemical shift for backbone amide proton of perturbed residues. Only residues with δ |0.05| were used for calculation. The δi information was translated into the ligand localization using spherical dot density representation, done by the program Jsurf.

[0075] MD Simulation. MD simulation of the CBP BRD was done using Gromacs 3.2.1 and GROMOS96 (v. 43al) force field (Lindahl et al., (2001) Journal of Molecular Modeling 7, 306- 317; van Gunsteren et al., (1996). The GROMOS96 manual and user guide (Zurich, Switzerland: Hochschulverlag AG an der ETH Zurich). As starting geometry, first model of NMR structures of the CBP BRD (PDB USP; without the peptide) was used. The protein was centered in a periodic cubic simulation box with a minimum protein-box distance of 1.0 nm, and a volume of 351.03 nm ³ . The box was filled with 10,969 SPC water molecules (Berendesen et al., (1981) Intermolecular Forces (Dordrecht, The Netherlands: Reidel). One Na ⁺ was added to ensure charge neutrality of simulation cell. The MD protocol used the LINCS method (Hess et al., (1997) Journal of Computational Chemistry 18, 1463-1472) to constrain covalent bond lengths. Temperature and pressure were kept constant separately by coupling the protein, ions and solvent to external temperature and pressure baths with respective coupling constant (τ) of 0.1 ps and 0.5 ps. The reference temperature was adjusted to 300 K. To relax the solvent configuration, a steepest descent minimization was adopted. The following step was position-restrained dynamics, which restrains atom positions of the protein while letting the solvent move in the simulation box to reach equilibrium before a full molecular dynamics simulation starts. Long- range electrostatics is calculated with the Particle-Mesh Ewald (PME) method (Essmann et al., (1995) Journal of Chemical Physics J 03, 8577-8593; Darden et al., (1993) Journal of Chemical Physics 98, 10089-10092). The MD time step was set to be 0.002 ps. After equilibration, the simulation time used was 5 ns.

[0076] Estimation of Backbone Amide S ² Order Parameters from the Protein Structure.

The S ² order parameter of protein backbone amide vectors was calculated from the protein 3D structure using an analytical relationship. The method relates S ² of amide vector of residue i to close contacts experienced by the amide proton and carbonyl oxygen of the preceding residue M with heavy atoms k:

where rP _{l k} is the distance between the carbonyl oxygen of residue /-1 to the heavy atom k, and

r" _k is the distance between the amide proton and the heavy atom k. The parameter b was set to -

0.1 considering that the value of order parameter for rigid protein region typically is about 0.9. A python-based script uses MMTK (Hinsen (2000) Journal of Computational Chemistry 21, 79-85) was used for the calculation (http://niTir.clarlcu.edu/software/S2/s2predict.html).

[0077] Fluorescence Binding Experiment. Fluorescence measurements were performed on ISS PCl photon counting spectro-fluorometer at room temperature. The concentration of the protein (calculated using the theoretical absorption coefficient of 24,750 M ^" 'cm ^' ' at 280 nm) was 5 μM in 100 mM phosphate buffer, pH 6.5, containing 5 mM DTT. Protein intrinsic fluorescence was measured at an excitation wavelength of 295 nm and emission was collected from 300-500 nm using 8nm band passes for both excitation and emission. The protein sample was titrated with a ligand MS7972 to a final concentration of 80 μM with 0.7 % final dilution.

[0078] In vitro Protein-Peptide Binding Assay. GST-fusion CBP BRD (10 μM) was incubated with an N-terminal biotinylated p53 AcK.382 peptide (50 μM) in a 50 mM Tris buffer, pH 7.5, containing 50 mM NaCl, 0.1% BSA and 1 mM DTT at 22 ⁰ C for 2 hours. Streptavidin-agarose

beads were added to the mixture and washed in the Tris buffer containing 500 mM NaCl and 0.1% NP-40. The CBP BRD eluted from the beads was run on SDS-PAGE, and visualized in western blots by anti-GST antibody and horseradish-peroxidase-conjugated goat anti-rabbit IgG. Small-molecule inhibition assay was performed by incubating the CBP BRD and the biotinylated p53 AcK382 peptide with increasing amount of small-molecule compound.

[0079] p53 Expression and Functional Assays. Wild-type p53 expressing U2OS cells were either incubated with DMSO or treated with 20 or 200 μM of various small-molecule compounds for about 16 hours. The cells were further incubated with 0.1 μg/ml doxorubicin for a specified time of 2 to 24 hours, and then cell lysates were subjected to immunoblotting analysis using specific antibodies for p53, phosphorylated Serl5 of p53, acetylated Lys382 of p53, ρ21 or Ku70. For the up-regulation of HIFl α, U2OS cells were incubated in the absence or presence of 200 μM of MS2126 for 16 hours, and then further incubated in either normoxic or hypoxic conditions for an additional 24 hours. Cell lysates were then subjected to immunoblotting with the specific antibodies for HIFl α and Ku70.

