WO2006083692A2 - Methods of identifying modulators of bromodomains - Google Patents

Methods of identifying modulators of bromodomains Download PDF

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WO2006083692A2
WO2006083692A2 PCT/US2006/002840 US2006002840W WO2006083692A2 WO 2006083692 A2 WO2006083692 A2 WO 2006083692A2 US 2006002840 W US2006002840 W US 2006002840W WO 2006083692 A2 WO2006083692 A2 WO 2006083692A2
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och
group
hydrogen
lower alkyl
aralkyl
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Ming-Ming Zhou
Lei Zeng
Zhiyong Wang
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Mount Sinai Schoool Of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/16—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/26—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing at least one six-membered aromatic ring
    • C07C211/29—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by halogen atoms or by nitro or nitroso groups
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/43—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C211/44—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring
    • C07C211/53—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring having the nitrogen atom of at least one of the amino groups further bound to a hydrocarbon radical substituted by amino groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/14—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring
    • C07C217/18—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring or condensed ring system containing that ring being further substituted
    • C07C217/20—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring or condensed ring system containing that ring being further substituted by halogen atoms, by trihalomethyl, nitro or nitroso groups, or by singly-bound oxygen atoms
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/18—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/40—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/42—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton with carboxyl groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by saturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/49—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C255/58—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/18—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted otherwise than in position 3 or 7

Definitions

  • the present invention provides novel compounds that interact with a histone acetyltransferase bromodomain.
  • the interaction between bromodomains and their binding partners play a crucial role in various cellular functions, including in the regulation/modulation of DNA transcription. Therefore, the present invention provides agents that can modulate the interaction of bromodomains and their binding partners.
  • intercellular signal transduction generally begins with an intercellular ligand binding the extracellular portion of a receptor of the plasma membrane. The bound receptor then either directly or indirectly initiates the activation of one or more cellular factors.
  • An activated cellular factor may act as transcription factor by entering the nucleus to interact with its corresponding genomic response element, or alternatively, it may interact with other cellular factors depending on the complexity of the process. In either case, one or more transcription factors ultimately bind to one or more specific genomic response elements.
  • Chromatin contains several highly conserved histone proteins including: H3, H4, H2A, H2B, and Hl. These histone proteins package eukaryotic DNA into repeating nucleosomal units that are folded into higher-order chromatin fibers [Luger and Richmond, Curr. Opin. Genet. Dev. 8:140-146 (1998)]. A portion of the histone that comprises roughly a quarter of the protein protrudes from the chromatin surface, and is thereby sensitive to proteolytic enzymes [van Holde, in Chromatin (Rich, A 5 . ed., Springer, New York ) pagesl 11-148 (1988); Hect et al, Cell 80:583-592 (1995)].
  • Histone tails tend to be free for protein-protein interaction, and are also the portion of the histone most prone to post-translational modification.
  • post-translational modification includes acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation [van Holde, in Chromatin (Rich, A,, ed., Springer, New York ) pagesl 11-148 (1988)].
  • histones are amongst the most susceptible to post- translational modification. Perhaps the best studied post-translational modification of histones is the acetylation of specific lysine residues [Grunstin, M., Nature, 389:349-352 (1997)]. Indeed, acetylation of histone lysine residues has been suggested to play a pivotal role in chromatin remodeling and gene activation. Consistently, distinct classes of enzymes, namely histone acetyltransferases (HATs) and histone deacetylases (HDACs), acetylate or de-acetylate specific histone lysine residues [Struhl, Genes D ev. 12:599-606 (1998)].
  • HATs histone acetyltransferases
  • HDACs histone deacetylases
  • bromodomains are found in a large number of chromatin-associated proteins and have now been identified in approximately 70 human proteins, often adjacent to other protein motifs [Jeanmougin et al, Trends in Biochemical Sciences, 22:151-153 (1997); Tamkun et al, Cell, 68:561-572 (1992): Hanes et al, Nucleic Acids Research, 20:2603 (1992)]. Proteins that contain a bromodomain often contain a second bromodomain. However, despite the wide occurrence of bromodomains and their likely role in chromatin regulation, their three-dimensional structure and binding partners heretofore have remained unknown.
  • the bromodomain, present in chromatin associated proteins and histone lysine acetyltransferases, 6a is an acetyl-lysine binding domain.
  • 6b Bromodomain/ AcK binding plays an important role in control of chromatin remodeling and gene transcription.
  • 60 BRDs adopt the highly conserved structural fold of a left-handed four- helix bundle ( ⁇ Z, ⁇ A, ⁇ B and ⁇ C), as first shown in the PCAF BRD (Zeng, et al, FEBS Letters (2002) 513, 124-8) ( Figure IA).
  • the ZA and BC loops at one end of the bundle form a hydrophobic pocket for AcK binding.
  • the structure of the PCAF BRD bound to a Tat-AcK50 peptide shows that AcK50 interacts with protein residues V752, Y802 and Y809, Y47(AcK-3) with V763, and R53(AcK+3) and Q54(AcK+4) with E756, conferring a specific intermolecular association.
  • the structures of CBP BRD/ p53-AcK382 and GCN5p BRD/H4-AcK16 complexes show that the residues in BRDs important for AcK recognition are largely conserved, whereas sequence variations in the ZA and BC loops enable discrimination of different binding targets (Mujtaba, et al, MoI Cell. (2004) 13, 251-63).
  • the ZA and BC loops contain significant sequence variations with amino acid deletion or insertion, supporting the notion that different sets of residues in the ZA and/or BC loops dictate BRD ligand specificity by interacting with residues flanking the acetyl-lysine in a target protein.
  • HIV human immunodeficiency virus
  • the current anti-HIV drugs specifically target the viral reverse transcriptase, protease and integrase (Garg, R et al, Chem. Rev. (1999) 99, 3525-601)
  • continuous viral production by chronically infected cells contributes to HIV-mediated ' immune dysfunction (Ho, et al, Nature. Med. (2000) 6, 757-61; Wei et al, Nature (1995) 373, 117-22) and there is still no cure for AIDS.
  • a rapid growing AIDS epidemic calls for new therapeutic strategies targeting different steps in the viral life cycle.
  • Tat binds to a viral RNA TAR and recruits cyclin Tl and cyclin-dependent kinase 9 that hyper-phosphorylates and enhances elongation efficiency of the RNA polymerase II (Keen et al., J. EMBO. J. (1997) 16, 5260-72; Karn, J. MoL Biol. (1999) 293, 235-54; Jones, Genes. Dev. (1997) 11, 2593-99; Kao, et al., Nature (1987) 330, 489-93).
  • Tat transactivation requires acetylation of its lysine 50 and recruitment of histone lysine acetyltransferase transcriptional coactivators for remodeling micleosome that contains the integrated proviral DNA (Ott et al., Curr. Biol. (1999) P 5 1489-92).
  • Tat coactivator recruitment requires its acetylated lysine 50 (AcK50) binding to the bromodomain (BRD) of the coactivator PCAF (Mujtaba, et al., MoL Cell. (2002) 9, 575-86), and microinjection of anti-PCAF BRD antibody blocks Tat transactivation (Dorr et al., EMBO. J. (2002) 21, 2715-33).
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2; NO 2j SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH 1 NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy; and
  • ii is an integer from O to 10;
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R 1 may be selected from the group consisting of hydrogen, lower alkyl, phenyl, and CN.
  • R 2 may be selected from the group consisting OfNH 3 + , carboxy, and alkoxy.
  • X may be selected from the group consisting of lower alkyl, NH, CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , and OH.
  • n may be 3.
  • the present invention features compounds of the following general formula(II) wherein:
  • R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH 2 , NH 3 NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R is selected from the group consisting of lower alkyl, aryl, SO2, NH,N ⁇ 2, , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH 3 , OH, SH, and halogen. Also iinn eess]pecially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
  • the present invention features compounds of the following general formula(III) wherein
  • R 1 R 2 , R 3 , R 4 R 5 , and R 6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 2 , NH 3 + , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2) NH(CH 2 ) 3 N(CH 3 ) 2 , NH(CH 2 ) 2 N(CH 3
  • R 2 is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 ,
  • R 3 is selected from the group consisting of hydrogen, OCH 2 CH 3 , and NHCOCH 3 ;
  • R 5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH 2 ) 3 N(CH 3 ) 2, NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3 , and NHCH 3 ; and
  • R 6 is selected from the group consisting of hydrogen, and NH 2.
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the present invention provides novel compounds that inhibit the binding of bromodomains to acetyl-lysine residues of proteins.
  • the present invention makes use of the three-dimensional structure of a bromodomain as well as the three-dimensional structure of a bromodomain-acetyl-histamine complex.
  • the present invention features a pharmaceutical composition comprising a compound of Formula I wherein
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NB 2 , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH 1 NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , and OH, carboxy, and alkoxy; and
  • n is an integer from O to 10;
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula (II) wherein:
  • R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH3 NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R is selected from the group consisting of lower alkyl, aryl, SO 2 , NH, NO 2 , , CH 3 , CH2CH3, OCH 3 , OCOCH 3 , CH2COCH3, OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH 3 , OH, SH, and halogen. Also iinn eessjpecially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula (III) wherein
  • R R 5 R 3 R R R , and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 2 ,NH 3 + , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ).:, OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CHS) 2 , NH(CH 2 ) 3 N(CH 3 )2, NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2
  • R 1 and R 4 are selected from the group consisting of hydrogen and OH;
  • R 2 is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2> OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 ,
  • R 3 is selected from the group consisting of hydrogen, OCH 2 CH 3, and NHCOCH 3 ;
  • R 5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH 2 ) 3 N(CH 3 ) 2j NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3 , and NHCH 3 ; and
  • R 6 is selected from the group consisting of hydrogen, and NH 2 .
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2, NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN and halogen;
  • R is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2, NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH 1 NO 2 , , O, carboxy, and alkoxy;
  • n is an integer from O to 10;
  • the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
  • R 1 R 2 and R 3 are independently selected from the group consisting of hydrogen; lower allcyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R 4 is selected from the group consisting of lower alkyl, aryl, SO 2 , NH 1 NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture 1 9 * ⁇ or in isolated form.
  • R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NHb , OH, SH, and halogen. Also in especially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
  • the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
  • R 1 R 2 , R 3 , R 4 R 5 , and R 6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 25 NH 3 + , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH2COCH3, OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 35 OCH2CONH2, OCOCH(CHs) 2 , OCH2CH2OH, OCH2CH2CH3, O(CH 2 ) 3 CH 3 , OCHCHsCOOCH 3 , OCH 2 CON(CHS) 2 , NH(CH 2 ) 3 N(CH 3 ) 2 , NH(CH 2 )2N(CH 3 )2 , NH(CHz) 2
  • R and R are selected from the group consisting of hydrogen and OH;
  • R is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CHs) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH2CONH2, OCOCH(CHs) 2 , OCH2CH2OH, OCH 2 CH 2 CH 3 , 0(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 )2;
  • R 3 is selected from the group consisting of hydrogen, OCH 2 CH 3 , and NHCOCH 3 ;
  • R 5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH 2 ) 3 N(CH 3 ) 2) NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3) and NHCH 3 ; and
  • R 6 is selected from the group consisting of hydrogen, and NH 2 .
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2 , NO 2 , SO 2 , O, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, arallcyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 21 NH 3 + NO 2 , SO 2 , O, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH, NO 2, , O, carboxy, and alkoxy;
  • n is an integer from O to 10;
  • the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
  • R 1 R 2 and R 3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R 4 is selected from the group consisting of lower alkyl, aryl, SO 2 , NH, NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R 1 R 2 and R 3 are independently selected from the group consisting of hydrogen, lower alkyl, NH 3 + , OH, SH, and halogen.
  • R 4 is selected from the group consisting of lower alkyl and aryl.
  • the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
  • R R 2 , R 3 , R R 5 , and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 21 NH 3 + , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2 , NH(CH 2 ) 3 N(CH 3 ) 2 , NH(CH 2 ) 2 N(CH 3 ) 2
  • R and R are selected from the group consisting of hydrogen and OH;
  • R is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CHs) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CHs) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 )sCHs, OCHCH 3 COOCH 3 , OCH 2 CON(CHs) 2 ;
  • R is selected from the group consisting of hydrogen, OCH 2 CH 3 , and NHCOCHs
  • R is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH 2 )sN(CH 3 ) 2 ,NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3 , and NHCH 3 ; and
  • R is selected from the group consisting of hydrogen, and NH 2 .
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the viral infection is HIV infection.
  • Fig. 1 Structure-based sequence alignment of a selected number of bromodomains. The sequences were aligned based on the NMR-derived structure of the P/CAF bromodomain, and the predicated four ⁇ -helices are shown in green boxes. Bromodomains are grouped on the basis of the sequence and/or functional similarities as described by Jeanmougin et ah, [Trends in Biochemical Sciences, 22:151-153 (1997)]. Residue numbers of the P/CAF bromodomain are indicated above its sequence. Three absolutely conserved residues, corresponding to Pro751, Pro767, and Asn803 in the P/CAF bromodomain, are shown in red. Highly conserved residues are colored in blue.
  • the residues of the P/CAF bromodomain that interact with acetyl-histamine, as determined by intermolecular NOEs, are indicated by asterisks.
  • the ZA loop, which is critical for acetyl-lysine binding, for each of the indicated bromodomains is also identified.
  • the underlined residues were changed individually by site-directed mutagenesis to Ala.
  • Figs. 2A-2H depict the structure of the P/CAF bromodomain.
  • Figs. 2A-2B shows the stereoview of the C ⁇ trace of 30 superimposed NMR-derived structures of the bromodomain (residues 722-830).
  • the N-terminal four residues (SKEP) which are structurally disordered are omitted for clarity.
  • the root-mean-square deviations (RMSDs) of the backbone and all heavy atoms are 0.63 ⁇ O.l lA and 1.15 ⁇ 0.12A for residues 723-830, respectively.
  • Figs. 2C-2D show the stereoview of the bromodomain structures from the bottom of the protein, which is rotated approximately 90° from the orientation in Figures 2A-2B.
  • Fig. 2E shows the Ribbons [Carson, M., J. Appl. Q ⁇ stallogr. 24:958-961 (1991)] depiction of the averaged minimized NMR structure of the P/CAF bromodomain.
  • the orientation of Fig. 2E is as shown in Figures 2A-2B.
  • Figures 2F-2G are schematic representations of the overall topology of the up-and-down four-helix bundle folds with the opposite handedness.
  • the left-handed fold is seen in bromodomain, cytochrome & 5 , and T4 lysozyme (left, Fig. 2F), whereas the right-handed four- helix bundles are observed in proteins such as hemerytlirin and cytochrome O 562 (right, Fig. 2G) [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. Sd. USA 86:6592-6596 (1989)].
  • the hydrophobic and aromatic residues (Tyr809, Tyr802, Tyr760, Ala757, and Val752) located between the ZA and BC loops are indicated.
  • Figs. 3A-3C show the binding of the P/CAF bromodomain to AcK.
  • Fig. 3A shows the superimposed region of the 2D 15 N-HSQC spectra of the bromodomain (approximately 0.5 mM) in its free form (red) and complexed to the AcK-containing H4 peptide (molar ratio 1 :6) (black).
  • Fig. 3B is the Ribbon and dotted-surface diagram of the bromodomain depicting the location of the lysine-acetylated H4 peptide binding site.
  • the color coding reflects the chemical shift changes ( ⁇ ) of the backbone amide 1 H and 15 N resonances upon binding to the AcK peptide as observed in the 15 N-HSQC spectra.
  • the backbone atoms are color-coded in red, yellow, or green for residues that have ⁇ m / ⁇ max of >0.6 (Tyr809, Glu808, Asn803, and Ala757), 0.2-0.6 (Ala813, Tyr802, Tyr760, and Val752), or ⁇ 0.2 (Cys812, Ser807, Cys799, Phe796, and Phe748), respectively.
  • the non-perturbed residues are shown in blue.
  • Fig. 3 C shows the chemical structures of acetyl-lysine, acetyl-histamine, and acetyl- histidine.
  • Fig. 4 depicts the acetyl-lysine binding pocket. This is the Ribbons [Carson, M., J. Appl. Cryistallogr. 24:958-961 (1991)] depiction of a portion of the P/CAF bromodomain complexed with the acetyl-histamine. The ligaiid is color-coded by atom type.
  • Fig. 5A-5B show the binding of various bromodomains from P/CAF, CBP and TIFIb to the N-terminal biotinylated and lysine-acetylated Tat peptide that was immobilized on streptavidin agarose.
  • Fig. 6A-6D shows the lysine-acetylated HIV-I Tat protein interactions with bromodomains using 2D 1H-15N-HSQC spectra of the P/CAF or CBP bromodomain in the presence (red) or absence (black) of the lysine-acetylated peptides.
  • Binding of the P/CAF bromodomain to the Tat AcK 50 peptide SYGR-AcK-KRRQRC (SEQ ID NO:50) is shown in Fig. 6A
  • to the Tat AcK 28 peptide TNCYCK-AcK-CCFH SEQ ID NO:58
  • Fig. 6B shows histone H4 AcK16 peptide S GRGKGGKGLGKGGA- AcK-RHRK (SEQ ID NO:59) is shown in Fig. 6C.
  • Fig. 6D shows the binding of the CBP of the bromodomain to the Tat AcK50 peptide.
  • AcK is an acetyl-lysine residue
  • Fig. 7 is a bar graph of the measurement of superinduction of Tat transactivation activity by P/CAF.
  • Tat-KK is the wild type Tat protein
  • Tat-RR is the double mutant Tat carrying lysine to arginine mutations at K50 and K51 positions.
  • Figs. 8A-8B show a western blot assay to detect P/CAF interaction with the Tat protein. Note that the protein-protein interaction was only observed with the wild type Tat but not with the Tat K50R/K51R mutant protein. The FLAG was joined to the Tat peptide, whereas the HA-tag was joined to P/CAF.
  • Fig. 9 depicts the structure of the P/CAF bromodomain in the complex with the lysine-acetylated Tat peptide (SYGR-AcK-KRRQRC, SEQ ID NO:50, where AcK is acetyl- lysine residue).
  • SYGR-AcK-KRRQRC SEQ ID NO:50, where AcK is acetyl- lysine residue.
  • the side chains of the amino acid residues on both the protein (green) and peptide (dark orange) that showed intermolecular NOEs in the NMR spectra are displayed.
  • Fig. 10A- 1OB shows the results of the mutational analyses of the P/CAF bromodomain binding to the HTV-I Tat.
  • Fig. 1OA shows the effects of the point mutation of the individual residues of the bromodomain to alanine on the protein binding to the lysine-acetylated Tat peptide.
  • Fig. 1OB is an assessment of the peptide residue mutation on its binding to the P/CAF bromodomain.
  • Fig. 11 depicts the chemical structure common to the acetyl-lysine analogs of the present invention.
  • R 1 , R 2 , and R 3 can be H, CH 3 , a halogen (e.g., F, Cl, Br, I etc.), OH, SH, or NH 3 + .
  • R4 can be an alkyl (including a peptide/protein attached thereto such as a peptide comprising an acetyl-lysine in which the "N" of the structure depicted is the epsilon nitrogen (i.e., N ⁇ of a lysyl residue), or an aryl group. See also Fig. 13 for examples.
  • Fig. 12 depicts examples of acetyl-lysine analogs.
  • Fig. 13 depicts inhibition of PCAF BRD/Tat AcK50 binding by 2.
  • 2 inhibits the biotinylated Tat AcK50 peptide immobilized on streptavidin agarose binding to the GST-PCAF BRD, as assessed by anti-GST Western blot.
  • Lower panel indicates an equal amount of BRD used in each assay.
  • Fig. 14 illustrates selective binding of the lead compound 1 by (A) the PCAF bromodomain but not (B) the CBP bromodomain and (C) the tandem PHD finger and bromodomain of TIFl ⁇ .
  • the 2D 1 H- 15 N HSQC spectra of the bromodomain show changes of backbone amide resonances of the protein in the presence (red) or absence (black) of the chemical ligand.
  • Fig. 15 represents the three-dimensional structure of the PCAF bromodomain, as determined by NMR.
  • A Superimposition of the backbone atoms (N, C ⁇ and C) of the final 20 NMR structures of the bromodomain in complex with the lead compound 2 (highlighted in red).
  • B Superimposition of the final representative structures of the bromodomain in the free (yellow) and when bound to the lead compound 2 (blue).
  • Fig. 16 demonstrates inhibition of Tat-mediated transactivation by the PCAF
  • FIG. 17 demonstrates the effect of PCAF BRD inhibitors on Tat transactivation.
  • HeLa cells were transfected with LTR-luciferase and 20 ng Tat-expression vector.
  • PCAF BRD inhibitors were added after transfection, and cells were harvested after 8 hours.
  • Fig. 18 demonstrates the effect of PCAF BRD inhibitors on viral infection.
  • T cells were infected with an LTR-Tat-IRES-GFP virus.
  • PCAF BRD inhibitors were added after overnight infection. The percentage of infection was monitored 48 hours later by FACS analysis.
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2 , NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH 1 NO 2, , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy; and
  • n is an integer from O to 10;
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R 1 may be selected from the group consisting of hydrogen, lower alkyl, phenyl, and CN.
  • R 2 may be selected from the group consisting OfNH 3 + , carboxy, and alkoxy.
  • X may be selected from the group consisting of lower alkyl, NH, CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , and OH.
  • n may be 3.
  • the present invention features compounds of the following general formula(II) wherein:
  • R 1 R 2 and R 3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R 4 is selected from the group consisting of lower alkyl, aryl, SO 2 , NH 1 NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R 1 R 2 and R 3 are independently selected from the group consisting of hydrogen, lower alkyl, NH 3 + , OH, SH, and halogen.
  • R 4 is selected from the group consisting of lower alkyl and aryl.
  • R R , R , R R 5 , and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 2 , NH 3 + , NO 2 , SO 2 , CH 3 , CH2CH3, OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH2CH3, OCH(CHs) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH2CONH2, OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH2CH2CH3, O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2 ,NH(CH 2 )SN(CH 3 ) 2 , NH(CH 2 ) 2 N(CH 3 )2 , NH(CH 2 )2 ⁇ H
  • R and R are selected from the group consisting of hydrogen and OH;
  • R is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH3, CH 2 COCH 3 , OCH 2 CH 3 , OCH(CHs) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CHs, O(CH 2 )sCHs, OCHCH 3 COOCH 3 , OCH 2 CON(CHs) 2 ;
  • R is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
  • R is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH 2 ) 3 N(CH 3 ) 2 ,NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CHs, and NHCH 3 ; and
  • R is selected from the group consisting of hydrogen, and NH 2 .
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the present invention provides novel compounds that inhibit the binding of bromodomains to acetyl-lysine residues of proteins.
  • the present invention makes use of the three-dimensional structure of a bromodomain as well as the three-dimensional structure of a bromodomain-acetyl-histamine complex.
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I wherein
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2 , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH 1 NO 2, , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , and OH, carboxy, and alkoxy; and
  • n is an integer from O to 10;
  • the present invention features a pharmaceutical composition comprising a compound of Formula (II) wherein:
  • R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH2, NH 3 NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R 4 is selected from the group consisting of lower alkyl, aryl, SO 2 , NH, NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture
  • R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH 3 , OH, SH, and halogen. Also in especially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula (III) wherein
  • R 1 R 2 , R 3 , R 4 R 5 , and R 6 are independently selected from the group consisting of hydrogen; lower allcyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 2 , NH 3 + , NO 2, SO 2, CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2; NH(CH 2 ) 3 N(CH 3 ) 2 , NH(CH 2 ) 2 N(CH 3 ) 2
  • R 1 and R 4 are selected from the group consisting of hydrogen and OH;
  • R 2 is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3, OCH(CH 3 ) 2, OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2 ;
  • R 3 is selected from the group consisting of hydrogen, OCH 2 CH 3 , and NHCOCH 3 ;
  • R 5 is selected from the group consisting of hydrogen, lower allcyl, aryl, phenyl, aralkyl, NH(CH 2 ) 3 N(CH 3 ) 2, NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3 , and NHCH 3 ; and
  • R 6 is selected from the group consisting of hydrogen, and NH 2 .
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2 , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 , NH 3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH 1 NO 2 , , O, carboxy, and alkoxy;
  • n is an integer from O to 10;
  • the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
  • R R and R are independently selected from the group consisting of hydrogen; lower allcyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2 ,NH3 + NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO 2 , , CH3, CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture
  • R R and R are independently selected from the group consisting of hydrogen, lower allcyl, NH 3 , OH, SH, and halogen. Also iinn eess]pecially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
  • the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
  • R 1 R 2 , R 3 , R 4 R 5 , and R 6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted arallcyl, heteroaryl, substituted heteroaryl, SO 2 , NH 21 NH 3 + , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2; N ⁇ (CH 2 ) 3 N(CH 3 ) 2 , NH(CH 2 ) 2 N(CH 3 )
  • R 1 and R 4 are selected from the group consisting of hydrogen and OH;
  • R 2 is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2 ;
  • R 3 is selected from the group consisting of hydrogen, OCH 2 CH 3 , and NHCOCH 3 ;
  • R 5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, arallcyl, NH(CH 2 ) 3 N(CH 3 ) 2, NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3 , and NHCH 3 ; and
  • R 6 is selected from the group consisting of hydrogen, and NH 2 .
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
  • R 1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO 2 , NH 2 , NO 2 , SO 2 , O, CN and halogen;
  • R 2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 2, NH 3 + NO 2 , SO 2 , O, halogen, carboxy, and alkoxy;
  • X is selected from the group consisting of lower alkyl, SO 2 , NH, NO 2 , , O, carboxy, and alkoxy;
  • n is an integer from O to 10;
  • the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
  • R 1 R 2 and R 3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO 2 , NH 21 NH 3 NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, SH, halogen, carboxy, and alkoxy;
  • R 4 is selected from the group consisting of lower alkyl, aryl, SO 2 , NH 1 NO 2 , , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, carboxy, and alkoxy; and their pharmaceutically acceptable salts of acids or bases;
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH 3 , OH, SH, and halogen. Also in especially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
  • the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
  • R R , R , R R , and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO 2 , NH 2 , NH 3 + , NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CHs) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CHs) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 )sCHs, OCHCH 3 COOCHS, OCH 2 CON(CHS) 2 , NH(CH 2 )SN(CHS) 2 , NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2
  • R and R are selected from the group consisting of hydrogen and OH;
  • R 2 is selected from the group consisting of hydrogen, OH, and CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , OCH 2 COOH, OCHCH 3 COOH, OCH 2 COCH 3 , OCH 2 CONH 2 , OCOCH(CH 3 ) 2 , OCH 2 CH 2 OH, OCH 2 CH 2 CH 3 , O(CH 2 ) 3 CH 3 , OCHCH 3 COOCH 3 , OCH 2 CON(CH 3 ) 2 ;
  • R 3 is selected from the group consisting of hydrogen, OCH 2 CH 3 , and NHCOCH 3 ;
  • R 5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH 2 ) 3 N(CH 3 ) 2, NH(CH 2 ) 2 N(CH 3 ) 2 , NH(CH 2 ) 2 OH, NH(CH 2 ) 3 CH 3; and NHCH 3 ; and
  • R 6 is selected from the group consisting of hydrogen, and NH 2
  • the general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the viral infection is HIV infection.
  • Compounds of the present in invention may function by modulating the stability of the binding complex formed between P/CAF and Tat that is acetylated at the lysine residue at position 50 of SEQ ID NO:45.
  • the method comprises contacting the bromodomain of P/CAF or a fragment thereof with a binding partner in the presence of the compound under conditions in which the bromodomain of P/CAF and the binding partner bind in the absence of the compound.
  • the stability of the bromodomain of P/CAF and the binding partner is then determined (e.g., measured).
  • the compound is identified as a modulator.
  • the binding partner is Tat that is acetylated at the lysine residue at position 50 of SEQ ED NO:45.
  • the binding partner is a fragment of Tat comprising an acetyl-lysine at position 50.
  • the binding partner is an analog of the fragment of Tat comprising an acetyl-lysine at position 50.
  • the compound is selected by performing rational drug design with the set of atomic coordinates obtained from one or more of Tables 1-5 and 10-14. More preferably the selection is performed in conjunction with computer modeling. Compounds identified by these methods are also part of the present invention.
  • the compound is an analog of acetyl-lysine. More preferably the compound is a small organic molecule not included in Fig. 13.
  • Compounds of the present invention may function by modulating the binding of P/CAF and Tat.
  • the agent is a small organic molecule.
  • the agent inhibits and/or destabilizes the binding of P/CAF with Tat.
  • the agent is an analog of acetyl-lysine. More preferably the agent is not included in Fig. 13.
  • Compounds of the present in invention are useful for modulating preventing, and/or retarding the progression and/or treating HIV infection in an individual.
  • One such method employs administering to the individual compounds that modulate the Tat-P/CAF complex selected by performing rational drug design with the set of atomic coordinates obtained from one or more of Tables 1-5 and 10-14.
  • the compound administered is an acetyl-lysine analog.
  • this compound is a small organic molecule contained in Fig. 13.
  • the compound either de-stabilizes or inhibits the Tat-P/CAF complex.
  • lower alkyl and lower alkoxy are understood as meaning straight or branched alkyl and alkoxy groups having from 1 to 8 carbon atoms;
  • aryl is understood as meaning an aromatic group selected from phenyl and naphthyl groups
  • 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 ringEj, including in its carbon skeleton from one to three heteroatoms selected from nitrogen, oxygen and sulphur;
  • lower aralkyl and “lower heteroaralkyl” are understood as meaning, in view of the definitions above, phenyl(Ci -C 8 )alkyl or naphthyl(d -Cs)alkyl and heteroar(C] -C 8 )alkyl respectively;
  • substituted concerning the terms aryl, aralkyl, phenyl, radical (f ⁇ ve-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: (C 1 -C 8 )alkyl, trifluoromethyl, (C 1 -C 8 )alkoxy, hydroxy, nitro, amino, (C 1 - Cs)alkylammo, di(d -C 8 )alkylamino, sulphoxyl, sulphonyl, sulphonamide, sulpho(d -C 8 )alkyl, carboxyl, carbalkoxyl, carbamide (it being possible for said (C 1 -C 8 )alkyl groups to be linear or branched) or substituted with one or more halogen atoms;
  • 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.
  • 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.
  • 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.
  • a bromodomain-acetyl-lysine binding complex is that formed between P/CAF with Tat (the "Tat- P/CAF complex") as exemplified below.
  • acetyl-lysine analog is used interchangeably with the term “analog of acetyl-lysine” and is a compound that contains the acetyl-amine-like structure as depicted in Fig. 12. Examples of acetyl-lysine analogs are included in Fig. 13.
  • 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 bromodomain of P/CAF is depicted in Fig. 2 A.
  • a ZA loop has between about 20 and 40 amino acids and preferably comprises the amino acid sequence of SEQ ID NO:3 and/or SEQ ID NO:48. More preferably the ZA loop comprises between about 23 to 34 amino acids.
  • the ZA loop has the amino acid sequence SEQ ID NO:43.
  • the amino acid sequence of the ZA loop for a representative number of individual bromodomains is shown in Fig. 1.
  • 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”.
  • 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.
  • 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.
  • agent 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.
  • a “small organic 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.
  • 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.
  • 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 ah, Cell, 50:667 (1987)]. Such proteins have sequence homology as reflected by their high degree of sequence similarity.
  • 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 a , supra).
  • sequence similarity when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.
  • 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 ah, 1989 supra; DNA Cloning, VoIs.
  • 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.
  • DNA and protein sequence percent identity can be determined using MacVector 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.
  • Compound 1 binds the PCAF BRD with an affinity comparable to that of the Tat-AcK50 peptide (see below)( Mujtaba, et al., MoI Cell. (2004) 13, 251-63). Importantly, these compounds do not bind the structurally similar BRDs from CBP and TIF l ⁇ at millimolar concentration.
  • the functional importance of the 2-nitro is further supported by the effects of changing the NH to an O linkage in the aniline, which severely compromises the binding to the PCAF BRD (12-17 vs. 1-5). Moreover, changing to a carbon linkage eliminates the binding (18 vs. 1).
  • the BRD prefers an amino three-carbon aliphatic chain in 1 - a four-carbon chain reduces the binding by 30-fold (19 vs. 1) and a two-carbon chain nearly loses the binding (20 vs. V). Alteration of 1 by two key elements, i.e. changing to a four-carbon chain and 4-nitro, abolishes the binding (21 vs. 1).
  • the terminal amine group is also an important functional moiety for the BRD binding (22-24 vs. 1).
  • the 2-nitro group of 2 possibly forms a hydrogen bond with the phenolic -OH of Y809 and/or Y802, and the terminal - NH 3 + interacts eletrostatically with the side-chain carboxylate of E750.
  • the functional importance of the 2-nitro and the aniline NH likely results from a possible six-member ring structure formed between these two groups. However, it is not clear why substitution of 2-nitro with 2-carboxylate abrogates the BRD binding (10 vs. 1).
  • the aromatic ring of 2 is sandwiched between the side-chains of Y802 and A757 on one side, and Y809 and E756 on the other side; and the propane carbon chain is surrounded by the hydrophobic portions of the side-chains of P747, E756 and V752.
  • the 4-methyl of 2 fills a small hydrophobic cavity formed by side-chains of A757, Y802 and Y809 ( Figure ID), contributing to a 3-fold increase over 1 in binding to the PCAF BRD.
  • the present invention therefore provides a class of novel small molecules that can effectively inhibit the PCAF BRD/Tat-AcK50 association in vitro by selectively binding to the BRD ( Figure 13).
  • the detailed SAR understanding of the lead compounds 1 and 2 will facilitate our efforts to optimize their affinity and selectivity by branching out to interact with the neighboring AcK binding pocket by the tethering techniques.
  • Such small-molecule inhibitors will help validate the novel anti-HIV/AIDS therapeutic strategy by targeting a cellular protein to block HTV transcription and replication.
  • Bromodomain utilizes detailed structural information regarding a bromodomain and a bromodomain complexed with its acetylated binding partner.
  • the present invention therefore provides the three-dimensional structure of the bromodomain and a bromodomain acetylated binding partner complex. Since the interaction of the bromodomain with a histone for example, can play a significant role in chromatin remodeling/regulation, the structural information provided herein can be employed in methods of identifying drugs that can modulate basic cell processes by modulating the transcription. In a particular embodiment, the three-dimensional structural information is used in the design of a small organic molecule for the treatment of cancer or as disclosed below, HIV-I infection and/or AIDs.
  • the present invention provides a critical structural feature for a class of inhibitors (acetyl-lysine analogs) of the interaction between bromodomains and their protein binding partners which contain an acetylated-lysine ⁇ e.g., Tat with P/CAF), see Fig. 11, as well as a compilation of compounds that share this critical feature, see Fig. 12.
  • bromodomain and lysine-acetylated protein interaction can now be implicated to play a causal role in the development of a number of diseases including cancers such as leukemia.
  • chromatin remodeling plays a central role in the etiology of viral infection and cancer [Archer and Hodin, Curr. Opin. Genet. Biol. 9:171-174 (1999); Jacobson and Pillus, Curr. Opin. Genet. Biol. 9:175-184 (1999)].
  • Both altered histone acetylation/ deacetylation and aberrant forms of chromatin-remodeling complexes are associated with human diseases.
  • chromosomal translocation of various cellular genes with those encoding HATs and subunits of chromatin remodeling complexes have been implicated in leukomogenesis.
  • the MOZ (monocytic leukemia zinc finger) and MLL/ ALL-I genes are frequently fused to the gene encoding the co-activator HAT CBP [Sobulo et al, Proc. Natl. Acad. ScL USA 94:8732-8737(1997)].
  • the resulting fusion protein MLL-CBP contains the tandem bromodomain-PHD finger-HAT domain of CBP.
  • HIV-I human immunodeficiency virus type 1 trans-activator protein
  • Tat is tightly regulated by lysine acetylation [Kiernan et ⁇ l, EMBO Journal 18:6106-6118 (1999)]. HIV-I Tat transcriptional activity is absolutely required for productive HIV viral replication [Jeang and Gatignol, Curr. Top. Microbiol. Immunol, 188:123- 144(1994)]. Therefore, the interaction of the acetyl-lysine of Tat with one or more bromodomain-containing proteins associated with chromatin remodeling could mediate gene transcription.
  • acetylated lysine50 of Tat specifically binds to the bromodomain of P/CAF. Therefore, this particular bromodomain/lysine-acetylated Tat interaction serves as a drug target for blocking HIV replication in cells.
  • an antibody raised specifically against a peptide from the ZA loop of the P/CALF bromodomain could also be effective for treating and/or preventing HIV infections including those that lead to AIDs.
  • 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.
  • Compounds may be active to bind to two nearby sites on the bromodomain.
  • 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).
  • 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.
  • a linked compound is synthesized in which the ligand for the first site and the ligand for the second site are linked.
  • 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.
  • the affinity of the linked compound with the bromodomain is determined monitoring the 15 N- or 1H-amide chemical shift changes in two dimensional 15 N-heteronuclear single-quantum correlation ( 15 N-HSQC) spectra upon the addition of the linked compound to the 15 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.
  • compositions may be for administration for injection, or for oral, pulmonary, nasal or other forms of administration.
  • 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, emulsif ⁇ ers, adjuvants and/or carriers.
  • 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.
  • 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.
  • 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 lyopliilized form.
  • 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.
  • 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).
  • 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.
  • oral dosage forms of the above derivatized 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.
  • 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 multi-particulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Sample preparation The bromodomain of P/CAF (residues 719-832 of SEQ ID NO:2) was subcloned into the pET14b expression vector (Novagen) and expressed in Escherichia coli BL21(DE3) cells. Uniformly 15 N- and 15 N/ 13 C-labelled proteins were prepared by growing bacteria in a minimal medium containing 15 NH 4 Cl with or without 13 C 6 -glucose. A uniformly 15 N/ 13 C-labelled and fractionally deuterated protein sample was prepared by growing the cells in 75% 2 H 2 O.
  • the bromodomain was purified by affinity chromatography on a nickel- ID A column (Invitrogen) followed by the removal of poly-His tag by thrombin cleavage. The final purification of the protein was achieved by size-exclusion chromatography.
  • the acetyl- lysine-containing peptides were prepared on a MilliGen 9050 peptide synthesizer (Perkin Elmer) using Fmoc/HBTU chemistry. Acetyl-lysine was incorporated using the reagent Fmoc-Ac-Lys with HBTU/DIPEA activation.
  • NMR samples contained approximately 1 mM protein in 10OmM phosphate buffer of pH 6.5 and 5mM perdeuterated DTT and 0.5mM EDTA in H 2 O/ 2 H 2 O (9/l) or 2 H 2 O.
  • NMR spectroscopy AU ⁇ MR spectra were acquired at 30 0 C on a Bruker DRX600 or DRX500 spectrometer.
  • the backbone assignments of the 1 H, 13 C, and 15 N resonances were achieved using deuterium-decoupled triple-resonance experiments of HNCACB and HN(CO)CACB ⁇ Ywuad ⁇ et al., J. Am. Chem. Soc. 116:11655-11666 (1994)] recorded using the uniformly 15 N/ 13 C-labeled and fractionally deuterated protein.
  • the side-chain atoms were assigned from 3D HCCH-TOCSY [Clore and Gronenborn, Meth. Enzymol.
  • the NOE-derived distance restraints were obtained from 15 N- or 13 C- edited 3D NOESY spectra, ⁇ -angle restraints were determined based on the JHN,H « coupling constants measured in a 3D HNHA spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249- 363 (1994)].
  • Slowly exchanging amide protons were identified from a series of 2D 15 N-HSQC spectra recorded after the H 2 O buffer was changed to a 2 H 2 O buffer.
  • a total of 1519 unambiguous and 590 ambiguous distance restraints were identified from the NOE data by ARIA, many of which were checked and confirmed manually.
  • the ARIA-assigned distance restraints were in agreement with the structures calculated using only the manually assigned NOE distance restraints, 28 hydrogen-bond distance restraints for 14 hydrogen bonds, and 54 ⁇ >-angle restraints.
  • the final structure calculations employed a total of 3515 NMR experimental restraints obtained from the manual and the ARIA-assisted assignments, 2843 of which were unambiguously assigned NOE-derived distance restraints that comprise of 1077 intra-residue, 621 sequential, 550 medium-range, and 595 long-range NOEs.
  • Ligand titration experiments were performed by recording a series of 2D 15 N- and 13 C-HSQC spectra on the uniformly 15 N-, and 15 N/ 13 C-labelled bromodomain ( ⁇ 0.3mM), respectively, in the presence of different amounts of ligand concentration ranging from 0 to approximately 2.0 mM.
  • the protein sample and the stock solutions of the ligands were all prepared in the same aqueous buffer containing 10OmM phosphate and 5mM perdeuterated DTT at pH 6.5.
  • the P/CAF bromodomain represents an extensive family of bromodomains (Fig. 1).
  • a large number of long-range nuclear Overhauser enhancement (NOE)-derived distance restraints were identified in the NMR data of the P/CAF bromodomain, yielding a well- defined three-dimensional structure (Figs. 2A -2D).
  • Table 2 shows the NMR chemical shift assignment of the P/CAF bromodomain.
  • Table 2 shows the Unambiguous NOE-derived distance restraints.
  • Table 4 shows the Ambiguous NOE-derived distance restraints.
  • Table 5 shows the Hydrogen bond restraints.
  • the NMR structure coordinates of the P/CAF bromodomain in the free and complexed to acetyl-histamine are shown in Tables 5 and 6, respectively.
  • the structure consists of a four-helix bundle (helices OL Z , OL A , CC B , and ⁇ c) with a left-handed twist, and a long intervening loop between helices ⁇ z and «A (termed the ZA loop, Fig. 2E).
  • the four amphipathic ⁇ -helices are packed tightly against one another in an antiparallel manner, with crossing angles for adjacent helices of ⁇ 16-20°.
  • the up-and-down four-helix bundle can adapt two topological folds with opposite handedness ( Figures 2F-2G).
  • the right-handed four-helix bundle fold occurs more commonly and is seen in proteins such as hemerythrin and cytochrome O 562 .
  • the left-handed fold of the bromodomain structure is less common, but also observed in proteins such as cytochrome bs and T4 lysozyme [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. ScL USA 86:6592-6596 (1989)].
  • This topological difference arises from the orientation of the loop between the first two helices (Fig. 2F-2G).
  • the right-handed four-helix bundle proteins have a relatively short hairpin-like connection between the first two helices, which makes the "preferred" turn to the right at the top of the first helix [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. Sd. USA 86:6592-6596 (1989); Weber and Salemme, Nature 287:82-84 (1980)].
  • proteins with the left-handed fold usually have a long loop after the first helix and often contain additional secondary structural elements at the base of the helix bundle [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. ScL USA 86:6592-6596 (1989)].
  • this long ZA loop has a defined conformation and is packed against the loop between helices CX B and ⁇ c (termed the BC loop) to form a hydrophobic pocket.
  • the hydrophobic pocket formed by loops ZA and BC is lined by residues Val752, Ala757, Tyr760, Val763, Tyr802 and Tyr809 (Fig. 2H), and appears to be a site for protein-protein interactions (see below).
  • the pocket is located at one end of the four-helix bundle, opposite to the N- and C-termini of the protein.
  • the ZA loop varies in length amongst different bromodomains, but almost always contains residues corresponding to Phe748, Pro751, Pro758, Tyr760, and Pro767 (Fig. 1).
  • the conservation of these residues within the ZA loop as well as residues within the ⁇ -helical regions implies a similar left-handed four-helix bundle structure for the large family of bromodomains.
  • the modular bromodomain structure supports the idea that bromodomain can act as a functional unit for protein-protein interactions.
  • the NMR titration of the P/CAF bromodomain were performed with a peptide (SGRGKGG- AcK-GLGK) derived from histone H4, in which Lys8 is acetylated (Lys8 is the major acetylation site in H4 for GCN5, a yeast homologue of P/CAF).
  • the bromodomain could indeed bind the AcK peptide.