Example 2

Protection of mammalian cells from UV radiation induced cell death/apopotosis

[0080] Radiation therapy is a widely used clinical treatment of human cancers including locally advanced cervix, lung, head and neck, rectal, esophagus, anal and prostate cancers, and it compares favorably to radical prostatectomy (DeVita, et ai, Cancer J, 2001. 7 Suppl I: S2-13; D'Amico, et al, JAMA, 1998. 280(1 1):969-74). However, high dose of ionizing radiation (IR) treatment often required for effective treatment often cause serve damage of normal issues via IR-induced cell death. As such, it is believed that concomitant chemotherapy and IR that would work synergistically should improve outcome compared to IR alone. It is well established that the human tumor suppressor p53 plays a pivotal role in directing a cell's response to extra-

cellular stress signals (See, Verdonk, et al., J MoI Biol, 2001. 307(3): 841-59; Levine, A. JCe//, 1997. 88:323-31 ; Prives, C. et al., J. Pathol, 1999. 187: p. 112-126; Ko, LJ. et al. Genes Dev., 1996. 10: p. 1054-1072). Under severe UV radiation treatment that results in cellular DNA damage, for instance, p53 directs cells to undergo cell cycle arrest or programmed cell death. A chemical agent that is capable of down-regulating p53's transcriptional activation could inhibit p53-induce programmed cell death. This idea was tested with a DUl 45 prostate cancer cell line using two lead compounds MS7972 and NS5557, which have been shown to inhibit lysine 382- acetylated p53 interaction with the bromodomain of the transcriptional co-activator CBP (Mujtaba, S., et al., MoI Cell, 2004. 13(2):251-63 and Sachchidanand, et al., Chem Biol, 2006. 13(l):δl-90). As shown in Figure 6, in a clonogenic assay, the percent of colonies surviving 2 Gy of ionizing radiation for DU145 cells is about 65%, whereas in the presence of MS7972 or MS5557 (either at 20 or 200 μM), the cell survival rate was significantly increased to about 85- 89%. Although DU- 145 has a mutation in p53, published data demonstrate that DUl 45 derived mutants are capable of inducing p21 after UV-induced DNA damage, that DNA damage agents etoposide and doxorubicin can induce p53 -dependent apopotosis and that DU145 mutations do not have a dominant-negative effect on p53 function (Gurova, K. V., et al., Cancer Res, 2003. 63(11):2905-12). These p53 inhibitors are also effective in additional cell lines including a wild- type p53 cell line LnCAP and a p53 deleted cell line PC-3. The p53 inhibitors capable of modulating p53 function in gene transcription activation are especially effective as therapeutic agents in combined chemotherapy or radiation therapy to minimize the deleterious effects of radiation damage to normal issues in cancer treatment.

[0081] A cell survival test by ionizing radiation treatment was performed using a procedure as described in a recently published study (Taneja, et al., J Biol Chem, 2004. 279(3):2273-80; Fernandez-Capetillo, et al., Nat Cell Biol, 2002. 4(12):993-7). Briefly, the DU145 cells were cultured in culture dishes and grown on glass coverslips in 24 well plates to about 50% confluence before irradiation. Ionizing radiation was performed with a ¹³⁷ Cs γ irradiator at 1.5 Gy/min to a dose ranging from 0-2 Gy. Cell death was measured by a clonogenic survival analysis using a colony formation assay that counts and compares cell colonies before and after IR treatment. Typically, at least assays were performed to assess relative radio-sensitivity of these cells.

Example 3

[0082] Muscle wasting (cachexia) is a severe complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery. As a key inflammatory cytokine associated with cachexia, tumor necrosis factor-alpha (TNFα) inhibits myogenic differentiation and skeletal muscle regeneration through downstream effectors of the p53-directed cell death pathway including PWl/Peg3, bax, and caspases (Figure 7). Recent data demonstrate that p53 is required for the TNFα-mediated inhibition of myogenesis in vitro and contributes to muscle wasting in response to tumor load in vivo, and suggests a novel role for p53 in mediating muscle stem cell behavior and muscle atrophy (Schwarzkopf, M., et al, Genes Dev, 2006. 20(24):3440- 52). We tested the effects of the p53 inhibitors on p53's function in TNFα inhibition myogenic differentiation in a myoblast cell cline C2C12. As illustrated in Figure 7, the myoblast cells undergo differentiation after shifting to low serum-medium within 2-4 days (top left panel). This process can be inhibited by TNFα (top left panel). Because the TNFα function is ρ53-deρendent, blocking p53 activity with the known p53 inhibitor Pifithrin allows C2C12 cells to differentiate also in the presence of TNFα. The p53 inhibitor MS7972, but not MS2126, could also effectively reduce the TNFα inhibition of myogenic differentiation of the myoblast cell line C2C12 by as much as 50% at 100 μM of the compound (Figure 8). This data is consistent with the previous observation that the compound MS7972 is capable of down-regulating p53's transcriptional activation in cells by blocking lysine 382-acetylated p53 interaction with the bromodomain of the co-activator CBP upon DNA damage induced by UV radiation or doxorubicin treatment (Ko, et al, Genes Dev., 1996. 10:1054-1072). Taken together, the p53 inhibitors of the instant invention could be beneficial for the therapeutic treatment of muscle wasting that is associated with chronic infection and cancers.

[0083] Differentiation studies were performed using procedures as described by Schwarzkopf, et al., Genes Dev, 2006. 20(24): p. 3440-52. Specifically, myogenic differentiation of C2C12 myoblast cell lines that were maintained in high-serum medium (GM) low-scrum medium within 2-4 days was performed (Megeney, et al., Genes Dev, 1996. 10(10):l 173-83; Coletti, et al, Genesis, 2005. 43(3): 120-8). Cells were then plated on gelatin-coated coverslips and treated for

24 hours with 30 μM Pifithrin (Calbiochem) or 50- 100 μM of the p53 compound. TNFα (Roche

Molecular Biochemicals) used in the low-serum medium was present at about 20 ng/mL. DMSO was used in the control experiment. The morphological changes of the C2C 12 cells were evaluated for cell differentiation under the microscope (Figure 7). Quantitative analysis of myogenic differentiation was performed by determining the number of nuclei in MF20-positive cells and expressing this as a percentage of the total number of nuclei in multiple microscopic fields (% differentiation) (Figure 8) (Schwarzkopf, et al, Genes Dev, 2006. 20(24):3440-52; Coletti, et al, Embo J, 2002. 21(4):631-42). Typically, 150-300 cells from randomly chosen fields in three independent experiments were analyzed.