  • this interaction appeared to be specific, based on the 15 N-HSQC spectra which showed that only a limited number of residues underwent chemical shift changes as a function of peptide concentration (Fig. 3A).
  • Tyr809, Tyr802, Tyr760, and Val752 in the bromodomain appear to be related to Phe220, Phe261, Val254, and Ile217 of Hatl, respectively, in their interactions with the acetyl moiety.
  • This observation may suggest an evolutionary convergent mechanism of acetyl-lysine recognition between bromodomains and histone acetyltransferases.
  • site-directed mutagenesis was used to alter residues Tyr809, Tyr802, Tyr760, and Val752 (Table 7).
  • HIV-I binds to CD4 protein of a host T cell or macrophage and with the aid of a chemokine receptor (e.g., CCR5 or CXCR4) enters the host cell.
  • a chemokine receptor e.g., CCR5 or CXCR4
  • the retrovirus, HIV-I is converted to a DNA by reverse transcriptase and the expression of the HIV-I genome is dependent on a complex series of events that are believed to be under the control of two viral regulatory proteins, Tat and Rev [Romano et al, J. CellBiochem. 75(3):357-368 (1999)].
  • Tat the trans-activator protein
  • the Tat protein transactivates the transcription of HIV-I starting at the 5' long terminal repeat (LTR) [Romano et al, J. CellBiochem. 75(3):357-368 (1999)] by recruiting one or more carboxyl- terminal domain kinases to the HIV-I promoter. More specifically, Tat stimulates transcription from the LTR at a hairpin element, the transactivation responsive region (TAR) [Kiernan et al, EMBO J.
  • LTR 5' long terminal repeat
  • TAR transactivation responsive region
  • P-TEFb is a muti-subunit kinase that minimally comprises a heterodimer consisting of the regulatory cyclin Tl and its corresponding catalytic subunit, cyclin-dependent kinase 9 (CDK9).
  • P-TEFb acts by phosphorylating the carboxyl-terminal domain of RNA polymerase II [Peng et al, J.Biol. Chem. 274 (49):34527-34530 (1999); Romano et al, J. CellBiochem. 75(3):357-368 (1999)].
  • HIV-I Tat transcription activity is regulated through lysine acetylation by, and association with the histone acetyltransferases (HATs) p300/CBP and the p300/CBP-associating factor (P/CAF), which specifically acetylate Lysine 50 (K50) and Lysine 28 (K28) of the Tat protein, respectively [Kiernan et al, EMBO J. 18:6106-6118 (1999); Ott et al, Curr. Biol 9:1489-1492 (1999)].
  • HATs histone acetyltransferases
  • P/CAF p300/CBP-associating factor
  • the acetylation of K50 by the transcriptional co-activator p300/CBP is on the C-terminal arginine-rich motif (ARM) of Tat, which is essential for its binding to the TAR RNA element and for nuclear localization, [Kiernan et al, EMBOJ. 18:6106-6118 (1999); Ott et al, Curr. Biol. 9:1489-1492 (1999)].
  • ARM arginine-rich motif
  • a uniformly 15 N/ 13 C-labeled and fractionally deuterated protein sample was prepared by growing the cells in 75% 2 H 2 O.
  • the bromodomain was purified by affinity chromatography on a nickel-IDA column (Invitrogen) followed by the removal of poly-His tag by thrombin cleavage.
  • the final purification of the protein was achieved by size-exclusion chromatography.
  • the acetyl-lysine-containing peptides were prepared on a MilliGen 9050 peptide synthesizer (Perkin Elmer) using Fmoc/HBTU chemistry. Acetyl-lysine was incorporated using the reagent Fmoc-Ac-Lys with HBTU/DIPEA activation.
  • NMR samples contained ⁇ 0.5 mM protein in complex with the lysine-acetylated Tat peptide in 100 mM phosphate buffer of pH 6.5 and 5mM perdeuterated DTT and 0.5mM EDTA in H 2 CV 2 H 2 O (9/1) or 2 H 2 O.
  • the bromodomain-containing constructs from P/CAF, CBP and TIF-l ⁇ were cloned into pGEX4T-3 vector (Pharmacia). These recombinant GST-fusion proteins were expressed in BL21 (DE3) codon plus cell line, and purified by using glutathione sepharose column.
  • NMR spectroscopy AU NMR spectra were acquired at 3O 0 C on a Broker DRX600 or DRX500 spectrometer.
  • the backbone assignments of the 1 H, 13 C, and 15 N resonances were achieved using deuterium-decoupled triple-resonance experiments of HNCACB and HN(CO)CACB [Yamazaki et ⁇ /., J. Am. Chem. Soc. 116:11655-11666 (1994)] recorded using the uniformly 15 N/ 13 C-labelled and fractionally deuterated protein.
  • the side-chain atoms were assigned from 3D HCCH-TOCSY [Clore and Gronenborn, Meth. Enzymol.
  • ⁇ -angle restraints were determined based on the 3 J HN1 H coupling constants measured in a 3D HNHA spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)].
  • Slowly exchanging amide protons were identified from a series of 2D 15 N-HSQC spectra recorded after the H 2 O buffer was changed to a 2 H 2 O buffer.
  • the intermolecular NOEs used in defining the structure of the bromodomain/Ac-histamine complex were detected in 13 C-edited (Fy), 13 Q 15 N- filtered (F 3 ) 3D NOESY spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)]. All NMR spectra were processed with the NMRPipe/NMRDraw programs and analyzed using NMRView [Johnson and Blevins, J. Biomol, NMR 4:603-614 (1994)].
  • Ligand titration experiments were performed by recording a series of 2D 15 N-HSQC spectra on the uniformly 15 N-labelled bromodomain (-0.3 mM), respectively, in the presence of different amounts of ligand concentration ranging from 0 to ⁇ 2.0mM.
  • the protein sample and the stock solutions of the ligands were all prepared in the same aqueous buffer containing 100 mM phosphate and 5mM perdeuterated DTT at pH 6.5.
  • Proteins were eluted from the argarose beads in SDS buffer and separated on a 14% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membrane (Pharmacia), and the membrane was blocked overnight with 5% non-fat milk in washing buffer of 20 mM Tris, pH 7.5, plus 150 mM NaCl and 0.1% Tween-20 at 4 0 C. Western blotting was performed with anti-GST antibody (Sigma) and goat anti-rabbit IgG conjugated with horseradish-peroxidase (Promega) and developed by chemiluminescence.
  • NMR binding studies further confirmed the specific interaction of the P/CAF bromodomain and lysine-acetylated Tat peptide. Because NMR resonances of amide protons are highly sensitive to local chemical environment and conformational change in a protein, two-dimensional 1 H- 15 N heteronuclear single quantum correlation (HSQC) spectrum can be used to detect even weak but specific interactions between a protein and its binding ligand. As shown in 2D HSQC spectra (Figs.
  • the bromodomain of P/CAF binds weakly to the lysine-acetylated peptides derived from known acetylation sites of K28 on Tat and of K16 on histone H4 by only interacting with the acetyl-lysine residue in the peptides (K d ⁇ 300 ⁇ M). This is reflected the relatively small chemical shift perturbation of the amide proton signals of the protein upon addition of ligand.
  • the P/CAF bromodomain interacts strongly with the Tat AcK50 peptide, which involves many protein residues in addition to those for acetyl-lysine binding with an estimated IQ of -20 ⁇ M.
  • Binding of peptide residues flanking the acetyl- lysine may explain the high specificity of the P/CAF bromodomain for the acetylated Tat.
  • the p300/CBP bromodomain did not bind the lysine-acetylated Tat peptide in a specific manner except its weak interaction with the acetyl-lysine residue in the peptide (Figs. 6A-6D). Together, these results demonstrate the P/CAF bromodomain can specifically recognize the lysine-acetylated Tat involving K50.
  • the three-dimensional structure was determined for the P/CAF bromodomain in complex with an 11 -residue Tat peptide containing AcK50. A total of 2,903 NMR-derived distance and dihedral angle restraints were used.
  • the structure of the bromodomain in the peptide-bound form consists of an up-and-down four-helix bundle (helices ccz, « A , c- B , and ⁇ c) with a left-handed twist, and a long intervening loop between helices a ⁇ and CCA (termed the ZA loop) (Fig. 9).
  • the overall structure of the complex is well defined, and similar to the structure of the free bromodomain [Dhalluin et al, Nature 399:491-496 (1999)] except that the ZA and BC loops, which compose the acetyl-lysine binding pocket, undergo local conformational changes in order to accommodate their interactions with the peptide residues.
  • the Tat AcK50 peptide adopts an extended conformation and lies between the ZA and BC loops (Fig. 9).
  • the acetyl-lysine side-chain intercalates deep into a preformed hydrophobic and aromatic cavity located between the ZA and BC loops opposite to the N- and C-termini, and interacts extensively with residues V752, Y760, 1764, Y802, and Y809.
  • mutant proteins were tested for binding to the biotinylated and lysine- acetylated Tat peptide that is immobilized onto streptavidin agarose (Fig. 10A).
  • proteins containing alanine point mutation at the residue Y809, Y802, V752, or F748, which interact directly with the acetyl-lysine residue showed nearly complete loss or significantly reduced binding to the Tat peptide.
  • the HIV-I Tat is a versatile protein and elicits many cellular functions.
  • this portion of arginine-rich motif (named ARM) has also been shown to interact with the TAR RNA element as well as protein nuclear localization, particularly involving arginine52 and arginine53.
  • the findings disclosed herein that are based on the detailed structural and mutational analyses indicate that the lysine-acetylated Tat specifically is associated with P/CAF via a bromodomain interaction in vivo, and that this interaction is important for transactivation activity of Tat in cells.
  • the data disclosed herein reveal that in addition to the acetylated-lysine (K50) the flanking residues, tyrosine (AcK-3) and glutamine at (AcK+4) positions in Tat are also uniquely important for the specificity of the Tat and P/CAF bromodomain recognition, but not with its other functions.
  • This new information is extremely useful in applying mutational analysis in in vivo studies to further elucidate the biological importance of the Tat-P/CAF association in molecular mechanisms by which Tat transactivates gene transcription of HIV-I via chromatin remodeling.
  • Example 3 Synthesis of the Compounds of Formula I [0115] Sample preparation. The PCAF bromodomain (residues 719-832) was expressed in E. coli BL21(D ⁇ 3) cells using the pET14b vector (Novagen) (Dhalluin, et al., Nature (1999) 399, 491-496). Isotope-labeled proteins were prepared from cells grown on a minimal medium containing 15 NH 4 Cl with or without 13 C 6 -glucose in either H 2 O or 75% 2 H 2 O. The protein was purified by affinity chromatography on a nickel-IDA column (Invitrogen), followed by the removal of poly-His tag by thrombin cleavage.
  • GST-fusion PCAF bromodomain was expressed in E. coli BL21 (DE3) codon plus cells using the pGEX4T-3 vector (Pharmacia), and purified with a glutathione sepharose column.
  • the lysine-acetylated peptide was ordered from Biosynthesis, Inc.
  • NMR samples contained the bromodomain (0.5 mM) in complex with a chemical ligand ( ⁇ 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 3O 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 13 C-edited 3D NOESY spectra.
  • the 3 JIi KHa coupling constants measured from 3D HNHA data were used to determine D -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 PCAF bromodomain/ligand complex were detected in 13 C-edited (F / ), 13 C/ 15 N- filtered (F 3 ) 3D NOESY spectra (Clore et al., Meth. Enzymol. (1994) 239, 249-363). Protein structures were calculated with a distance geometry-simulated annealing protocol with X-PLOR (Brunger A.T., X-PLOR Version 3.1: A system for X-Ray crystallography and NMR. version 3.1 ed. 1993, 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., Prog. NMR Spectroscopy (1998) 32, 107-139). Structure quality was assessed by Procheck-NMR (Laskowski et al, J. Biomol. NMR (1996) 8, 477-486).
  • the structure of the protein/ligand complex was determined using intermolecular NOE-derived distance restraints.
  • the plate was washed and blocked with PBS buffer containing 10% BSA and 1% Tween-20.
  • the biotinylated HIV Tat-AcK50 peptide (Biotin-GISYGR-AcK-KKRRQRRRP) (5 ⁇ M) and increasing concentrations of a given compound were added and allowed to bind to the PCAF bromodomain overnight at 4 0 C.
  • bromodomain -bound peptide was determined by incubating 100 ⁇ L of a neutravidin-conjugated HRP (Pierce) solution (0.1 ⁇ g/ml) for 1 hour at room temperature, followed by washes and incubation with 100 ⁇ L of tetramethyl benzidine (Pierce) as an HRP substrate. The reaction was stopped by addition of 100 ⁇ L of 2.0 M sulfuric acid. The absorbance of the colored product was measured at 450 nm. Absorbance in each well was corrected for the blank obtained in a corresponding well subjected to the complete procedure but containing no PCAF bromodomain.
  • HRP neutravidin-conjugated HRP
  • a Reagents and conditions (a) N-(3-bromopropyl)phthalimide, (Et) 3 N, 12O 0 C; (b) (i) NH 2 NH 2 -H 2 O, EtOH, reflux, 6 h; (ii) HCl, EtOH.
  • Nr(3-nitro-biphenyl-4-yl)-propane-l,3-diamine monohydrochloride [0125] Nr(3-nitro-biphenyl-4-yl)-propane-l,3-diamine monohydrochloride. mp >190 0 C; FTIR
  • FIG. 16 demonstrates inhibition of Tat-mediated transactivation by the PCAF BRD inhibitor.
  • A Schematic diagram of the microinjection assay.
  • B Effect of a lead compound 765 on Tat-mediated transactivation as compared to DMSO control.
  • Fifiure 17 demonstrates the effect of PCAF BRD inhibitors on Tat transactivation.
  • HeLa cells were transfected with LTR-luciferase and 20 ng Tat-expression vector.
  • PCAF BRD inhibitors were added after transfection, and cells were harvested after 8 hours.

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Abstract

The present invention features compounds of the following general formula (I) wherein R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH2, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH, CN and halogen; R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2 NH2NH3+NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH, halogen, carboxy, and alkoxy; X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH, carboxy, and alkoxy; and n is an integer from O to 10 and their pharmaceutically acceptable salts of acids or bases as well as pharmaceutical compositions comprising these compounds. The invention further provides methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins and methods for treating HIV infection and HW related disease. The present invention also features compounds of the following general formula (II) wherein: R1R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3+ NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy; and R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy as well as pharmaceutical compositions comprising these compounds. The invention further provides methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins and methods f treating HW infection and HIY related disease. The present invention also features compounds of formula (III) wherein R1, R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2 NH2, NH3+, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2,NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2, NH(CH2)2OH, NH(CH2)3CH3, NHCH3, SH, halogen, carboxy, and alkoxy as well as pharmaceutical compositions comprising these compounds. The invention further provides methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins and methods for treating HIV infection and HIV related disease.

Description

METHODS OF IDENTIFYING MODULATORS OF BROMODOMAINS
FIELD OF THE INVENTION
[0001] The present invention provides novel compounds that interact with a histone acetyltransferase bromodomain. The interaction between bromodomains and their binding partners play a crucial role in various cellular functions, including in the regulation/modulation of DNA transcription. Therefore, the present invention provides agents that can modulate the interaction of bromodomains and their binding partners.
BACKGROUND OF THE INVENTION
[0002] In recent years great strides have been made in the elucidation of the steps involved in intercellular and intracellular signaling. Indeed, the individual steps of the cascade of events involved in a number of cellular signal transduction processes have been determined. For example, intercellular signal transduction generally begins with an intercellular ligand binding the extracellular portion of a receptor of the plasma membrane. The bound receptor then either directly or indirectly initiates the activation of one or more cellular factors. An activated cellular factor may act as transcription factor by entering the nucleus to interact with its corresponding genomic response element, or alternatively, it may interact with other cellular factors depending on the complexity of the process. In either case, one or more transcription factors ultimately bind to one or more specific genomic response elements. This binding plays a crucial role in the up and/or down regulation of the transcription of the specific genes that are under the control of these genomic response elements. However, the process of re-organizing the chromatin of eukaryotic cells, which is a prerequisite for the binding of the transcription factor to the genomic response elements, has remained a mystery.
[0003] Chromatin contains several highly conserved histone proteins including: H3, H4, H2A, H2B, and Hl. These histone proteins package eukaryotic DNA into repeating nucleosomal units that are folded into higher-order chromatin fibers [Luger and Richmond, Curr. Opin. Genet. Dev. 8:140-146 (1998)]. A portion of the histone that comprises roughly a quarter of the protein protrudes from the chromatin surface, and is thereby sensitive to proteolytic enzymes [van Holde, in Chromatin (Rich, A5. ed., Springer, New York ) pagesl 11-148 (1988); Hect et al, Cell 80:583-592 (1995)]. This portion of the histone is known as the "histone tail". Histone tails tend to be free for protein-protein interaction, and are also the portion of the histone most prone to post-translational modification. Such post-translational modification includes acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation [van Holde, in Chromatin (Rich, A,, ed., Springer, New York ) pagesl 11-148 (1988)].
[0004] Of all classes of proteins, histones are amongst the most susceptible to post- translational modification. Perhaps the best studied post-translational modification of histones is the acetylation of specific lysine residues [Grunstin, M., Nature, 389:349-352 (1997)]. Indeed, acetylation of histone lysine residues has been suggested to play a pivotal role in chromatin remodeling and gene activation. Consistently, distinct classes of enzymes, namely histone acetyltransferases (HATs) and histone deacetylases (HDACs), acetylate or de-acetylate specific histone lysine residues [Struhl, Genes D ev. 12:599-606 (1998)].
[0005] Nearly all known nuclear HATs contain an approximately 110 amino acid sequence known as the bromodomain [Jeanmougin et al, Trends in Biochemical Sciences, 22:151-153 (1997)], a protein motif that was initially discovered in Drosophila brahma protein. Bromodomains are found in a large number of chromatin-associated proteins and have now been identified in approximately 70 human proteins, often adjacent to other protein motifs [Jeanmougin et al, Trends in Biochemical Sciences, 22:151-153 (1997); Tamkun et al, Cell, 68:561-572 (1992): Hanes et al, Nucleic Acids Research, 20:2603 (1992)]. Proteins that contain a bromodomain often contain a second bromodomain. However, despite the wide occurrence of bromodomains and their likely role in chromatin regulation, their three-dimensional structure and binding partners heretofore have remained unknown.
[0006] The bromodomain, present in chromatin associated proteins and histone lysine acetyltransferases,6a is an acetyl-lysine binding domain.6b Bromodomain/ AcK binding plays an important role in control of chromatin remodeling and gene transcription.60 BRDs adopt the highly conserved structural fold of a left-handed four- helix bundle (αZ, αA, αB and αC), as first shown in the PCAF BRD (Zeng, et al, FEBS Letters (2002) 513, 124-8) (Figure IA). The ZA and BC loops at one end of the bundle form a hydrophobic pocket for AcK binding. The structure of the PCAF BRD bound to a Tat-AcK50 peptide shows that AcK50 interacts with protein residues V752, Y802 and Y809, Y47(AcK-3) with V763, and R53(AcK+3) and Q54(AcK+4) with E756, conferring a specific intermolecular association. The structures of CBP BRD/ p53-AcK382 and GCN5p BRD/H4-AcK16 complexes show that the residues in BRDs important for AcK recognition are largely conserved, whereas sequence variations in the ZA and BC loops enable discrimination of different binding targets (Mujtaba, et al, MoI Cell. (2004) 13, 251-63). Notably, as compared to the other parts of the protein, the ZA and BC loops contain significant sequence variations with amino acid deletion or insertion, supporting the notion that different sets of residues in the ZA and/or BC loops dictate BRD ligand specificity by interacting with residues flanking the acetyl-lysine in a target protein.
[0007] Therefore, there is a need to identify a binding partner for a bromodomain. In addition, there is a need to identify agonists or antagonists to the bromodomain-binding partner complex. Since a preferred method of drug-screening relies on structure based drug design, there is also a need to determine the three-dimensional structure of a bromodomain. In this case, once the three dimensional structure of bromodomain is determined, potential agonists and/or potential antagonists can be designed with the aid of computer modeling [Bugg et al, Scientific American, Dec.:92-98 (1993); West et al, TIPS, 16:67-74 (1995); Dunbrack et al, Folding & Design, 2:27-42 (1997)]. However, heretofore the three-dimensional structure of the bromodomain has remained unknown. Therefore, there is a need for obtaining a form of the bromodomain that is amenable for NMR analysis and/or X-ray crystallographic analysis. Furthermore, there is a need for the determination of the three-dimensional structure of the bromodomain. Finally, there is a need for procedures for related structural based drug design predicated on such structural data.
[0008] The replication cycle of the human immunodeficiency virus (HIV) presents several viable targets for anti-HIV chemotherapy. The current anti-HIV drugs specifically target the viral reverse transcriptase, protease and integrase (Garg, R et al, Chem. Rev. (1999) 99, 3525-601) However, because of the development of viral drag resistance from mutations in the targeted proteins, continuous viral production by chronically infected cells contributes to HIV-mediated ' immune dysfunction (Ho, et al, Nature. Med. (2000) 6, 757-61; Wei et al, Nature (1995) 373, 117-22) and there is still no cure for AIDS. A rapid growing AIDS epidemic calls for new therapeutic strategies targeting different steps in the viral life cycle. Therapeutic intervention at the stage of HIV gene expression can complement the existing therapy to interfere with virus production. Transcription of the integrated HIV provirus is regulated by the concerted action between cellular transcription factors and a unique viral trans-activator Tat. Tat binds to a viral RNA TAR and recruits cyclin Tl and cyclin-dependent kinase 9 that hyper-phosphorylates and enhances elongation efficiency of the RNA polymerase II (Keen et al., J. EMBO. J. (1997) 16, 5260-72; Karn, J. MoL Biol. (1999) 293, 235-54; Jones, Genes. Dev. (1997) 11, 2593-99; Kao, et al., Nature (1987) 330, 489-93).
[0009] Tat transactivation requires acetylation of its lysine 50 and recruitment of histone lysine acetyltransferase transcriptional coactivators for remodeling micleosome that contains the integrated proviral DNA (Ott et al., Curr. Biol. (1999) P5 1489-92). Our recent study shows that Tat coactivator recruitment requires its acetylated lysine 50 (AcK50) binding to the bromodomain (BRD) of the coactivator PCAF (Mujtaba, et al., MoL Cell. (2002) 9, 575-86), and microinjection of anti-PCAF BRD antibody blocks Tat transactivation (Dorr et al., EMBO. J. (2002) 21, 2715-33). These data suggest that Tat/ PCAF recruitment via a BRD-AcK binding is essential for HIV transcription, and this interaction serves as a new therapeutic target for intervening HIV replication.
[0010] Small molecule inhibitors that block Tat/PCAF binding by targeting the BRD of PCAF are needed. Targeting a host cell protein essential for viral reproduction, rather than a viral protein, may minimize the problem of drug resistance due to mutations of the viral counterpart as observed with protease inhibitors. Here we report the development of a novel class of Nl-aryl- propane-1,3- diamine compounds using a structure-based approach that bind the PCAF BRD selectively over other structurally similar BRDs. SUMMARY OF THE INVENTION [0011] The present invention features compounds of the following general formula(I) wherein:
/ Ra
^(CH 2)n
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2;NO2j SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy; and
ii is an integer from O to 10;
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.
[0012] In preferred embodiments, R1 may be selected from the group consisting of hydrogen, lower alkyl, phenyl, and CN. Also, in preferred embodiments, R2 may be selected from the group consisting OfNH3 +, carboxy, and alkoxy. In yet other preferred embodiments, X may be selected from the group consisting of lower alkyl, NH, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH. In other preferred embodiments, n may be 3. [0013] In other preferred embodiments, the present invention features compounds of the following general formula(II) wherein:
Figure imgf000008_0001
R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R is selected from the group consisting of lower alkyl, aryl, SO2, NH,Nθ2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases.
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. In especially preferred embodiments, R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH3 , OH, SH, and halogen. Also iinn eess]pecially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
[0014] In yet other preferred embodiments, the present invention features compounds of the following general formula(III) wherein
Figure imgf000008_0002
R1 R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 +, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2)NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3) NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments, R1 and R4 are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3,
OCH2CON(CH3)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0015] The present invention provides novel compounds that inhibit the binding of bromodomains to acetyl-lysine residues of proteins. The present invention makes use of the three-dimensional structure of a bromodomain as well as the three-dimensional structure of a bromodomain-acetyl-histamine complex. [0016] In a second aspect, the present invention features a pharmaceutical composition comprising a compound of Formula I wherein
-(CH2),,'-
R1- "
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NB2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases;
together with a pharmaceutically acceptable carrier.
[0017] In other preferred embodiments, the present invention features a pharmaceutical composition comprising a compound of Formula (II) wherein:
Figure imgf000010_0001
R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. In especially preferred embodiments, R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH3 , OH, SH, and halogen. Also iinn eessjpecially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
[0018] In yet other preferred embodiments, the present invention features a pharmaceutical composition comprising a compound of Formula (III) wherein
Figure imgf000011_0001
R R 5 R 3 R R , and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2,NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3).:, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CHS)2, NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments,
R1 and R4 are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2> OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3,
OCH2CON(CH3)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2jNH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0019] In a third aspect, the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
s(CH2)n-
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen; R is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH1NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases;
[0020] In other preferred embodiments, the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
Figure imgf000013_0001
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower allcyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture 1 9 *} or in isolated form. In especially preferred embodiments, R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NHb , OH, SH, and halogen. Also in especially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
[0021] In yet other preferred embodiments, the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
Figure imgf000014_0001
R1 R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH25NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH35 OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCHsCOOCH3, OCH2CON(CHS)2, NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CHz)2OH, NH(CH2)3 CH3 ,NHCHs, SH5 halogen, carboxy, and alkoxy. In especially preferred embodiments,
R and R are selected from the group consisting of hydrogen and OH;
R is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CH3, 0(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2; R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2)NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3) and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0022] In a fourth aspect, the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
Figure imgf000015_0001
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, O, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, arallcyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH21NH3 +NO2, SO2, O, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases. [0023] In other preferred embodiments, the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
Figure imgf000016_0001
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. In especially preferred embodiments, R1 R2 and R3 are independently selected from the group consisting of hydrogen, lower alkyl, NH3 +, OH, SH, and halogen. Also in especially preferred embodiments, R4 is selected from the group consisting of lower alkyl and aryl.
[0024] In yet other preferred embodiments, the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
Figure imgf000017_0001
R R2, R3, R R5, and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH21NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2, NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)SCH3, NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments,
R and R are selected from the group consisting of hydrogen and OH;
R is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)sCHs, OCHCH3COOCH3, OCH2CON(CHs)2;
R is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCHs;
R is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)sN(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R is selected from the group consisting of hydrogen, and NH2. The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0025] In some preferred embodiments, the viral infection is HIV infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Fig. 1. Structure-based sequence alignment of a selected number of bromodomains. The sequences were aligned based on the NMR-derived structure of the P/CAF bromodomain, and the predicated four α-helices are shown in green boxes. Bromodomains are grouped on the basis of the sequence and/or functional similarities as described by Jeanmougin et ah, [Trends in Biochemical Sciences, 22:151-153 (1997)]. Residue numbers of the P/CAF bromodomain are indicated above its sequence. Three absolutely conserved residues, corresponding to Pro751, Pro767, and Asn803 in the P/CAF bromodomain, are shown in red. Highly conserved residues are colored in blue. The residues of the P/CAF bromodomain that interact with acetyl-histamine, as determined by intermolecular NOEs, are indicated by asterisks. The ZA loop, which is critical for acetyl-lysine binding, for each of the indicated bromodomains is also identified. The underlined residues were changed individually by site-directed mutagenesis to Ala.
[0027] Figs. 2A-2H depict the structure of the P/CAF bromodomain. Figs. 2A-2B shows the stereoview of the Cα trace of 30 superimposed NMR-derived structures of the bromodomain (residues 722-830). The N-terminal four residues (SKEP) which are structurally disordered are omitted for clarity. For the final 30 structures, the root-mean-square deviations (RMSDs) of the backbone and all heavy atoms are 0.63 ± O.l lA and 1.15 ± 0.12A for residues 723-830, respectively. The RMSDs of the backbone and all heavy atoms for the four α-helices (residues 727-743, 770-776, 785-802, and 807-827), are 0.34 ± 0.04A and 0.87 ± 0.06A, respectively. Figs. 2C-2D show the stereoview of the bromodomain structures from the bottom of the protein, which is rotated approximately 90° from the orientation in Figures 2A-2B. Fig. 2E shows the Ribbons [Carson, M., J. Appl. Qγstallogr. 24:958-961 (1991)] depiction of the averaged minimized NMR structure of the P/CAF bromodomain. The orientation of Fig. 2E is as shown in Figures 2A-2B. Figures 2F-2G are schematic representations of the overall topology of the up-and-down four-helix bundle folds with the opposite handedness. The left-handed fold is seen in bromodomain, cytochrome &5, and T4 lysozyme (left, Fig. 2F), whereas the right-handed four- helix bundles are observed in proteins such as hemerytlirin and cytochrome O562 (right, Fig. 2G) [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. Sd. USA 86:6592-6596 (1989)]. Fig. 2H is a molecular surface representation of the electrostatic potential (blue = positive; red = negative) of the bromodomain calculated in GRASP [Nicholls et ah, Biophys. J. 64:166-170 (1993)]. The hydrophobic and aromatic residues (Tyr809, Tyr802, Tyr760, Ala757, and Val752) located between the ZA and BC loops are indicated.
[0028] Figs. 3A-3C show the binding of the P/CAF bromodomain to AcK. Fig. 3A shows the superimposed region of the 2D 15N-HSQC spectra of the bromodomain (approximately 0.5 mM) in its free form (red) and complexed to the AcK-containing H4 peptide (molar ratio 1 :6) (black). Fig. 3B is the Ribbon and dotted-surface diagram of the bromodomain depicting the location of the lysine-acetylated H4 peptide binding site. The color coding reflects the chemical shift changes (Δδ) of the backbone amide 1H and 15N resonances upon binding to the AcK peptide as observed in the 15N-HSQC spectra. The normalized weighted average of the chemical shift changes was calculated by Δav/Δmax= [Δ (52 NH + Δ(?^l25)l2]λl2IΔmπx, where Δmax is the maximum weighted chemical shift difference observed for Tyr809 (0.16ppm). The backbone atoms are color-coded in red, yellow, or green for residues that have Δm/Δmax of >0.6 (Tyr809, Glu808, Asn803, and Ala757), 0.2-0.6 (Ala813, Tyr802, Tyr760, and Val752), or <0.2 (Cys812, Ser807, Cys799, Phe796, and Phe748), respectively. The non-perturbed residues are shown in blue. Fig. 3 C shows the chemical structures of acetyl-lysine, acetyl-histamine, and acetyl- histidine.
[0029] Fig. 4 depicts the acetyl-lysine binding pocket. This is the Ribbons [Carson, M., J. Appl. Cryistallogr. 24:958-961 (1991)] depiction of a portion of the P/CAF bromodomain complexed with the acetyl-histamine. The ligaiid is color-coded by atom type. [0030] Fig. 5A-5B show the binding of various bromodomains from P/CAF, CBP and TIFIb to the N-terminal biotinylated and lysine-acetylated Tat peptide that was immobilized on streptavidin agarose.
[0031] Fig. 6A-6D shows the lysine-acetylated HIV-I Tat protein interactions with bromodomains using 2D 1H-15N-HSQC spectra of the P/CAF or CBP bromodomain in the presence (red) or absence (black) of the lysine-acetylated peptides. Binding of the P/CAF bromodomain to the Tat AcK 50 peptide SYGR-AcK-KRRQRC (SEQ ID NO:50) is shown in Fig. 6A, to the Tat AcK 28 peptide TNCYCK-AcK-CCFH (SEQ ID NO:58) is shown in Fig. 6B, and to histone H4 AcK16 peptide S GRGKGGKGLGKGGA- AcK-RHRK (SEQ ID NO:59) is shown in Fig. 6C. Fig. 6D shows the binding of the CBP of the bromodomain to the Tat AcK50 peptide. AcK is an acetyl-lysine residue
[0032] Fig. 7 is a bar graph of the measurement of superinduction of Tat transactivation activity by P/CAF. Tat-KK is the wild type Tat protein, and Tat-RR is the double mutant Tat carrying lysine to arginine mutations at K50 and K51 positions.
[0033] Figs. 8A-8B show a western blot assay to detect P/CAF interaction with the Tat protein. Note that the protein-protein interaction was only observed with the wild type Tat but not with the Tat K50R/K51R mutant protein. The FLAG was joined to the Tat peptide, whereas the HA-tag was joined to P/CAF.
[0034] Fig. 9 depicts the structure of the P/CAF bromodomain in the complex with the lysine-acetylated Tat peptide (SYGR-AcK-KRRQRC, SEQ ID NO:50, where AcK is acetyl- lysine residue). The side chains of the amino acid residues on both the protein (green) and peptide (dark orange) that showed intermolecular NOEs in the NMR spectra are displayed.
[0035] Fig. 10A- 1OB shows the results of the mutational analyses of the P/CAF bromodomain binding to the HTV-I Tat. Fig. 1OA shows the effects of the point mutation of the individual residues of the bromodomain to alanine on the protein binding to the lysine-acetylated Tat peptide. Fig. 1OB is an assessment of the peptide residue mutation on its binding to the P/CAF bromodomain.
[0036] Fig. 11 depicts the chemical structure common to the acetyl-lysine analogs of the present invention. R1, R2, and R3 can be H, CH3, a halogen (e.g., F, Cl, Br, I etc.), OH, SH, or NH3 +. R4 can be an alkyl (including a peptide/protein attached thereto such as a peptide comprising an acetyl-lysine in which the "N" of the structure depicted is the epsilon nitrogen (i.e., N^ of a lysyl residue), or an aryl group. See also Fig. 13 for examples.
[0037] Fig. 12 depicts examples of acetyl-lysine analogs.
[0038] Fig. 13 depicts inhibition of PCAF BRD/Tat AcK50 binding by 2. In this assay, 2 inhibits the biotinylated Tat AcK50 peptide immobilized on streptavidin agarose binding to the GST-PCAF BRD, as assessed by anti-GST Western blot. Lower panel indicates an equal amount of BRD used in each assay.
[0039] Fig. 14 illustrates selective binding of the lead compound 1 by (A) the PCAF bromodomain but not (B) the CBP bromodomain and (C) the tandem PHD finger and bromodomain of TIFl β. The 2D 1H-15N HSQC spectra of the bromodomain show changes of backbone amide resonances of the protein in the presence (red) or absence (black) of the chemical ligand.
[0040] Fig. 15 represents the three-dimensional structure of the PCAF bromodomain, as determined by NMR. (A) Superimposition of the backbone atoms (N, Cα and C) of the final 20 NMR structures of the bromodomain in complex with the lead compound 2 (highlighted in red). (B) Superimposition of the final representative structures of the bromodomain in the free (yellow) and when bound to the lead compound 2 (blue).
[0041] Fig. 16 demonstrates inhibition of Tat-mediated transactivation by the PCAF
BRD inhibitor. (A) Schematic diagram of the microinjection assay. (B) Effect of a lead compound 765 on Tat-mediated transactivation as compared to DMSO control. [0042] Fig. 17 demonstrates the effect of PCAF BRD inhibitors on Tat transactivation.
HeLa cells were transfected with LTR-luciferase and 20 ng Tat-expression vector. PCAF BRD inhibitors were added after transfection, and cells were harvested after 8 hours.
[0043] Fig. 18 demonstrates the effect of PCAF BRD inhibitors on viral infection. Jurkat
T cells were infected with an LTR-Tat-IRES-GFP virus. PCAF BRD inhibitors were added after overnight infection. The percentage of infection was monitored 48 hours later by FACS analysis.
DETAILED DESCRIPTION OF THE INVENTION [0044] The present invention features compounds of the following general formula(I) wherein:
. R2
"(CH2Jn-
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy; and
n is an integer from O to 10;
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.
[0045] In preferred embodiments, R1 may be selected from the group consisting of hydrogen, lower alkyl, phenyl, and CN. Also, in preferred embodiments, R2 may be selected from the group consisting OfNH3 +, carboxy, and alkoxy. In yet other preferred embodiments, X may be selected from the group consisting of lower alkyl, NH, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH. In other preferred embodiments, n may be 3.
[0046] In other preferred embodiments, the present invention features compounds of the following general formula(II) wherein:
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. In especially preferred embodiments, R1 R2 and R3 are independently selected from the group consisting of hydrogen, lower alkyl, NH3 +, OH, SH, and halogen. Also in especially preferred embodiments, R4 is selected from the group consisting of lower alkyl and aryl. [0047] In yet other preferred embodiments, the present invention features compounds of the following general formula(III) wherein
Figure imgf000024_0001
R R , R , R R5, and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2,NH(CH2)SN(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2θH, NH(CH2)3CH3, NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments,
R and R are selected from the group consisting of hydrogen and OH;
R is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CHs, O(CH2)sCHs, OCHCH3COOCH3, OCH2CON(CHs)2;
R is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CHs, and NHCH3; and
R is selected from the group consisting of hydrogen, and NH2. The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0048] The present invention provides novel compounds that inhibit the binding of bromodomains to acetyl-lysine residues of proteins. The present invention makes use of the three-dimensional structure of a bromodomain as well as the three-dimensional structure of a bromodomain-acetyl-histamine complex.
[0049] In a second aspect, the present invention features a pharmaceutical composition comprising a compound of Formula I wherein
, R,
"(CH2)
R, -
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 +NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, and OH, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases;
together with a pharmaceutically acceptable carrier. [0050] In other preferred embodiments, the present invention features a pharmaceutical composition comprising a compound of Formula (II) wherein:
Figure imgf000026_0001
1 9 ^
R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture
1 9 ^ or in isolated form. In especially preferred embodiments, R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH3 , OH, SH, and halogen. Also in especially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
[0051] In yet other preferred embodiments, the present invention features a pharmaceutical composition comprising a compound of Formula (III) wherein
Figure imgf000026_0002
R1 R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower allcyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2;NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3) NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments,
R1 and R4 are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower allcyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. [0052] In a third aspect, the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
^R2
"(CH2Jn
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 +NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH1NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases;
[0053] In other preferred embodiments, the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
Figure imgf000029_0001
I 1 3
R R and R are independently selected from the group consisting of hydrogen; lower allcyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2,NH3+NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
^4 .
R is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture
1 2 3 or in isolated form. In especially preferred embodiments, R R and R are independently selected from the group consisting of hydrogen, lower allcyl, NH3 , OH, SH, and halogen. Also iinn eess]pecially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
[0054] In yet other preferred embodiments, the present invention features methods for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
Figure imgf000029_0002
R1 R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted arallcyl, heteroaryl, substituted heteroaryl, SO2, NH21NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2;NΗ(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments,
R1 and R4 are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, arallcyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0055] In a fourth aspect, the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
Figure imgf000030_0001
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, O, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, O, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases.
[0056] In other preferred embodiments, the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
Figure imgf000031_0001
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH21NH3 NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH1NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy; and their pharmaceutically acceptable salts of acids or bases;
The general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. In especially preferred embodiments, R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH3 , OH, SH, and halogen. Also in especially preferred embodiments, R is selected from the group consisting of lower alkyl and aryl.
[0057] In yet other preferred embodiments, the present invention features methods for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
Figure imgf000032_0001
R R , R , R R , and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)sCHs, OCHCH3COOCHS, OCH2CON(CHS)2, NH(CH2)SN(CHS)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CHs, NHCH3, SH, halogen, carboxy, and alkoxy. In especially preferred embodiments,
R and R are selected from the group consisting of hydrogen and OH; R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3; and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2
The general formula (III) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
[0058] In some preferred embodiments, the viral infection is HIV infection.
[0059] Compounds of the present in invention may function by modulating the stability of the binding complex formed between P/CAF and Tat that is acetylated at the lysine residue at position 50 of SEQ ID NO:45. In one such embodiment the method comprises contacting the bromodomain of P/CAF or a fragment thereof with a binding partner in the presence of the compound under conditions in which the bromodomain of P/CAF and the binding partner bind in the absence of the compound. The stability of the bromodomain of P/CAF and the binding partner is then determined (e.g., measured). When there is a change in the stability of the binding complex between the bromodomain of P/CAF and the binding partner in the presence of the compound, the compound is identified as a modulator. In one embodiment of this type the binding partner is Tat that is acetylated at the lysine residue at position 50 of SEQ ED NO:45. In a preferred embodiment the binding partner is a fragment of Tat comprising an acetyl-lysine at position 50. In still another embodiment the binding partner is an analog of the fragment of Tat comprising an acetyl-lysine at position 50. When the stability of the bromodomain of P/CAF for the binding partner increases in the presence of the compound, the compound is identified as a stabilizing agent, whereas when the stability of the bromodomain of P/CAF for the binding partner decreases in the presence of the compound, the compound is identified as an inhibitor of the Tat-P/CAF complex. In a preferred embodiment the compound is selected by performing rational drug design with the set of atomic coordinates obtained from one or more of Tables 1-5 and 10-14. More preferably the selection is performed in conjunction with computer modeling. Compounds identified by these methods are also part of the present invention. Preferably the compound is an analog of acetyl-lysine. More preferably the compound is a small organic molecule not included in Fig. 13.
[0060] Compounds of the present invention may function by modulating the binding of P/CAF and Tat. In a preferred embodiment the agent is a small organic molecule. Preferably the agent inhibits and/or destabilizes the binding of P/CAF with Tat. Preferably the agent is an analog of acetyl-lysine. More preferably the agent is not included in Fig. 13.
[0061] Compounds of the present in invention are useful for modulating preventing, and/or retarding the progression and/or treating HIV infection in an individual. One such method employs administering to the individual compounds that modulate the Tat-P/CAF complex selected by performing rational drug design with the set of atomic coordinates obtained from one or more of Tables 1-5 and 10-14. In a preferred embodiment the compound administered is an acetyl-lysine analog. In a particular embodiment this compound is a small organic molecule contained in Fig. 13. Preferably the compound either de-stabilizes or inhibits the Tat-P/CAF complex.
[0062] 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 ringEj, including in its carbon skeleton from one to three heteroatoms selected from nitrogen, oxygen and sulphur;
the terms "lower aralkyl" and "lower heteroaralkyl" are understood as meaning, in view of the definitions above, phenyl(Ci -C8)alkyl or naphthyl(d -Cs)alkyl and heteroar(C] -C8)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: (C1 -C8)alkyl, trifluoromethyl, (C1 -C8)alkoxy, hydroxy, nitro, amino, (C1 - Cs)alkylammo, di(d -C8)alkylamino, sulphoxyl, sulphonyl, sulphonamide, sulpho(d -C8)alkyl, carboxyl, carbalkoxyl, carbamide (it being possible for said (C1 -C8)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.
[0063] 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. [0064] 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 as depicted in Fig. 12. Examples of acetyl-lysine analogs are included in Fig. 13.
[0065] 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 bromodomain of P/CAF is depicted in Fig. 2 A. As used herein, however, a ZA loop has between about 20 and 40 amino acids and preferably comprises the amino acid sequence of SEQ ID NO:3 and/or SEQ ID NO:48. More preferably the ZA loop comprises between about 23 to 34 amino acids. In a specific embodiment the ZA loop has the amino acid sequence SEQ ID NO:43. The amino acid sequence of the ZA loop for a representative number of individual bromodomains is shown in Fig. 1.
[0066] 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".
[0067] 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.
[0068] 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. [0069] As used herein a "small organic 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.
[0070] 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.
[0071] 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 ah, Cell, 50:667 (1987)]. Such proteins have sequence homology as reflected by their high degree of sequence similarity.
[0072] 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 a , 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.
[0073] 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 ah, 1989 supra; DNA Cloning, VoIs. I & II, supra; Nucleic Acid Hybridization, supra., and Sambrook and Russell, 2001] [0074] 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 MacVector 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.
[0075] To develop selective small-molecule inhibitors for blocking Tat/ PCAF association, we conducted NMR-based chemical screening for the BRD by monitoring ligand-induced protein signal changes in 2D 15N-HSQC spectra (Hajduk, et al., Q. Rev. Biophys. (1999) 32, 211-40). We placed an emphasis on identifying compounds that bind selectively the BRD near but not just the AcK binding pocket, as the former may be more selective for this BRD. From screening of a few thousands of small-molecules from commercial libraries, we discovered several compounds including 1 that meet this criterion. Compound 1 binds the PCAF BRD with an affinity comparable to that of the Tat-AcK50 peptide (see below)( Mujtaba, et al., MoI Cell. (2004) 13, 251-63). Importantly, these compounds do not bind the structurally similar BRDs from CBP and TIF lβ at millimolar concentration.
[0076] We next synthesized a series of analogs of compound 1 to probe the structure- activity relationship (SAR) (Table 1). We assessed their binding to the PCAF BRD by measuring an IC50 in an ELISA assay, in which a compound competes against a biotinylated Tat-AcK50 peptide for binding to the GST-fusion BRD immobilized to glutathione-coated 96-well microtiter plate. The SAR study reveals salient features of BRD recognition of 1. First, the BRD prefers a 4-methyl group on the aniline ring, which improves IC50 by 3-fold to 1.6 μM (2 vs. V). While substitution of a 4-ethyl, 3- or 5-methyl group on the aniline ring slightly weakens the binding (3-5 vs. 1), addition of a 4-phenyl group nearly abolishes the binding (6 vs. 1). Adding a 4- or 5-cyano group weakens the binding by ~7- 12-fold (7 and 8 vs. 1). Second, a 2-nitro group on the aniline ring is vital for the binding. Swapping of 2-nitro and 5 -methyl causes a 7-fold reduction in binding (9 vs. 5). Surprisingly, substitution of 2-nitro with 2-caroxylate or 2-caroxyl ester abrogates the binding (10 and 11 vs. 1). Third, the functional importance of the 2-nitro is further supported by the effects of changing the NH to an O linkage in the aniline, which severely compromises the binding to the PCAF BRD (12-17 vs. 1-5). Moreover, changing to a carbon linkage eliminates the binding (18 vs. 1). Fourth, the BRD prefers an amino three-carbon aliphatic chain in 1 - a four-carbon chain reduces the binding by 30-fold (19 vs. 1) and a two-carbon chain nearly loses the binding (20 vs. V). Alteration of 1 by two key elements, i.e. changing to a four-carbon chain and 4-nitro, abolishes the binding (21 vs. 1). Finally, the terminal amine group is also an important functional moiety for the BRD binding (22-24 vs. 1).
Tablel. Structure- Activity Relationship Data for Derivatives of 1
Figure imgf000040_0001
Cmpd R1 X n R2 IC50 (μM)
1 2-NO2 NH 3 -NH3 + 5 1 ± 0 1
2 2-NO2, 4-CH3 NH 3 -NH3 + 1 6 ± 0 1
3 2-NO2, 4-CH2-CH3 NH 3 -NH3 + 7 2 ± 0.1
4 2-NO2, 3-CH3 NH 3 -NH3 + 5 9 ± 0.1
5 2-NO2, 5-CH3 NH 3 -NH3 + 10 8 ± 0 1
6 2-NO2, 4-Ph NH 3 -NH3 + >10,000
7 2-NO2, 4-CN NH 3 -NH3 + 34 9 ± O 1
8 2-NO2, 5-CN NH 3 -NH3 + 634 ± O 6
9 2-CH3, 5-NO2 NH 3 -NH3 + 77 8 ± O 4
10 2-COO NH 3 -NH3 + >10,000
11 2-COOCH3 NH 3 -NH3 + >10,000
12 2-NO2 O 3 -NH3 + 125 6 ± 0 6
13 2-NO2, 4-CH3 O 3 -NH3 + 180 O ± 0 5
14 2-NO2, 4-CH3O O 3 -NH3 + 1027 ± 04
15 2-NO2, 4-CI O 3 -NH3 + 215 1 ± 0 6
16 2-NO2, 5-CH3 O 3 -NH3 + 203 6 ± 0 6
17 2-NO2, 3-CH3 O 3 -NH3 + 164 8 ± 0 8
18 2-NO2 CH2 3 -NH3 + >10,000
19 2-NO2 NH 4 -NH3 + 145 9 ± 0 7
20 4-NO2 NH 2 -NH3 + >2,000
21 4-NO2 NH 4 -NH3 + >10,000
22 3-NH2, 4-NO2 NH 3 -COO >2,000
23 2-NO2, 4-CI NH 2 -(OH)CH3 >10,000
24 2-CI, 4-NO2 NH 2 -(OH)CH3 >10,000
[0077] To understand ligand selectivity of the PCAF BRD, we solved the 3D structures of the protein bound to 1 and 2. The two ligands are bound in the protein structure in nearly the same manner. For clarity, only the 2-bound structure is reported here, which is similar to the free structure except for the ZA and BC loops that move closer to each other by clamping onto the ligand (Figure IB). 2 is engulfed by residues in the ZA and BC loops outside the AcK binding pocket, blocking BRD binding to AcK of a target protein (Figure 1C). The 2-nitro group of 2 possibly forms a hydrogen bond with the phenolic -OH of Y809 and/or Y802, and the terminal - NH3 + interacts eletrostatically with the side-chain carboxylate of E750. The functional importance of the 2-nitro and the aniline NH likely results from a possible six-member ring structure formed between these two groups. However, it is not clear why substitution of 2-nitro with 2-carboxylate abrogates the BRD binding (10 vs. 1). The aromatic ring of 2 is sandwiched between the side-chains of Y802 and A757 on one side, and Y809 and E756 on the other side; and the propane carbon chain is surrounded by the hydrophobic portions of the side-chains of P747, E756 and V752. Finally, the 4-methyl of 2 fills a small hydrophobic cavity formed by side-chains of A757, Y802 and Y809 (Figure ID), contributing to a 3-fold increase over 1 in binding to the PCAF BRD. Notably, out of the ligand- interacting residues, only Y802 is among the conserved residues at the AcK binding site in BRDs, thus explaining the selective binding by this class of compounds to the PCAF BRD over the structurally similar CBP and TIFl β BRDs.
[0078] The present invention therefore provides a class of novel small molecules that can effectively inhibit the PCAF BRD/Tat-AcK50 association in vitro by selectively binding to the BRD (Figure 13). The detailed SAR understanding of the lead compounds 1 and 2 will facilitate our efforts to optimize their affinity and selectivity by branching out to interact with the neighboring AcK binding pocket by the tethering techniques. Such small-molecule inhibitors will help validate the novel anti-HIV/AIDS therapeutic strategy by targeting a cellular protein to block HTV transcription and replication.
[0079] Bromodomain. The present invention utilizes detailed structural information regarding a bromodomain and a bromodomain complexed with its acetylated binding partner. The present invention therefore provides the three-dimensional structure of the bromodomain and a bromodomain acetylated binding partner complex. Since the interaction of the bromodomain with a histone for example, can play a significant role in chromatin remodeling/regulation, the structural information provided herein can be employed in methods of identifying drugs that can modulate basic cell processes by modulating the transcription. In a particular embodiment, the three-dimensional structural information is used in the design of a small organic molecule for the treatment of cancer or as disclosed below, HIV-I infection and/or AIDs. In addition, the present invention provides a critical structural feature for a class of inhibitors (acetyl-lysine analogs) of the interaction between bromodomains and their protein binding partners which contain an acetylated-lysine {e.g., Tat with P/CAF), see Fig. 11, as well as a compilation of compounds that share this critical feature, see Fig. 12.
[0080] 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 including cancers such as leukemia. For example, chromatin remodeling plays a central role in the etiology of viral infection and cancer [Archer and Hodin, Curr. Opin. Genet. Biol. 9:171-174 (1999); Jacobson and Pillus, Curr. Opin. Genet. Biol. 9:175-184 (1999)]. Both altered histone acetylation/ deacetylation and aberrant forms of chromatin-remodeling complexes are associated with human diseases. Furthermore, chromosomal translocation of various cellular genes with those encoding HATs and subunits of chromatin remodeling complexes have been implicated in leukomogenesis. The MOZ (monocytic leukemia zinc finger) and MLL/ ALL-I genes are frequently fused to the gene encoding the co-activator HAT CBP [Sobulo et al, Proc. Natl. Acad. ScL USA 94:8732-8737(1997)]. The resulting fusion protein MLL-CBP contains the tandem bromodomain-PHD finger-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 human acute leukemia. 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.
[0081] In addition, it now known that the human immunodeficiency virus type 1 (HIV-I) trans-activator protein, Tat, is tightly regulated by lysine acetylation [Kiernan et αl, EMBO Journal 18:6106-6118 (1999)]. HIV-I Tat transcriptional activity is absolutely required for productive HIV viral replication [Jeang and Gatignol, Curr. Top. Microbiol. Immunol, 188:123- 144(1994)]. Therefore, the interaction of the acetyl-lysine of Tat with one or more bromodomain-containing proteins associated with chromatin remodeling could mediate gene transcription. More particularly, it is disclosed herein that acetylated lysine50 of Tat specifically binds to the bromodomain of P/CAF. Therefore, this particular bromodomain/lysine-acetylated Tat interaction serves as a drug target for blocking HIV replication in cells. As indicated above, an antibody raised specifically against a peptide from the ZA loop of the P/CALF bromodomain could also be effective for treating and/or preventing HIV infections including those that lead to AIDs. [0082] In addition, based on the new structural information disclosed herein, 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.
[0083] 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 15N- or 1H-amide chemical shift changes in two dimensional 15N-heteronuclear single-quantum correlation (15N-HSQC) spectra upon the addition of the linked compound to the 15N-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. [0084] Pharmaceutical Compositions. In yet another aspect of the present invention, provided are pharmaceutical compositions of the compounds of Formula I. Such pharmaceutical compositions may be 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, emulsifϊers, 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 lyopliilized form.
[0085] 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 multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation 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.
[0086] 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. 5,008,110, 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.
[0087] 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. CHn. Invest., 84:1145-1146 (1989) (α-1 -proteinase); Oswein et 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
[0088] The following examples are put 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. Structure and Ligand of a Histone Acetyltransferase Bromodomain
[0089] Sample preparation: The bromodomain of P/CAF (residues 719-832 of SEQ ID NO:2) was subcloned into the pET14b expression vector (Novagen) and expressed in Escherichia coli BL21(DE3) cells. Uniformly 15N- and 15N/13C-labelled proteins were prepared by growing bacteria in a minimal medium containing 15NH4Cl with or without 13C6-glucose. A uniformly 15N/13C-labelled and fractionally deuterated protein sample was prepared by growing the cells in 75% 2H2O. The bromodomain was purified by affinity chromatography on a nickel- ID A column (Invitrogen) followed by the removal of poly-His tag by thrombin cleavage. The final purification of the protein was achieved by size-exclusion chromatography. The acetyl- lysine-containing peptides were prepared on a MilliGen 9050 peptide synthesizer (Perkin Elmer) using Fmoc/HBTU chemistry. Acetyl-lysine was incorporated using the reagent Fmoc-Ac-Lys with HBTU/DIPEA activation. NMR samples contained approximately 1 mM protein in 10OmM phosphate buffer of pH 6.5 and 5mM perdeuterated DTT and 0.5mM EDTA in H2O/2H2O (9/l) or 2H2O.
[0090] NMR spectroscopy: AU ΝMR spectra were acquired at 300C on a Bruker DRX600 or DRX500 spectrometer. The backbone assignments of the 1H, 13C, and 15N resonances were achieved using deuterium-decoupled triple-resonance experiments of HNCACB and HN(CO)CACB \Ywuadά et al., J. Am. Chem. Soc. 116:11655-11666 (1994)] recorded using the uniformly 15N/13C-labeled and fractionally deuterated protein. The side-chain atoms were assigned from 3D HCCH-TOCSY [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)] and (H)C(CO)NH-TOCSY [Logan et al, J. Biohnol. NMR 3:225-231 (1993)] data collected on the uniformly 15N/13C-labeled protein. Stereospecific assignments of methyl groups of the VaI and Leu residues were obtained using a fractionally 13C-labeled sample [Neri et al., Biochemistry 28:7510-7516 (1989)]. The NOE-derived distance restraints were obtained from 15N- or 13C- edited 3D NOESY spectra, ø-angle restraints were determined based on the JHN,H« coupling constants measured in a 3D HNHA spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249- 363 (1994)]. Slowly exchanging amide protons were identified from a series of 2D 15N-HSQC spectra recorded after the H2O buffer was changed to a 2H2O buffer. The intermolecular NOEs used in defining the structure of the bromodomain/Ac-histamine complex were detected in 13C- edited (F7), 13C/15N-filtered (F3) 3D NOESY spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)]. AU NMR spectra were processed with the NMRPipe/NMRDraw programs and analyzed using NMRView [Johnson and Blevins, J. Biomol, NMR 4:603-614 (1994)].
[0091] Structure calculations: Structures of the bromodomain were calculated with a distance geometry/simulated annealing protocol using the X-PLOR program [Brunger, X-PLOR Version 3.1: A system for X-Ray crystallography and NMR, Yale University Press, New Haven, CT, (1993)]. A total of 1324 manually assigned NOE-derived distance restraints were obtained from the 15N- and 13C-edited NOE spectra. Further analysis of the NOE spectra was carried out by the iterative automated assignment procedure using ARIA [Nilges and O'Donoghue, Prog. NMR Spectroscopy 32:107-139 (1998)], which integrates with X-PLOR for structure calculations. A total of 1519 unambiguous and 590 ambiguous distance restraints were identified from the NOE data by ARIA, many of which were checked and confirmed manually. The ARIA-assigned distance restraints were in agreement with the structures calculated using only the manually assigned NOE distance restraints, 28 hydrogen-bond distance restraints for 14 hydrogen bonds, and 54^>-angle restraints. The final structure calculations employed a total of 3515 NMR experimental restraints obtained from the manual and the ARIA-assisted assignments, 2843 of which were unambiguously assigned NOE-derived distance restraints that comprise of 1077 intra-residue, 621 sequential, 550 medium-range, and 595 long-range NOEs. For the ensemble of the final 30 structures, no distance and torsional angle restraints were violated by more than 0.3 A and 5°, respectively. The total, distance violation, and dihedral violation energies were 178.7 ± 2.4 kcal mol"1, 41.6 ± 0.9 kcal mol"1, and 0.50 ± 0.06 kcal mol"1, respectively. The Lennard- Jones potential which was not used during any refinement stage, was - 526.2 ± 16.8 kcal mol"1 for the final structures. Ramachandran plot analysis of the final structures (residues 727-828) with Procheck-NMR [Laskowski et al, J. Biolmol. NMR 8:477-486 (1996)] showed that 71.0 ± 0.6%, 23.8 ± 0.6%, 3.5 ± 0.2%, and 1.7 ± 0.2% of the non-Gly and non-Pro residues were in the most favorable, additionally allowed, generously allowed, and disallowed regions, respectively. The corresponding values for the residues in the four α-helices (residues 727-743, 770-776, 785-802, and 807-827) were 88.9 ± 0.4%, 11.0 ± 0.4%, 0.1 ± 0.1%, and 0.0 ± 0.0%, respectively. The structure of the bromodomain/acetyl-histamine complex was determined using the free form structure and additional 25 intermolecular and 5 intra-ligand NOE-derived distance restraints.
[0092] Site-directed mutagenesis: Mutant proteins were prepared using the QuickChange site-directed mutagenesis kit (Stratagene). The presence of appropriate mutations was confirmed by DNA sequencing.
[0093] Ligand titration: Ligand titration experiments were performed by recording a series of 2D 15N- and 13C-HSQC spectra on the uniformly 15N-, and 15N/13C-labelled bromodomain (~0.3mM), respectively, in the presence of different amounts of ligand concentration ranging from 0 to approximately 2.0 mM. The protein sample and the stock solutions of the ligands were all prepared in the same aqueous buffer containing 10OmM phosphate and 5mM perdeuterated DTT at pH 6.5.
[0094] The full length nucleic acid sequence of the human p300/CBP-associated factor (P/CAF) was obtained from GenBank. Accession No: U57317.2 (SEQ ID NO:l).The full length protein sequence of the human p300/CBP-associated factor (P/CAF) was obtained from GenBank. Accession No: U57317.2, (SEQ ID NO:2).
[0095] Results. The P/CAF bromodomain represents an extensive family of bromodomains (Fig. 1). A large number of long-range nuclear Overhauser enhancement (NOE)-derived distance restraints were identified in the NMR data of the P/CAF bromodomain, yielding a well- defined three-dimensional structure (Figs. 2A -2D). Table 2 shows the NMR chemical shift assignment of the P/CAF bromodomain. Table 2 shows the Unambiguous NOE-derived distance restraints. Table 4 shows the Ambiguous NOE-derived distance restraints. Table 5 shows the Hydrogen bond restraints. The NMR structure coordinates of the P/CAF bromodomain in the free and complexed to acetyl-histamine are shown in Tables 5 and 6, respectively. The structure consists of a four-helix bundle (helices OLZ, OLA, CCB, and αc) with a left-handed twist, and a long intervening loop between helices αz and «A (termed the ZA loop, Fig. 2E). The four amphipathic α-helices are packed tightly against one another in an antiparallel manner, with crossing angles for adjacent helices of ~16-20°. The up-and-down four-helix bundle can adapt two topological folds with opposite handedness (Figures 2F-2G). The right-handed four-helix bundle fold occurs more commonly and is seen in proteins such as hemerythrin and cytochrome O562. The left-handed fold of the bromodomain structure is less common, but also observed in proteins such as cytochrome bs and T4 lysozyme [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. ScL USA 86:6592-6596 (1989)]. This topological difference arises from the orientation of the loop between the first two helices (Fig. 2F-2G). The right-handed four-helix bundle proteins have a relatively short hairpin-like connection between the first two helices, which makes the "preferred" turn to the right at the top of the first helix [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. Sd. USA 86:6592-6596 (1989); Weber and Salemme, Nature 287:82-84 (1980)]. In contrast, proteins with the left-handed fold usually have a long loop after the first helix and often contain additional secondary structural elements at the base of the helix bundle [Richardson, J., Adv.Protein Chem., 34:167-339 (1989); Presnell and Cohen, Proc. Natl. Acad. ScL USA 86:6592-6596 (1989)]. In the bromodomain structure, this long ZA loop has a defined conformation and is packed against the loop between helices CXB and αc (termed the BC loop) to form a hydrophobic pocket. These tertiary interactions between the two loops appear to favor the left turn of the ZA loop, resulting in the left-handed four-helix bundle fold of the bromodomain. The hydrophobic pocket formed by loops ZA and BC is lined by residues Val752, Ala757, Tyr760, Val763, Tyr802 and Tyr809 (Fig. 2H), and appears to be a site for protein-protein interactions (see below). The pocket is located at one end of the four-helix bundle, opposite to the N- and C-termini of the protein. Interestingly, the ZA loop varies in length amongst different bromodomains, but almost always contains residues corresponding to Phe748, Pro751, Pro758, Tyr760, and Pro767 (Fig. 1). The conservation of these residues within the ZA loop as well as residues within the α-helical regions implies a similar left-handed four-helix bundle structure for the large family of bromodomains.
[0096] The modular bromodomain structure supports the idea that bromodomain can act as a functional unit for protein-protein interactions. The observation that bromodomains are found in nearly all known nuclear HATs (A-type) that are known to promote transcription-related acetylation of histones on specific lysine residues, but not present in cytoplasmic HATs (B-type), prompted the determination of whether bromodomains can interact with acetyl-lysine (AcK). The NMR titration of the P/CAF bromodomain were performed with a peptide (SGRGKGG- AcK-GLGK) derived from histone H4, in which Lys8 is acetylated (Lys8 is the major acetylation site in H4 for GCN5, a yeast homologue of P/CAF). Remarkably, the bromodomain could indeed bind the AcK peptide. Moreover, this interaction appeared to be specific, based on the 15N-HSQC spectra which showed that only a limited number of residues underwent chemical shift changes as a function of peptide concentration (Fig. 3A). Conversely, the NMR titration of the bromodomain with a non-acetylated, but otherwise identical H4 peptide, showed no noticeable chemical shift changes, demonstrating that the interaction between the bromodomain and the lysine-acetylated H4 peptide was dependent upon acetylation of lysine. The dissociation constant (/CD) for the AcK peptide was estimated to be 346 ± 54 μM. This binding is likely reinforced through additional interactions between bromodomam-containing proteins and target proteins. Notably, many chromatin-associated proteins contain two or multiple bromodomains (Fig. 1). Indeed, binding with another lysine-acetylated peptide (RKSTGG-ACK- APRKQ) derived from the major acetylation site on histone H3 (residues 9-20) was also observed. Together, these data demonstrate that the P/CAF bromodomain has the ability to bind AcK peptides in an acetylation dependent manner.
[0097] Intriguingly, the bromodomain residues that exhibited the most significant 1H and 15N chemical shift changes on peptide binding are located near the hydrophobic pocket between the ZA and BC loops (Fig. 3B). Because a similar pattern of amide chemical shift changes was observed with the two different AcK-containing peptides, it was surmised that the hydrophobic cavity is the primary binding site for AcK. This hypothesis was further supported by titration with acetyl-histamine, which mimics the chemical structure of the AcK side-chain (Fig. 3C). Both 15N- and 13C-HSQC spectra showed that interaction with acetyl-histamine was also acetylation-dependent, involving the same set of residues that showed chemical shift perturbations with similar concentration dependence. It should be noted that the bromodomain did not bind to the amino acids acetyl-lysine or acetyl-histidine alone, possibly due to the presence of the charged amino, carboxyl, or caboxylate group adjacent to the acetyl moiety (Fig. 3C). Taken together, these results strongly suggest that the P/CAF bromodomain can interact with acetyl-lysine-containing proteins in a specific manner, and that this interaction is localized to the bromodomain hydrophobic cavity.
[0098] To identify the key residues involved in bromodomain- AcK recognition, the NMR structure of the P/CAF bromodomain in complex with acetyl-histamine was elucidated. As anticipated, the acetylated moiety binds in the bromodomain hydrophobic pocket (Fig. 4). The intermolecular interactions are largely hydrophobic in nature, with the methyl group of acetyl- histamine making extensive contacts with the side-chains of Val752, Ala757, and Tyr760, and the methylene groups of acetyl-histamine displaying specific NOEs to Val752, Ala757, Tyr760, Tyr802, and Tyr809. No intermolecular NOEs were observed for the imidazole ring of acetyl- histamine. From the spectral analysis it is clear that the structure of the bromodomain is very similar in both the free and complex forms. It is worth noting that the bromodomain-AcK recognition is reminiscent of the interactions between the histone acetyltransferase Hatl and acetyl-CoA. Although the binding pockets of these two otherwise structurally unrelated proteins are composed of different secondary structural elements, the nature of acetyl-lysine recognition has striking similarities. In particular, Tyr809, Tyr802, Tyr760, and Val752 in the bromodomain appear to be related to Phe220, Phe261, Val254, and Ile217 of Hatl, respectively, in their interactions with the acetyl moiety. This observation may suggest an evolutionary convergent mechanism of acetyl-lysine recognition between bromodomains and histone acetyltransferases. To determine the relative contributions of residues within the hydrophobic cavity in bromodomain- AcK binding, site-directed mutagenesis was used to alter residues Tyr809, Tyr802, Tyr760, and Val752 (Table 7).
Table 2. Structural and Functional Analysis of the P/CAF Bromodomain Mutants
Figure imgf000053_0001
The effects of mutations on the structural integrity of the bromodomain were assessed by using the 15N-HSQC spectra. The amide 1HZ15N resonances of the mutant proteins were compared to those of the wild-type bromodomain to determine if the particular mutations lead to global or local structure disruption. Severe line-broadening of the amide resonances would indicate protein conformational exchange due to a decrease of structure stability resulting from point mutations. Structural integrity of the mutant proteins is expressed here relative to that of the wild-type, using the signs of "++++" for as stable as the wild-type, "+++" for mildly destabilized, "++" for moderately destabilized, and "-" for completely unfolded. b The ligand binding affinity (KD) of the bromodomain proteins was estimated by following chemical shift changes of amide peaks in the 15N-HSQC spectra as a function of the ligand concentration. c No detectable ligand binding observed in the NMR titration. d Ligand binding affinity was significantly reduced and beyond the limit for reliable measurements by NMR titration.
[0099] Substitution of Ala for Tyr809 completely abrogated the bromodomain binding to the lysine-acetylated H4 peptide, while the Tyr802Ala, Tyr760Ala, and Val752Ala mutants had significantly reduced ligand binding affinity. To assess whether these mutations disrupted the overall bromodomain fold, the 15N-HSQC spectra of the mutants was compared to that of the wild-type protein. For the Tyr809Ala mutant, the amide chemical shifts were only affected for a few residues near the mutation site. However, mutations of the other residues in the hydrophobic binding pocket perturbed the local protein conformation to greater extents, particularly the ZA loop (Table 7). Thus, the NMR structural analysis and the mutagenesis studies show that Tyr809, which is structurally supported by Trp746 and Asn803 (Fig. 4), is essential for the bromodomain interaction with the acetyl group of acetyl-lysine, while residues of Tyr802, Tyr760, and Val752 likely play both structural and functional roles in the recognition. These residues are highly conserved throughout the bromodomain family (Fig. 1), suggesting that recognition of acetyl-lysine may be a feature of bromodomains, in general. Therefore, Val752, Ala757, Tyr760, Tyr802, Asn803, and Tyr809 are key amino acid residues for the P/CAF bromodomain binding to acetyl-lysine.
Table 3: Amino Acid Sequences of Bromodomains Identified in Figure 1
Figure imgf000054_0001
Example 2. Structural Insights Into HIV-I TAT Trans activation via P/CAF
[0100] Whereas the life cycle of HIV is still being elucidated, it is currently accepted that HIV binds to CD4 protein of a host T cell or macrophage and with the aid of a chemokine receptor (e.g., CCR5 or CXCR4) enters the host cell. Once in the host cell, the retrovirus, HIV-I, is converted to a DNA by reverse transcriptase and the expression of the HIV-I genome is dependent on a complex series of events that are believed to be under the control of two viral regulatory proteins, Tat and Rev [Romano et al, J. CellBiochem. 75(3):357-368 (1999)]. Rev controls post-translational events, whereas, Tat (the trans-activator protein) functions to stimulate the production of full-length HIV transcripts and viral replication in infected cells. The Tat protein transactivates the transcription of HIV-I starting at the 5' long terminal repeat (LTR) [Romano et al, J. CellBiochem. 75(3):357-368 (1999)] by recruiting one or more carboxyl- terminal domain kinases to the HIV-I promoter. More specifically, Tat stimulates transcription from the LTR at a hairpin element, the transactivation responsive region (TAR) [Kiernan et al, EMBO J. 18 : 6106-6118 ( 1999)] at least in part by interacting with and thereby recruiting the carboxyl-terminal domain kinase, i.e., the positive transcriptional elongation factor (P-TEFb) to the TAR RNA element [Garber et al, Mol.Cell.Biol. 20(18):6958-6969 (2000)]. P-TEFb is a muti-subunit kinase that minimally comprises a heterodimer consisting of the regulatory cyclin Tl and its corresponding catalytic subunit, cyclin-dependent kinase 9 (CDK9). P-TEFb acts by phosphorylating the carboxyl-terminal domain of RNA polymerase II [Peng et al, J.Biol. Chem. 274 (49):34527-34530 (1999); Romano et al, J. CellBiochem. 75(3):357-368 (1999)].
[0101] Recently, it has been shown that HIV-I Tat transcription activity is regulated through lysine acetylation by, and association with the histone acetyltransferases (HATs) p300/CBP and the p300/CBP-associating factor (P/CAF), which specifically acetylate Lysine 50 (K50) and Lysine 28 (K28) of the Tat protein, respectively [Kiernan et al, EMBO J. 18:6106-6118 (1999); Ott et al, Curr. Biol 9:1489-1492 (1999)]. Notably, the acetylation of K50 by the transcriptional co-activator p300/CBP is on the C-terminal arginine-rich motif (ARM) of Tat, which is essential for its binding to the TAR RNA element and for nuclear localization, [Kiernan et al, EMBOJ. 18:6106-6118 (1999); Ott et al, Curr. Biol. 9:1489-1492 (1999)]. Acetylation of K28 of Tat by P/CAF enhances Tat binding to P-TEFb, whereas acetylation of K50 of Tat by P300/CBP promotes the dissociation of Tat from the TAR RNA element. This dissociation of Tat from the TAR RNA element occurs during early transcription elongation [Kiernan et al, EMBO J. 18:6106-6118 (1999)]. However, heretofore, little else was known regarding the relationship of these HATs with Tat after the acetylation has occurred. [0102] Sample preparation: The bromodomain of P/CAF (residues 719-832) was subcloned into the pET14b expression vector (Novagen) and expressed in Escherichia coli BL21(DE3) cells. Uniformly 15N- and 15N/13C-labeled proteins were prepared by growing bacteria in a minimal medium containing 15NH4Cl with or without 13C6-glucose. A uniformly 15N/13C-labeled and fractionally deuterated protein sample was prepared by growing the cells in 75% 2H2O. The bromodomain was purified by affinity chromatography on a nickel-IDA column (Invitrogen) followed by the removal of poly-His tag by thrombin cleavage. The final purification of the protein was achieved by size-exclusion chromatography. The acetyl-lysine-containing peptides were prepared on a MilliGen 9050 peptide synthesizer (Perkin Elmer) using Fmoc/HBTU chemistry. Acetyl-lysine was incorporated using the reagent Fmoc-Ac-Lys with HBTU/DIPEA activation. NMR samples contained ~0.5 mM protein in complex with the lysine-acetylated Tat peptide in 100 mM phosphate buffer of pH 6.5 and 5mM perdeuterated DTT and 0.5mM EDTA in H2CV2H2O (9/1) or 2H2O. The bromodomain-containing constructs from P/CAF, CBP and TIF-lβ were cloned into pGEX4T-3 vector (Pharmacia). These recombinant GST-fusion proteins were expressed in BL21 (DE3) codon plus cell line, and purified by using glutathione sepharose column.
[0103] NMR spectroscopy: AU NMR spectra were acquired at 3O0C on a Broker DRX600 or DRX500 spectrometer. The backbone assignments of the 1H, 13C, and 15N resonances were achieved using deuterium-decoupled triple-resonance experiments of HNCACB and HN(CO)CACB [Yamazaki et α/., J. Am. Chem. Soc. 116:11655-11666 (1994)] recorded using the uniformly 15N/13C-labelled and fractionally deuterated protein. The side-chain atoms were assigned from 3D HCCH-TOCSY [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)] and (H)C(CO)NH-TOCSY [Logan et al, J Biolmol. NMR 3:225-231 (1993)] data collected on the uniformly 15N/13C-labeled protein. Stereospecific assignments of methyl groups of the valine and leucine residues were obtained using a fractionally 3C-labeled sample [Neri et ah, Biochemistry 28:7510-7516 (1989)]. The NOE-derived distance restraints were obtained from 15N- or 13C-edited 3D NOESY spectra [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)]. <^-angle restraints were determined based on the 3JHN1H coupling constants measured in a 3D HNHA spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)]. Slowly exchanging amide protons were identified from a series of 2D 15N-HSQC spectra recorded after the H2O buffer was changed to a 2H2O buffer. The intermolecular NOEs used in defining the structure of the bromodomain/Ac-histamine complex were detected in 13C-edited (Fy), 13Q15N- filtered (F3) 3D NOESY spectrum [Clore and Gronenborn, Meth. Enzymol. 239:249-363 (1994)]. All NMR spectra were processed with the NMRPipe/NMRDraw programs and analyzed using NMRView [Johnson and Blevins, J. Biomol, NMR 4:603-614 (1994)].
[0104] Ligand titration experiments were performed by recording a series of 2D 15N-HSQC spectra on the uniformly 15N-labelled bromodomain (-0.3 mM), respectively, in the presence of different amounts of ligand concentration ranging from 0 to ~2.0mM. The protein sample and the stock solutions of the ligands were all prepared in the same aqueous buffer containing 100 mM phosphate and 5mM perdeuterated DTT at pH 6.5.
[0105] Structure calculations. Structures of the bromodomain were calculated with a distance geometry/simulated annealing protocol using the X-PLOR program [Brunger, X-PLOR Version 3.1: A system for X-Ray crystallography and NMR, Yale University Press, New Haven, CT, (1993)]. A total of 1324 manually assigned NOE-derived distance restraints were obtained from the 15N- and 13C-edited NOE spectra. Further analysis of the. NOE spectra was carried out by the iterative automated assignment procedure by using ARIA [Nilges and O'Donoghue, Prog. NMR Spectroscopy 32:107-139 (1998)], which integrates with X-PLOR for structure calculations. The ARIA-assigned distance restraints were in agreement with the structures calculated using only the manually assigned NOE distance restraints, hydrogen-bond distance restraints, and 54 φ- angle restraints. The final structure calculations employed a total of 2903 NMR experimental restraints obtained from the manual and the ARIA-assisted assignments. For the ensemble of the final 30 structures, no distance and torsional angle restraints were violated by more than 0.3A and 5A, respectively. The Lemiard- Jones potential which was not used during any refinement stage, and stereochemistry of the final structures was validated with Ramachandran plot analysis by using Procheck-NMR [Laskowski et ah, J. Biolmol. NMR 8:477-486 (1996)]. [0106] Site directed mutagenesis. Site directed mutagenesis was performed on selected residues of P/CAF Bromodomain using quick-change kit (Stratagene). The mutants were confirmed by sequencing and proteins were expressed and purified as above.
[0107] Peptide binding assay. Equal amount (10 μM) of GST, GST-P/CAF bromodomain and its mutant proteins, as well as various GST-fusion bromodomains from CBP and TIF lβ were incubated for at least two hours at room temperature with the N-terminal biotinylated and lysine- acetylated Tat peptide (50 μM) in a 50 mM Tris buffer of pH 7.5, containing 50 mM NaCl, 0.1% BSA and 1 mM DTT. Streptavidin agarose (10 μL) was added to mixture and the beads were washed twice in the Tris buffer with 500 mM NaCl and 0.1% NP -40. Proteins were eluted from the argarose beads in SDS buffer and separated on a 14% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose membrane (Pharmacia), and the membrane was blocked overnight with 5% non-fat milk in washing buffer of 20 mM Tris, pH 7.5, plus 150 mM NaCl and 0.1% Tween-20 at 40C. Western blotting was performed with anti-GST antibody (Sigma) and goat anti-rabbit IgG conjugated with horseradish-peroxidase (Promega) and developed by chemiluminescence. Peptide competition experiments were performed by incubating various non-biotinylated and mutant Tat peptide with the P/CAF bromodomain and the biotinylated and wild type Tat peptide. The molar ratio of the wild type and mutant Tat peptides in the mixture were kept at 1:2. The binding results were analyses by using the procedure as described above. The full length protein sequence of the Human Immunodeficiency Virus type 1 Tat was obtained from GenBank, Accession No: AAA83395 (SEQ ID NO:45).
[0108] Results. To test whether or not the bromodomains of these HATs can bind to the lysine-acetylated Tat, in vitro binding assays were performed by using recombinant and purified bromodomains and lysine-acetylated peptides derived from the acetylation sites in Tat. While the bromodomains of CBP and TIF lβ did not show any binding, the P/CAF bromodomain binds tightly only to the Tat peptide containing AcK50 (where AcK stands for an Ne-acetyl lysine residue) (Figs. 5A-5B). NMR binding studies further confirmed the specific interaction of the P/CAF bromodomain and lysine-acetylated Tat peptide. Because NMR resonances of amide protons are highly sensitive to local chemical environment and conformational change in a protein, two-dimensional 1H-15N heteronuclear single quantum correlation (HSQC) spectrum can be used to detect even weak but specific interactions between a protein and its binding ligand. As shown in 2D HSQC spectra (Figs. 6A-6D), the bromodomain of P/CAF binds weakly to the lysine-acetylated peptides derived from known acetylation sites of K28 on Tat and of K16 on histone H4 by only interacting with the acetyl-lysine residue in the peptides (Kd <300 μM). This is reflected the relatively small chemical shift perturbation of the amide proton signals of the protein upon addition of ligand. On the other hand, the P/CAF bromodomain interacts strongly with the Tat AcK50 peptide, which involves many protein residues in addition to those for acetyl-lysine binding with an estimated IQ of -20 μM. Binding of peptide residues flanking the acetyl- lysine may explain the high specificity of the P/CAF bromodomain for the acetylated Tat. Furthermore, the p300/CBP bromodomain did not bind the lysine-acetylated Tat peptide in a specific manner except its weak interaction with the acetyl-lysine residue in the peptide (Figs. 6A-6D). Together, these results demonstrate the P/CAF bromodomain can specifically recognize the lysine-acetylated Tat involving K50.
[0109] To determine how the P/CAF binding affects Tat function in vivo, transactivation activity of Tat was measured. Superinduction of Tat transactivation activity exhibited as much as a 30-fold increase upon P/CAF stimulation (Fig. 7). This profound P/CAF effect requires acetylation at K50 on Tat, as a double mutant of K50 and K51 substituted with arginines resulted in a nearly two-thirds reduction of the enhancement. Further, specific interaction between P/CAF and wild type Tat in cells was also detected, but not with the Tat double mutant containing K50R/K51R (Figs. 8A-8B). Taken together, these results confirm that P/CAF can directly interact via its bromodomain with the lysine-acetylated Tat, which possibly regulates Tat transactivation activity.
[0110] To further understand the molecular basis of the P/CAF bromodomain recognition of the lysine-acetylated Tat, the three-dimensional structure was determined for the P/CAF bromodomain in complex with an 11 -residue Tat peptide containing AcK50. A total of 2,903 NMR-derived distance and dihedral angle restraints were used. The structure of the bromodomain in the peptide-bound form consists of an up-and-down four-helix bundle (helices ccz, «A, c-B, and αc) with a left-handed twist, and a long intervening loop between helices a∑ and CCA (termed the ZA loop) (Fig. 9). The overall structure of the complex is well defined, and similar to the structure of the free bromodomain [Dhalluin et al, Nature 399:491-496 (1999)] except that the ZA and BC loops, which compose the acetyl-lysine binding pocket, undergo local conformational changes in order to accommodate their interactions with the peptide residues.
Table 4. NMR Structural Statistics of the P/CAF Bromodomain/Tat Peptide Complex
Total Experimental Restraints 2903
Distance Restraints a 2822
Total Ambiguous 122
Total Unambiguous 2700
Intra-residue (i = j) 1118 (41.40%)
Hydrogen Bond Restraints 28
Dihedral Angle Restraints 53
Final Energies (kcalmol"1)
Eτotal 366.35 ± 31.11
Ramachandran Plot (%) Protein/Peptide Complex Secondary Structure
Most Favorable Region 72.06 ± 2.29 91.95 ± 3.04
RMSDs of Atomic Coordinates (A) Protein/Peptide Complex Secondary Structure
Protein (aa 9-116)
Backbone 0.66 ± 0.14 0.39 ± 0.05
Heavy atoms 1.25 ± 0.18 0.96 ± 0.07
Peptide (aa 202-206, 208-209)
Backbone 0.50 ± 0.16
Heavy atoms 1.83 ± 0.50
Complex (aa 9-116, 202-206, 208-209)
Backbone 0.72 ± 0.15 0.54 ± 0.09
Heavy atoms 1.39 ± 0.20 1.24 ± 0.16 a Of the total 2903 NOE-derived distance restraints, only 341 were obtained by using ARIA program, of which 122 are classified as ambiguous NOEs. The latter resonance signals in the spectra match with more than one proton atom in both the chemical shift assignment and the final NMR structures. b The Lennard- Jones potential was not used during any refinement stage. c None of these final structures exhibit NOE-derived distance restraint violations greater than 0.5 A or dihedral angle restraint violations greater than 5°.
[0111] The Tat AcK50 peptide adopts an extended conformation and lies between the ZA and BC loops (Fig. 9). The acetyl-lysine side-chain intercalates deep into a preformed hydrophobic and aromatic cavity located between the ZA and BC loops opposite to the N- and C-termini, and interacts extensively with residues V752, Y760, 1764, Y802, and Y809. While the peptide residues S(AcK-4), K(AcK+l), R(AcK+2), R(AcK+5) do not interact directly with the protein, the residues Y(AcK-3), G(AcK-2), R(AcK-I), R(AcK+3), and Q(AcK+4) showed numerous intermolecular NOEs with the protein. Particularly, Y(AcK-3) and Q(AcK+4) form extensive contacts with V763 and E756, respectively, suggesting that these two residues contribute significantly to specificity of the bromodomain/Tat recognition.
[0112] To identify the amino acid residues of the P/CAF bromodomain that are important for complex formation, mutant proteins were tested for binding to the biotinylated and lysine- acetylated Tat peptide that is immobilized onto streptavidin agarose (Fig. 10A). As expected, proteins containing alanine point mutation at the residue Y809, Y802, V752, or F748, which interact directly with the acetyl-lysine residue, showed nearly complete loss or significantly reduced binding to the Tat peptide. Moreover, when the residue V763 or E756 was mutated to alanine, a nearly complete loss in binding to the Tat AcK50 peptide was observed, indicating that these two amino acid residues provide essential contributions to the Tat recognition by interacting with the residues flanking the acetyl-lysine. The results from the mutational analysis agree with the observations of intermolecular NOEs in the NMR spectra.
[0113] To further determine Tat sequence preference for P/CAF interaction, various mutant peptides were synthesized and their binding to the P/CAF bromodomain tested in a competition assay by using a western blot with the antibody against the GST-fusion bromodomain (Fig. 10B). Because of high sensitivity of this detection method, the binding assay was performed at protein concentration (~10 μM) much lower than that in the NMR binding studies, which ensured specificity of protein-peptide interactions. In agreement with the binding results described above (e.g., see Figs. 5A-5B, 6A-6D, 7, and 8A-8B), lysine-acetylated peptides derived from acetylation sites at K50 or K28 in Tat, or from histone H4 at Kl 6 showed almost no competition with the Tat AcK50 peptide in binding to the P/CAF bromodomain, confirming that the latter interaction is tight and specific. Additionally, while substitution of residue R(AcK-I), K(AcK+l), R(AcK+2), or R(AcK+3) to alanine slightly weakened Tat peptide binding to the bromodomain, mutation of Y(AcK-3) or Q(AcK+4) resulted in significant loss in binding to the protein. These data can be explained by the observation of extensive pair-wise interactions between Y(AcK-3) and V763, and between Q(AcK+4) and E756, which agrees perfectly with the site-directed mutatagenesis results obtained with the protein (Fig. 10A). Together, these results demonstrate that the specificity of P/CAF bromodomain and acetylated Tat complex formation is achieved through specific interactions with acetyl-lysine as well as amino acid residues at (AcK-3) and (AcK+4) positions.
[0114] The HIV-I Tat is a versatile protein and elicits many cellular functions. In addition to its lysine-acetylation and interaction with P/CAF as disclosed herein, this portion of arginine-rich motif (named ARM) has also been shown to interact with the TAR RNA element as well as protein nuclear localization, particularly involving arginine52 and arginine53. The findings disclosed herein that are based on the detailed structural and mutational analyses indicate that the lysine-acetylated Tat specifically is associated with P/CAF via a bromodomain interaction in vivo, and that this interaction is important for transactivation activity of Tat in cells. Further, the data disclosed herein reveal that in addition to the acetylated-lysine (K50) the flanking residues, tyrosine (AcK-3) and glutamine at (AcK+4) positions in Tat are also uniquely important for the specificity of the Tat and P/CAF bromodomain recognition, but not with its other functions. This new information is extremely useful in applying mutational analysis in in vivo studies to further elucidate the biological importance of the Tat-P/CAF association in molecular mechanisms by which Tat transactivates gene transcription of HIV-I via chromatin remodeling.
Example 3. Synthesis of the Compounds of Formula I [0115] Sample preparation. The PCAF bromodomain (residues 719-832) was expressed in E. coli BL21(DΕ3) cells using the pET14b vector (Novagen) (Dhalluin, et al., Nature (1999) 399, 491-496). Isotope-labeled proteins were prepared from cells grown on a minimal medium containing 15NH4Cl with or without 13C6-glucose in either H2O or 75% 2H2O. The protein was purified by affinity chromatography on a nickel-IDA column (Invitrogen), followed by the removal of poly-His tag by thrombin cleavage. GST-fusion PCAF bromodomain was expressed in E. coli BL21 (DE3) codon plus cells using the pGEX4T-3 vector (Pharmacia), and purified with a glutathione sepharose column. The lysine-acetylated peptide was ordered from Biosynthesis, Inc.
[0116] Protein structure determination by NMR. NMR samples contained the bromodomain (0.5 mM) in complex with a chemical ligand (~2 mM) in 100 mM phosphate buffer of pH 6.5, containing 5 mM perdeuterated DTT and 0.5 mM EDTA in H2O/2H2O (9/1) or 2H2O. AU NMR spectra were acquired at 3O0C on a Bruker 500 or 600 MHz NMR spectrometer. The backbone 1H, 13C and 15N 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 15N- or 13C-edited 3D NOESY spectra. The 3JIiKHa coupling constants measured from 3D HNHA data were used to determine D -angle restraints. Slowly exchanging amide protons were identified from a series of 2D 15N-HSQC spectra recorded after H2O/2H2O exchange. The intermolecular NOEs used in defining the structure of the PCAF bromodomain/ligand complex were detected in 13C-edited (F/), 13C/15N- filtered (F3) 3D NOESY spectra (Clore et al., Meth. Enzymol. (1994) 239, 249-363). Protein structures were calculated with a distance geometry-simulated annealing protocol with X-PLOR (Brunger A.T., X-PLOR Version 3.1: A system for X-Ray crystallography and NMR. version 3.1 ed. 1993, 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., Prog. NMR Spectroscopy (1998) 32, 107-139). Structure quality was assessed by Procheck-NMR (Laskowski et al, J. Biomol. NMR (1996) 8, 477-486). The structure of the protein/ligand complex was determined using intermolecular NOE-derived distance restraints.
[0117] Chemical screening by NMR. A chemical library was constructed with small-molecules obtained from Chembridge Corp. (San Diego, CA), which were selected on the basis of molecular weight (<250 Da), non-reactivity, drug-like chemical framework (i.e. ring systems, linker atoms, side-chain atoms and framework), functional moieties (for hydrogen-bond or electrostatic interactions, but not reactive) and good solubility in aqueous solution. All the stock solutions of the chemical compounds are prepared in predeuterated DMSO. NMR-based screening was conducted with compounds (~1 mM) and the PCAF bromodomain (50-200 μM) using methods including ID NOE-pumping (Chen et al., J. Am. Chem. Soc. (1998) 120, 10258- 10259) and saturation transfer difference (Kwak et al., J. Biol. Chem. (1995) 270, 1156-1160; Klein et al., J. Am. Chem. Soc. (1999) 121, 5336-5337), as well as 2D 15N-HSQC spectra (Hajduk et al., Quarterly Reviews of Biophysics (1999) 32, 211-240; Moore, Curr. Opin. in Biotech. (1999) 10, 54-58). The latter is particularly helpful for selective screening to identify compounds that bind to a specific site of the target protein.
[0118] Ligand binding to the PCAF bromodomain. The ELISA assay was carried on a 96-well microplate (Nunc) that was pre-coated with anti-GST antibody (Sigma- Aldrich) overnight at 40C in 100 μL carbonate/bicarbonate buffer, and then washed with PBS buffer supplemented with 1% Tween-20. Non-specific binding sites were minimized by treatment with PBS buffer containing 2% BSA and 1% Tween-20 for 2 hours at room temperature. GST-PCAF bromodomain (1 μg per well) was added to the plate and incubated for two hours at room temperature for binding to anti-GST antibody. The plate was washed and blocked with PBS buffer containing 10% BSA and 1% Tween-20. The biotinylated HIV Tat-AcK50 peptide (Biotin-GISYGR-AcK-KKRRQRRRP) (5 μM) and increasing concentrations of a given compound were added and allowed to bind to the PCAF bromodomain overnight at 40C. Plate was washed with washing buffer, and bromodomain -bound peptide was determined by incubating 100 μL of a neutravidin-conjugated HRP (Pierce) solution (0.1 μg/ml) for 1 hour at room temperature, followed by washes and incubation with 100 μL of tetramethyl benzidine (Pierce) as an HRP substrate. The reaction was stopped by addition of 100 μL of 2.0 M sulfuric acid. The absorbance of the colored product was measured at 450 nm. Absorbance in each well was corrected for the blank obtained in a corresponding well subjected to the complete procedure but containing no PCAF bromodomain.
[0119] Chemistry. Melting points were recorded on an XT-4 micro-melting point apparatus and were uncorrected. 1H and 13C NMR spectra were recorded on a 300 MHz Bruker NMR spectrometer using tertramethylsilane as internal standard and the data were reported as the following: chemical shifts in ppm (δ), number of protons, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet), coupling constants in hertz. IR spectra were measured on Bruker EQUTNOX55 spectrometer. Mass spectra were recorded on HRMS [LC-TOF spectrometer (micromass)] (EI/CI). Elemental analyses were recorded on Elementar Vario EL-III spectrometer. All chromatographic purifications were performed with silica gel (100-200 mesh). AU purchased materials were used without further purifications. AU solvents were reagent grades.
Scheme 1 "
Figure imgf000065_0001
.NH-(CH2Jn-NH2-HCl fl
Ks
1, 2, 4-6, 19-21
R=H, CH3, Ph, NO2; R=H, NO2; n=2, 3, 4 a Reagents and conditions: (a) NaNO2, HBF4, 0~5°C; (b) SiO2/heat; (c) (i) 1,3- diaminopropane, or 1,2- diaminoethane, or 1,4-diaminobutane, 12O0C; (ii) concentrated HCl, EtOH
Scheme 2 "
Figure imgf000065_0002
5a, b 7, 8 1 Reagents and conditions: (a) (i) 1,3-diaminopropane; (ii) concentrated HCl, EtOH.
Scheme 3 a
Figure imgf000066_0001
10 a Reagents and conditions: (a) potassium carbonate; (b) N-(3-bromopropyl)phthalimide, H2O, reflux, 5h; (c) 30% NaOH, reflux, 6 h, HCl, 500C, 6h.
Scheme 4 a
Figure imgf000066_0002
8a-c 9a-c
Figure imgf000066_0003
3, 9, 11
R=C2H5, NO2; R =NO2, CH3, COOCH3. a Reagents and conditions: (a) N-(3-bromopropyl)phthalimide, (Et)3N, 12O0C; (b) (i) NH2NH2-H2O, EtOH, reflux, 6 h; (ii) HCl, EtOH.
Scheme 5 "
Figure imgf000067_0001
HCl
Figure imgf000067_0002
12a-f 12-17
R=H, CH3, CH3O, Cl, a Reagents and conditions: (a) 1,3-dibromopropane, NaOH, H2O, reflux; (b) potassium phthalimide, DMF, 90~95°C, 2-3 h; (c) NH2NH2-H2O, EtOH, reflux, 6h, or concentrated HCl, HOAc (V/V=l:l), reflux, several days.
Scheme 6 "
Figure imgf000067_0003
13a 18
" Reagents and conditions: (a) (i) (CH3CO)2O, 60 0C, Ih; (ii) HNO3 (d=1.5), 0-10 0C; (b) (i) H2SO4/H2O, reflux, 5h; (ii) HCl, EtOH, yield 8%.
[0120] N i-(2-nitro-phenyl)-propane-l ,3-diamine monohydrochloride. Yield: 78%; mp >169°C;
FTIR (KBr) ϋ cm"1: 2988.48, 1614.95, 1592.93, 1530.24, 1499.85, 1382.35, 1347.25, 1224.06, 1154.59, 1134.19, 1004.51, 918.02, 858.90, 789.80, 747.36; 1H NMR (D2O, 4.79) δ: 7.80 (IH, ra, Ar-H), 7.35 (IH, m, Ar-H), 6.76 (IH, m, Ar-H), 6.50 (IH, m, Ar-H), 3.31 (2H, t, J = 6.90 Hz, 3-CH2), 3.05 (2H, t, J = 7.40 Hz5 1-CH2), 1.96 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3):30.60] δ: 145.28, 137.35, 130.64, 126.44, 116.27, 114.44, 39.87, 37.64, 26.48; HRMS(CI) calculated for C9H13N3O2: (M+l) 196.1086, found 196.1080.
[0121] N i-(4-methyl-2-nitro-phenyl)-propane-l ,3-diamine monohydrochloride. Yield: 60.8%; mp 184- 1860C; FTIR (KBr) ϋ cm"1: 2964.50, 136.57, 1566.58, 1526.08, 1398.59, 1351.62, 1229.25, 1167.67, 1059.48, 851.53, 814.01, 764.74; 1H NMR (D2O, 4.79) δ: 7.79 (IH, s, 3-Ar- H), 7.36 (IH, d, J = 8.69 Hz, 5-Ar-H), 6.88 (IH, d, J = 8.67 Hz, 6-Ar-H), 3.47 (2H, t, J = 6.62 Hz, 3-CH2), 3.15 (2H, t, J = 7.43 Hz, 1-CH2), 2.21 (3H, s, -CH3), 2.08 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3):30.60] δ: 144.06, 139.14, 130.62, 126.21, 125.53, 114.43, 39.81, 37.67, 26.65, 19.38; HRMS(CI) calculated for C10H15N3O2: (MH-I) 210.1243, found 210.1251.
[0122] N r(4-ethyl-2-nitro-phenyl)-propane-l ,3-diamine monohydrochloride. Yield: 72.7%; mp
183- 1850C; FTIR(KBr) ϋ cm"!:2965.79, 1634.14, 1569.30, 1523.56, 1407.80, 1351.13, 1269.12,
1225.26, 1165.22, 1069.81, 817.62, 762.33; 1H NMR (D2O, 4.79)δ7.76 (IH, s, 3-Ar-H), 7.37 (IH, dd, J=7.01, 1.83Hz, 5-Ar-H), 6.87 (IH, d, J=8.87Hz, 6-Ar-H), 3.44 (2H, t, J=6.82Hz, NH- CH2), 3.12 (2H, t, J=7.57Hz, CH2-NH2), 2.48 (2H, q, J=7.53Hz, Ar-CH2), 2.05 (2H, m, 2-CH2), 1.14 (3H, s, -CH3); 13C NMR [D2O+acetone, acetone (CH3):30.60] 6144.21, 138.16, 132.47, 130.58, 124.08, 114.52, 39.82, 37.65, 27.04, 26.68, 14.59; HRMS(CI) Calcd for C11H17N3O2: (MH-I) 224.1399, found 224.1398.
[0123] Ni-(3-methyl-2-nitro-phenyl)-propane-l,3-diamine monohydrochloride. Yield: 81.5%; mp 180- 1820C; FTIR (KBr) ϋ cm"1: 2972.09, 1606.43, 1576.89, 1534.85, 1501.40, 1351.61, 1250.77, 1161.47, 1064.62, 1033.92, 852.11, 796.63, 769.27; 1H NMR (D2O, 4.79) 6: 7.35 (IH, t, J - 7.88 Hz, 5-Ar-H), 6.86 (IH, d, J = 8.50 Hz, 4-Ar-H), 6.70 (IH, d, J = 7.36 Hz, 6-Ar-H), 3.38 (2H, t, J = 6.85 Hz, 3-CH2), 3.11 (2H, t, J = 7.41 Hz, 1-CH2), 2.36 (3H, s, -CH3), 2.03 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3):30.60] 5141.92, 136.58, 135.56, 134.11, 121.53, 113.10, 41.22, 37.66, 26.27, 20.34; HRMS(CI) calculated for Ci0H15N3O2: (M+l) 210.1243, found 210.1251.
[0124] Ni-(5-methyl-2-nitro-phenyl)-propane-l,3-diamine monohydrochloride. Yield: 67.1%; mp >215 0C; FTIR (KBr) ϋ cm'1 : 2925.75, 1628.10, 1577.27, 1488.41, 1408.98, 1338.69, 1252.10, 1217.79, 1182.52, 1060.46, 752.22; 1H NMR (D2O, 4.79) δ: 7.99 (IH, m, 3-Ar-H), 6.83 (IH, s, 6-Ar-H), 6.58 (IH, d, J = 6.76 Hz, 4-Ar-H), 3.52 (2H, t, J = 6.64 Hz, 3-CH2), 3.17 (2H, t, J = 7.60 Hz, 1-CH2), 2.36 (3H, s, -CH3), 2.10 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3):30.60] δ: 150.00, 145.91, 129.12, 126.62, 118.14, 113.71, 39.64, 37.65, 26.59, 21.68; HRMS(CI) calculated for C10H15N3O2: (M+l) 210.1243, found 210.1234.
[0125] Nr(3-nitro-biphenyl-4-yl)-propane-l,3-diamine monohydrochloride. mp >190 0C; FTIR
(KBr) ϋ 0-0^3435.0I, 2971.02, 1635.79, 1561.44, 1404.99, 1359.55, 1241.99, 1222.08, 1169.70, 158.97, 698.40; 1H NMR (D2O, 4.79) δ: 8.34 (IH, d, J=2.01Hz, Ar-H), 7.83 (IH, m, Ar-H), 7.64 (2H, d, J=7.26Hz, Ar-H), 7.50 (2H, m, Ar-H), 7.42 (IH, m, Ar-H), 7.08 (IH, d, J=9.02Hz, Ar-H), 3.53 (2H, t, J=6.96Hz, 3-CH2), 3.13 (2H, t, J=7.59Hz, 1-CH2), 2.08 (2H, m, 2- CH2); 13C NMR (DMSO-d6) δ: 144.19, 144.06, 138.05, 134.84, 131.49, 129.07, 127.15, 127.11, 125.80, 123.36, 115.45, 36.44, 36.35, 26.16; HRMS(CI) calculated for Ci5H17N3O2: (M+l) 272.1399, found 272.1390.
[0126] N i-(4-cyano-2-nitro-phenyl)-propane-l ,3-diamine monohydrochloride. mp >230°C;
FTIR (KBr) ϋ cm"1: 3364.13, 2929.80, 2222.94, 1625.89, 1561.22, 1524.98, 1410.25, 1364.46, 1261.01, 1176.10, 921.75, 819.69; 1H NMR (D2O, 4.79) δ: 8.58 (IH, d, J=I.54Hz, 3-Ar-H), 7.73 (IH, dd, J=1.70, 9.13Hz, 5-Ar-H), 7.12 (IH, d, J=9.15Hz, 6-Ar-H), 3.58 (2H, t, J=6.91Hz, 3- CH2), 3.13 (2H, t, J=8.01Hz, 1-CH2), 2.08 (2H, m, 2-CH2); 13C NMR (DMSO-d6) δ: 146.68, 137.58, 131.90, 131.01, 118.22, 115.90, 96.29, 36.06, 29.78, 25.77; HRMS(CI) calculated for C10H12N4O2: (M+l) 221.1039, found 221.1040.
[0127] N i-(5-cyano-2-nitro-phenyl)-propane-l ,3-diamine monohydrochloride. mp >185°C;
FTIR (KBr) ϋ cm"1 : 3234.94, 2228.70, 1602.69, 1470.61, 1308.98, 1266.12, 1158.97, 1070.74,
874.89, 829.64, 750.00; 1H NMR (D2O, 4.79) δ: 8.20 (IH, d, J=8.73Hz, 3-Ar-H), 7.91 (IH, brs, Ai-NH), 7.66 (IH, s, 6-Ar-H), 7.04 (IH, dd, J=1.38, 8.76Hz, 4-Ar-H), 3.52 (2H, t, J=6.39Hz, 3- CH2), 2.86 (2H, brs, 1-CH2), 1.86 (2H, m, 2-CH2); 13C NMR (DMSO-d6) δ: 144.15, 133.43, 127.68, 119.35, 118.11, 117.65, 116.81; 36.43, 30.61, 25.96; HRMS(CI) calculated for C10H12N4O2: (M+l) 221.1039, found 221.1045. [0128] Ni-(2-methyl-5-nitro-phenyl)-propane-l,3-diamine monohydrochloride. Yield: 81%; mp
>152 0C; FTIR(KBr) ϋ cm'1: 3246.44, 2984.42, 1627.55, 1534.15, 1510.90, 1349.34, 1167.14, 1130.54, 917.28, 853.18, 737.04; 1H NMR (D2O, 4.79)67.77 (IH, dd, J=2.15, 8.24Hz, 4-Ar-H), 7.70 (IH, d, J=1.94Hz, 6-Ar-H), 7.38 (IH, d, J=8.27Hz, 3-Ar-H), 3.43 (2H, t, J=7.27Hz, 3-CH2), 3.14 (2H, t, J=7.51Hz, 1-CH2), 2.32 (3H, s, -CH3), 2.10 (2H, m, 2-CH2); 13C NMR [D2O+acetone, acetone (CH3):30.60] 5146.78, 137.91, 132.96, 121.03, 114.10, 45.80, 37.24, 24.59, 17.36; HRMS(CI) calculated for C10H15N3O2: (M+l) 210.1243, found 210.1238.
[0129] Ni-(2-carboxyl-phenyl)-propane-l,3-diamine monohydrochloride. Yield: 51%; mp
>135°C; FTIR (KBr) ϋ cm"1: 3328.13, 3045.98, 1687.79, 1605.86, 1579.99, 1379.37, 1198.87, 1158.54, 755.76; 1H NMR (DMSO-d6) 57.69 (IH, d, J=7.73Hz, 3-Ar-H), 7.41 (IH, t, J=7.35, 8.02Hz, 5-Ar-H), 7.15 (IH, d, J=8.02Hz, 6-Ar-H), 7.08 (IH, t, J=7.73, 7.35Hz, 4-Ar-H), 3.47 (2H, t, J=6.27Hz, 3-CH2), 3.31 (2H, t, J=4.89Hz, 1-CH2), 1.69 (2H, m, 2-CH2); 13C NMR (DMSO-d6) 5167.28, 131.92, 128.45, 122.23, 121.14, 116.86, 114.13, 58.59, 36.50, 32.19; HRMS(CI) calculated for Ci0Hi4N2O2: (M+l) 195.1134, found 195.1119.
[0130] Ni-(2-carboxymethyl-phenyl)-propane-l,3-diamine monohydrochloride. mp 141-1430C;
FTIR(KBr) ϋcm4:3344.52, 2952.20, 1697.49, 1605.34, 1574.36, 1508.79, 1438.17, 1258.09, 1222.79, 1169.82, 1088.74, 751.55, 706.79; 1H NMR (D2O, 4.79)57.95 (IH, dd, J=4.49, 8.03Hz, 3-Ar-H), 7.52 (IH, t, J=8.53Hz, 5-Ar-H), 6.92 (IH, d, J=8.45Hz, 4-Ar-H), 3.89 (3H, s, -OCH3), 3.40 (2H, t, J=6.83Hz, 3-CH2), 3.14 (2H, t, J=7.64Hz, 1-CH2), 2.05 (2H, m, 2-CH2); 13C NMR [D2O+acetone, acetone (CH3): 30.60] 5170.31, 150.60, 135.69, 132.17, 116.13, 112.46, 110.80, 52.26, 39.78, 37.83, 26.72; HRMS(CI) calculated for CnHi6N2O2: (M+) 208.1212, found 208.1228.
[0131] 3-(2-nitro-phenoxy)-l-propylamine monohydrochloride. Yield 79%; mp 160-1620C;
FTIR (KBr) ϋ cm'' :2957.52, 1612.32, 1581.01, 1525.19, 1398.45, 1339.86, 1271.26, 1254.75, 1167.97, 1059.83, 854.61, 740.78; 1H NMR (D2O, 4.79) δ: 8.06 (IH, d, J = 8.19 Hz, 3-Ar-H), 7.75 (IH, t, J = 8.28 Hz, 5-Ar-H), 7.34 (IH, d, J = 8.51 Hz, 4-Ar-H), 7.21 (IH, t, J = 7.99 Hz, 6- Ar-H), 4.42 (2H, t, J = 5.45 Hz, 3-CH2), 3.34 (2H, t, J = 6.40 Hz, 1-CH2), 2.30 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ: 152.26, 138.47, 136.33, 126.42, 121.41, 115.20, 68.13, 38.36, 26.47; HRMS(EI) Calcd for C9H12N2O3: (M+l) 197.0926, found 197.0928.
[0132] 3-(4-methyl-2-nitro-phenoxy)-l-propylaτnine monohydrochloride. Yield 83%; mp 158-
16O0C; FTIR(KBr) ϋcm"1: 2952.95, 1628.51, 1572.03, 1533.89, 1399.08, 1340.97, 1265.53,
1249.95, 1162.89, 1060.92, 910.02809.57, 792.09; 1H NMR (D2O, 4.79) δ: 7.85 (IH, t, J = 1.5 Hz, 3-Ar-H), 7.55 (IH, tt, J = 1.64, 6.33Hz, 5-Ar-H), 7.20 (IH, d, J = 8.66 Hz, 6-Ar-H), 4.36 (2H, t, J = 5.53 Hz,3-CH2), 3.34 (2H, t, J = 6.52 Hz, 1-CH2), 2.36 (3H, s, Ar-CH3), 2.28 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ: 150.21, 138.38, 136.71, 131.59, 126.03, 115.31, 68.03, 38.19, 26.65, 19.61; HRMS(EI) calculated for Ci0H14N2O3: (M+l) 211.1083, found 211.1076.
[0133] 3-(4-methoxy-2-nitro-phenoxy)-l -propylamine monohydrochloride. Yield 73.5%; mp
154-156 0C; FTIR (KBr) ϋcm"1: 2950.19, 1576.28, 1342.42, 1286.70, 1265.19, 1222.72,
1157.96, 1059.63, 1040.04, 871.77, 823.40, 792.10; 1H NMR (D2O, 4.79) δ: 7.45 (IH, d, J = 2.94Hz, 3-Ax-H), 7.22-7.06 (2H, m, 5,6-Ar-H), 4.22 (2H, t, J = 5.58Hz, 3-CH2), 3.75 (3H, s, - OCH3), 3.21 (2H, t, J = 6.39Hz, 1-CH2), 2.15 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ: 152.86, 146.75, 138.38, 122.40, 116.89, 110.63, 68.59, 56.38, 38.30, 26.62; HRMS(EI) calculated for C10H14N2O4: (M+l) 227.1032, found 227.1027.
[0134] 3-(4-chloro-2-nitro-phenoxy)-l-propylamine monohydrochloride. Yield 82%; mp 184-
1860C; FTIR (KBr) ϋcm"1: 2970.91, 1614.90, 1530.26, 1343.08, 1291.04, 1266.11, 1161.89, 1063.10, 883.30, 818.43; 1H NMR (D2O, 4.79) δ: 8.11 (IH, t, J = 2.61 Hz, 3-Ar-H), 7.76-7.71 (IH, m, 5-Ar-H),7.34-7.30 (IH, m, 6-Ar-H), 4.40 (2H, t, J = 5.49 Hz, 3-CH2), 3.32 (2H, t, J = 6.51 Hz, 1-CH2), 2.29 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ: 151.22, 138.67, 135.78, 125.94, 125.54, 116.83, 68.47, 38.20, 26.48; HRMS(EI) calculated for C9HnClN2O3: (M+l) 231.0536, found 231.0528.
[0135] 3-(5-methyl-2-nitro-phenoxy)-l-propylamine monohydrochloride. Yield 80.6%; mp 211-
213 0C; FTIR (KBr) ϋcm"1 :2954.26, 1611.32, 1589.03, 1513.38, 1341.30, 1272.89, 1182.24, 1059.49, 897.54, 817.55; 1H NMR (D2O, 4.79) δ: 7.93 (IH, d, J = 8.39Hz, 2-Ar-H), 7.11 (IH, s, 6-Ar-H), 6.96 (IH, d, J = 8.28 Hz, 4-Ar-H), 4.34 (2H, t, J = 5.20 Hz, 3-CH2), 3.30 (2H, t, J = 5.69 Hz, 1-CH2), 2.41 (3H, s, Ar-CH3), 2.24 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ: 152.83, 149.43, 135.89, 126.90, 122.39, 115.66, 68.53, 38.83, 26.71, 21.74; HRMS(EI) Calcd for Ci0Hi4N2O3: (M+l) 211.1083, found 211.1080.
[0136] 3-(3-methyl-2-nitro-phenoxy)-l-propylamine monohydrochloride. Yield 77%; mp 160-
1620C; FTIR (KBr) ϋcm 1: 2892.96, 1628.38, 1581.93, 1534.27, 1476.82, 1369.63, 1279.42, 1091.30, 852.71, 776.96, 747.35; 1H NMR (D2O, 4.79) δ: 7.45 (IH, t, J = 7.94Hz, 5-Ar-H), 7.08 (IH, d, J = 8.23 Hz, 4-Ar-H), 7.01 (IH, d, J = 7.38 Hz, 6-Ar-H), 4.28 (2H, t, J = 5.34Hz, 3-CH2), 3.22 (2H, t, J = 6.84Hz, 1-CH2), 2.19 (2H, m, 2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ: 148.93, 140.68, 131.36, 130.72, 122.77, 111.06, 66.36, 36.79, 25.95, 15.70; HRMS (EI) Calcd for Ci0Hi4N2O3: (M+l) 211.1083, found 211.1084.
[0137] 4-(2-nitro-phenyl)-butylamine monohydrochloride. Yield 8%; oil; FTIR (film) ϋcm" ':2927.59, 1526.99, 1455.22, 1346.86, 1276.80, 1123.55, 1076.11, 779.06; 1H NMR (D2O, 4.79) δ:7.97 (IH, d, J=6.16Hz, 3-Ar-H), 7.68 (IH, t, J=5.67Hz, 5-Ar-H), 7.52-7.45 (2H, m, Ar-H), 3.08 (2H, t, J=5.16Hz, -CH2NH2), 2.93(2H, t, J=5.73Hz, Ar-CH2), 1.78 (4H, m, CH2-CH2); 13C NMR [D2O + acetone, acetone (CH3): 30.60] δ:148.80, 136.83, 133.90, 132.19, 127.55, 124.85, 39.42, 31.72, 26.91, 26.71; HRMS (CI) Calcd for Ci0Hi4N2O2: (M+l) 195.1089, found 195.1085.
[0138] N i~(2-nitro-phenyl)-butane-l ,4-diamine monohydrochloride. Yield: 50.4%; mp 173-
1760C; FTIR (KBr) ϋ αn ^SβO^, 2938.61, 1625.67, 1573.05, 1516.63, 1417.23, 1358.38, 1256.09, 1229.27, 1158.77, 1036.31, 732.93; 1H NMR (D2O, 4.79) 68.11 (IH, m, Ar-H), 7.52 (IH, m, Ar-H), 7.01 (IH, m, Ar-H), 6.72 (IH, m, Ar-H), 3.43 (2H, m, 4-CH2), 3.06 (2H, m, 1- CH2), 1.80 (4H, m, 2,3-CH2); 13C NMR [D2O+acetone, acetone (CH3): 30.60] 6146.12, 137.56, 130.82, 126.69, 116.05, 114.65, 42.20, 39.66, 25.66, 24.84; HRMS (CI) Calcd for Ci0Hi5N3O2: (M+l) 210.1234, found 210.1237. [0139] Ni-(4-nitro-phenyl)-ethane-l,2-diamine monohydrochloride. Yield: 67%; mp 1980C;
FTIR (KBr) ϋ cm"1:3272.73, 3069.16, 1601.46, 1543.80, 1504.85, 1469.89, 1328.32, 1118.56, 934.92, 835.07, 803.03, 750.56; 1H NMR (D2O, 4.79) 57.99 (2H, dd, J=1.90, 5.37Hz, 3,5-Ar-H), 6.66 (2H, dd, J=I.98, 5.29Hz, 2, 6-Ar-H), 3.61 (2H, t, J=5.96Hz, 2-CH2), 3.27 (2H, t, J=6.07Hz, 1-CH2); 13C NMR [D2O+acetone, acetone (CH3):30.60] 5154.33, 137.09, 126.97, 111.68, 40.09, 38.57; HRMS (CI) Calcd for C8HnN3O2: (M+l) 182.0930, found 182.0922.
[0140] Nj-(4-nitro-phenyl)-butane-l,4-diamine monohydrochloride. Yield: 70.5%; mp 183-
1850C; FTIR (KBr) ϋ cm"1: 3273.67, 2954.13, 1605.10, 1543.48, 1500.61, 1434.47, 1329.48, 1118.68, 896.13, 833.95, 754.75; 1H NMR (D2O, 4.79) 58.06 (2H, d, J=9.28Hz, 3,5-Ar-H), 6.68 (2H, d, JM9.38HZ, 2, 6-Ar-H), 3.29 (2H, t, J=6.44Hz, 4-CH2), 3.05 (2H, t, J=7.16Hz, 1-CH2), 1.76 (4H, m, 2,3-CH2); 13C NMR [D2O+acetone, acetone (CH3): 30.60] 5154.66, 136.54, 127.16, 111.95, 42.67, 39.59, 25.37, 24.77; HRMS (CI) Calcd for Ci0H15N3O2: (M+l) 210.1234, found 210.1242.
[0141] Structure coordinates: Coordinates for the three-dimensional NMR structures of the PCAF bromodomain in complex with the lead compounds Ni-(2-nitro-phenyl)-propane-l,3- diamine monohydrochloride or Ni -(4-methyl-2-nitro-phenyl)-propane- 1,3 -diamine monohydrochloride have been deposited in the Brookhaven Protein Data Bank under accession numbers IWUM and IWUG, respectively.
Table 5. NMR Structural Statistics for the PCAF BRD/Ligand Complexes
BRD/1 complex BRD/2 complex
Total Experimental Restraints 3371 3410
Total NOE Distance Restraints 3269 3308
Total Ambiguous 206 112
Total Unambiguous 3063 3196
Manually assigned 2895 3072
ARIA assigned 168 124
Intra-residue 1242 1263
Inter-residue 1821 1933
Sequential (|i-j| = l) 608 615
Medium (2<|i-j|<4) 555 584
Long range (|i - j] > 4) 658 734
Intermolecular 51 54
Hydrogen Bond Restraints 44 44
Dihedral Angle Restraints 58 58
Final Energies (kcal-mol"1) a
Eτotal 270.1 ±23.1 264.2 ±16.1
ENOE 43.6 ±12.5 39.5 ±10.7
-^Dihedral 0.0 ± 0.0 0.01 ±0.03
EL-J b -614.4 ±15.6 -600.9 ± 20.0
Secondary Secondary
Ramachandran Plot (%) Full Protein Full Protein Structure Structure
Most Favorable Region
83.7±3.0 97.1 ±1.8 82.4 ±2.0 95.7 ±1.6 Additionally Allowed Region 14.6 ±2.8 2.9 ±1.8 14.7 ±1.9 4.3 ±1.6
1.6 ±0.8 0.0 ±0.0 2.5 ±0.9 0.0 ±0.0
Generously Allowed region 0.2 ±0.4 0.0 ±0.0 0.4 ±0.7 0.0 ±0.0 Disallowed Region Cartesian coordinate RMSDs (A)c
Backbone atoms (N, CD, C)
0.44 ± 0.09 0.32 ± 0.05 0.48 ± 0.07 0.33 ± 0.06
Heavy atoms 0.96 ± 0.07 0.84 ± 0.05 0.97 ± 0.05 0.81 ± 0.05
Heavy atoms d 1.04 ± 0.07 1.05 ± 0.05
Notes: a None of these final structures exhibit NOE-derived distance restraint violations greater than 0.3 A or dihedral angle restraint violations greater than 5°. b The Lennard- Jones potential was not used during any refinement stage. c Protein residues 723-830. The residues in the secondary structure of the protein are 727-741, 772-777, 785-802 and 808-826. d Protein and ligand complex.
Example 4. Synthesis of the Compounds of Formula III
[0142] The following compounds represented in Table 6 were prepared according to synthesis techniques well known to those of skill in the art.
Table 6.
Figure imgf000076_0001
ID Ri R2 R3 R4 R5 R6 Kc (μM)
5110065 -OH H H -CH3 H
OH 149 + 22
CM1 -OH H H -CH2-CH2-CH3 H
OH 147 + 6
7910894 -OH H H -(CH2)2-CH3 H
OH 225 ± 20
6163501 -OH H H
OH -Ph H 335 + 35
6148450 -OH H H -cyclopentane H
OH 327 ± 12
5102141 H -OH H H -CH3 H 222 + 17
5752339 H -OH H H -Cπ2~CH2"Cπ3 H 204 ± 17
5771728 H -OH H -OH ~Cπ2~Cri2"GH3 H 367 ± 40
6147652 H -CH3 H -OH -CH2"CH2~CH3 H 819 + 75
6141880 H -CH3 H -OH -CH2-CH3 H 2,294
6140532 H -0-CH3 H H -CH2~Cri3 H 357 ± 33
5768797 H -0-CH3 H H "Cπ2"CH2~Crl3 H 400 + 74
6153095 H -0-CH2-CH3 H H -Cr^-CHs H 523 ± 34
5754756 H -0-CH2-CH3 H H -Cπ2"CH2~CH3 H 337 ± 37
6147035 H -O-CH(CH3)2 H H -CH3 H 545 + 50
5754213 H -0-CO-CH3 H H -Cπ2~CH2"Cπ3 H 183 ± 12
6155412 H -0-CH2-CO-OH H H -CH2-CH3 H 176 ± 12
5749750 H -0-CH2-CO-OH H H -(CH2)2-CH3 H 119 + 14
5757535 -0-CH(CH3)-
H H H -(CH2)2"CH3 H CO-OH 184 ± 14
6144139 H -0-CH2-CO-CH3 H H -GH2-CH3 H 316 ± 20 6139643 H -0-CH2-CO-NH2 H H -CH2~C-/H3 H 765 ± 50
56 H -0-CO-CH3 H H -CH3 H 65 ± 5
-O-CO-
5279012 H H H -CH3 H 321 ± 10 CH(CHs)2
6085633 H -0-CH2-CO-CH3 H H -CH3 H 155 ± 9
5807139 H -0-CH2-CH2-OH H H -CH3 H 167 + 16
-0-CH2-CH2-
6141510 H H H -CH3 H 426 ± 25 CH3
-0-CH2-CH2-
6146310 H H H H H 415 ± 30 CH3
6094981 H -O-(CH2)3-CH3 H H H H 678 ± 50
-0-CH(CH3)-
6155336 H H H H H 271 ± 22 CO-OCH3
-0-CH2-CO-
6149123 H H H H H 214 ± 19 N(CH3)2
6238869 H -O-CH(CH3)2 H H -cyclophentane H 2,000
5313498 H -0-CH2-CH3 -0-CH2-CH3 H -CH3 H 190 ± 15
6153309 H -CH3 -NH-CO-CH3 H -CH3 H 365 ± 20
5244700 -NH-(CH2J3-
H H H H -NH2 662 ± 45 N(CH3)2
-NH-(CH2)2-
5244722 H H H H -NH2 205 ± 14 N(CH3)2
5354863 H H H H -NH-(CH2)2-OH -NH2 238 ± 27
5162478 H H H H -NH-(CH2)3-CH3 -NH2 4,476
5162468 H H H H -NH-CH3 -NH2 156 ± 8
5162465 H H H H -NH2 -NH2 109 + 8
Example 5. Efficacy of the small-molecule inhibitors of PCAF bromodomain (BRD)/HIV-1 Tat-AcK50 complex on HIV Tat-mediated trans activation
[0143] Inhibition of Tat-mediated transactivation by small-molecule inhibitors that block the PCAF bromodomain interaction with HIV-I Tat-AcK50. The effect was assessed by a microinjection study as described previously by Dorr et al. (EMBO J. 21; 2715-2723, 2002). In this microinjection assay, HeLa-T at cells were grown on Cellocate coverslips and microinjected at room temperature with an automated injection system (Carl Zeiss). Samples were prepared as a 20 μl injection mix containing the LTR-luciferase (lOO ng/ml) and CMV-GFP (50 ng/ml) constructs together with 5 mg/ml a chemical compound or pre-immune IgGs. Live cells were examined on a Zeiss Axiovert microscope to determine the number of GFP -positive cells. Four hours after injection, cells were washed in cold phosphate buffer and processed for luciferase assays (Promega). Figure 16 demonstrates inhibition of Tat-mediated transactivation by the PCAF BRD inhibitor. (A) Schematic diagram of the microinjection assay. (B) Effect of a lead compound 765 on Tat-mediated transactivation as compared to DMSO control.
[0144] Effect of PCAF bromodomain inhibitors on Tat transactivation. Fifiure 17 demonstrates the effect of PCAF BRD inhibitors on Tat transactivation. HeLa cells were transfected with LTR-luciferase and 20 ng Tat-expression vector. PCAF BRD inhibitors were added after transfection, and cells were harvested after 8 hours.
[0145] Effect of the PCAF Bromodomain inhibitors on viral infection, using a procedure described by Pagans et al. (PLoS Biol. 3(D: e41. 2005V Figure 18 demonstrates the effect of PCAF BRD inhibitors on viral infection. Jurkat T cells were infected with an LTR-Tat-IRES- GFP virus. PCAF BRD inhibitors were added after overnight infection. The percentage of infection was monitored 48 hours later by FACS analysis.

Claims

WHAT IS CLAIMED IS:
1. A compound according to the following general formula(I) wherein:
^ R2
"(CH2)n
Rr
R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases thereof.
2. A compound according to claim 1 wherein R1 is selected from the group consisting of hydrogen, lower alkyl, phenyl, and CN.
3. A compound according to claim 1 wherein R2 is selected from the group consisting of NH3 +, carboxy, and alkoxy.
4. A compound according to claim 1 wherein X is selected from the group consisting of lower alkyl, NH, and O.
5. A compound according to claim 1 wherein n is 3.
6. A pharmaceutical composition comprising a compound of Formula I
"(CH2).'
FV
wherein R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH21NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases;
together with a pharmaceutically acceptable carrier.
7. A pharmaceutical composition according to claim 6 wherein R1 is selected from the group consisting of hydrogen, lower alkyl, phenyl, and CN.
8. A pharmaceutical composition according to claim 6 wherein R2 is selected from the group consisting OfNH3 +, carboxy, and alkoxy.
9. A pharmaceutical composition according to claim 6 wherein X is selected from the group consisting of lower alkyl, NH, and O.
10. A pharmaceutical composition according to claim 6 wherein n is 3.
11. A method for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a compound of the following general formula(I)
Figure imgf000081_0001
wherein R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, arallcyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases.
12. A method of preventing or treating viral infection in an individual comprising administering to the individual a compound of Formula I: NCH2Jn'
wherein R1 is selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, SO2, NH2, NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, CN and halogen;
R2 is selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, halogen, carboxy, and alkoxy;
X is selected from the group consisting of lower alkyl, SO2, NH, NO2, , O, carboxy, and alkoxy; and
n is an integer from O to 10;
and their pharmaceutically acceptable salts of acids or bases;
13. The method of claim 12 wherein the viral infection is HIV infection.
14. A compound of the following general formula(II) wherein:
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy; and their pharmaceutically acceptable salts of acids or bases.
15. A compound according to claim 14 wherein R1 R2 and R3 are independently selected from the group consisting of hydrogen, lower alkyl, NH3 +, OH, SH, and halogen.
16. A compound according to claim 14 wherein R4 is selected from the group consisting of lower alkyl and aryl.
17. A pharmaceutical composition comprising a compound of Formula (II) wherein:
Figure imgf000083_0001
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases.
18. A pharmaceutical composition according to claim 17 wherein R1 R2 and R3 are independently selected from the group consisting of hydrogen, lower alkyl, NH3 +, OH, SH, and halogen.
19. A pharmaceutical composition according to claim 17 wherein R4 is selected from the group consisting of lower alkyl and aryl.
20. A method for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein:
Figure imgf000084_0001
R1 R2 and R3 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH2, NH3 + NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R4 is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases.
21. A method according to claim 20 wherein R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH3 +, OH, SH, and halogen.
22. A method according to claim 20 wherein R4 is selected from the group consisting of lower alkyl and aryl.
23. A method for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (II) wherein: R R and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, SO2, NH21NH3 +NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, SH, halogen, carboxy, and alkoxy;
R is selected from the group consisting of lower alkyl, aryl, SO2, NH, NO2, , CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OH, carboxy, and alkoxy;
and their pharmaceutically acceptable salts of acids or bases.
1 2. ^
24. A method according to claim 23 wherein R R and R are independently selected from the group consisting of hydrogen, lower alkyl, NH3 , OH, SH, and halogen.
25. A method according to claim 23 wherein R is selected from the group consisting of lower alkyl and aryl.
26. A method according to claim 23 wherein the viral infection is HIV infection.
27. A compound of the following general formula (III) wherein
R1 R , R , R4 R5, and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH5 OCH2CH2CH3, 0(CH2)SCH3, OCHCH3COOCH3, OCH2CON(CH3)2,NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)SCH3, NHCH3, SH, halogen, carboxy, and alkoxy.
28. A compound according to claim 27 wherein R and R are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CHs, OCHs, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH5 OCHCH3COOH5 OCH2COCH35 OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH5 OCH2CH2CH3, 0(CH2)SCHs, OCHCHSCOOCHS5 OCH2CON(CHs)2;
R is selected from the group consisting of hydrogen, OCH2CHs, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)SN(CHs)2, NH(CH2^N(CHs)2 , NH(CHf)2OH5 NH(CH2)SCH3, and NHCHs; and
R is selected from the group consisting of hydrogen, and NH2
29. A pharmaceutical composition comprising a compound of Formula (III) wherein
Figure imgf000086_0001
R1 R2, R3, R R . and R are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHb)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCHsCOOCHs, OCH2CON(CHS)2, NH(CH2)SN(CHS)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CHs, NHCHs, SH, halogen, carboxy, and aUcoxy.
30. A pharmaceutical composition according to claim 29 wherein R1 and R4 are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CHs)2, OCH2COOH, OCHCH3COOH, OCH2COCHs, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CHs, O(CH2)sCHs, OCHCHsCOOCHs, OCH2CON(CHs)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)sN(CHs)2, NH(CH2)2N(CHs> , NH(CH2)2OH, NH(CH2)sCHs, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
31. A method for preventing or inhibiting the binding of bromodomains to acetyl-lysine residues of proteins comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
Figure imgf000087_0001
R1 R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2) SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2) OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2, NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, NHCH3, SH, halogen, carboxy, and alkoxy.
32. A method according to claim 31 wherein
R1 and R4 are selected from the group consisting of hydrogen and OH;
R2 is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CH3)2;
R3 is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3;
R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, aralkyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
33. A method for treating viral infection comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the following general formula (III) wherein
Figure imgf000089_0001
R1 R2, R3, R4 R5, and R6 are independently selected from the group consisting of hydrogen; lower alkyl, aryl, phenyl, aralkyl; substituted aralkyl, heteroaryl, substituted heteroaryl, SO2, NH2, NH3 + , NO2, SO2, CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CH3)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CHa)2, NH(CH2)3N(CH3)2, NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, NHCH3, SH, halogen, carboxy, and alkoxy.
34. A method according to claim 33 wherein
R and R are selected from the group consisting of hydrogen and OH;
R is selected from the group consisting of hydrogen, OH, and CH3, CH2CH3, OCH3, OCOCH3, CH2COCH3, OCH2CH3, OCH(CH3)2, OCH2COOH, OCHCH3COOH, OCH2COCH3, OCH2CONH2, OCOCH(CHs)2, OCH2CH2OH, OCH2CH2CH3, O(CH2)3CH3, OCHCH3COOCH3, OCH2CON(CHa)2;
R is selected from the group consisting of hydrogen, OCH2CH3, and NHCOCH3; R5 is selected from the group consisting of hydrogen, lower alkyl, aryl, phenyl, arallcyl, NH(CH2)3N(CH3)2,NH(CH2)2N(CH3)2 , NH(CH2)2OH, NH(CH2)3CH3, and NHCH3; and
R6 is selected from the group consisting of hydrogen, and NH2.
35. A method according to claim 33 wherein the viral infection is HIV infection.
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